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https://openalex.org/W4292791816
https://zenodo.org/records/7016918/files/A-20.pdf
Russian
null
WAYS TO ORGANIZE AND DEVELOP MARKETING RESEARCH IN THE LABOR MARKET
Zenodo (CERN European Organization for Nuclear Research)
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cc-by
4,194
ПУТИ ОРГАНИЗАЦИИ И РАЗВИТИЯ МАРКЕТИНГОВЫХ ИССЛЕДОВАНИЙ НА РЫНКЕ ТРУДА Аннотация. В данной статье в целях разработки правильного плана развития техникумов разработана сегментация и расположение покупателей образовательных услуг, разработан стратегический план развития образовательного учреждения. Ключевые слова: рынок труда, образовательная услуга, покупатель, сегмент, развитие, стратегия, образование. WAYS TO ORGANIZE AND DEVELOP MARKETING RESEARCH IN THE LABOR MARKET Musayeva Shoira Azimovna Professor of Samarkand Institute of Economic and Service https://doi.org/10.5281/zenodo.7016918 Abstract. In this article, in order to develop the right plan for the development of technical schools, the segmentation and location of the buyers of educational services, and the development of a strategic plan for the development of the educational institution have been developed. Keywords: labor market, educational service, buyer, segment, development, strategy, education. INTRODUCTION The modern conditions of the activity of the educational institution are characterized by increasing instability, increasing uncertainty in the external environment, reduction of budget funds and increasing competition in the market of educational services. According to many scientists, an important tool for the successful operation of an educational institution can be the use of strategic planning. Most of the experts are concerned about the spread of existing strategic plans and strategiesemphasizes that it is not sufficiently adapted to the changing market conditions, that the main attention is mainly focused on solving internal problems, and that the interaction with the consumers of educational services is weak. In order to improve the university activity planning system, in our opinion, it is necessary to use marketing tools more actively in making management decisions. The most important marketing tool for making management decisions in an educational institution is marketing research of the educational services market. In order to unify information in the analytical stage of developing a strategic plan for the development of an educational institution, several existing methods of studying the market of educational services should be supplemented with the analysis of factors affecting the actions and decisions of two market subjects - consumers. SCIENCE AND INNOVATION INTERNATIONAL SCIENTIFIC JOURNAL VOLUME 1 ISSUE 5 UIF-2022: 8.2 | ISSN: 2181-3337 2) In the second stage, according to the research concept, it is necessary to analyze the market of educational services: to assess the size of the market, its capacity, "corporate" structure, the level and seriousness of competition, and to identify the main trends. The task of a comprehensive study of the educational services market is to obtain complete information about the market, its characteristics and development trends. An important stage of the research can be an expert survey, which allows not only subjective, but also objective analysis of the situation in the market of educational services, its development prospects, factors affecting consumers and purchases. Heads of educational institutions, employees of the region's education, youth policy and others can participate as experts. 3) At the third stage of research, it is necessary to analyze the actions of consumers of educational services: life goals, interests, evaluations, desires, motivations for actions, preferences and aspirations related to their internal value system. By studying their consumers, their wishes and privileges, Technical Colleges can offer them exactly what they need and do it better than their competitors. The main task in studying the consumer is to identify the factors affecting their behavior and to analyze the purchasing behavior models (the decision-making process and motives for choosing educational services). In order to successfully compete in the market of educational services, the management of Pasdargom Service Technical College should know in advance the changes in the wishes of consumers, if not in the educational program, to make timely changes in the equipment of the educational process, organization of consumers. This information is the communication of the educational institutionIt is especially important to develop a marketing and pricing policy, to form a brand. It is necessary to know exactly what is valuable and important for the consumers in shaping the advertising message, developing the advertising activity. In most markets, less expensive purchases are made or the consumer and buyer can act as one person, and this information may be sufficient to make management decisions. In the market of educational services, it is necessary to analyze the actions of another target audience - buyers of educational services. Studying buyers is one of the most important types of research, because it is they who bring the main benefit to the educational institution by influencing the choice of the consumer and purchasing the educational service. MATERIALS AND METHODS Our proposed methodology for studying the educational services market includes several stages. 1) At the first stage of the research, a marketing research concept is developed, which is a document reflecting the main idea of the research. The concession must justify the relevance of the research, determine its goals and tasks, the object and subject of the research, and indicate the time. Choosing and justifying methods, providing research with information is of great importance. A marketing research program is being developed based on the research concept. 99 SCIENCE AND INNOVATION INTERNATIONAL SCIENTIFIC JOURNAL VOLUME 1 ISSUE 5 UIF-2022: 8.2 | ISSN: 2181-3337 74 professional educational institutions in the region from 1998 to today received 26 billion 822 million soums from the Republic and local budgets, including 15 billion 866 million soums from loans from foreign countries, a total of 42 billion 708 million soums for educational laboratories, computer equipment, cars and tractors technique, equipped with mini workshop and workshop equipment. These devices are used in the training process. SCIENCE AND INNOVATION INTERNATIONAL SCIENTIFIC JOURNAL VOLUME 1 ISSUE 5 UIF-2022: 8.2 | ISSN: 2181-3337 Therefore, regardless of its size and specific features of its activity, the customer is the center of attention of any successful organization. In order to develop the right plan for the development of the technical school, it is necessary to identify the most attractive segments in order to segment the buyers of educational services and determine their location. It is necessary to evaluate the level of influence of various factors at the stages of the purchase decision-making process. It is necessary to analyze the price and other assumptions of buyers. 4) On the basis of data analysis, it is necessary to create a detailed portrait of the consumer of educational services, to distinguish the types and factors of the actions of consumers in the market of educational services. The result of the analysis of the buying behavior should be the segmentation of buyers and the evaluation of the factors of buying behavior in the market. 5) The first stage of research is the development of recommendations for the development of a strategic plan for the development of an educational institution. Also, it is necessary to analyze the research conducted at this stage, the difficulties and mistakes encountered, and develop recommendations for further marketing research. RESULTS 100 SCIENCE AND INNOVATION INTERNATIONAL SCIENTIFIC JOURNAL VOLUME 1 ISSUE 5 UIF-2022: 8.2 | ISSN: 2181-3337 74 professional educational institutions in the region from 1998 to today received 26 billion 822 million soums from the Republic and local budgets, including 15 billion 866 million soums from loans from foreign countries, a total of 42 billion 708 million soums for educational laboratories, computer equipment, cars and tractors technique, equipped with mini workshop and workshop equipment. These devices are used in the training process. DISCUSSION Brief information about educational institutions 2022 as of January No Educational institutions TM number TM strength Number of available students (2020/2021 academic year) Filling coefficient 1 Vocational schools 37 24190 17479 0.7 2 Technical schools 23 20220 15688 0.8 3 Colleges 14 11605 8664 0.8 Total 74 56015 4181 0.8 Brief information about educational institutions Brief information about educational institutions In Samarkand region, the potential demand for secondary specialized and professional education services is 41,831 people, but this indicator is constantly decreasing. This is explained by the increasing coverage of family education on the one hand, and the fact that middle school education is paid for on the other hand. The level of migration cannot significantly improve the situation, because the level of emigration is quite high. Against the backdrop of steady decline in demand, the obvious imbalance of this market is an increase in supply. According to statistical data, the offer in the educational services market of Samarkand region in January 2022 amounted to 56,015 people. The price equivalent to the offer in the market is the capacity of the educational services market in the region. A reduction in market opportunities leads to a fiercely competitive environment, which is particularly evident in the field of vocational training. This is also related to the increase in the number of non-state universities. Analyzing the competitive environment of the province gave an opportunity to evaluate it according to two main indicators: the material and technical base of technical schools (the presence of their own territory equipped for the educational process) and the number of students. From the point of view of the material and technical base of technical schools, the market of educational services is described as medium concentration. SCIENCE AND INNOVATION INTERNATIONAL SCIENTIFIC JOURNAL VOLUME 1 ISSUE 5 UIF-2022: 8.2 | ISSN: 2181-3337 In recent years, the analysis of the state of the market of educational services in the Samarkand region made it possible to identify the main trends in the development of this market: The market of educational services in the region is represented by a number of educational institutions of primary, secondary and professional education. The activities of all institutions are interconnected to a certain extent: consumers of primary vocational education may become consumers of secondary and secondary specialized and professional education in the future. the future. Brief information about educational institutions 2022 as of January No Educational institutions TM number TM strength Number of available students (2020/2021 academic year) Filling coefficient 1 Vocational schools 37 24190 17479 0.7 2 Technical schools 23 20220 15688 0.8 3 Colleges 14 11605 8664 0.8 Total 74 56015 4181 0.8 In Samarkand region, the potential demand for secondary specialized and professional education services is 41,831 people, but this indicator is constantly decreasing. This is explained by the increasing coverage of family education on the one hand, and the fact that middle school education is paid for on the other hand. The level of migration cannot significantly improve the situation, because the level of emigration is quite high. Against the backdrop of steady decline in demand, the obvious imbalance of this market is an increase in supply. According to statistical data, the offer in the educational services market of Samarkand region in January 2022 amounted to 56,015 people. The price equivalent to the offer in the market is the capacity of the educational services market in the region. A reduction in market opportunities leads to a fiercely competitive environment, which is particularly evident in the field of vocational training. This is also related to the increase in the number of non-state universities. Analyzing the competitive environment of the province gave an opportunity to evaluate it according to two main indicators: the material and technical base of technical schools (the presence of their own territory equipped for the educational process) and the number of students. From the point of view of the material and technical base of technical schools, the market of educational services is described as medium concentration. DISCUSSION Analyzing the portfolio of educational services of regional market entities showed that the specialties required in technical schools are healthcare (67%), engineering (11.7%), business and management (3.7%), agriculture, forestry and fisheries (2.9), architecture and construction. 101 SCIENCE AND INNOVATION INTERNATIONAL SCIENTIFIC JOURNAL VOLUME 1 ISSUE 5 UIF-2022: 8.2 | ISSN: 2181-3337 (2.7%), veterinary medicine (2.7%), computer technologies and informatics (2.6%), service and transport (3.3%), law (1.2%), environmental protection and agricultural machinery (0.8%), production and communication, information (1.2%). Medical and engineering specialties (nursing, paramedic-midwifery, pharmaceutics, installation of water and sewage systems, installation of gas networks, real estate registration) make up the largest share in the training of middle-level specialists. (2.7%), veterinary medicine (2.7%), computer technologies and informatics (2.6%), service and transport (3.3%), law (1.2%), environmental protection and agricultural machinery (0.8%), production and communication, information (1.2%). Medical and engineering specialties (nursing, paramedic-midwifery, pharmaceutics, installation of water and sewage systems, installation of gas networks, real estate registration) make up the largest share in the training of middle-level specialists. middle level specialists. SCIENCE AND INNOVATION INTERNATIONAL SCIENTIFIC JOURNAL VOLUME 1 ISSUE 5 UIF-2022: 8.2 | ISSN: 2181-3337 fisheries - 610,000 8 6 1 14 Agricultural machinery - 620,000 91 65 26 29 23 6 62 4 2 20 15 Veterinary medicine - 630,000 387 217 170 135 135 0 25 2 8 2 170 16 Agricultural irrigation and melioration - 640,000 0 0 0 0 0 0 0 0 0 17 Healthcare - 710,000 9777 609 9168 4600 441 4159 51 77 1 6 8 500 9 18 Social security - 720,000 0 0 0 0 0 0 0 0 0 19 Service - 810,000 264 195 69 93 36 57 17 1 1 5 9 12 20 Transport - 820,000 215 88 127 119 63 56 96 2 5 71 21 Environment - 830,000 24 15 9 14 11 3 10 4 6 22 Security of vital activities - 840,000 0 0 0 0 0 0 0 0 0 There is a significant gender difference in the selection of specialties, which is especially noticeable in the selection of a profession in secondary and higher educational institutions. So, among the students studying in education, healthcare, culture and art, economy and management, women made up 74-80%. Males predominate (79-92%) among the students studying higher and secondary engineering-technical education in such specialties as motor vehicles, geology, mineral exploration and exploitation, energy, energy and electrical engineering, metallurgy, mechanical engineering and material processing, automation. In the labor market, graduates with primary and higher vocational education are in greater demand than those with secondary vocational education. There is a significant gender difference in the selection of specialties, which is especially noticeable in the selection of a profession in secondary and higher educational institutions. So, among the students studying in education, healthcare, culture and art, economy and management, women made up 74-80%. Males predominate (79-92%) among the students studying higher and secondary engineering-technical education in such specialties as motor vehicles, geology, mineral exploration and exploitation, energy, energy and electrical engineering, metallurgy, mechanical engineering and material processing, automation. In the labor market, graduates with primary and higher vocational education are in greater demand than those with secondary vocational education. 5 professional educational institutions in the 2020-2021 academic year194 junior specialists graduated.The address of residence, passport information of the graduates was collected and a list of names was created. Today, 185 of the 194 graduates of professional educational institutions (95.4%) have been employed. SCIENCE AND INNOVATION INTERNATIONAL SCIENTIFIC JOURNAL VOLUME 1 ISSUE 5 UIF-2022: 8.2 | ISSN: 2181-3337 Of them, 181 people (93.3%) were employed, 4 people (2.1.0%) entered family educational institutions. Among the employed graduates, 101 are employed in enterprises and organizations, 25 are working as sole entrepreneurs, 22 are running family businesses, and 33 are engaged in real estate business. SCIENCE AND INNOVATION INTERNATIONAL SCIENTIFIC JOURNAL VOLUME 1 ISSUE 5 UIF-2022: 8.2 | ISSN: 2181-3337 About the number of students studying full-time education at technical schools in Samarkand region INFORMATION T/ r Number of educational fields Total educa tion the numb er of childr en so: From the total number of students run lat gran t pay ment acco unt canc er 1st step 2nd stage Tota l gran t cont ract To tal gr a nt con trac t Total by region: 14599 2325 1227 4 6773 1306 5395 77 14 9 9 2 672 2 1 Pedagogy - 110,000 1 1 0 1 1 0 0 0 0 2 Art - 210,000 0 0 0 0 0 0 0 0 0 3 Humanities - 220,000 0 0 0 0 0 0 0 0 0 4 Journalism and social information - 310,000 0 0 0 0 0 0 0 0 0 5 Business and management - 320,000 552 142 410 217 85 132 33 5 5 7 278 6 Rights - 330,000 184 27 157 72 0 0 0 0 0 7 Natural sciences - 410,000 0 0 0 0 0 0 0 0 0 8 Engineering work - 510,000 1720 286 1434 938 162 776 78 2 1 2 4 658 9 Computer technologies and informatics - 520,000 384 188 196 151 107 44 23 3 8 1 152 10 Production and processing industries - 530,000 133 78 55 66 39 27 67 3 9 28 11 Architecture and construction - 540,000 395 131 264 178 85 93 21 7 4 6 171 12 Communication and information, telecommunication technologies - 550,000 41 9 32 17 5 12 24 4 20 13 Agriculture, forestry and 431 274 157 143 113 30 28 1 127 About the number of students studying full-time education at technical schools in Samarkand region INFORMATION bout the number of students studying full-time education at technical schools in Samarkand region 102 102 REFERENCES 1. 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In secondary vocational education - 103 SCIENCE AND INNOVATION INTERNATIONAL SCIENTIFIC JOURNAL VOLUME 1 ISSUE 5 UIF-2022: 8.2 | ISSN: 2181-3337 automation and management, culture and art, education and pedagogy, economy and management, construction and architecture, vehicles. The specialties most in demand in the labor market in higher professional education are metallurgy, mechanical engineering, material processing, vehicles, informatics and computer technologies, instrumentation and optics engineering, automation and control, construction and architecture, education and pedagogy, culture and art, energy, energy. and electrical engineering. automation and management, culture and art, education and pedagogy, economy and management, construction and architecture, vehicles. The specialties most in demand in the labor market in higher professional education are metallurgy, mechanical engineering, material processing, vehicles, informatics and computer technologies, instrumentation and optics engineering, automation and control, construction and architecture, education and pedagogy, culture and art, energy, energy. and electrical engineering. REFERENCES Development Prospects of Business Subjects in the Republic of Uzbekistan //Web of Scholars: Multidimensional Research Journal. – 2022. – Т. 1. – №. 4. – С. 13-19. 16. Azimovna M. S., Ilkhomovna U. D., Shokhrukhovich U. F. The Concept of Marketing Policy in Trade and Service Enterprises //CENTRAL ASIAN JOURNAL OF INNOVATIONS ON TOURISM MANAGEMENT AND FINANCE. – 2022. – Т. 3. – №. 8. – С. 1-5. 104 SCIENCE AND INNOVATION INTERNATIONAL SCIENTIFIC JOURNAL VOLUME 1 ISSUE 5 UIF-2022: 8.2 | ISSN: 2181-3337 17. Azimovna M. S., Shokhrukhovich U. F. 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Azimovna M. S., Shohrukhovich U. S. THE ROLE OF AGRICULTURAL PRODUCTS IN THE FOREIGN TRADE TURNOVER OF THE REPUBLIC OF UZBEKISTAN //BARQARORLIK VA YETAKCHI TADQIQOTLAR ONLAYN ILMIY JURNALI. – 2022. – Т. 2. – №. 6. – С. 110-112. 20. Azimovna M. S. IMPROVING THE STUDY OF CONSUMER BEHAVIOR //Gospodarka i Innowacje. – 2022. – С. 109-112. 20. Azimovna M. S. IMPROVING THE STUDY OF CONSUMER BEHAVIOR //Gospodarka i Innowacje. – 2022. – С. 109-112. 21. Azimovna M. S. et al. Analysis of the main economic and marketing indicators of FE" DAKA-TEX" LLC //ASIA PACIFIC JOURNAL OF MARKETING & MANAGEMENT REVIEW ISSN: 2319-2836 Impact Factor: 7.603. – 2022. – Т. 11. – №. 06. – С. 4-7. 21. Azimovna M. S. et al. Analysis of the main economic and marketing indicators of FE" DAKA-TEX" LLC //ASIA PACIFIC JOURNAL OF MARKETING & MANAGEMENT REVIEW ISSN: 2319-2836 Impact Factor: 7.603. – 2022. – Т. 11. – №. 06. – С. 4-7. p 22. Azimovna M. S., Ilkhomovna U. D. 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https://openalex.org/W4385196905
https://bmcresnotes.biomedcentral.com/counter/pdf/10.1186/s13104-023-06429-5
English
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Evaluation of levels of uric acid and lipid profile in hospitalized patients with diabetes
BMC research notes
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cc-by
5,042
© 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. Abstract Objective  Diabetes is the most common metabolic disorder that leads to various complications, and among these complications, disruption in the lipid profile and serum uric acid (SUA) is one of the significant cases that can lead to the deterioration of the health status of patients with diabetes. So, we aimed to evaluate the level of SUA and lipid profiles in patients with diabetes. A total of 230 patients with diabetes who were admitted to Razi Hospital, Rasht, Iran, were enrolled in this study. Demographical data and clinical characteristics of the patients include gender, body mass index (BMI), duration of diabetes, history of smoking, FBS, HbA1c, SUA, Creatinine (Cr), Cholesterol (Chol), low- density lipoprotein (LDL), high-density lipoprotein (HDL), triglycerides (TG), retinopathy, hypertension, ischemic heart disease (IHD), and microalbuminuria were recorded. All data were analyzed using the SPSS version 21 by a significant level < 0.05. Result  According to our results, 70 were male, and 160 were female, with a mean age of 57.36 ± 8.05 years and a mean BMI of 28.10 ± 4.62. The most frequent comorbidities were hypertension, 67%. The serum level of FBS, HBA1c, SUA, Cr, Chol, LDL, HDL, and TG were 191.47 ± 71.66 mg/dL, 7.94 ± 1.21 mg/dL, 5.65 ± 1.95 mg/dL, 0.94 ± 0.16 mg/dL, 167.28 ± 45.22 mg/dL, 95.91 ± 37.03 mg/dL, 39.78 ± 10.44 mg/dL, and 186.75 ± 76.65 mg/dL, respectively. Only UA had a significant relationship with TG level (P < 0.05). Keywords  Triglyceride, Serum uric acid, Lipids, Diabetics, Lipid profile BMC Research Notes BMC Research Notes Fayazi et al. BMC Research Notes (2023) 16:154 https://doi.org/10.1186/s13104-023-06429-5 Open Access Introduction cardiovascular complication, and metabolic syndrome [3]. Some diseases, such as metabolic syndrome and type 2 diabetes mellitus (T2DM), are often considered lifestyle-related diseases due to their pathological basis [4–6]. Diabetes has become an epidemic over the past few decades as one of the most significant global health emergencies of the 21st century. According to a report by the International Diabetes Federation, diabetes mel­ litus prevalence is likely to increase to 642  million by 2040 [7]. Despite the high prevalence of diagnosed dia­ betes, almost half of diabetic individuals are unaware of their disease. Diabetes is contributed to a higher rate of morbidity and mortality in the future half of them occur due to cardiovascular complications [8]. Pieces of evi­ dence have shown that an abnormal lipid profile, along Uric acid (UA) is a final enzymatic product in humans’ degradation of purine nucleosides and free bases. The pathway of purine catabolism in humans is shorter than in other primates. Consequently, humans have to deal with higher levels of UA in the blood and are sus­ ceptible to hyperuricemia and gout [1, 2]. High serum UA (SUA) levels, and hyperuricemia, are associated with gout, hypertension, kidney stones, kidney disease, *Correspondence: Maryam Yaseri Maryam.yaseri1400@gmail.com 1Department of Internal Medicine, School of Medicine, Razi Hospital, Guilan University of Medical Sciences, Rasht, Iran © 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. Fayazi et al. BMC Research Notes (2023) 16:154 Page 2 of 5 Page 2 of 5 Fayazi et al. BMC Research Notes with T2D, has a close relationship with insulin resistance, which is the major component of other metabolic disor­ ders [9, 10]. Insulin resistance is associated with a high level of very-low-density lipoprotein (VLDL), high con­ centrations of serum triglycerides (TG), and serum high- density lipoprotein (HDL). (BMI), duration of diabetes, history of smoking, retinop­ athy, hypertension, ischemic heart disease (IHD), micro­ albuminuria, and also laboratory findings that included HbA1c, FBS, UA, Creatinine (Cr), Cholesterol (Chol), LDL, HDL, and TG, which were analyzed by auto-ana­ lyzer (BS-800, Mindray, UK, Ltd). The cut-off values for hypercholesterolemia (≥ 240  mg/dL), hypertriglyc­ eridemia (≥ 200  mg/dL), and low HDL-cholesterolemia (≤ 40 mg/dL). Consequently, the lipid profile is highlighted in nearly all follow-up plans of T2DM and assists as a severe risk factor [11, 12]. Dyslipidemia is frequently associated with obesity, T2DM, and metabolic syndrome. Dyslipidemia includes elevated TG, normal or slightly elevated low- density lipoprotein cholesterol (LDL), and decreased lev­ els of HDL [13]. By changing the worldwide lifestyle, the prevalence of dyslipidemia has grown, especially in devel­ oping countries [14]. Elevated SUA levels or hyperurice­ mia are significantly associated with dyslipidemia [15].f Results According to our result, among 230 participants, 70 were male, and 160 were female, with a mean age of 57.36 ± 8.05 years and a mean BMI of 28.10 ± 4.62. The average duration of diabetes existence was 11.68 ± 7.13 years. The level of FBS, HBA1c, UA, Cr, Chol, LDL, HDL, and TG were 191.47 ± 71.66  mg/dL, 7.94 ± 1.21  mg/dL, 5.65 ± 1.95 mg/dL, 0.94 ± 0.16 mg/dL, 167.28 ± 45.22 mg/ dL, 95.91 ± 37.03  mg/dL, 39.78 ± 10.44  mg/dL, and 186.75 ± 76.65  mg/dL, respectively. The most frequent comorbidity in the patients was hypertension 67% (154 n) (Table 1). Due to Table 2, only UA had a statistically significant relationship with TG (P = 0.03), so 55% of patients with SUA level under 6.8 mg/dL represented 150 mg/dL ≤ TG, and 77% of patients with SUA level above 6.8  mg/dL, had 150  mg/dL < TG. Only 5.3% of patients with SUA levels under 6.8 mg/dL and 8.2% with SUA levels above 6.8  mg/dL had 240  mg/dL ≤ Chol level. Also, among patients with SUA levels under 6.8  mg/dL, 6.5% had 160  mg/dL ≤ LDL and 83.4% had HDL < 40  mg/dL (in males) and HDL < 50  mg/dL (in females). In the group of patients with SUA levels above 6.8 mg/dL, about 4.9% had 160 mg/dL ≤ LDL and 78.7% had HDL < 40 mg/dL (in males), and HDL < 50 mg/dL (in females). Statistical analysis Categorical variables are frequency and percentage, while continuous variables are Mean ± S.D. The chi-square test or exact Fisher test was applied to evaluate the associa­ tion between two categorical variables. Statistical cal­ culations were performed using IBM SPSS Statistics for Windows, version 21, with a statistically significant level of less than 0.05. i By considering gender differences in some complica­ tions and diseases, it is still not clear whether the rela­ tionship between SUA levels and dyslipidemia bares consistent across different genders, which merits con­ sideration. A study reported a strong positive associa­ tion between SUA levels and dyslipidemia in the male gender but not in females [16]. Only a few studies were conducted on the association between lipid profile and SUA in patients with diabetes, who are usually engaged with those two dysregulations in Iran. Early preven­ tion of hyperuricemia and dyslipidemia may help reduce further complications in patients with diabetes. In this regard, we aimed to conduct this study to evaluate the level of SUA and dyslipidemia in patients with diabetes in North Iran and evaluate the demographical and clinical characteristics. Patients and methods Study design, categorization of patients, and sample size The data of this cross-sectional study were collected from 230 adult patients with diabetes, selected by cen­ sus method, and referred to Razi Hospital, Rasht, Iran, from March 2019 to March 2020. Diabetes in these patients was confirmed by specialists with a level of hemoglobin A1c (HbA1c) > 6.5% and fasting blood sugar (FBS) > 126 mg/dl [17] through laboratory results or who were on blood glucose control medication. Individuals with a history of kidney disease, pregnant ones, patients who have recently undergone surgery, and patients with incomplete information were excluded from the study. Before the study, the informed consent form was fulfilled by patients. The study design was approved by the ethical committee of Guilan University of Medical Sciences (IR. GUMS.REC.1399.171). Discussionh *Chi-square test or exact Fisher test were applied to evaluate the association between two categorical variables. The statistical significance was evaluated at the level of 0.05 Table 2  Comparison of the frequency of hyperlipidemia in patients with normal and abnormal Uric acid level Qualitative variables are described as frequency and percentage, while continuous variables are as Mean ± S.D Qualitative variables are described as frequency and percentage, while continuous variables are as Mean ± S.D (quantitative and qualitative), with a reported prevalence of 24–40% [18–20].i Low-density lipoprotein (LDL); High-density lipoprotein (HDL). *Chi-square test or exact Fisher test were applied to evaluate the association between two categorical variables. The statistical significance was evaluated at the level of 0.05 *Chi-square test or exact Fisher test were applied to evaluate the association between two categorical variables. The statistical significance was evaluated at the level of 0.05 IN THEIR STUDY, Kodama S et al. reported signifi­ cant hyperuricemia in patients with T2DM. They also suggested that UA had a crucial role in worsening insulin resistance by inhibiting the bioavailability of nitric oxide, which is essential for insulin-stimulated glucose uptake [21]. Another study represented that high UA levels are significantly associated with prediabetes, indicating that UA has a significant role in glucose metabolism. Also, they reported a significant positive association between dyslipidemia and serum UA [22]. HbA1c and UA were thus found in two study groups compared to the healthy control group [24]. Due to our data, retinopathy was reported in 39.6% of the study population. The association of dyslipidemia with diabetic retinopathy is also a subject of considerable debate. In many studies. Dyslipidemia has not been reported to be associated with diabetic retinopathy [25–27]. Although, some other studies have illustrated a significant association between hypercholesterolemia and LDL with the severity of reti­ nal hard exudates [28, 29]. Also, as demonstrated in our study, a higher frequency of hypertension was the most comorbidities among the study population. In line with our results, in some other studies, it has been reported that dyslipidemia is associated with an increased risk of hypertension [30, 31]. Gaubert et al. Discussionh Qualitative variables are described as frequency and percentage, while continuous variables are as Mean ± S.D Table 2  Comparison of the frequency of hyperlipidemia in patients with normal and abnormal Uric acid level Uric acid n(%) Total P-Value* < 6.8(mg/ dL) 6.8(mg/ dL)< Tri­ glyc­ er­ ide < 150 mg/dL 76 (45%) 14 (23%) 90 (39.1%) 0.003 150 mg/dL ≥ 93 (55%) 47 (77%) 140 (60.9%) Cho­ les­ terol < 200 mg/dL 130 (76.9%) 44 (72.1%) 174 (75.6%) 0.659 200 mg/ dL ≥ Chol < 240 mg/ dL 30 (17.8%) 12 (19.7%) 42 (18.3%) 240 mg/dL ≥ 9 (5.3%) 5 (8.2%) 14 (6.1%) LDL < 100 mg/dL 99 (58.6%) 38 (62.3%) 137 (59.6%) 0.840 100 mg/ dL ≥ LDL < 160 mg/ dL 59 (34.9%) 20 (32.8%) 79 (34.3%) 160 mg/dL ≥ 11 (6.5%) 3 (4.9%) 14 (6.1%) HDL < 40 mg/dL in male < 50 mg/dL in female 141 (83.4%) 48 (78.7%) 189 (82.2%) 0.407 40 mg/ dL < HDL < 100 mg/ dL in male 50 mg/ dL < HDL < 110 mg/ dL in female 28 (16.6%) 13 (21.3%) 41 (17.8) Total 169 (73.5%) 61 (26.5%) - Low-density lipoprotein (LDL); High-density lipoprotein (HDL). *Chi-square test or exact Fisher test were applied to evaluate the association between two categorical variables. The statistical significance was evaluated Table 2  Comparison of the frequency of hyperlipidemia in patients with normal and abnormal Uric acid level Uric acid n(%) Total P-Value* < 6.8(mg/ dL) 6.8(mg/ dL)< Tri­ glyc­ er­ ide < 150 mg/dL 76 (45%) 14 (23%) 90 (39.1%) 0.003 150 mg/dL ≥ 93 (55%) 47 (77%) 140 (60.9%) Cho­ les­ terol < 200 mg/dL 130 (76.9%) 44 (72.1%) 174 (75.6%) 0.659 200 mg/ dL ≥ Chol < 240 mg/ dL 30 (17.8%) 12 (19.7%) 42 (18.3%) 240 mg/dL ≥ 9 (5.3%) 5 (8.2%) 14 (6.1%) LDL < 100 mg/dL 99 (58.6%) 38 (62.3%) 137 (59.6%) 0.840 100 mg/ dL ≥ LDL < 160 mg/ dL 59 (34.9%) 20 (32.8%) 79 (34.3%) 160 mg/dL ≥ 11 (6.5%) 3 (4.9%) 14 (6.1%) HDL < 40 mg/dL in male < 50 mg/dL in female 141 (83.4%) 48 (78.7%) 189 (82.2%) 0.407 40 mg/ dL < HDL < 100 mg/ dL in male 50 mg/ dL < HDL < 110 mg/ dL in female 28 (16.6%) 13 (21.3%) 41 (17.8) Total 169 (73.5%) 61 (26.5%) - Low-density lipoprotein (LDL); High-density lipoprotein (HDL). Discussionh The results of the present study can be summarized as follows; First, hypertension was the most frequent comorbid in patients with diabetes. Secondly, abnor­ mal SUA was associated with a significantly higher TG level. In addition, abnormal microalbuminuria was pres­ ent in almost half of T2DM patients. Patients with dia­ betes are more susceptible to developing dyslipidemia Demographical data and clinical characteristics were collected from patients’ medical records in the hospital archive. They included gender, gender, body mass index Fayazi et al. BMC Research Notes (2023) 16:154 Page 3 of 5 Fayazi et al. BMC Research Notes Page 3 of 5 Table 1  Frequency of demographical and clinical data of patients Variables Number Percentage Gender Male 70 30.4% Female 160 69.6% History of smoking No 187 81.3% Yes 43 18.7% Retinopathy No 139 60.4% Yes 91 39.6% Hypertension No 76 33% Yes 154 67% Ischemic heart disease No 173 75.2% Yes 57 24.8% Microalbuminuria No 124 53.9% Yes 106 46.1% Mean ± SD Age 57.36 ± 8.05 BMI 28.10 ± 4.62 Duration of diabetes 11.68 ± 7.13 FBS 191.47 ± 71.66 HBA1C 7.94 ± 1.21 Uric Acid 5.65 ± 1.95 Creatinine 0.94 ± 0.16 Cholesterol 167.28 ± 45.22 LDL 95.91 ± 37.03 HDL 39.78 ± 10.44 Triglyceride 186.75 ± 76.65 Low-density lipoprotein (LDL); High-density lipoprotein (HDL). Qualitative variables are described as frequency and percentage, while continuous variables are as Mean ± S.D (quantitative and qualitative), with a reported prevalence of 24–40% [18–20]. IN THEIR STUDY, Kodama S et al. reported signifi­ cant hyperuricemia in patients with T2DM They also Table 1  Frequency of demographical and clinical data of patients Variables Number Percentage Gender Male 70 30.4% Female 160 69.6% History of smoking No 187 81.3% Yes 43 18.7% Retinopathy No 139 60.4% Yes 91 39.6% Hypertension No 76 33% Yes 154 67% Ischemic heart disease No 173 75.2% Yes 57 24.8% Microalbuminuria No 124 53.9% Yes 106 46.1% Mean ± SD Age 57.36 ± 8.05 BMI 28.10 ± 4.62 Duration of diabetes 11.68 ± 7.13 FBS 191.47 ± 71.66 HBA1C 7.94 ± 1.21 Uric Acid 5.65 ± 1.95 Creatinine 0.94 ± 0.16 Cholesterol 167.28 ± 45.22 LDL 95.91 ± 37.03 HDL 39.78 ± 10.44 Triglyceride 186.75 ± 76.65 Low-density lipoprotein (LDL); High-density lipoprotein (HDL). Competing interests p g The authors reported no potential conflict of interest. p g The authors reported no potential conflict of interest. Received: 11 December 2022 / Accepted: 12 July 2023 Received: 11 December 2022 / Accepted: 12 July 2023 Received: 11 December 2022 / Accepted: 12 July 2023 Consent for publicationfi The authors affirm that participants provided informed consent for the study’s publication. References l According to our results, a higher level of SUA was reported among diabetic patients with a higher level of TG. Also, conducting the follow-up cohort study can be helpful to achieve more robust results to help physicians monitor patients with diabetes to make an early diagno­ sis of either Elevated SUA or dyslipidemia, which can be helpful to prevent other adverse consequences. 1. Glantzounis GK, Tsimoyiannis EC, Kappas AM, Galaris DA. Uric acid and oxida­ tive stress. Curr Pharm Des. 2005;11:4145–51. 2. Johnson RJ, Titte S, Cade JR, Rideout BA, Oliver WJ. Uric acid, evolution and primitive cultures. Semin Nephrol. 2005;25:3–8. 3. Johnson RJ, Bakris GL, Borghi C, Chonchol MB, Feldman D, Lanaspa MA, et al. Hyperuricemia, Acute and chronic kidney Disease, Hypertension, and Cardio­ vascular Disease: report of a Scientific Workshop Organized by the national kidney Foundation. Am J kidney Dis Off J Natl Kidney Found. 2018;71:851–65. 4. Watanabe S, Kang D-H, Feng L, Nakagawa T, Kanellis J, Lan H et al. Uric acid, hominoid evolution, and the pathogenesis of salt-sensitivity. Hypertens (Dal­ las, Tex 1979). 2002;40:355–60. 5. Powers AC, Niswender KD, Rickels MR. Diabetes Mellitus: management and therapies. In: Jameson JL, Fauci AS, Kasper DL, Hauser SL, Longo DL, Loscalzo J, editors. Redakteurs. Harrison’s principles of Internal Medicine, 20e. New 1. Glantzounis GK, Tsimoyiannis EC, Kappas AM, Galaris DA. Uric acid and oxida­ tive stress. Curr Pharm Des. 2005;11:4145–51. 2. Johnson RJ, Titte S, Cade JR, Rideout BA, Oliver WJ. Uric acid, evolution and primitive cultures. Semin Nephrol. 2005;25:3–8. 3. Johnson RJ, Bakris GL, Borghi C, Chonchol MB, Feldman D, Lanaspa MA, et al. Hyperuricemia, Acute and chronic kidney Disease, Hypertension, and Cardio­ vascular Disease: report of a Scientific Workshop Organized by the national kidney Foundation. Am J kidney Dis Off J Natl Kidney Found. 2018;71:851–65. 4. Watanabe S, Kang D-H, Feng L, Nakagawa T, Kanellis J, Lan H et al. Uric acid, hominoid evolution, and the pathogenesis of salt-sensitivity. Hypertens (Dal­ las, Tex 1979). 2002;40:355–60. Ethics approval and consent to participate Informed consent to participate was obtained from each study participant. All methods were carried out following relevant guidelines and regulations. The study was approved by the ethical committee at the Guilan University of Medical Sciences, Rasht, Iran [IR.GUMS.REC.1399.171]. Data Availability h d d y The datasets used and analyzed during the current study are available from the corresponding author on reasonable request. Funding g y High lipid profile, except the LDL, was reported in the group with 6.8 mg/dL < SUA in comparison to the group with SUA < 6.8 mg/dL, in which this elevation has repre­ sented no statistically significant differences (P > 0.05). As reported in Liu et al.‘ study, the proportion of individu­ als with abnormal levels was higher in the hyperuricemia group than in the normal group [39]. A study by Vekic et al. reported that the highest SUA was associated with significantly smaller sizes of LDL and HDL [40]. In our study, comparing LDL levels among the two groups illus­ trated the same result for LDL, although it represented no statistically significant differences. Several authors concluded elevated SUA reflects insulin resistance [41, 42]. However, it is controversial whether hyperuricemia plays a fundamental role in diabetes. Lu et al. confirmed no causal association between hyperuricemia and dia­ betes [43]. Elevated SUA accelerates but does not cause diabetes because SUA itself is insufficient to induce dia­ betes, although it can damage glucose tolerance and lead to insulin resistance [43]. 1. Glantzounis GK, Tsimoyiannis EC, Kappas AM, Galaris DA. Uric acid and oxida­ tive stress. Curr Pharm Des. 2005;11:4145–51. 2. Johnson RJ, Titte S, Cade JR, Rideout BA, Oliver WJ. Uric acid, evolution and primitive cultures. Semin Nephrol. 2005;25:3–8. 3. Johnson RJ, Bakris GL, Borghi C, Chonchol MB, Feldman D, Lanaspa MA, et al. Hyperuricemia, Acute and chronic kidney Disease, Hypertension, and Cardio­ vascular Disease: report of a Scientific Workshop Organized by the national kidney Foundation. Am J kidney Dis Off J Natl Kidney Found. 2018;71:851–65. 4. Watanabe S, Kang D-H, Feng L, Nakagawa T, Kanellis J, Lan H et al. Uric acid, hominoid evolution, and the pathogenesis of salt-sensitivity. Hypertens (Dal­ las, Tex 1979). 2002;40:355–60. 5. Powers AC, Niswender KD, Rickels MR. Diabetes Mellitus: management and therapies. In: Jameson JL, Fauci AS, Kasper DL, Hauser SL, Longo DL, Loscalzo J, editors. Redakteurs. Harrison’s principles of Internal Medicine, 20e. New Discussionh illustrated the important role of high SUA levels in hypertension-asso­ ciated morbidities and should be considered in patients Some studies compare SUA levels with lipid profiles in patients with diabetes; as reported in a study by Moham­ medsaeed, dyslipidemia (high level of LDL, TG, and low level of HDL) and HbA1c were highly correlated with UA in patients with diabetes [23]. Moreover, Agrawal et al. reported a significantly lower mean of HDL in patients with diabetes, with and without retinopathy, than in healthy individuals. Mean HbA1c and mean FBS signifi­ cantly correlated with UA (P = 0.01 and P = 0.02, respec­ tively) in both groups. A deranged HDL profile and a significant correlation between glycemic control and Fayazi et al. BMC Research Notes (2023) 16:154 Page 4 of 5 Page 4 of 5 with chronic hyperuricemia [32]. Moreover, about 24.8% of patients with diabetes had a history of IHD. As reported in some studies, dyslipidemia and hyperglyce­ mia can play a key role in cardiovascular dysfunction in at-risk adolescents with diabetes [33, 34]. Microalbuminuria was reported at 46.1% in patients with diabetes due to our results. According to the result of a study, hypoalbuminemia rates in the non-survivors and survivor groups were 76.7% and 57.8%, respectively (P = 0.019). Hyperuricemia rates were 74.4% and 57.1%, respectively (P = 0.033) [35]. In patients with T2DM, it was reported that SUA levels correlated with urinary albumin excretion, which was associated with microalbu­ minuria in these patients [36, 37]. In T2DM patients with abnormal albuminuria, the lipid profile was characterized by lower Chol levels and a tendency toward higher TG levels than those with normal albuminuria. 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Prevalence of dyslip­ idemia and achievement of low-density lipoprotein cholesterol targets in chi­ nese adults: a nationally representative survey of 163,641 adults. Int J Cardiol. 2018;260:196–203. 36. Tseng C-H. Correlation of uric acid and urinary albumin excretion rate in patients with type 2 diabetes mellitus in Taiwan. Kidney Int. 2005;68:796–801. 15. McFarlane SI, Banerji M, Sowers JR. Insulin resistance and cardiovascular disease. J Clin Endocrinol Metab. 2001;86:713–8. 37. Fukui M, Tanaka M, Shiraishi E, Harusato I, Hosoda H, Asano M, et al. Serum uric acid is associated with microalbuminuria and subclinical atherosclerosis in men with type 2 diabetes mellitus. Metabolism. 2008;57:625–9. 16. Sowers JR. Recommendations for special populations: diabetes mellitus and the metabolic syndrome. Am J Hypertens. 2003;16(11 Pt 2):41S–5. 38. Kajingulu FM, Lepira FB, Mbutiwi FI, Makulo JR, Sumaili EK, Bukabau JB, et al. Albuminuria status and patterns of dyslipidemia among type 2 diabetes black patients managed at a tertiary health-care hospital: a Post hoc analysis. Limitationsh Saudi J Kidney Dis Transpl. 2018;29:649–57. 17. Diagnosis and classification of diabetes mellitus. Diabetes Care. 2010;33(1 Suppl 1):62–9. 18. Maahs DM, Ogden LG, Dabelea D, Snell-Bergeon JK, Daniels SR, Hamman RF, et al. Association of glycaemia with lipids in adults with type 1 diabetes: modification by dyslipidaemia medication. Diabetologia. 2010;53:2518–25. 39. Liu N, Sun Q, Xu H, Yu X, Chen W, Wei H, et al. Hyperuricemia induces lipid disturbances mediated by LPCAT3 upregulation in the liver. FASEB J. 2020;34:13474–93. i y y 19. Petitti DB, Imperatore G, Palla SL, Daniels SR, Dolan LM, Kershnar AK, et al. Serum lipids and glucose control: the SEARCH for diabetes in Youth study. Arch Pediatr Adolesc Med. 2007;161:159–65. 40. Yu W, Cheng J-D. Uric Acid and Cardiovascular Disease: an update from molecular mechanism to clinical perspective. Front Pharmacol. 2020;11:582680. 20. Loh KC, Thai AC, Lui KF, Ng WY. High prevalence of dyslipidaemia despite adequate glycaemic control in patients with diabetes. Ann Acad Med Singa­ pore. 1996;25:228–32. 41. Yoo TW, Sung KC, Shin HS, Kim BJ, Kim BS, Kang JH, et al. Relationship between serum uric acid concentration and insulin resistance and metabolic syndrome. Circ J. 2005;69:928–33. 21. Kodama S, Saito K, Yachi Y, Asumi M, Sugawara A, Totsuka K, et al. Association between serum uric acid and development of type 2 diabetes. Diabetes Care. 2009;32:1737–42. https://doi.org/10.2337/dc09-0288. 42. Rho YH, Woo J-H, Choi SJ, Lee YH, Ji JD, Song GG. Association between serum uric acid and the adult treatment panel III–defined metabolic syndrome:: results from a single hospital database. Metabolism. 2008;57:71–6. 22. Alqahtani SAM, Awan ZA, Alasmary MY, Al Amoudi SM. Association between serum uric acid with diabetes and other biochemical markers. J Fam Med Prim care. 2022;11:1401–9. 43. Lu J, He Y, Cui L, Xing X, Liu Z, Li X, et al. Hyperuricemia predisposes to the Onset of Diabetes via promoting pancreatic β-Cell death in uricase-deficient male mice. Diabetes. 2020;69:1149–63. 23. Mohammedsaeed W. Uric acid levels in saudi females with type 2 diabetes mellitus. Med Sci. 2020;24:4019–25. 24. Agrawal P, Sethi S, Reddy V, Lamba S. Serum uric acid and lipid profile in diabetic retinopathy in rural Haryana, India. Int J Adv Med. 2016;3:309–12. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 25. He B-B, Wei L, Gu Y-J, Han J-F, Li M, Liu Y-X, et al. Factors associated with diabetic retinopathy in chinese patients with type 2 diabetes mellitus. Int J Endocrinol. 2012;2012:157940. 26. Tapp RJ, Shaw JE, Harper CA, de Courten MP, Balkau B, McCarty DJ, et al. The prevalence of and factors associated with diabetic retinopathy in the austra­ lian population. Diabetes Care. 2003;26:1731–7.
https://openalex.org/W2142327394
https://seer.sis.puc-campinas.edu.br/seer/index.php/oculum/article/download/2709/1935
Portuguese
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O enigma do capital e as crises do capitalismo
Oculum Ensaios
2,015
cc-by
1,373
O ENIGMA DO CAPITAL E AS CRISES DO CAPITALISMO De David Harvey São Paulo: Boitempo Editorial, 2011 AS ESFERAS DE ATIVIDADE O capital se movimenta em busca de lucro, por meio de diferentes esferas de atividade. Segundo Harvey, as esferas de atividade são as seguintes: tecnologias e formas de organização; relações sociais; arranjos institucionais e administrativos; processos de produção e trabalho; relações com a natureza; reprodução da vida cotidiana e da espécie; e “concepções mentais do mundo”. O autor conceitua a formação das crises em termos de tensões e antagonismos que surgem entre as diferentes esferas de atividade. O capital não pode circular ou acumu‑ lar‑se sem tocar em cada uma ou em todas as esferas de alguma forma. A dinâmica geográfica que surge da esfera da reprodução da vida cotidiana é simul‑ taneamente autônoma e profundamente afetada por suas relações com as outras esferas. A dinâmica geográfica que surge da esfera da reprodução da vida cotidiana é simul‑ taneamente autônoma e profundamente afetada por suas relações com as outras esferas. INTRODUÇÃO O texto de David Harvey analisa as crises na evolução do capitalismo, explicando o processo pelo qual o capital realimenta sua expansão e acumulação, sempre com novos arranjos temporais e espaciais. A questão central do livro é o entendimento da representatividade do fluxo do capital. Harvey identifica os mecanismos pelos quais o capitalismo sobrevive; expli‑ cando os determinantes da crise de 2008, o autor estuda as condições necessárias para a acumulação do capital e como estas têm superado antigas barreiras. Ao tratar do entendimento de como o poder do capital formata o mundo, Harvey argumenta em defesa das ideias de Karl Marx. Para tanto, o autor realiza um profundo diagnóstico sobre as inter‑relações entre as “esferas de atividades” humanas, as quais devem ser pensadas de forma interdependente. Harvey contextualiza e apresenta as esferas de atividade na trajetória evolutiva do capitalismo. O autor mostra as razões para a propensão do capital a crises, assim como os riscos que a reprodução do capital representa para a vida do planeta, fundamentado na tese central de que o capitalismo — modo de produção voltado para a acumulação e o lucro —, necessita de contínua expansão e inovação. Pensa o desenvolvimento capitalista inicialmente, sem considerar sua orga‑ nização espacial evolutiva na dinâmica geográfica e seus impactos e constrangi‑ mentos ambientais. O estudo dos “riscos sistêmicos” traduz as contradições funda‑ mentais da acumulação de capital. O autor conclui que o capital nunca resolve sua tendência a crises. Oculum ens. | Campinas | 12(1) | 185-187 | Janeiro-Junho 2015 Oculum ens. | Campinas | 12(1) | 185-187 | Janeiro-Junho 2015 186 O ENIGMA DO CAPITAL revelado | Resenha 186 O ENIGMA DO CAPITAL revelado | Resenha 187 empreendimento especulativo, muitas vezes com a conivência e a cumplicidade, senão com a ativa colaboração do aparelho do Estado. A reorganização drástica da paisagem geográfica da produção, da distribuição e do consumo, com as mudanças nas relações de espaço, não é apenas uma ilustração dramá‑ tica da tendência do capitalismo para a aniquilação do espaço no decorrer do tempo, mas também implica ataques ferozes de destruição criativa. Existe um campo de contradições dentro da tendência de criar um mundo sem barreiras espaciais. Segundo Harvey as estratégias políticas, diplomáticas, econômicas e militares mobilizadas pelo aparelho de Estado em seu próprio interesse constituem a lógica ter‑ ritorial. Essas estratégias objetivam controlar e gerenciar as atividades da população no território e acumular poder e riqueza dentro das fronteiras do Estado. A lógica capitalista, por outro lado, coloca em foco a maneira pela qual o poder do dinheiro flui por e dentro do espaço na busca da acumulação sem fim. Essa lógica é mais processual e molecular do que territorial. O desenvolvimento geográfico desigual é fun‑ damental para a reprodução do capitalismo. No entanto, os capitalistas não podem seguir barreiras geográficas — nem espaciais nem ambientais —, e estão engajados em uma luta perpétua para burlá‑las ou transcendê‑las. A GEOGRAFIA DA ACUMULAÇÃO DO CAPITAL E A DESTRUIÇÃO CRIATIVA DA TERRA As conexões entre a urbanização, a acumulação do capital e a formação de crises são ana‑ lisadas por Harvey cuidadosamente. Desde seus primórdios, as cidades dependeram da disponibilidade de alimentos e trabalhos excedentes. O desenvolvimento geográfico desigual é fundamental para sua reprodução. Por outro lado, os capitalistas não podem seguir barreiras geográficas de qualquer espécie — nem espacial nem ambiental —, e estão engajados em uma luta perpétua para burlá‑las ou transcendê‑las. O desenvolvimento geográfico desigual é fundamental para sua reprodução. Por outro lado, os capitalistas não podem seguir barreiras geográficas de qualquer espécie — nem espacial nem ambiental —, e estão engajados em uma luta perpétua para burlá‑las ou transcendê‑las. Harvey analisa como a acumulação de capital impacta as cidades e como a segre‑ gação se reproduz no espaço urbano, avaliando que a geografia do desenvolvimento e da subsequente crise tem sido desigual. A paisagem geográfica é igualmente moldada por uma perpétua tensão entre as economias de centralização, de um lado, e os lucros poten‑ cialmente maiores que vêm da descentralização e da dispersão, por outro lado. A produção do espaço em geral e da urbanização em particular tornou‑se um grande negócio no capitalismo, sendo um dos principais meios de absorver o excesso de capital. Ao longo da história tem havido uma tendência para a redução geral das barreiras espaciais e a aceleração da acumulação de capital. O autor aponta dois grandes processos de destruição criativa na história da huma‑ nidade: um é a reconstrução de Paris por Haussmann, outro é a Segunda Guerra Mundial seguida pelas intervenções de Moses em New York. O autor evidencia que atualmente a urbanização da China é parcialmente a fonte primária de estabilização do capitalismo mundial como epicentro de um processo de urbanização que se tornou global, ajudado pela integração mundial dos mercados financeiros. A destruição criativa da terra é entendida no texto de Harvey como produção e reprodução da geografia através dos principais agentes sistêmicos — o Estado e o capital. Os capitalistas e seus agentes se envolveram na produção de uma segunda natureza, a pro‑ dução ativa de sua geografia, da mesma maneira como produzem todo o resto: como um Oculum ens. | Campinas | 12(1) | 185-187 | Janeiro-Junho 2015 Oculum ens. | Campinas | 12(1) | 185-187 | Janeiro-Junho 2015 O ENIGMA DO CAPITAL revelado | Resenha OS PROTAGONISTAS DA TRANSFORMAÇÃO Há tempos o sonho de muitos no mundo é que uma alternativa à (ir)racionalidade capi‑ talista possa ser definida e alcançada racionalmente por meio da mobilização das paixões humanas na busca coletiva de uma vida melhor para todos, trabalhando incessantemente para produzir um futuro diferente do que anuncia o capitalismo. O desenvolvimento desigual das práticas capitalistas ao redor do mundo tem pro‑ duzido movimentos anticapitalistas por toda parte, gerando descontentamentos diferen‑ tes em comparação com a agitação antineoliberal das lutas que ocorrem em boa parte da América Latina. O problema central é que, na totalidade, não há movimento anticapitalista sufi‑ cientemente unificado e decidido capaz de desafiar de modo adequado a reprodução da classe capitalista e a perpetuação do seu poder no cenário mundial. A primeira lição que deve ser aprendida é que um capitalismo ético, sem exploração e socialmente justo que beneficie a todos é impossível. Enfim, esclarecer o enigma do capital, tornando transparente o que o poder político sempre quer manter opaco, é crucial para qualquer estratégia revolucionária. SONIA ROHLING SOARES | Universidade Federal de Santa Catarina | Faculdade de Arquitetura e Urbanismo | Pós‑Graduação em Arquitetura e Urbanismo | Campus Trindade, Caixa Postal 476, 88040‑900, Florianópolis, SC, Brasil | E‑mail: <sonia_rohling@hotmail.com>. Recebido em 30/6/2014 e aprovado em 5/8/2014. SONIA ROHLING SOARES | Universidade Federal de Santa Catarina | Faculdade de Arquitetura e Urbanismo | Pós‑Graduação em Arquitetura e Urbanismo | Campus Trindade, Caixa Postal 476, 88040‑900, Florianópolis, SC, Brasil | E‑mail: <sonia_rohling@hotmail.com>. Recebido em 30/6/2014 e aprovado em 5/8/2014. SONIA ROHLING SOARES | Universidade Federal de Santa Catarina | Faculdade de Arquitetura e Urbanismo | Pós‑Graduação em Arquitetura e Urbanismo | Campus Trindade, Caixa Postal 476, 88040‑900, Florianópolis, SC, Brasil | E‑mail: <sonia_rohling@hotmail.com>. 30/6/2014 e aprovado em 5/8/2014. Oculum ens. | Campinas | 12(1) | 185-187 | Janeiro-Junho 2015 Oculum ens. | Campinas | 12(1) | 185-187 | Janeiro-Junho 2015
https://openalex.org/W2887316076
https://journals.openedition.org/pontourbe/pdf/3875
Portuguese
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As ruínas do castelo: contra-narrativa quilombola sobre o patrimônio cultural de Pelotas, RS
Ponto Urbe
2,018
cc-by
2,402
Núcleo de Antropologia Urbana da Universidade de São Paulo Núcleo de Antropologia Urbana da Universidade de São Paulo As ruínas do castelo: contra-narrativa quilombola sobre o patrimônio cultural de Pelotas, RS Patrícia dos Santos Pinheiro, Guilherme Rodrigues de Rodrigues, Claudia Turra Magni e Simone Assis Alves Roberto Ponto Urbe Revista do núcleo de antropologia urbana da USP  22 | 2018 Ponto Urbe 22 Ponto Urbe Revista do núcleo de antropologia urbana da USP  22 | 2018 Ponto Urbe 22 Refêrencia eletrónica Patrícia dos Santos Pinheiro, Guilherme Rodrigues de Rodrigues, Claudia Turra Magni e Simone Assis Alves Roberto, «As ruínas do castelo: contra-narrativa quilombola sobre o patrimônio cultural de Pelotas, RS», Ponto Urbe [Online], 22 | 2018, posto online no dia 15 agosto 2018, consultado o 09 agosto 2024. URL: http://journals.openedition.org/pontourbe/3875 ; DOI: https://doi.org/10.4000/ pontourbe.3875 pontourbe.3875 Este documento foi criado de forma automática no dia 9 de agosto de 2024. Apenas o texto pode ser utilizado sob licença CC BY 4.0. Outros elementos (ilustrações, anexos importados) são "Todos os direitos reservados", à exceção de indicação em contrário. As ruínas do castelo: contra-narrativa quilombola sobre o patrimônio cultural... As ruínas do castelo: contra-narrativa quilombola sobre o patrimônio cultural... 1 1 As ruínas do castelo: contra-narrativa quilombola sobre o patrimônio cultural... The castell ruins: quilombo counternarrative on cultural heritage in Pelotas, RS Patrícia dos Santos Pinheiro, Guilherme Rodrigues de Rodrigues, Claudia Turra Magni e Simone Assis Alves Roberto 1 Aos 65 anos, Dona Eva Maria Furtado Mourão é matriarca de uma Comunidade Quilombola situada na zona rural do município de São Lourenço do Sul, Rio Grande do Sul. Em 2016, retornou ao local vivido na infância, o outrora luxuoso Castelo Simões Lopes, situado no centro urbano do município vizinho, Pelotas1. Tombado como patrimônio histórico, foi construído em 1922, pelo Dr. Augusto Simões Lopes, intendente de Pelotas na década de 1920 e posteriormente senador. 1 Aos 65 anos, Dona Eva Maria Furtado Mourão é matriarca de uma Comunidade Quilombola situada na zona rural do município de São Lourenço do Sul, Rio Grande do Sul. Em 2016, retornou ao local vivido na infância, o outrora luxuoso Castelo Simões Lopes, situado no centro urbano do município vizinho, Pelotas1. Tombado como patrimônio histórico, foi construído em 1922, pelo Dr. Augusto Simões Lopes, intendente de Pelotas na década de 1920 e posteriormente senador. 2 Dona Eva reencontra um castelo em ruínas, diferente daquele que seus pais testemunharam, com grandes bailes e nomes importantes para a política regional e nacional. Essa lembrança se vincula a um passado em que imperavam barões e coronéis neste que foi um importante centro econômico fornecedor de charque (um tipo de carne conservada com sal), usado como alimento de escravos das províncias brasileiras. O charque movimentou um intenso sistema produtivo na região especialmente ao longo dos séculos XVIII e XIX, englobando desde estâncias agropastoris até os estabelecimentos industriais de produção de charque, atividades movidas por mãos cativas (Osório 2007). 3 Se no discurso patrimonial sobre a cidade impera a visão da elite pelotense, herdeira de imponentes casarões e hábitos refinados, na narrativa de D. Eva vislumbra-se outra perspectiva, aquela herdada de seus pais, que, oriundos de comunidades negras de São Lourenço e de Canguçu, foram empregados no castelo. Naquele período pós- abolicionista, a distinção racial ainda imperava e muitos trabalhadores marcados pelas relações de escravidão migraram para territórios negros nos arredores de Pelotas, onde 3 Se no discurso patrimonial sobre a cidade impera a visão da elite pelotense, herdeira de imponentes casarões e hábitos refinados, na narrativa de D. Eva vislumbra-se outra perspectiva, aquela herdada de seus pais, que, oriundos de comunidades negras de São Lourenço e de Canguçu, foram empregados no castelo. As ruínas do castelo: contra- narrativa quilombola sobre o patrimônio cultural de Pelotas, RS The castell ruins: quilombo counternarrative on cultural heritage in Pelotas, RS The castell ruins: quilombo counternarrative on cultural heritage in Pelotas, RS Naquele período pós- abolicionista, a distinção racial ainda imperava e muitos trabalhadores marcados pelas relações de escravidão migraram para territórios negros nos arredores de Pelotas, onde Ponto Urbe, 22 | 2018 As ruínas do castelo: contra-narrativa quilombola sobre o patrimônio cultural... 2 atualmente existem pelo menos 43 comunidades quilombolas, como o Rincão das Almas, onde Dona Eva foi morar na infância e acabou constituindo família. 4 Na visita que lhe propusemos ao Castelo, Dona Eva narra histórias que ouviu e viveu em contextos de exploração e discriminação, e, através de sua sensibilidade e dom espiritual, dá a ver o sofrimento, tantas vezes abafado e invisibilizado, dos negros que ergueram esse patrimônio, dentre outras obras da cidade aristocrática. Atualmente, ela é uma referência para a cura física e espiritual das tristezas que ainda se fazem presentes nesse contexto, como heranças perversas da escravidão no extremo sul do Brasil. 4 Na visita que lhe propusemos ao Castelo, Dona Eva narra histórias que ouviu e viveu em contextos de exploração e discriminação, e, através de sua sensibilidade e dom espiritual, dá a ver o sofrimento, tantas vezes abafado e invisibilizado, dos negros que ergueram esse patrimônio, dentre outras obras da cidade aristocrática. Atualmente, ela é uma referência para a cura física e espiritual das tristezas que ainda se fazem presentes nesse contexto, como heranças perversas da escravidão no extremo sul do Brasil. 5 Os efeitos duradouros desse processo surgem no percurso tortuoso que fizemos nas ruínas do castelo, guiados por Dona Eva. O objetivo deste ensaio fotográfico é mostrar uma contranarrativa ao discurso hegemônico sobre o patrimônio cultural pelotense, tendo por base as memórias apócrifas transmitidas pelos seus pais, assim como suas experiências espirituais, visões e sonhos. Ao entrar no Castelo com a missão de cura, Dona Eva quebra hierarquias instituídas nesse contexto e propõe uma expansão das narrativas históricas, convidando-nos a refletir sobre perspectivas epistêmicas que se constroem pela alteridade étnico-racial. 5 Os efeitos duradouros desse processo surgem no percurso tortuoso que fizemos nas ruínas do castelo, guiados por Dona Eva. O objetivo deste ensaio fotográfico é mostrar uma contranarrativa ao discurso hegemônico sobre o patrimônio cultural pelotense, tendo por base as memórias apócrifas transmitidas pelos seus pais, assim como suas experiências espirituais, visões e sonhos. The castell ruins: quilombo counternarrative on cultural heritage in Pelotas, RS Ao entrar no Castelo com a missão de cura, Dona Eva quebra hierarquias instituídas nesse contexto e propõe uma expansão das narrativas históricas, convidando-nos a refletir sobre perspectivas epistêmicas que se constroem pela alteridade étnico-racial. “Saí daqui com dois anos de idade. Não tenho lembrança de nada, mas as histórias que meu pai contava... [...] Ele era jardineiro e a minha mãe era tipo governanta, com outras, nesse castelo. E eles trabalharam bastante. [...] E meu pai era um preto velho muito sábio, inteligente, ele servia o dono aqui e ele prestava bastante atenção em tudo o que conversavam”. Foto: Guilherme Rodrigues, 2016. “Saí daqui com dois anos de idade. Não tenho lembrança de nada, mas as histórias que meu pai contava... [...] Ele era jardineiro e a minha mãe era tipo governanta, com outras, nesse castelo. E eles trabalharam bastante. [...] E meu pai era um preto velho muito sábio, inteligente, ele servia o dono aqui e ele prestava bastante atenção em tudo o que conversavam”. Foto: Guilherme Rodrigues, 2016. Ponto Urbe, 22 | 2018 As ruínas do castelo: contra-narrativa quilombola sobre o patrimônio cultural... 3 “[...] Na juventude a gente, moça, sonha com um casamento feliz, constrói um castelinho, e se torna um castelinho de areia. Mas fazer o quê? A gente tem que se erguer e se tornar o castelo, de verdade. [...] Lá mesmo no quilombo, o que nós temos? Tem branco, negro, tudo misturado. […] Veio a mistura no tempo da escravidão. […] O negro é coragem, é força e é uma mistura, entende? Porque Deus ama a todos”. Foto: Guilherme Rodrigues, 2016. “[...] Na juventude a gente, moça, sonha com um casamento feliz, constrói um castelinho, e se torna um castelinho de areia. Mas fazer o quê? A gente tem que se erguer e se tornar o castelo, de verdade. [...] Lá mesmo no quilombo, o que nós temos? Tem branco, negro, tudo misturado. […] Veio a mistura no tempo da escravidão. […] O negro é coragem, é força e é uma mistura, entende? Porque Deus ama a todos”. Foto: Guilherme Rodrigues, 2016. “As pessoas lêem, falam da escravidão, mas sentir o que eu sinto… Mas o que é o escravo? Escravos que me procuravam, eles vinham chorando, né? Morriam no tronco, com brilho [...]. Mas aquilo foi no passado. [...] E o negro brilhando, mesmo morrendo no tronco, acorrentado, mas ele morria valente. The castell ruins: quilombo counternarrative on cultural heritage in Pelotas, RS Diz que é para orar ali, sentir uma paz de espírito, talvez até para mim”. Foto: Patrícia Pinheiro, 2016. “Eu vou orar aqui. Aconteceu algo triste por aqui. [...] O Senhor me mostrou essa peça, Senhor. Até os desenhos da janela. Só o Senhor sabe o porquê, eu não sei. Sou tão pequena, meu Deus. Eu te peço que neste momento, Senhor, se algo aconteceu aqui, que o Senhor dê o descanso eterno a todas as almas, que aqui fizeram talvez o que não deviam, Senhor, de errado. Meu Deus, toma conta deste Castelo, Pai. Faz com que, meu Deus, neste momento, que aonde eles tiverem, eles recebam o descanso eterno do Senhor. Meu Deus, o Senhor me botou sobre a Terra, Pai, com esse dom que só o Senhor sabe. O dom da profecia, Pai [...]”. Foto: Patrícia Pinheiro, 2016. “Por que eu tive uma visão. Eu vi lá naquela casa, me mostraram uma peça. Diz que é para orar ali, sentir uma paz de espírito, talvez até para mim”. Foto: Patrícia Pinheiro, 2016. “Por que eu tive uma visão. Eu vi lá naquela casa, me mostraram uma peça. Diz que é para orar ali, sentir uma paz de espírito, talvez até para mim”. Foto: Patrícia Pinheiro, 2016. “Eu vou orar aqui. Aconteceu algo triste por aqui. [...] O Senhor me mostrou essa peça, Senhor. Até os desenhos da janela. Só o Senhor sabe o porquê, eu não sei. Sou tão pequena, meu Deus. Eu te peço que neste momento, Senhor, se algo aconteceu aqui, que o Senhor dê o descanso eterno a todas as almas, que aqui fizeram talvez o que não deviam, Senhor, de errado. Meu Deus, toma conta deste Castelo, Pai. Faz com que, meu Deus, neste momento, que aonde eles tiverem, eles recebam o descanso eterno do Senhor. Meu Deus, o Senhor me botou sobre a Terra, Pai, com esse dom que só o Senhor sabe. O dom da profecia, Pai [...]”. Foto: Patrícia Pinheiro, 2016. “Eu vou orar aqui. Aconteceu algo triste por aqui. [...] O Senhor me mostrou essa peça, Senhor. Até os desenhos da janela. Só o Senhor sabe o porquê, eu não sei. Sou tão pequena, meu Deus. Eu te peço que neste momento, Senhor, se algo aconteceu aqui, que o Senhor dê o descanso eterno a todas as almas, que aqui fizeram talvez o que não deviam, Senhor, de errado. The castell ruins: quilombo counternarrative on cultural heritage in Pelotas, RS Um brilho no corpo, do suor, porque o negro, quando brilha, como uma estrela, brilhava, misturado com sangue, com chicote. Dali eles desencarnavam para a luz […]”. Foto: Guilherme Rodrigues, 2016. “As pessoas lêem, falam da escravidão, mas sentir o que eu sinto… Mas o que é o escravo? Escravos que me procuravam, eles vinham chorando, né? Morriam no tronco, com brilho [...]. Mas aquilo foi no passado. [...] E o negro brilhando, mesmo morrendo no tronco, acorrentado, mas ele morria valente. Um brilho no corpo, do suor, porque o negro, quando brilha, como uma estrela, brilhava, misturado com sangue, com chicote. Dali eles desencarnavam para a luz […]”. Foto: Guilherme Rodrigues, 2016. Ponto Urbe, 22 | 2018 As ruínas do castelo: contra-narrativa quilombola sobre o patrimônio cultural... As ruínas do castelo: contra-narrativa quilombola sobre o patrimônio cultural... Ruínas. Foto: Guilherme Rodrigues, 2016. “Ele me chamava ‘negrinha’. Eu tinha uns nove meses. Ele era uma pessoa de idade, diz que ele enxergava coisas, se assustava […]. Então ele carregava eu no colo, a minha mãe disse que às vezes me procurava, ele tava numa cadeirinha de balanço, sentado, comigo no colo. [...] A minha mãe perguntava: 'mas o que o senhor quer com essa criança no colo?', e ele dizia: 'com ela no colo, eles não vêm me pegar'. E a minha mãe começou a notar algo em mim”. Foto: Simone Assis, 2016. R í F G ilh R d i 2016 Ruínas. Foto: Guilherme Rodrigues, 2016. Ruínas. Foto: Guilherme Rodrigues, 2016. Ruínas. Foto: Guilherme Rodrigues, 2016. “Ele me chamava ‘negrinha’. Eu tinha uns nove meses. Ele era uma pessoa de idade, diz que ele enxergava coisas, se assustava […]. Então ele carregava eu no colo, a minha mãe disse que às vezes me procurava, ele tava numa cadeirinha de balanço, sentado, comigo no colo. [...] A minha mãe perguntava: 'mas o que o senhor quer com essa criança no colo?', e ele dizia: 'com ela no colo, eles não vêm me pegar'. E a minha mãe começou a notar algo em mim”. Foto: Simone Assis, 2016. Ponto Urbe, 22 | 2018 As ruínas do castelo: contra-narrativa quilombola sobre o patrimônio cultural... As ruínas do castelo: contra-narrativa quilombola sobre o patrimônio cultural... 5 “Por que eu tive uma visão. Eu vi lá naquela casa, me mostraram uma peça. BIBLIOGRAFIA OSÓRIO, H.. 2007. O império português no sul da América: estancieiros, lavradores e comerciantes. Porto Alegre: Editora da Ufrgs. OSÓRIO, H.. 2007. O império português no sul da América: estancieiros, lavradores e comerciantes. Porto Alegre: Editora da Ufrgs. The castell ruins: quilombo counternarrative on cultural heritage in Pelotas, RS Meu Deus, toma conta deste Castelo, Pai. Faz com que, meu Deus, neste momento, que aonde eles tiverem, eles recebam o descanso eterno do Senhor. Meu Deus, o Senhor me botou sobre a Terra, Pai, com esse dom que só o Senhor sabe. O dom da profecia, Pai [...]”. Foto: Patrícia Pinheiro, 2016. Ponto Urbe, 22 | 2018 As ruínas do castelo: contra-narrativa quilombola sobre o patrimônio cultural... As ruínas do castelo: contra-narrativa quilombola sobre o patrimônio cultural... NOTAS 1. Uma primeira composição relativa a essa visita foi realizada em uma exposição fotográfica feita em 2016, no VIII Visualidades, em Sobral/CE, com os mesmos autores deste ensaio. Ponto Urbe, 22 | 2018
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https://www.frontiersin.org/articles/10.3389/fpls.2014.00196/pdf
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The activity of SnRK1 is increased in Phaseolus vulgaris seeds in response to a reduced nutrient supply
Frontiers in plant science
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INTRODUCTION may cause either loss of ABA function and/or disconnection between metabolic signals and ABA, resulting in the prolonged expression of genes related to cell proliferation (Radchuk et al., 2006). Seed development is a complex and highly resource-demanding process that requires large amounts of C and N. Because of struc- tural restrictions, legume embryos grow in an environment in which O2 represents only 0.4% of the atmospheric concentration, resulting in low ATP levels (Rolletschek et al., 2003). The adapta- tions of becoming green and photosynthetically active provide the O2 and energy required to increase biosynthetic fluxes. Early in development (pre-storage phase), embryos contain high levels of glucose (Weber et al., 1995). Later, sucrose becomes more abun- dant, and the switch from hexoses to sucrose is accompanied by cell differentiation and the synthesis of storage products (Borisjuk et al., 1995). In cotyledons of Vicia faba, high glucose concen- trations are found in non-differentiated regions, and glucose concentrations are particularly low in mature starch-accumulating regions (Borisjuk et al., 1998). By contrast, starch-accumulating cells contain the highest sucrose concentrations (Borisjuk et al., 2002). ATP distribution is also important in regulating the accu- mulation of storage products. In V. faba cotyledons, protein accumulation occurs in the foremost regions, where ATP is more readily available, and starch synthesis is more active in the internal sections, where ATP levels are lower (Borisjuk et al.,2003). Sucrose non-fermenting related kinase 1 (SnRK1) is also a modulator of abscisic acid (ABA) functions, linking nutrient and/or energy state to ABA-regulated responses, and reduction in SnRK1 activity Variations in environmental factors (drought, high temper- ature, disease caused by pathogens, etc.) affect photosynthetic activity and may produce a significant reduction in the supply of nutrients required for seed development. Plants use different strategies to cope with conditions of stress, including nutrient remobilisation (Yang et al., 2001a). This process is normally asso- ciated with leaf senescence, where most of the available nutrients are transported to developing seeds (Yang et al., 2001b), but mate- rials accumulated in stems and pods are also important for seed development (Schiltz et al., 2005). Seeds of Phaseolus vulgaris can continue their development at the expense of pod reserves when the fruits are removed from the plant at 15–25 days after flow- ering (DAF; Fountain et al., 1989). ORIGINAL RESEARCH ARTICLE published: 16 May 2014 Patricia Coello and Eleazar Martínez-Barajas* Departamento de Bioquímica, Facultad de Química, Universidad Nacional Autónoma de México, Ciudad de México, México Phaseolus vulgaris seeds can grow and develop at the expense of the pod reserves after the fruits have been removed from the plant (Fountain et al., 1989). Because this process involves sensing the reduction of nutrients and the remobilisation of pod reserves, we investigated the effect on sucrose non-fermenting related kinase 1 (SnRK1) activity during this process. Bean fruits removed from the plant at 20 days after flowering (DAF) demonstrated active remobilisation of nutrients from the pod to the seeds. After 5 days, the pod dry weight was reduced by 50%.The process was characterized by a rapid degradation of starch, with the greatest decrease observed on day 1 after the fruits were removed. The pod nutrients were insufficient for the needs of all the seeds, and only some seeds continued their development.Those seeds exhibited a transient reduction in sucrose levels on day 1 after the fruits were removed. However, the normal level of sucrose was recovered, and the rate of starch synthesis was identical to that of a seed developed under normal conditions. Removing the fruits from the plant had no effect on the activity of SnRK1 in the pods, whereas in the seeds, the activity was increased by 35%. Simultaneously, a large reduction in seed sucrose levels was observed. The increase in SnRK1 activity was observed in both the cotyledon and embryo axes, but it was higher in the cotyledon. At 20– 25 DAF, cotyledons actively accumulate storage materials. It is possible that the increase in SnRK1 activity observed in seeds developed in fruits that have been removed from the plant is part of the mechanism required for nutrient remobilisation under conditions of stress. Keywords: nutrient remobilisation, SnRK1, bean seed development Keywords: nutrient remobilisation, SnRK1, bean seed development Edited by: y Matthew Paul, Rothamsted Research, UK Matthew Paul, Rothamsted Research, UK The activity of SnRK1 is increased in Phaseolus vulgaris seeds in response to a reduced nutrient supply Patricia Coello and Eleazar Martínez-Barajas* Keywords: nutrient remobilisation, SnRK1, bean seed development Reviewed by: Frederik Börnke, Leibniz-Institute for Vegetable and Ornamental Crops, Germany Pierre Crozet, Fundação Calouste Gulbenkian – Instituto Gulbenkian de Ciência, Portugal Frederik Börnke, Leibniz-Institute for Vegetable and Ornamental Crops, Germany Frederik Börnke, Leibniz-Institute for Vegetable and Ornamental Crops, Germany Pierre Crozet, Fundação Calouste Gulbenkian – Instituto Gulbenkian de Ciência, Portugal *Correspondence: *Correspondence: Eleazar Martínez-Barajas, Departamento de Bioquímica, Facultad de Química, Universidad Nacional Autónoma de México, Ciudad de México, D.F. 04510, México e-mail: emtz@unam.mx Keywords: nutrient remobilisation, SnRK1, bean seed development RESULTS Bean seeds (P. vulgaris cv V8025) were grown in a greenhouse at 25◦C under a 14 h natural light/10 h dark regime in 3 L plastic potswithagrolite. Theywereirrigateddailywith150mLHoagland solution (Jones, 1982). Flowers were tagged at anthesis, and fruits were removed from the plant at 20 DAF and incubated at 25◦C in darkness. In fruits removed from the plant only seeds that were able to continuing their development were analysed. By 20–25 DAF, pods have reached their final size (Fountain et al., 1989), and fruit growth is mainly associated with the active accu- mulation of storage materials in seeds (Figure 1A). In fruits removed from the plant at 20 DAF,seeds can growth at the expense of the nutrients provided for the pod. In the 5 day period after the fruits were detached, the pods lost 50% of their dry weight, whereas a significant dry weight increase was observed in the seeds (Figure 1A). However, the material transported from the pod is not sufficient to fully cover the needs of the seeds. Figure 1B shows that after 5 days, only some seeds from the removed fruit were able to continue development, and a large dispersion in the size of indi- vidual seeds is observed (Figure 1C). Bean pods have a significant amount of starch, and approximately one third of it is normally degraded in the period from 20 to 25 DAF. In detached fruits, the process was accelerated, and two-thirds of the starch was hydrol- ysed during the first day after fruit removal (Figure 2). The levels of glucose, fructose and sucrose remained almost constant, and no changes were observed between the pods of fruits developed in the plant and the pods of fruits that were removed (Figure 2). Under normal conditions, sucrose tends to be reduced as seed matures. However, in the seeds of fruits that were removed from the plant, an important reduction was observed at day 1. Sucrose levels recovered gradually, and 3 days after the fruits were removed, the levels were identical to those in seeds developed under normal conditions (Figure 3). In seeds that were able to continue devel- oping in removed fruits, the rate of starch accumulation decreased during the first 3 days and then returned to values similar to those observed in seeds developed under normal conditions (Figure 3). SUGAR DETERMINATION Pod and seed samples (200 mg) were homogenized in 3 mL 80% ethanol and extracted twice at 80◦C for 15 min. The soluble frac- tion was used to determine glucose, fructose, and sucrose. Starch was measured from the insoluble pellet using procedures previ- ously reported (Bernal et al., 2005). Two fruits from three different plants were analyzed. SnRK1 ASSAY The SnRK1 activity was assayed following a procedure previously reported (Zhang et al., 2009) in 25 μl in microtiter plate wells at 30◦C. Assay medium was 40 mM Hepes-NaOH, pH 7.5, 5 mM MgCl2, 200 μM ATP containing 0.337 μCi[γ33P]ATP (Perkin Elmer), 200 μM AMARA peptide (AMARAASAAALARRR), 5 mM DTT, 1X protease inhibitor cocktail (Sigma, Mexico) and phosphatase inhibitors (5 mM sodium fluoride, 2.5 mM β- glycerophosphate and 0.2 mM sodium orthovanadate). Assays were started with extract (5 μg protein) and after 6 min, 15 μl was transferred to 4 cm2 Whatman P81 phosphocellulose paper, immediately immersed in 1% phosphoric acid, then washed with three800mlvolumesof 1%phosphoricacid,immersedinacetone, dried, and transferred to liquid scintillation vials. RESULTS SnRK1 activity in pods showed a tendency to decrease from 20 to 25 DAF, and a similar response was observed in fruits removed INTRODUCTION SnRK1 has been identified as an important component in the mechanism that allows plants to respond to C and energy deficiencies (Schluepmann et al., 2012). SnRK1 kinases function as heterotrimeric complexes composed of one catalytic subunit (α) and two regulatory subunits (β- and γ-type subunits; Polge and Thomas, 2007). These kinases can phosphorylate and inactivate important enzymes, such as 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA) reductase, sucrose phosphate synthase (SPS),and nitrate reductase May 2014 | Volume 5 | Article 196 | 1 www.frontiersin.org Coello and Martínez-Barajas Nutrient remobilisation (NR; Sugden et al., 1999). SnRK1 also phosphorylates trehalose phosphate synthase (TPS5; Harthill et al., 2006), fructose-6- phosphate, 2-kinase/fructose-2,6-bisphosphatase (F2KP; Kulma et al., 2004) and non-phosphorylating glyceraldehyde 3-P dehy- drogenase (Piattoni et al., 2011) and promote their association to 14-3-3 proteins. SnRK1 kinase activity is also required for the redox regulation of ADP glucose PPase (Tiessen et al., 2003). In pea cotyledons, SnRK1 coordinates and adjusts the physiologi- cal and metabolic demands of growth (Radchuk et al., 2010), and by mediating transcriptional reprograming in the cells, SnRK1 (AKIN10) also helps plants to survive adverse-energy deplet- ing conditions (Baena-González et al., 2007). SnRK1 activity is under complex regulation, it increases in response to the phos- phorylation of the catalytic subunit mediated for GRIK kinases (Shen et al., 2009) and is inhibited by trehalose 6-P (T6P), glu- cose 6-P, and glucose 1-P (Toroser et al., 2000; Zhang et al., 2009; Nunes et al., 2013b). T6P inhibition of SnRK1 is impor- tant in young tissue, and the dissociation constant (Ki) for the SnRK1-T6P complex is calculated to be closer to 4 (μM; Nunes et al., 2013a). In this context, the main objective of this work is to investigate how SnRK1 activity is affected by the reduc- tion in nutrient supply when bean fruits are removed from the plant. (Sigma, Mexico), and the desalted material was used for the SDS- PAGE (Laemmli, 1970). Proteins were visualized by staining with coomassie blue. Specific antibodies for the SnRK1 catalytic sub- unit were used for western-blot according to previously reported procedures (Fragoso et al., 2009). The phosphorylation of the cat- alytic subunit was evaluated with with an anti-phospho-AMPKα (T172) antibody (Cell Signaling). Densitometric analysis was per- formed using Image Lab software (Bio-Rad), and the blot signal was normalized by the amount of protein detected by coomassie blue staining. PREPARATION OF PLANT EXTRACTS Experiments were replicated three times with identical results. in a large variation in individual seed dry weight (C). Bars represent an average of the analysis of 25 fruits ± SD, * and ** denote significant differences at p < 0.05 and 0.01 by ANOVA, respectively. Experiments were replicated three times with identical results. in a large variation in individual seed dry weight (C). Bars represent an average of the analysis of 25 fruits ± SD, * and ** denote significant differences at p < 0.05 and 0.01 by ANOVA, respectively. Experiments were replicated three times with identical results. FIGURE 1 |Transference of pod material to seeds. Fruits were removed from the plant at 20 DAF; 5 days later (20 + 5R), there was a 50% reduction in pod dry weight and a significant increment in dry weight of seeds (A). Only some seeds in the removed pods continued their development (B), resulting in a large variation in individual seed dry weight (C). Bars represent an average of the analysis of 25 fruits ± SD, * and ** denote significant differences at p < 0.05 and 0.01 by ANOVA, respectively. Experiments were replicated three times with identical results. FIGURE 2 | Effect of fruit removal on pod glucose, fructose, starch, and sucrose levels. Bean fruits were removed from the plant at 20 DAF (□), and carbohydrate levels were compared to fruits developed attached to the plant (■). Data points represent the average of three fruits from different plants ± SD, **p < 0.01 by ANOVA. Experiments were replicated three times with identical results. FIGURE 2 | Effect of fruit removal on pod glucose, fructose, starch, and sucrose levels. Bean fruits were removed from the plant at 20 DAF (□), and carbohydrate levels were compared to fruits developed attached to the plant (■). Data points represent the average of three fruits from different plants ± SD, **p < 0.01 by ANOVA. Experiments were replicated three times with identical results. FIGURE 2 | Effect of fruit removal on pod glucose, fructose, starch, and sucrose levels. Bean fruits were removed from the plant at 20 DAF (□), and carbohydrate levels were compared to fruits developed attached to the plant (■). Data points represent the average of three fruits from different plants ± SD, **p < 0.01 by ANOVA. Experiments were replicated three times with identical results. PREPARATION OF PLANT EXTRACTS Plant material was frozen with liquid nitrogen and soluble protein was extracted at 4◦C in homogenisation buffer containing 100 mM Tricine-NaOH (pH 8.0), 5 mM DTT, 0.5 mM EGTA, 0.5 mM EDTA, 10% glycerol, 0.02% Brij 35 and 1 mM benzamidine. Prior to the homogenisation 1 mM PMSF,1X protease inhibitor cocktail (Sigma, Mexico), phosphatase inhibitors (5 mM sodium fluoride, 2.5 mM β-glycerophosphate and 0.2 mM sodium orthovanadate) and insoluble polyvinylpyrrolidone (2% w/v) were added. The homogenate was transferred to microfuge tubes, and insoluble material was removed by centrifugation (13,000 × g) at 4◦C for 20 min. The supernatant was desalted using an NAP-5 column (GE Healthcare) that was previously equilibrated with homogenisation buffer. Protein content was determined using Bradford reagent May 2014 | Volume 5 | Article 196 | 2 Frontiers in Plant Science | Plant Physiology Coello and Martínez-Barajas Nutrient remobilisation FIGURE 1 |Transference of pod material to seeds. Fruits were removed from the plant at 20 DAF; 5 days later (20 + 5R), there was a 50% reduction in pod dry weight and a significant increment in dry weight of seeds (A). Only some seeds in the removed pods continued their development (B), resulting in a large variation in individual seed dry weight (C). Bars represent an average of the analysis of 25 fruits ± SD, * and ** denote significant differences at p < 0.05 and 0.01 by ANOVA, respectively. Experiments were replicated three times with identical results. FIGURE 2 | Effect of fruit removal on pod glucose, fructose, starch, and sucrose levels. Bean fruits were removed from the plant at 20 DAF (□), and carbohydrate levels were compared to fruits developed attached to the plant (■). Data points represent the average of three fruits from different plants ± SD, **p < 0.01 by ANOVA. Experiments were replicated three times with identical results. FIGURE 1 |Transference of pod material to seeds. Fruits were removed from the plant at 20 DAF; 5 days later (20 + 5R), there was a 50% reduction in pod dry weight and a significant increment in dry weight of seeds (A). Only some seeds in the removed pods continued their development (B), resulting in a large variation in individual seed dry weight (C). Bars represent an average of the analysis of 25 fruits ± SD, * and ** denote significant differences at p < 0.05 and 0.01 by ANOVA, respectively. DISCUSSION At 20 DAF, bean pods have completed their development, and the period from 20 to 25 DAF is characterized by the active growth of the seeds (Figure 1A). Later in development, pods can transfer some materials to support seed needs (Schiltz et al., 2005), how- ever, according to previous research (Fountain et al., 1989), when bean fruits are removed from the plant within this period, the pod is converted to provide nutrients for seed development. The present study found that this process is characterized by the rapid reduction of starch, and almost two-thirds of the initially present in pod tissue was degraded during the first day after the fruit was detached (Figure 2). In addition to starch, the degradation of other materials may also contribute to seed development. Bean pods also contain galactose-rich pectin polymers that are degraded at the maturation of the fruits (Stolle-Smits et al., 1999), and pro- teins stored in soybean pods make a significant contribution to the pool of nutrients mobilized for developing seeds (Staswick, 1989). It has been estimated that in Pisum sativum, the N remobilised from the pods contributes to 20% of the seed N (Schiltz et al., 2005). By removing the fruits from the plant, both pod and seeds were subjected to a severe nutritional deficiency. This nutrient loss was partially compensated by the acceleration of starch degrada- tion in the pods (Figure 2). It is also possible that some seeds are sacrificed to increase the probability to develop some viable seeds (Figure 1B). However, the reserves transferred from the pods are insufficient to fully supply the needs of all seeds (Figure 1), and a largereductioninsucrosewasobservedat1dayafterthefruitswere detached. Sucrose gradually increased, and after 3 days returned to those levels observed in normally developing seeds, when the rate of starch accumulation was also recovered (Figure 3). SnRK1 con- trols the early steps of cotyledon growth and differentiation and is required to transmit a nutrient-derived signal that stimulates the gene expression involved in nutrient partitioning (Radchuk et al., 2010). SnRK1 also mediates low-energy stress responses (Baena- González and Sheen,2008). Its activity in the pods was not affected by removing the fruits from the plant (Figure 4A). However, in seeds that were able to develop in detached fruits a 35% increase was observed 1 day after its removal (Figure 4B). DISCUSSION It coincided with the largest reduction in sucrose levels detected in the seeds, and the difference disappeared as soon as the sucrose levels were restored (Figures 3 and 4B). Sugar starvation and ABA transcriptionally activates SnRK1 (Radchuk et al., 2010). It might contribute to the higher level of catalytic subunit observed in seeds of detached fruits (Figure 4D and Supplementary Figure 1). However, the increment in SnRK1 activity observed in those seeds was associ- ated with larger proportion of the catalytic subunit that appears phosphorylated (Figure 4E and Supplementary Figure 1). In bean seeds, SnRK1 activity reaches its highest point around 20 DAF (Coello and Martínez-Barajas, 2014), and it peaks at 18 FIGURE 3 | Effect of fruit removal on the levels of sucrose and starch in seeds. Bean fruits were removed from the plant at 20 DAF (□), and carbohydrate levels were compared to fruits developed attached to the plant (■). Data points represent the average of three fruits from different plants ± SD, *p < 0.05 and **p < 0.01 by ANOVA, respectively. Experiments were replicated three times with identical results. from the plant (Figure 4A). In seeds, fruit removal resulted in a 35% increase in SnRK1 activity at day 1 (Figure 4B). The increase in SnRK1 activity coincided with the largest reduction in sucrose (Figure 3). The abundance of total and phosphorylated SnRK1 catalytic subunit was also analyzed. In normally developed seeds, SnRK1 catalytic subunit showed a tendency to decrease after 20 DAF, while in the seeds developed in removed fruits the pro- cess is slower (Figure 4D and Supplementary Figure 1). In the period from 20 to 25 DAF the phosphorylation of the catalytic subunit is low in seeds developed normally, but it increased at 1–3 days after the fruits were removed from the plant (Figure 4E and Supplementary Figure 1). SnRK1 activity was also measured in cotyledons and embryo axes of seeds of 21 DAF and in seeds of 20 DAF fruits the day after removal from the plant. In both cases, removing the fruits from the plant increased the activity with a larger increase observed for cotyledons (Figure 5A). The western-blot analysis shows that the increment in SnRK1 activ- ity was not associated with changes in the abundance of catalytic subunit (Figure 5C). PREPARATION OF PLANT EXTRACTS (■) l May 2014 | Volume 5 | Article 196 | 3 www.frontiersin.org Coello and Martínez-Barajas Nutrient remobilisation FIGURE 3 | Effect of fruit removal on the levels of sucrose and starch in seeds. Bean fruits were removed from the plant at 20 DAF (□), and carbohydrate levels were compared to fruits developed attached to the plant (■). Data points represent the average of three fruits from different plants ± SD, *p < 0.05 and **p < 0.01 by ANOVA, respectively. Experiments were replicated three times with identical results. DISCUSSION T6P concentration is highly variable, depending on tissue type and environmental conditions (Martinez-Barajas et al., 2011; Nunes et al., 2013a), and generally, T6P levels correlate well with levels May 2014 | Volume 5 | Article 196 | 4 Frontiers in Plant Science | Plant Physiology Coello and Martínez-Barajas Nutrient remobilisation from fruits of 20 to 25 DAF developed under normal conditions (control) or in fruits removed at 20 DAF (removed) and analyzed 1–5 days after (C). Western-blot for total (D) and phosphorylated (E) SnRK1 catalytic subunit. Experiments were repeated two times with identical results. from fruits of 20 to 25 DAF developed under normal conditions (control) or in fruits removed at 20 DAF (removed) and analyzed 1–5 days after (C). Western-blot for total (D) and phosphorylated (E) SnRK1 catalytic subunit. Experiments were repeated two times with identical results. FIGURE 4 | Effect of fruit removal on the activity of SnRK1 in pods (A) and seeds (B) from fruits removed from the plant at 20 DAF (□) and developed normally (■). Data points represent the average of three fruits from different plants ± SD, **p < 0.01 by ANOVA. SDS-PAGE stained with coomassie blue of seed proteins from fruits of 20 to 25 DAF developed under normal conditions (control) or in fruits removed at 20 DAF (removed) and analyzed 1–5 days after (C). Western-blot for total (D) and phosphorylated (E) SnRK1 catalytic subunit. Experiments were repeated two times with identical results. from fruits of 20 to 25 DAF developed under normal conditions (control) or in fruits removed at 20 DAF (removed) and analyzed 1–5 days after (C). Western-blot for total (D) and phosphorylated (E) SnRK1 catalytic subunit. Experiments were repeated two times with identical results. FIGURE 4 | Effect of fruit removal on the activity of SnRK1 in pods (A) and seeds (B) from fruits removed from the plant at 20 DAF (□) and developed normally (■). Data points represent the average of three fruits from different plants ± SD, **p < 0.01 by ANOVA. SDS-PAGE stained with coomassie blue of seed proteins The highest concentrations of T6P have been reported early in wheat seed development, in which SnRK1 activity is also high (Martinez-Barajas et al., 2011). There is no information available regarding T6P levels in P. vulgaris seeds. DISCUSSION However, large increment in the phosphory- lation of the catalytic subunit was observed in cotyledon extracts, while in embryo axe the phosphorylation of the catalytic subunit is reduced (Figure 5D). from the plant (Figure 4A). In seeds, fruit removal resulted in a 35% increase in SnRK1 activity at day 1 (Figure 4B). The increase in SnRK1 activity coincided with the largest reduction in sucrose (Figure 3). The abundance of total and phosphorylated SnRK1 catalytic subunit was also analyzed. In normally developed seeds, SnRK1 catalytic subunit showed a tendency to decrease after 20 DAF, while in the seeds developed in removed fruits the pro- cess is slower (Figure 4D and Supplementary Figure 1). In the period from 20 to 25 DAF the phosphorylation of the catalytic subunit is low in seeds developed normally, but it increased at 1–3 days after the fruits were removed from the plant (Figure 4E and Supplementary Figure 1). SnRK1 activity was also measured in cotyledons and embryo axes of seeds of 21 DAF and in seeds of 20 DAF fruits the day after removal from the plant. In both cases, removing the fruits from the plant increased the activity with a larger increase observed for cotyledons (Figure 5A). The western-blot analysis shows that the increment in SnRK1 activ- ity was not associated with changes in the abundance of catalytic subunit (Figure 5C). However, large increment in the phosphory- lation of the catalytic subunit was observed in cotyledon extracts, while in embryo axe the phosphorylation of the catalytic subunit is reduced (Figure 5D). In bean seeds, SnRK1 activity reaches its highest point around 20 DAF (Coello and Martínez-Barajas, 2014), and it peaks at 18 DAF in pea seeds (Radchuk et al., 2010). Eventhoug SnRK1 activ- ity is close to the highest value that can be reached in bean seed development, sugar deprivation can produce further increments (Figure 4B). It has been estimated that in vivo, up to 80% or more of SnRK1 activity is inhibited by T6P (Nunes et al., 2013a). DISCUSSION However, since we did not observed changes in sensitivity of SnRK1 activity to T6P in the seeds of detached fruits (data not shown), we cautiously spec- ulate that a reduction in T6P when sucrose declines (Figure 3), of sucrose in plant tissues (Lunn et al., 2006; Martinez-Barajas et al., 2011; Wingler et al., 2012; Nunes et al., 2013a). It has been suggested that the inhibition of SnRK1 activity by T6P allows the cells to initiate the anabolic processes required for growth. When carbon availability decreases, T6P is also reduced, and active SnRK1 participates in the processes required to make car- bon available to sink cells into growth (Schluepmann et al., 2012). FIGURE 5 | Analysis of SnRK1 activity in cotyledon and embryo axe from seeds developed under normal conditions for 21 DAF (21) or in fruits removed from the plant at 20 DAF and analyzed at day 1 after (20 + 1R). Bars represent the average of three independent SnRK1 activity determinations ± SD, *p < 0.05 and **p < 0.01 by ANOVA, respectively (A). SDS-PAGE stained with coomassie blue (B) and western-blot for total (C) and phosphorylated (D) SnRK1 catalytic subunit. Experiments were repeated two times with identical results. www.frontiersin.org May 2014 | Volume 5 | Article 196 | 5 FIGURE 5 | Analysis of SnRK1 activity in cotyledon and embryo axe from seeds developed under normal conditions for 21 DAF (21) or in fruits removed from the plant at 20 DAF and analyzed at day 1 after (20 + 1R). Bars represent the average of three independent SnRK1 activity determinations ± SD, *p < 0.05 and **p < 0.01 by ANOVA, respectively (A). SDS-PAGE stained with coomassie blue (B) and western-blot for total (C) and phosphorylated (D) SnRK1 catalytic subunit. Experiments were repeated two times with identical results. May 2014 | Volume 5 | Article 196 | 5 www.frontiersin.org Nutrient remobilisation Coello and Martínez-Barajas will increase the relevance of SnRK1 activity to promote seed development under nutrient deficiency. The data presented here suggest that it is complemented by the changes in the phosphory- lation status of the catalytic subunit (Figure 4E). The regulation of the phosphorylation of the catalytic subunit could be impor- tant to promote differential responses in cotyledon and embryo axe to the nutrient deprivation. Finally, plants provide a number of nutrients to support seed development (sugars, amino acids, minerals, water, among others). REFERENCES Nunes, C., Primavesi, L. F., Patel, M. K., Martinez-Barajas, E., Powers, S. J., Sagar, R., et al. (2013b). Inhibition of SnRK1 by metabolites: tissue-dependent effects and cooperative inhibition by glucose 1-phosphate in combination with trehalose 6- phosphate. Plant Physiol. Biochem. 63, 89–98. doi: 10.1016/j.plaphy.2012.11.011 Baena-González, E., Rolland, F., Thecelein, J. M., and Sheen, J. (2007). A central integrator of transcription networks in plant stress and energy signalling. Nature 448, 938–942. doi: 10.1038/nature06069 Baena-González, E., and Sheen, J. (2008). Convergent energy and stress signalling. 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ATP distribution withing Vicia faba embryos is developmentally regulated and correlated with photosynthetic capacity. Plant J. 36, 318–329. doi: 10.1046/j.1365- 313X.2003.01879.x Radchuk, R., Emery, R. J. N., Weier, D., Vigeolas, H., Geigenberger, P., Lunn, J. E., et al. (2010). Sucrose non-fermenting kinase 1 (SnRK1) coordinates metabolic and hormonal signals during pea cotyledon growth and differentiation. Plant J. 61, 324–338. doi: 10.1111/j.1365-313X.2009.04057.x Borisjuk, L., Walenta, S., Roelletschek, H., Mueller-Klieser, W., Wobus, U., and Weber, H. (2002). Spatial analysis of plant development: sucrose imaging whiting Vicia faba cotyledons reveals specific development patterns. Plant J. 15, 583–591. doi: 10.1046/j.1365-313X.1998.00214.x Radchuk, R., Radchuk, V., Weschke, W., Borisjuk, L., and Weber, L. (2006). Repress- ing the expression of the sucrose nonfermenting-1-related protein kinase gene in pea embryo causes pleiotropic defects of maturation similar to an abscisic acid insensitive phenotype. Plant Physiol. 140, 263–278. doi: 10.1104/pp.105. 071167 Borisjuk, L., Walenta, S., Weber, H., Mueller-Klieser, W., and Wobus, W. (1998). High-resolution histographical mapping of glucose concentrations in developing cotyledons of Vicia faba in relation to mitotic activity and storage process: glucose as a possible developmental trigger. Plant J. 15, 583–591. doi: 10.1046/j.1365- 313X.1998.00214.x Rodrigues, A., Adamo, M., Crozet, P., Margalha, L., Confraria, A., Marthino, C., et al. (2013). ACKNOWLEDGMENTS The skilful technical assistance provided by Alejandra Ávila and Laurel Fabila is deeply appreciated. We thank to Dr. Jorge Acosta- Gallegos (INIFAP) for providing bean seeds. This research was supported by DGAPA-UNAM (IN217811) and FQ-UNAM (PAIP 429014). Lunn, J. E., Feil, R., Hendriks, J. H. M., Gibon, Y., Morcuende, R., Osuna, D., et al. (2006). Sugar-induced increases in trehalose 6-phosphate are correlated with redox activation of ADPglucose pyrophosphorylase and higher rates of starch in Arabidopsis thaliana. Biochem. J. 397, 139–148. doi: 10.1042/BJ20060083 idopsis thaliana. Biochem. J. 397, 139–148. doi: 10.1042/BJ2006008 Martinez-Barajas, E., Delatte, T., Schluepmann, H., de Jong, G. J., Somsen, G. W., Nunes, C., et al. (2011). Wheat grain development is characterized by remarkable trehalose 6-phosphate accumulation pregrain filling: tissue distribution and rela- tionship to SNF1-related protein kinase 1 activity. Plant Physiol. 156, 373–381. doi: 10.1104/pp.111.174524 AUTHOR CONTRIBUTIONS Jossier, M., Bouly, J.-P., Meimoun, P., Arjmand, A., Lessard, P., Hawley, S., et al. (2009). SnRK1 (SNF1-related kinase 1) has a central role in sugar and ABA signalling in Arabidopsis thaliana. Plant J. 59, 316–328. doi: 10.1111/j.1365- 313X.2009.03871.x Conceived and designed the experiments Patricia Coello and Eleazar Martínez-Barajas. Performed the experiments Eleazar Martínez-Barajas. Analyzed the data Patricia Coello and Eleazar Martínez-Barajas. Wrote the paper Patricia Coello and Eleazar Martínez-Barajas. Kulma, A., Vikkadsen, D., Campbell, D. G., Meek, S. E. M., Harthill, J. E., Nielsen, T. H., et al. (2004). Phosphorylation and 14-3-3 binding of Arabidop- sis 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Plant J. 37, 654–667. doi: 10.1111/j.1365-313X.2003.01992.x Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head bacteriophage T4. Nature 227, 680–685. doi: 10.1038/227680a0 SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: http://www.frontiersin.org/Journal/10.3389/fpls.2014.00196/ abstract Nunes, C., O’Hara, L. E., Primavesi, L. F., Delatte, T. L., Schluepmann, H., Somsen, G.W.,et al. (2013a). The trehalose 6-phosphate/SnRK1 signalling pathway primes growth recovery following relief of sink limitation. Plant Physiol. 162, 1720–1732. doi: 10.1104/pp.113.220657 DISCUSSION On the other hand, SnRK1 is a modulator of ABA functions, linking nutrient and/or energy state to ABA-regulated responses (Finkelstein et al., 2002; Rad- chuk et al., 2006; Jossier et al., 2009; Rodrigues et al., 2013). It will be important to investigate how the individual nutrients and ABA contribute to the responses observed in seeds of detached fruits. Coello, P., and Martínez-Barajas, E. (2014). SnRK1 is differentially regulated in the cotyledon and embryo axe of bean (Phaseolus vulgaris L) seeds. Plant Physiol. Biochem. 80, 153–159. doi: 10.1016/j.plaphy.2014.03.033 Finkelstein, R. R., Gampala, S. S., and Rock, C. D. (2002). Abscisic acid signalling in seeds and seedlings. Plant Cell 14(Suppl.), S15–S45. doi: 10.1105/tpc.010441 Fountain, D. W., Outred, H. A., Holdsworth, J. M., and Thomas, R. G. (1989). Seed development in Phaseolus vulgaris L. cv Seminole: I. Develop- mental Independence of seed maturation. Plant Physiol. 89, 333–340. doi: 10.1104/pp.89.1.333 Fragoso, S., Espindola, L., Paez-Valencia, J., Gamboa, A., Camacho, Y., Martinez- Barajas, E., et al. (2009). SnRK1 isoforms AKIN10 and AKIN11 are differentially regulated in Arabidopsis plants under phosphate starvation. Plant Physiol. 149, 1906–1916. doi: 10.1104/pp.108.133298 Harthill, J. E., Meek, S. E. M., Morrice, N., Peggie, M. W., Borch, J., Wong, B. H. C., et al. (2006). Phosphorylation and 14-3-3 binding of Arabidopsis trehalose- phosphate synthase 5 in response to 2-deoxyglucose. Plant J. 47, 211–223. doi: 10.1111/j.1365-313X.2006.02780.x Jones, J. B. (1982). Hydroponics: it history and use in plant nutrition studies. J. Plant Nutr. 5, 1003–1030. doi: 10.1080/01904168209363035 REFERENCES ABI1 and PP2CA phosphatases are negative regulators of Snf1- related protein kinase 1 signaling in Arabidopsis. Plant Cell 25, 3871–3884. doi: 10.1105/tpc.113.114066 Borisjuk, L.,Weber, H., Panitz, R., Manteuffel, R., and Wobus, U. (1995). Embryoge- nesis of Vicia faba L: histodiferentiation in relation to starch and storage protein synthesis. J. Plant Physiol. 147, 203–218. doi: 10.1016/S0176-1617(11)81507-5 Rolletschek, H., Weber, H., and Borijuk, L. (2003). Energy status and its control on embryogenesis of legumes. Embryo photosynthesis contributes to oxygen May 2014 | Volume 5 | Article 196 | 6 Frontiers in Plant Science | Plant Physiology Nutrient remobilisation Coello and Martínez-Barajas cloning of cDNAs and cell type-specific expression. Plant Cell 7, 1835–1846. doi: 10.1105/tpc.7.11.1835 supply and is coupled to biosynthetic fluxes. Plant Physiol. 132, 1196–1206. doi: 10.1104/pp.102.017376 cloning of cDNAs and cell type-specific expression. Plant Cell 7, 1835–1846. doi: 10.1105/tpc.7.11.1835 supply and is coupled to biosynthetic fluxes. Plant Physiol. 132, 1196–1206. doi: 10.1104/pp.102.017376 Schiltz, S., Munier-Jolain, N., Jeudy, C., Burstin, J., and Salon, C. (2005). Dynamics of exogenous nitrogen partitioning and nitrogen remobilization from vegetative organs in pea revealed by 15N in vivo labelling throughout seed filling. Plant Physiol. 137, 1463–1473. doi: 10.1104/pp.104.056713 Wingler, A., Delatte, T. L., O’Hara, L. E., Primavesi, L. F., Jhurreea, D., Paul, M. J., et al. (2012). Trehalose 6-phosphate is required for the onset of leaf senes- cence associated with high carbon availability. Plant Physiol. 158, 1241–1251. doi: 10.1104/pp.111.191908 Schluepmann, H., Berke, L., and Sanchez-Perez, G. F. (2012). Metabolic control over growth: a case for trahalose-6-phosphate in plants. J. Exp. Bot. 63, 3379–3390. doi: 10.1093/jxb/err311 Yang, J., Zhang, J., Wang, Z., and Zhu, Q. (2001a). Activities of starch hydrolytic enzymes and sucrose-phosphate synthase in the stems of rice subjected to water stress during grain filling. J. Exp. Bot. 52, 2169–2179. Shen, W., Reyes, M. I., and Hanley-Bowdoin, L. (2009). Arabidopsis protein kinases GRIK1 and GRIK2 specifically activate SnRK1 by phosphorylating its activation loop. Plant Physiol. 150, 996–1005. doi: 10.1104/pp.108.132787 Yang, J., Zhang, J., Wang, Z., Zhu, Q., and Liu, L. (2001b). Water deficit-induced senescenceanditsrelationshiptotheremobilizationof pre-storedcarboninwheat during grain filling. Agron. J. 93, 196–206. doi: 10.2134/agronj2001.931196x Staswick, P. E. (1989). Preferential loss of an abundant storage protein from soybean pods during seed development. Plant Physiol. 90, 1252–1255. doi: 10.1104/pp.90.4.1252 Zhang, Y., Primavesi, L. F., Jhurreea, D., Andralojc, P. J., Mitchell, R. A., Powers, S. J., et al. (2009). REFERENCES Inhibition of SNF1-related protein kinase 1 activity and regulation of metabolic pathways by trehalose 6-phosphate. Plant Physiol. 149, 1860–1871. doi: 10.1104/pp.108.133934 Stolle-Smits, T., Beekhuizen, J. G., Kok, M. T. C., Pijnenburg, M., Recourt, K., Derksen, J., et al. (1999). Changes in cell wall polysaccharides of green bean during development. Plant Physiol. 121, 363–372. doi: 10.1104/pp.121. 2.363 Conflict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Conflict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Sugden, C., Donaghy, P. G., Halford, N. G., and Hardie, D. G. (1999). Two SNF1- related protein kinases from spinach leaf phosphorylate and inactivate 3-hydroxy- 3-methylglutaryl-coenzymeAreductase,nitratereductase,andsucrosephosphate synthase. Plant Physiol. 120, 257–274. doi: 10.1104/pp.120.1.257 Received: 25 January 2014; accepted: 23 April 2014; published online: 16 May 2014. Citation: Coello P and Martínez-Barajas E (2014) The activity of SnRK1 is increased in Phaseolus vulgaris seeds in response to a reduced nutrient supply. Front. Plant Sci. 5:196. doi: 10.3389/fpls.2014.00196 Tiessen, A., Preschaa, K., Branscheid, A., Palacios, N., McKibbin, R., Halford, N. G., et al. (2003). Evidence that SNF1-related kinase and hexokinase are involved in separated sugar-signalling pathways modulating post-translational redox activa- tion of ADP-glucose pyrophosphorylase in potato tubers. Plant J. 35, 490–500. doi: 10.1046/j.1365-313X.2003.01823.x This article was submitted to Plant Physiology, a section of the journal Frontiers in Plant Science. Copyright © 2014 Coello and Martínez-Barajas. This is an open-access article dis- tributed 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)orlicensorarecreditedandthattheoriginalpublicationinthisjournaliscited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Copyright © 2014 Coello and Martínez-Barajas. This is an open-access article dis- tributed 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)orlicensorarecreditedandthattheoriginalpublicationinthisjournaliscited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Toroser, D., Plaut, Z., and Huber, S. C. (2000). Regulation of plant SNF1- related protein kinase by glucose-6-phosphate. Plant Physiol. 123, 403–412. May 2014 | Volume 5 | Article 196 | 7 REFERENCES doi: 10.1104/pp.123.1.403 Weber, H., Borisjuk, L., Heim, L., Buchner, P., and Wobus, U. (1995). Seed-coat associated invertases of Fava bean control both unloading and storage functions: May 2014 | Volume 5 | Article 196 | 7 www.frontiersin.org www.frontiersin.org
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Equatorial Pacific forcing of western Amazonian precipitation during Heinrich Stadial 1
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Equatorial Pacific forcing of western Amazonian precipitation during Heinrich Stadial 1 Yancheng Zhang1, Xu Zhang2, Cristiano M. Chiessi3, Stefan Mulitza1, Xiao Zhang1,†, Gerrit Lohmann1,2, Matthias Prange1, Hermann Behling4, Matthias Zabel1, Aline Govin5, André O. Sawakuchi6, Francisco W. Cruz6 & Gerold Wefer1 Yancheng Zhang1, Xu Zhang2, Cristiano M. Chiessi3, Stefan Mulitza1, Xiao Zhang1,†, Gerrit Lohmann1,2, Matthias Prange1, Hermann Behling4, Matthias Zabel1, Aline Govin5, André O. Sawakuchi6, Francisco W. Cruz6 & Gerold Wefer1 received: 17 June 2016 accepted: 07 October 2016 Published: 25 October 2016 received: 17 June 2016 accepted: 07 October 2016 Published: 25 October 2016 Abundant hydroclimatic evidence from western Amazonia and the adjacent Andes documents wet conditions during Heinrich Stadial 1 (HS1, 18–15 ka), a cold period in the high latitudes of the North Atlantic. This precipitation anomaly was attributed to a strengthening of the South American summer monsoon due to a change in the Atlantic interhemispheric sea surface temperature (SST) gradient. However, the physical viability of this mechanism has never been rigorously tested. We address this issue by combining a thorough compilation of tropical South American paleorecords and a set of atmosphere model sensitivity experiments. Our results show that the Atlantic SST variations alone, although leading to dry conditions in northern South America and wet conditions in northeastern Brazil, cannot produce increased precipitation over western Amazonia and the adjacent Andes during HS1. Instead, an eastern equatorial Pacific SST increase (i.e., 0.5–1.5 °C), in response to the slowdown of the Atlantic Meridional Overturning Circulation during HS1, is crucial to generate the wet conditions in these regions. The mechanism works via anomalous low sea level pressure over the eastern equatorial Pacific, which promotes a regional easterly low-level wind anomaly and moisture recycling from central Amazonia towards the Andes. Amazonia, host of the richest terrestrial biomes on Earth1–3, plays a fundamental role in the tropical water cycle4. Future possible changes of Amazonian precipitation that bear direct consequences on Amazon ecosystems5,6 and carbon storage7–9 are of great concern10. Analysis of observational data demonstrated a strong dependence of western Amazonian precipitation (e.g., the 2005 drought) on the Atlantic meridional sea surface tempera- ture (SST) gradient11, but equatorial Pacific climate anomalies have also been related to Amazonian droughts and floods12,13. Potential decreases in the strength (by ca. 20–40%14) of the Atlantic Meridional Overturning Circulation (AMOC) under climate warming, which involve variations in both the Atlantic meridional SST gradient15 and the tropical eastern Pacific SST16, are rationally expected to affect Amazonian precipitation in the future. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Scientific Reports | 6:35866 | DOI: 10.1038/srep35866 Equatorial Pacific forcing of western Amazonian precipitation during Heinrich Stadial 1 Past intervals when the AMOC underwent substantial reduction, such as Heinrich Stadial 1 (HS1, 18-15 ka before present, BP), provide valuable information on the response of South American precipitation to a weakened AMOC.i HS1 was characterized as the strongest AMOC perturbation over the last glacial period17 and significantly influenced tropical South American climate18–21. For example, a southward migration of the Intertropical Convergence Zone (ITCZ) during HS1, if compared to the Last Glacial Maximum (LGM, 23-19 ka BP), resulted in a considerable decrease of precipitation over northernmost South America22,23 and a substantial increase over northeastern (NE) Brazil24–26. To explain wet conditions in the Andes27–29 and southeastern (SE) Brazil20 dur- ing HS1, some authors proposed a strengthening of the South American summer monsoon (SASM) (Fig. 1a). Various freshwater-hosing experiments with climate models of different complexity levels (under both LGM30 1MARUM – Center for Marine Environmental Sciences, University of Bremen, Germany. 2Alfred Wegener Institute Helmholtz Centre for Polar and Marine Research, Bremerhaven, Germany. 3School of Arts, Sciences and Humanities, University of São Paulo, São Paulo, Brazil. 4Department of Palynology and Climate Dynamics, Albrecht-von-Haller- Institute for Plant Sciences, University of Göttingen, Germany. 5IPSL/LSCE, Laboratoire des Sciences du Climat et de l′​Environnement (CEA-CNRS-UVSQ), Université Paris Saclay, Gif sur Yvette, France. 6Institute of Geosciences, University of São Paulo, São Paulo, Brazil. †Present address: School of Atmospheric Science, Nanjing University of Information Science and Technology, Nanjing, China. Correspondence and requests for materials should be addressed to Y.Z. (email: yzhang@marum.de) Scientific Reports | 6:35866 | DOI: 10.1038/srep35866 1 www.nature.com/scientificreports/ p Figure 1. Precipitation and low level atmospheric circulation (a) and paleorecords compilation for trop South America (b). (a) Long-term (1981–2010) averaged terrestrial precipitation (color scale) from the University of Delaware (http://climate.geog.udel.edu/~climate/) and 850 hPa wind field (vectors) from NOAA/OAR/ESRL PSD (http://www.esrl.noaa.gov/psd/) during austral summer (December-January-F DJF) Thi k k h S h A i l l l j (SALLJ) (b) C il i f h d li Figure 1. Precipitation and low level atmospheric circulation (a) and paleorecords compilation for tropical South America (b). (a) Long-term (1981–2010) averaged terrestrial precipitation (color scale) from the University of Delaware (http://climate.geog.udel.edu/~climate/) and 850 hPa wind field (vectors) from the NOAA/OAR/ESRL PSD (http://www.esrl.noaa.gov/psd/) during austral summer (December-January-February, DJF). Thick green arrow marks the South American low level jet (SALLJ). Scientific Reports | 6:35866 | DOI: 10.1038/srep35866 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 2. Results of the atmospheric model sensitivity experiments. Differences of simulated (ECHAM5) annual mean climate variables between Heinrich Stadial 1 and the Last Glacial Maximum for the (a) Atlantic (ATL) SST experiment, (b) eastern equatorial Pacific (EEP) SST experiment, (c) combined ATL +​ EEP experiment and (d) global (GLB) SST experiment (see Supplementary Information). Climate variables include rainfall (shaded, mm/day), 850 hPa wind field (vectors, m/s) and sea level pressure (contours, Pa). This map was plotted by using Grid Analysis and Display System (GrADS, Version 2.0.2, http://cola.gmu.edu/grads/grads.php). Figure 2. Results of the atmospheric model sensitivity experiments. Differences of simulated (ECHAM5) annual mean climate variables between Heinrich Stadial 1 and the Last Glacial Maximum for the (a) Atlantic (ATL) SST experiment, (b) eastern equatorial Pacific (EEP) SST experiment, (c) combined ATL +​ EEP experiment and (d) global (GLB) SST experiment (see Supplementary Information). Climate variables include rainfall (shaded, mm/day), 850 hPa wind field (vectors, m/s) and sea level pressure (contours, Pa). This map was plotted by using Grid Analysis and Display System (GrADS, Version 2.0.2, http://cola.gmu.edu/grads/grads.php). (AGCM) to evaluate the impacts of HS1 (relative to the LGM) SST anomalies on tropical South American precip- itation (see Materials and Methods). Our results show that SST changes over the eastern equatorial Pacific rather than the Atlantic are responsible for the increased precipitation over western Amazonia and the adjacent Andes during HS1. Scientific Reports | 6:35866 | DOI: 10.1038/srep35866 Equatorial Pacific forcing of western Amazonian precipitation during Heinrich Stadial 1 (b) Compilation of hydroclimate records, expressed as the difference between Heinrich Stadial 1 (HS1, 18-15 ka) and the Last Glacial Maximum (LGM, 23-19 ka). Symbol color indicates drier (red), wetter (blue), similar (grey) and unclear (white) conditions during HS1 in comparison to the LGM. Symbol size denotes the quality of the age model based on the chronological reliability index (CRI) (see Supplementary Information). Paleoclimate records with CRI values >​ 1 are numbered (Supplementary Table S1). Black dashed lines mark the schematic location of the Intertropical Convergence Zone (ITCZ) during austral summer (DJF) and austral winter (June-July-August, JJA). The Amazon River drainage basin is outlined by the black solid line in both panels (a,b). The map was plotted by using the ArcGIS software (version 10, https://software.zfn.uni-bremen.de/software/arcgis/). Figure 1. Precipitation and low level atmospheric circulation (a) and paleorecords compilation for tropical South America (b). (a) Long-term (1981–2010) averaged terrestrial precipitation (color scale) from the University of Delaware (http://climate.geog.udel.edu/~climate/) and 850 hPa wind field (vectors) from the NOAA/OAR/ESRL PSD (http://www.esrl.noaa.gov/psd/) during austral summer (December-January-February, DJF). Thick green arrow marks the South American low level jet (SALLJ). (b) Compilation of hydroclimate records, expressed as the difference between Heinrich Stadial 1 (HS1, 18-15 ka) and the Last Glacial Maximum (LGM, 23-19 ka). Symbol color indicates drier (red), wetter (blue), similar (grey) and unclear (white) conditions during HS1 in comparison to the LGM. Symbol size denotes the quality of the age model based on the chronological reliability index (CRI) (see Supplementary Information). Paleoclimate records with CRI values >​ 1 are numbered (Supplementary Table S1). Black dashed lines mark the schematic location of the Intertropical Convergence Zone (ITCZ) during austral summer (DJF) and austral winter (June-July-August, JJA). The Amazon River drainage basin is outlined by the black solid line in both panels (a,b). The map was plotted by using the ArcGIS software (version 10, https://software.zfn.uni-bremen.de/software/arcgis/). and modern31 boundary conditions) successfully simulated the Atlantic ITCZ shift, but exhibited a large spread of rainfall patterns across western Amazonia. In addition, a growing number of studies also suggested a correlation between increased precipitation along the Andes and the El Niño Southern Oscillation (ENSO) during HS132–34. In this study, we integrate (i) a quality-flagged compilation of 107 published hydroclimate records from trop- ical South America and (ii) a suite of sensitivity experiments in an Atmospheric General Circulation Model Scientific Reports | 6:35866 | DOI: 10.1038/srep35866 2 Discussion During HS1, a stronger SASM associated with a change in the Atlantic interhemispheric SST gradient was com- monly assumed to have triggered increased precipitation over the Amazonian Andes27–29. By contrast, our ATL SST experiment shows that the change in Atlantic interhemispheric SST gradient actually weakens the northeast trade winds over central Amazonia and the SALLJ (Fig. 2a), such that less moisture is transported from the tropi- cal Atlantic towards western Amazonia and the adjacent Andes (Fig. 2a). Decreased precipitation over these areas as reproduced by the ATL SST experiment (Fig. 2a), however, conflicts with the prevailing wet conditions derived from our compilation (Fig. 1b). Thus, the Atlantic interhemispheric SST gradient alone is insufficient to explain the wet conditions over western Amazonia and the adjacent Andes during HS1, and contributions from other oceanic regions (e.g., tropical Pacific) should be taken into account.h g g pi The EEP SST experiment demonstrates that positive climatological SST anomalies over the eastern equatorial Pacific (Supplementary Fig. S4) are able to cause increased precipitation over western Amazonia and the adjacent Andes during HS1, probably in relation to enhanced northeast trade winds over central Amazonia and the SALLJ (Fig. 2b). Intensified northeast trade winds over central Amazonia, importantly, are also clearly identified in the ATL +​ EEP SST experiment (Fig. 2c). Remarkably, the wind field pattern over the western tropical Atlantic from the ATL +​ EEP SST experiment rather resembles that of the ATL SST experiment than of the EEP SST experiment (Fig. 2a,c). This result implies that in the ATL +​ EEP SST experiment, western Amazonia and the adjacent Andes still experienced an increased rainfall, although less equatorial Atlantic moisture was transported towards the Andes. These features agree well with the overall characteristics of our compilation (Fig. 1b), in particular with the presence of dry conditions over central Amazonia during HS1. p y g If the Atlantic meridional SST gradient was not the only driver for increased rainfall over the Amazonian Andes37–39, other processes must be involved. We turn to the SST increases of around 0.5-1.5 °C in the eastern equatorial Pacific during HS1, with the exception of minor SST decreases over coastal regions40,41 (Supplementary Fig. S4 and Table S2). Discussion These SST variations tend to yield low-pressure anomalies over the eastern equatorial Pacific, which then deepens the zonal sea level pressure (SLP) gradient between the Atlantic and the Pacific and strengthens the easterly flow anomaly over western Amazonia and the adjacent Andes (Fig. 2b–d). Such easterly wind anomalies together with the northeast trade winds over central Amazonia subsequently promote mois- ture recycling from central Amazonia towards the Andes, enhancing the evaporation-condensation along its pathway42 (as sketched in Supplementary Fig. S5). In fact, this mechanism was previously suggested to account for the wet Andean conditions during the LGM42, with a particular consideration of the Andes topography (Supplementary Fig. S9). The extent to which enhanced moisture recycling contributed to the wet conditions over the Amazonian Andes remains elusive, but our interpretation coincides with abundant evidence across the cen- tral Andes that substantiated increased proportions of regional-sourced moisture over HS1 and the LGM32–35,43–46.it p p g Seasonal-scale SST changes in the eastern equatorial Pacific (Supplementary Figs S6 and S7) were often assigned to ENSO activity47. Because reconstructions of the ENSO variability across HS1 and the LGM were so far not well established from both numerical simulations48,49 and proxy data50–52, it is difficult to quantify the ENSO impact on South American precipitation during HS1. For instance, rainfall over NE Brazil and SE Brazil, which are today typically in strong negative and positive relationship with El Niño events53, indeed experienced similar wet patterns during HS1 (Fig. 1b). Analyses of instrumental data also suggested that climatological conditions over the eastern equatorial Pacific (e.g., related to ENSO52) may be linked to Atlantic climate forcing54,55. Eastern equatorial Pacific SST variations, probably a response to the weakened AMOC during HS1, are, nevertheless, crucial for triggering wet conditions over western Amazonia and the adjacent Andes (Fig. 1b). gg g j g Our ATL and ATL +​ EEP SST experiments (Fig. 2a,c) are unable to produce increased SE Brazilian rainfall as seen in the paleodata during HS1 (Fig. 1b). Interestingly, the GLB SST experiment (Fig. 2d), although forced by global SST anomalies (Supplementary Fig. S4), still cannot capture the wet conditions over SE Brazil. The SALLJ is weakened in both the GLB SST and the ATL +​ EEP SST experiments (relative to the EEP SST experiment), and thus seems unlikely to transport equatorial Atlantic moisture via western Amazon towards SE Brazil. Results il Compilation of hydroclimate records. Our compilation of paleomoisture difference between HS1 and the LGM indicates dry conditions to the north of the equator, but widespread wet conditions over the Andes, western Amazonia, NE and SE Brazil (Fig. 1b). Enhanced precipitation (or moisture) extends from the Ecuadorian Andes (e.g., Santiago Cave at ca. 3°S27) to the northern Chilean Andes (e.g., central Atacama Desert at 22°S–24°S35). The few available records from central Amazonia, characterized by low values of the chronological reliability index (CRI), exhibited in general dry climate during HS1 (Fig. 1b) (see Supplementary Information). Atmosphere model sensitivity experiments. The sensitivity experiments in this study were performed by using an atmosphere general circulation model (AGCM), the ECHAM5 (see Materials and Methods for a detailed design of model simulations). Driving the AGCM with global HS1 SST anomalies (see Supplementary Fig. S4) in the global SST experiment (Fig. 2d) shows comparable rainfall regimes to the ones simulated by the fully coupled atmosphere-ocean model36 (Supplementary Fig. S3). The ATL SST experiment that was forced by only Atlantic HS1 SST anomalies simulates a southward migration of the ITCZ, as evidenced by decreased rainfall over northernmost South America and increased rainfall over NE Brazil (Fig. 2a), but apparently fails to generate the wet conditions over western Amazonia. The EEP SST experiment (by applying only eastern equatorial Pacific HS1 SST anomalies) produces enhanced rainfall over western Amazonia together with the intensification of the northeast trade winds over central Amazonia and the South American Low Level Jet (SALLJ) (Fig. 2b), while the ITCZ displays no evident shift. The ATL +​ EEP SST experiment, in which we superimposed the eastern equatorial Pacific SST anomalies upon the Atlantic interhemispheric SST gradient, exhibits increased rainfall and easterly wind anomalies over western Amazonia (Fig. 2c), although dry conditions over SE Brazil are in contradiction to the GLB SST and the EEP SST experiments (Fig. 2b,d) as well as to our hydroclimate compilation (Fig. 1b). Scientific Reports | 6:35866 | DOI: 10.1038/srep35866 3 www.nature.com/scientificreports/ Discussion In a recent paper, Kageyama et al.30 compared eleven freshwater-hosing experiments (under the LGM conditions) with six different fully coupled climate models, none of which, notably, showed increased rainfall over SE Brazil. Therefore, additional investigations on paleoclimate records and model simulations are necessary to clarify this point. Conclusion Comparing a compilation of hydroclimate records and atmosphere model sensitivity experiments provides a deeper understanding of the influence of glacial North Atlantic climate on South American precipitation dur- ing HS1. An anomalous Atlantic meridional SST gradient, due to AMOC slowdown, drove a southward ITCZ shift leading to decreased precipitation over northernmost South America and increased precipitation over NE Brazil. The concomitant variations in eastern equatorial Pacific SST produced a negative SLP anomaly over the eastern tropical Pacific, which then deepened the SLP gradient between the Atlantic and the Pacific. As a result, it strengthened the northeasterly winds over the central and western Amazonia, enhancing moisture recycling over western Amazonia and the adjacent Andes.i Our results highlight that future changes in the eastern equatorial Pacific SST, as the AMOC weakens, will be of vital importance to affect western Amazonian precipitation. Depending on the magnitude of the AMOC slowdown under different global warming scenarios10,14, consideration of both the eastern equatorial Pacific and Atlantic SST variations may allow more accurate insights into the possible changes of Amazonian precipitation in the future. Scientific Reports | 6:35866 | DOI: 10.1038/srep35866 4 www.nature.com/scientificreports/ References Increasing risk of Amazonian drought due to decreasing aerosol pollution. Nature 453, 212–215 (2008).l g g g p 12. Marengo, J. A. & Espinoza, J. C. Extreme seasonal droughts and floods in Amazonia: causes, trends and impacts. Int. J. Climatol. 36, 1033–1050 (2016).h 3. Lopes, A. V., Chiang, J. C. H., Thompson, S. A. & Dracup, J. A. Trend and uncertainty in spatial-temporal patterns of hydrologica droughts in the Amazon basin. Geophys. Res. Lett. 43, 3307–3316 (2016). g p y 14. Weaver, A. J. et al. Stability of the Atlantic meridional overturning circulation: A model intercomparison. Geophys. Res. 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Materials and Methodsf Paleomoisture (precipitation) difference between HS1 and the LGM over tropical South America was determined by compiling 107 published terrestrial hydroclimate records between 30°S–10°N and 80°W-35°W, including 53 lacustrine sediment cores, 10 alluvial deposits, 9 speleothems, 9 moraine landforms, 9 fauna remains, 7 shoreline deposits, 5 paleosol sequences, 3 paleodune profiles as well as 2 ice cores. Chronologies and proxies of all these paleorecords were used directly from their original references. To evaluate the dating quality of the selected hydroclimate records, we applied a chronological reliability index (CRI)56 based on (i) age model properties and (ii) sampling resolution of each record (detailed description is given in Supplementary Information). Higher CRI values indicate more reliable hydroclimate records (Supplementary Fig. S1). By referring to interpretations of proxies in each record individually, we identified four types of paleomoisture (precipitation) anomalies between HS1 and the LGM as: drier, wetter, similar or unclear (Supplementary Fig. S2, Table S1 and Supplementary Information).f ) To evaluate different regional contributions of climatological SST changes to South American precipitation anomalies between the LGM and HS1 (Supplementary Figs S3–S5), an atmospheric general circulation model (AGCM), the ECHAM5 (L19/T31, i.e., 19 vertical levels and 3.75° ×​ 3.75° horizontal resolution)57 was employed. Since freshwater perturbation was the major forcing of the AMOC slowdown during HS130,58, we used the LGM boundary conditions (i.e., orbital parameters, topography land-sea mask, ice sheet and greenhouse gas concen- trations) to operate the experiments in this study. The LGM and HS1 control runs in the AGCM were forced by climatology monthly mean SST and sea ice cover from experiment LGMW and hosing experiment LGMW-0.2 Sv of the fully coupled general circulation model COSMOS (see ref. 36 for further details), respectively. To investi- gate the individual contributions of SST changes over different basins to South American precipitation anomalies during HS1, we conducted another three sensitivity experiments in which regional SST fields from the experi- ment LGMW−​0.2 Sv36 were imposed upon the LGMW SST background, such as the Atlantic basin (30°S–80°N) (ATL), the eastern equatorial Pacific (180°E–~70°W, 25°S–25°N) (EEP, Supplementary Fig. S8) and a combination of ATL and EEP (ATL +​ EEP). The atmosphere model was integrated for 50 years for each model experiment and the last 30 years were taken to calculate climatological fields. References 1. Silva, J. M. C., Rylands, A. B. & Fonseca, G. A. B. The fate of the Amazonian areas of endemism. 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C.M.C. was supported by FAPESP (grant 2012/17517-3) and CAPES (grants 1976/2014 and 564/2015). A.O.S. acknowledges financial supported from FAPESP (grant 2011/06609-1). The compilation of paleodata presented in this paper will be archived in PANGAEA (www.pangaea.de). References Palaeoclimatic insights into forcing and response of monsoon rainfall. Nature 533, 191–199 (2016). g g g p y ( ) 21. Mohtadi, M., Prange, M. & Steinke, S. Palaeoclimatic insights into forcing and response of monsoon rainfall. Nature 533, 191–199 (2016). 22. Peterson, L. C., Haug, G. H., Hughen, K. A. & Röhl, U. Rapid changes in the hydrologic cycle of the tropical Atlantic during the Last Glacial. Science 290, 1947–1951 (2000). 23. Deplazes, G. et al. Links between tropical rainfall and North Atlantic climate during the last glacial period. Nat. Geosci. 6, 213 (2013). ( ) 24. Wang, X. et al. Northeastern Brazil wet periods linked to distant climate anomalies and rainforest boundary changes. 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Lett. 432, 493–500 (2015). 27. Mosblech, N. A. S. et al. North Atlantic forcing of Amazonian precipitation during the last ice age. Nat. Geosci. 5, 817–820 (2012). g y 7. Mosblech, N. A. S. et al. North Atlantic forcing of Amazonian precipitation during the last ice age. Nat. Geosci. 5, 817–820 (2012). Scientific Reports | 6:35866 | DOI: 10.1038/srep35866 5 Scientific Reports | 6:35866 | DOI: 10.1038/srep35866 How to cite this article: Zhang, Y. et al. Equatorial Pacific forcing of western Amazonian precipitation during Heinrich Stadial 1. Sci. Rep. 6, 35866; doi: 10.1038/srep35866 (2016). Additional Information Supplementary information accompanies this paper at http://www.nature.com/srep Competing financial interests: The authors declare no competing financial interests. 6 Scientific Reports | 6:35866 | DOI: 10.1038/srep35866 www.nature.com/scientificreports/ How to cite this article: Zhang, Y. et al. Equatorial Pacific forcing of western Amazonian precipitation during Heinrich Stadial 1. Sci. Rep. 6, 35866; doi: 10.1038/srep35866 (2016). This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ © The Author(s) 2016 Scientific Reports | 6:35866 | DOI: 10.1038/srep35866 7
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Nonoperative management of blunt renal trauma: Is routine early follow-up imaging necessary? Address: 1Department of Urology, University of Tennessee Health Science Center, Memphis, Tennessee, USA, 2Department of Radiology, University of Tennessee Health Science Center, Memphis, Tennessee, USA and 3Urology Service, Elvis Presley Memorial Trauma Center, Regional Medical Center at Memphis, Tennessee, USA Email: John B Malcolm - john.b.malcolm@gmail.com; Ithaar H Derweesh* - iderweesh@utmem.edu; Reza Mehrazin - reza.mehrazin@gmail.com; Christopher J DiBlasio - cdiblasio76@yahoo.com; David D Vance - dvance1@utmem.edu; Salil Joshi - sjoshi@utmem.edu; Robert W Wake - rwake@utmem.edu; Robert Gold - rgold@utmem.edu Email: John B Malcolm - john.b.malcolm@gmail.com; Ithaar H Derweesh* - iderweesh@utmem.edu; Reza Mehrazin - reza.mehrazin@gmail.com; Christopher J DiBlasio - cdiblasio76@yahoo.com; David D V Salil Joshi - sjoshi@utmem.edu; Robert W Wake - rwake@utmem.edu; Robert Gold - rgold@utmem.edu * Corresponding author †Equal contributors Received: 20 May 2008 Accepted: 3 September 2008 Received: 20 May 2008 Accepted: 3 September 2008 Published: 3 September 2008 Published: 3 September 2008 BMC Urology 2008, 8:11 doi:10.1186/1471-2490-8-11 This article is available from: http://www.biomedcentral.com/1471-2490/8/11 This article is available from: http://www.biomedcentral.c © 2008 Malcolm et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. BioMed Central BioMed Central BMC Urology Open Access Abstract Background: There is no consensus on the role of routine follow-up imaging during nonoperative management of blunt renal trauma. We reviewed our experience with nonoperative management of blunt renal injuries in order to evaluate the utility of routine early follow-up imaging. Methods: We reviewed all cases of blunt renal injury admitted for nonoperative management at our institution between 1/2002 and 1/2006. Data were compiled from chart review, and clinical outcomes were correlated with CT imaging results. Results: 207 patients were identified (210 renal units). American Association for the Surgery of Trauma (AAST) grades I, II, III, IV, and V were assigned to 35 (16%), 66 (31%), 81 (39%), 26 (13%), and 2 (1%) renal units, respectively. 177 (84%) renal units underwent routine follow-up imaging 24– 48 hours after admission. In three cases of grade IV renal injury, a ureteral stent was placed after serial imaging demonstrated persistent extravasation. In no other cases did follow-up imaging independently alter clinical management. There were no urologic complications among cases for which follow-up imaging was not obtained. Conclusion: Routine follow-up imaging is unnecessary for blunt renal injuries of grades I-III. Grade IV renovascular injuries can be followed clinically without routine early follow-up imaging, but urine extravasation necessitates serial imaging to guide management decisions. The volume of grade V renal injuries in this study is not sufficient to support or contest the need for routine follow-up imaging. salvage and decreased morbidity compared to primary surgical management [1]. The nonoperative management scheme is not standardized amongst all urologists, but Results Patient demographics are shown in Table 1. 207 patients (mean age 35 years, 120 male/87 female) were admitted for nonoperative management of 210 blunt renal injuries (3 bilateral) between 1/2002 and 1/2006. Table 2 shows radiographic findings and clinical outcomes. AAST grades I, II, III, IV, and V were assigned to 35 (16%), 66 (31%), 81 (39%), 26 (13%), and 2 (1%) renal units, respectively. Among grade IV injuries, 19 (73%) were renovascular injuries (segmental infarcts) and 7 (27%) involved col- lecting system injury (urinary extravasation). Average BMI among patients with low-grade injury (grades I, II, and III) was 26.6, compared to 27.0 among patients with high- grade injury (grades IV and V) (p = 0.81). 177 (84%) renal injuries underwent routine follow-up imaging 24–48 hours after admission. Among the 33 (16%) renal injuries that were not re-imaged, 17 (52%), 9 (27%), and 6 (18%) were of injury grades I, II, and III, respectively. One patient with a grade V renal injury was not stable enough Background Background Nonoperative management has become the rule for the majority of blunt renal injuries, with higher rates of renal Page 1 of 6 (page number not for citation purposes) Page 1 of 6 (page number not for citation purposes) http://www.biomedcentral.com/1471-2490/8/11 BMC Urology 2008, 8:11 gross hematuria resolved, and follow-up CT imaging 24– 48 hours after admission. gross hematuria resolved, and follow-up CT imaging 24– 48 hours after admission. typically involves a period of bed rest, monitoring of vital signs and serial hematocrit measurements, with either selective or routine use of early follow-up imaging. Our center has previously advocated routine follow-up imag- ing 2 to 4 days after blunt renal trauma to identify patients that may require intervention for delayed complications [2]. However, noting in recent years that the vast majority of follow-up CT scans do not alter clinical management, we have elected to reevaluate our previously proposed management strategy. We reviewed our contemporary experience with nonoperative management of blunt renal injuries in order to reassess the utility of routine early fol- low-up imaging. After compiling data from chart review, we noted the rate of clinically significant new findings on repeat imaging and attempted to correlate clinical outcomes with repeat imaging results. Student's t-test was used to compare demographic subsets in our series. Fisher's exact test was used to compare re-imaging outcomes between patients with low-grade (I,II,III) and high-grade (IV,V) injuries. Methods Af i After receiving approval from the Institutional Review Board of the University of Tennessee Health Science Center, Memphis, Tennessee, we performed a retrospec- tive chart review of all patients admitted with blunt renal injury and primary nonoperative management at the Elvis Presley Memorial Trauma Center between 1/2002 and 1/ 2006. Data collected from chart review included patient age and gender, race, body mass index (BMI, kg/m2), Glas- cow coma score (GCS), mechanism of injury, side of injury, grade of injury, vital signs, serial hematocrit meas- urements, results of follow-up imaging, complications, and delayed interventions. All injuries were diagnosed at the time of admission using contrasted CT imaging in the cortical and delayed excre- tory phases. Imaging for the diagnosis of renal trauma was obtained based on standard indications for the adult trauma patient: gross hematuria, microscopic hematuria with hypotension, or high suspicion of renal injury based on the mechanism of trauma [3,4]. Injuries were graded by a staff radiologist according to the American Associa- tion for the Surgery of Trauma (AAST) organ injury scale [5]. Injuries were also independently evaluated and graded by the managing urologist. Where discrepancies in grading were noted on chart review, the imaging studies were reread to verify accurate injury grading. However, all films were not uniformly reread at the time of chart review. Table 1: Demographics and clinical presentations Variable Number of patients 207 Age (years) Mean (range) 35 (15–80) BMI (kg/m2) Mean (range) 26.7 (17.8–45.6) Gender Female 87 (42%) Male 120 (58%) Glascow Coma Score (GCS) Mean (range) 13.6 (3–15) Mechanism of Injury MVA 173 (84%) Pedestrian Struck 15 (7%) Fall 13 (6%) Assault 6 (3%) Race African American 79 (38%) Caucasian/other 128 (62%) Side of Injury Left 108 (52%) Right 96 (46%) Bilateral 3 (2%) Table 1: Demographics and clinical presentations Table 1: Demographics and clinical presentations Our renal trauma database captured all patients admitted with blunt renal injury and primary nonoperative man- agement. It did not include the rare patient who under- went primary operative management or those with grade I injuries that were deemed appropriate for outpatient management by the trauma service. Methods Af i Patients who were hemodynamically stable at the time of presentation were managed according to a nonoperative protocol with bed rest, serial measurement of vital signs and hematocrit every 6 hours until stable over a 24-hour period or until http://www.biomedcentral.com/1471-2490/8/11 BMC Urology 2008, 8:11 Table 2: Radiographic and clinical outcomes Grade N (%) F/U Imaging Injury D/G* Injury U/G** %D/G* %U/G** Complications Interventions Low Grade I 35 (16) 19 (54%) 4 (21%) 0 12% 3% 0 0 II 66 (31) 57 (86%) 5 (9%) 2 (4%) 0 0 III 81 (39) 75 (93%) 9 (12%) 2 (3%) 1 (1%) 0 High Grade IV 26 (13) 25 (96%) 1 (4%) 0 4% P = 0.32*** 0 P = 1.00*** 3 (12%) 3 (endoscopic ureteral stent) V 2 (1) 1 (50%) 0 0 1 (50%) 0 Total 210 177 (84%) 19 (11%) 4 (2%) 5 (2.4%) 3 (1%) * D/G = Downgrade ** U/G = Upgrade *** Fisher's exact test Table 2: Radiographic and clinical outcomes for transport to radiology for follow-up imaging, and he ultimately succumbed to multiple traumatic injuries. patients. One patient with a grade III renal injury devel- oped a febrile urinary tract infection that was successfully managed with IV antibiotics. There were no cases in which repeat imaging results independently prompted urologic intervention. There were no urologic complications among cases for which follow-up imaging was not obtained. We noted low rates of altered injury grading after follow- up imaging. After early re-imaging, renal injuries were downgraded in 4 (21%), 5 (9%), 9 (12%), 1 (4%), and 0 cases of grade I, II, III, IV, and V injury, respectively. Grade I injuries were downgraded when subcapsular hematoma was not evident on follow-up imaging; higher grade inju- ries were downgraded when lacerations appeared smaller or fewer in number on follow-up imaging compared to initial imaging. Renal injuries were upgraded in 0, 2 (4%), 2 (3%), 0, and 0 cases of grade I, II, III, IV, and V injury, respectively. Overall, the rate of injury downgrading was 12% for low-grade injury and 4% for high-grade injury (p = 0.32). The rate of injury upgrading was 3% for low-grade injuries and 0% for high-grade injuries (p = 1.00). There was no significant difference in the rates of altered injury grading on follow-up imaging between low and high- grade injuries. Discussion The incidence of traumatic renal injuries in the United States is approximately 5 per 100,000 persons [6], or 15,000 per year nationwide. The majority of renal injuries can be managed nonoperatively, with few absolute indi- cations for surgical intervention [7]. CT imaging results factor prominently in the initial management strategy for blunt renal trauma, allowing for reliable injury grading that has been shown to correlate well with the need for surgical intervention [8,9]. However there is little consen- sus on the role of routine re-imaging once a nonoperative management course has been selected. Our institution previously reported a retrospective review of 48 cases of blunt renal injury and primary nonoperative management, noting that one in ten patients with a grade II or higher blunt renal injury had a delayed urologic com- plication detected by follow-up CT scan that ultimately required invasive intervention [2]. Following publication of our previous institutional experience, we have main- tained a protocol of nonoperative management that includes routine re-imaging of all blunt renal injuries 24– 48 hours after admission. We elected to reevaluate this protocol because in our contemporary experience it has seemed that few, if any, routine re-imaging studies have independently altered clinical management. At a cost of approximately $700.00 per imaging evaluation (based on Medicare 2005 reimbursement rate for CT abdomen w/ wo contrast [74170] and CT pelvis w/wo contrast [72194]), more selective use of CT imaging in the nonop- erative management of blunt renal trauma could offer Of note, two cases of grade III injury were upgraded to grade IV on follow-up imaging. In the first case, the initial CT was performed with suboptimal delayed excretory phase imaging; urinary extravasation that was not appar- ent on the initial CT was demonstrated on the follow-up CT with appropriately timed delayed excretory phase imaging. The second case of grade III injury upgrade involved a patient with two devascularized segments on follow-up imaging in addition to a stable 1.5 cm lacera- tion noted on the initial CT scan. This patient was man- aged without surgical intervention, and there were no delayed urologic complications. Complications and delayed interventions were uncom- mon in this series. Page 3 of 6 (page number not for citation purposes) http://www.biomedcentral.com/1471-2490/8/11 Among patients with low-grade renal injury, there were no instances where early re-imag- ing detected or prevented a urologic complication. Of some concern, a single patient was found to have urinary extravasation on follow-up imaging not appreciated on initial CT. However, in this case the initial CT scan was of suboptimal diagnostic quality due to poorly timed delayed excretory phase imaging. This illustrates the importance of high-quality imaging from the outset of patient management, particularly in a management scheme that excludes routine early re-imaging. Neverthe- less, after demonstration of limited urinary extravasation on follow-up imaging, this patient was managed nonop- eratively and additional imaging 5 days later revealed res- olution of the urine leak. We are prospectively evaluating a revised management strategy (Figure 1), and future study will test our current conclusion that routine re-imaging of grade I-IV renal injuries is unnecessary. Since reviewing our experience with blunt renal trauma management from 2002 to 2006, we have abandoned routine early re-imaging for blunt renal injuries of grades I-III and grade IV renal injuries without urinary extravasation. We now use re-imaging studies selectively for patients with grade IV injuries with demonstrated urinary extravasation, patients with multi- ple comorbidities who are putatively at increased risk for complications from renal trauma, patients with severe injuries involving multiple organ systems, and patients with clinical signs (hemodynamic instability, decreasing hematocrit, fever) that may herald progressing complica- tions from blunt renal injury. We continue to routinely re- image the rare patient who meets criteria for nonoperative management of a grade V renal injury. Our experience with this management algorithm will be reported as a sizeable experience accrues. Weaknesses of this study include its retrospective design, with the inherent limitations and biases of a retrospective analysis. Furthermore, we do not have long-term follow- up data for the majority of the patients in this cohort, so we are unable to evaluate the impact of routine re-imaging on long term renal functional outcomes, development of hypertension, or other renal injury sequellae. We suspect that the impact of routine re-imaging on such parameters is minimal. Additionally, we have reviewed the use of rou- tine re-imaging 24–48 hours after blunt renal injury. http://www.biomedcentral.com/1471-2490/8/11 http://www.biomedcentral.com/1471-2490/8/11 http://www.biomedcentral.com/1471-2490/8/11 http://www.biomedcentral.com/1471-2490/8/11 BMC Urology 2008, 8:11 mum screening protocol for urologic complications in nonoperatively managed blunt renal injury should rely primarily on objective clinical signs and symptoms to the exclusion of routine, repeat, radiographic imaging. substantial cost-containment benefit. In the series pre- sented, routine use of early re-imaging amounted to a cost of $121,800 (174 × $700), which proved to be, by and large, an unnecessary expense. If early re-imaging had been used selectively (only grade IV collecting system injuries and grade V injuries), as is our current practice, the cost would have been $7700 (11 × $700), realizing a cost reduction of almost 94%. Furthermore, the clinical benefit of reducing unnecessary radiation exposure is likely to be significant. substantial cost-containment benefit. In the series pre- sented, routine use of early re-imaging amounted to a cost of $121,800 (174 × $700), which proved to be, by and large, an unnecessary expense. If early re-imaging had been used selectively (only grade IV collecting system injuries and grade V injuries), as is our current practice, the cost would have been $7700 (11 × $700), realizing a cost reduction of almost 94%. Furthermore, the clinical benefit of reducing unnecessary radiation exposure is likely to be significant. Our series of 207 patients (210 renal units) includes 32 patients (33 renal injuries) who did not undergo repeat imaging. The majority of patients in this subset had a grade I or grade II injury that was managed by the trauma surgery service without consultation by the urology serv- ice. Excluding one patient with a grade V renal injury (early mortality), there were no urologic complications among these patients. Admittedly, this group has limited statistical significance given its diminutive power. Our contemporary retrospective review includes 175 patients (177 renal units) who underwent routine early follow-up imaging during nonoperative management of a blunt renal injury. The majority of these renal units (151/ 85%) suffered a grade I, II, or III injury. It is noteworthy that the proportion of grade I injuries was significantly smaller than other published blunt renal trauma series (16% vs. 64% [6] and 86% [8]). It is probable that a sig- nificant proportion of patients with grade I renal injuries were deemed appropriate for outpatient management by the trauma surgery service, and were therefore not cap- tured in our database. Page 4 of 6 (page number not for citation purposes) Discussion In three cases of grade IV renal injury with collecting system insult, a ureteral stent was placed after serial imaging demonstrated persistent extravasa- tion; endoscopic management proved definitive in these Page 3 of 6 (page number not for citation purposes) Page 3 of 6 (page number not for citation purposes) http://www.biomedcentral.com/1471-2490/8/11 It has been shown that many of the delayed complications from blunt renal trauma (delayed bleed, AVF, infected uri- noma, abscess) occur at least 1–3 weeks after the injury occurs [7], so it is possible that routine re-imaging of blunt renal injuries would yield more clinically useful results if performed at a longer time-interval post injury, i.e. 1–3 weeks. Ultimately, we feel that such a manage- ment scheme is not practicable, and if 2–3 week follow-up is achievable we feel that more cost-effective and efficient The goals of nonoperative management of blunt renal injury are to identify, manage, and limit associated com- plications – including urinary extravasation, urinoma, infection, bleeding, and, most importantly, loss of renal function or unnecessary nephrectomy. Such complica- tions have been reported in 3% to 33% of patients after renal trauma [10]. Clinical management of such compli- cations is directed primarily by objective clinical signs and symptoms (i.e., hemodynamic instability, increasing pain, fever and leukocytosis, decreasing hematocrit and blood transfusion requirement) and not by imaging results [11]. Even in cases where imaging results demon- strate known harbingers of urologic complications (devascularized segments, urinary extravasation), contin- ued nonoperative management has proven practicable, with intervention based on clinical rather than radio- graphic criteria [11]. Page 5 of 6 (page number not for citation purposes) http://www.biomedcentral.com/1471-2490/8/11 It is our contention that the opti- Page 4 of 6 (page number not for citation purposes) Page 4 of 6 (page number not for citation purposes) BMC Urology 2008, 8:11 http://www.biomedcentral.com/1471-2490/8/11 Blunt Abdominal Trauma Gross Hematuria, Microhematuria with Hypotension, or Suspicion of Renal Injury Based on Mechanism Microhematuria without Hypotension CT Scan* Hemodynamically Stable Hemodynamically Unstable Grade I-III Renal Injury Grade IV Renal Injury, Segmental Vascular** Grade IV Renal Injury, Collecting System Grade V Renal Injury Observation; Re-image only when Clinically Indicated*** Observation, Early Repeat CT Scan* (48 Hours) Hemodynamically Stable Hemodynamically Unstable Surgical Exploration Surgical Exploration * CT scan with and without IV contrast and delayed images ** Segmental Infarct *** Hemodynamic instability, fever or progressive leukocytosis, marked change in physical exam findings Repeat Urinalysis in 6 Weeks Blunt Abdominal Trauma Hemodynamically Unstable Hemodynamically Stable Gross Hematuria, Microhematuria with Hypotension, or Suspicion of Renal Injury Based on Mechanism Microhematuria without Hypotension Repeat Urinalysis in 6 Weeks Surgical Exploration Grade I-III Renal Injury Grade V Renal Injury Grade IV Renal Injury, Collecting System Grade IV Renal Injury, Segmental Vascular** Hemodynamically Stable Hemodynamically Unstable Observation; Re-image only when Clinically Indicated*** Observation, Early Repeat CT Scan* (48 Hours) Surgical Exploration Page 5 of 6 (page number not for citation purposes) http://www.biomedcentral.com/1471-2490/8/11 BMC Urology 2008, 8:11 BMC Urology 2008, 8:11 ML: Organ injury scaling: spleen, liver, and kidney. J Trauma 1989, 29:1664-1666. screening for delayed complications can be achieved by physical exam, vital signs, and simple laboratory tests (hematocrit and serum creatinine). One additional com- plication of this study lies in the grading system used for blunt renal injuries. The AAST renal injury scale is straight- forward and has proven reliability. However we com- monly encounter renal injuries that are not explicitly accounted for in the AAST Organ Injury Scale, e.g., renal injuries with segmental devascularization (segmental artery injuries without main renal artery injuries) or mul- tiple cortical lacerations >1 cm in a single renal unit. Such injuries are classified as grade IV at our trauma center; it is these types of grade IV injuries for which we have aban- doned routine repeat imaging, and we continue to re- image grade IV injuries with demonstrated urinary extravasation. 6. 6. Wessells H, Suh D, Porter JR, Rivara F, MacKenzie EJ, Jurkovich GJ, Nathens AB: Renal injury and operative management in the United States: results of a population-based study. J Trauma 2003, 54:423-430. 7. g None. No sources of funding. Publish with BioMed Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp BioMedcentral Page 6 of 6 (page number not for citation purposes) Publish with BioMed Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp BioMedcentral Publish with BioMed Central and every scientist can read your work free of charge Conclusion Routine follow-up imaging is unnecessary in the nonop- erative management of blunt renal injuries of grades I-III. Grade IV renovascular injuries can be followed clinically without routine follow-up imaging, but urine extravasa- tion necessitates serial imaging to guide management decisions. The volume of grade V renal injuries in this study is not sufficient to support or contest the need for routine follow-up imaging, however we maintain a prac- tice of routine follow-up imaging of nonoperatively man- aged grade V renal injuries. Ongoing prospective study will test these conclusions. Pre-publication history The pre-publication history for this paper can be accessed here: The pre-publication history for this paper can be accessed here: http://www.biomedcentral.com/1471-2490/8/11/prepub Competing interests The authors declare that they have no competing interests. http://www.biomedcentral.com/1471-2490/8/11 Santucci RA, Wessells H, Bartsch G, Descotes J, Heyns CF, McAninch JW, Nash P, Schmidlin F: Evaluation and management of renal injuries: consensus statement of the renal trauma subcom- mittee. BJU Int 2004, 93:937-954. J 8. Santucci RA, McAninch JW, Safir M, Mario LA, Service S, Segal MR: Validation of the American Association for the Surgery of Trauma organ injury severity scale for the kidney. J Trauma 2001, 50:195-200. 9. Shariat SF, Roehrborn CG, Karakiewicz PI, Dhami G, Stage KH: Evi- dence-based validation of the predictive value of the Ameri- can Association for the Surgery of Trauma kidney injury scale. J Trauma 2007, 62:933-939. J 10. Al-Qudah HS, Santucci RA: Complications of renal trauma. Urol Clin North Am 2006, 33:41-53. 11. Moudouni SM, Patard JJ, Manunta A, Guiraud P, Guille F, Lobel B: A conservative approach to major blunt renal lacerations with urinary extravasation and devitalized renal segments. BJU Int 2001, 87:290-294. Acknowledgements None. No sources of funding. g None. No sources of funding. References 1. Santucci RA, Fisher MB: The literature increasingly supports expectant (conservative) management of renal trauma – a systematic review. J Trauma 2005, 59:493-503. y J 2. Blankenship JC, Gavant ML, Cox CE, Chauhan RD, Gingrich JR: Importance of delayed imaging for blunt renal trauma. World J Surg 2001, 25:1561-1564. J g 3. Brandes SB, McAninch JW: Urban free falls and patterns of renal injury: a 20-year experience with 396 cases. J Trauma 1999, 47:643-650. 4. Miller KS, McAninch JW: Radiographic assessment of renal trauma: our 15-year experience. J Urol 1995, 154:352-355. p J 5. Moore EE, Shackford SR, Pachter HL, McAninch JW, Browner BD, Champion HR, Flint LM, Gennarelli TA, Malangoni MA, Ramenofsky Authors' contributions JM and ID conceived of the study, participated in design and coordination, and drafted the manuscript. RM and DV participated in study design, data acquisition and analysis. SJ and RG oversaw interpretation of radiographic information. CD and RW helped with manuscript drafting and critical revision. All authors read and approved the final manuscript.
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Thermal plasticity of the miRNA transcriptome during Senegalese sole development
BMC genomics
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RESEARCH ARTICLE Open Access Campos et al. BMC Genomics 2014, 15:525 http://www.biomedcentral.com/1471-2164/15/525 Campos et al. BMC Genomics 2014, 15:525 http://www.biomedcentral.com/1471-2164/15/525 Thermal plasticity of the miRNA transcriptome during Senegalese sole development Catarina Campos1,2,3, Arvind YM Sundaram2, Luisa MP Valente1, Luis EC Conceição3,4, Sofia Engrola3 and Jorge MO Fernandes2* © 2014 Campos et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. * Correspondence: jorge.fernandes@uin.no 2Faculty of Biosciences and Aquaculture, University of Nordland, Bodø 8049, Norway Full list of author information is available at the end of the article Abstract Background: Several miRNAs are known to control myogenesis in vertebrates. Some of them are specifically expressed in muscle while others have a broader tissue expression but are still involved in establishing the muscle phenotype. In teleosts, water temperature markedly affects embryonic development and larval growth. It has been previously shown that higher embryonic temperatures promoted faster development and increased size of Senegalese sole (Solea senegalensis) larvae relatively to a lower temperature. The role of miRNAs in thermal-plasticity of growth is hitherto unknown. Hence, we have used high-throughput SOLiD sequencing to determine potential changes in the miRNA transcriptome in Senegalese sole embryos that were incubated at 15°C or 21°C until hatching and then reared at a common temperature of 21°C. Results: We have identified 320 conserved miRNAs in Senegalese sole, of which 48 had not been previously described in teleosts. mir-17a-5p, mir-26a, mir-130c, mir-206-3p, mir-181a-5p, mir-181a-3p and mir-199a-5p expression levels were further validated by RT- qPCR. The majority of miRNAs were dynamically expressed during early development, with peaks of expression at pre-metamorphosis or metamorphosis. Also, a higher incubation temperature (21°C) was associated with expression of some miRNAs positively related with growth (e.g., miR-17a, miR-181-5p and miR-206) during segmentation and at hatching. Target prediction revealed that these miRNAs may regulate myogenesis through MAPK and mTOR pathways. Expression of miRNAs involved in lipid metabolism and energy production (e.g., miR-122) also differed between temperatures. A miRNA that can potentially target calpain (miR-181-3p), and therefore negatively regulate myogenesis, was preferentially expressed during segmentation at 15°C compared to 21°C. Conclusions: Temperature has a strong influence on expression of miRNAs during embryonic and larval development in fish. Higher expression levels of miR-17a, miR-181-5p and miR-206-3p and down-regulation of miR-181a-3p at 21°C may promote myogenesis and are in agreement with previous studies in Senegalese sole, which reported enhanced growth at higher embryonic temperatures compared to 15°C. Moreover, miRNAs involved in lipid metabolism and energy production may also contribute to increased larval growth at 21°C compared to 15°C. Taken together, our data indicate that miRNAs may play a role in temperature-induced phenotypic plasticity of growth in teleosts. Keywords: Solea senegalensis, miRNA, Embryonic temperature, Myogenesis, Growth, Epigenetics Background of miR-206, miR-26a and miR-214 throughout skeletal muscle development in the common carp (Cyprinus carpio), points to an active role of these miRNAs in the myogenic process [14]. MicroRNAs (miRNAs) are short non-coding regulatory RNAs that regulate gene expression post-transcriptionally. Mature miRNAs are often conserved across a wide range of species, and mutations in proteins required for miRNA function or biogenesis have shown to impair animal de- velopment [1]. They are involved in the majority of phy- siological processes, including stem cell differentiation, cell lineage specification, haematopoiesis, neurogenesis, immune response and myogenesis [1]. MiRNA-mediated gene regulation involves binding to the 3’-UTR of their mRNA target and sequestration of mRNA by the miRNA/ RISC complex (RNA-induced silencing complex) in the cytoplasmic processing bodies, which results in repres- sion of translation initiation or mRNA degradation re- viewed in [2]. miRNAs can also modulate protein abundance and/or phosphorylation status of key signaling components of the insulin pathway. Expression of miR-122 [15] and miR-33 [16] is inversely correlated with expression and/ or phosphorylation status of the α-subunit of the me- tabolic sensor AMPK. In fact, miR-122, which is highly abundant in mammalian [17] and fish liver [18], exerts a stimulatory role on lipogenesis and cholesterol synthe- sis, and inhibits fatty-acid oxidation and therefore energy production [15]. In fish, changes in miRNA expression have been do- cumented during early ontogeny [19,20] or in response to food ingestion [18]. However, the potential effect of water temperature on the miRNA transcriptome has never been investigated in teleosts, in spite of its pro- found impact on larval and juvenile growth [21]. The flatfish Senegalese sole (Solea senegalensis) is a species of commercial interest for marine aquaculture, particu- larly in Southern Europe, and it can be exposed to large thermal variations in natural and aquaculture conditions [22]. In a previous study it was shown that higher embry- onic temperatures (18 and 21°C) promoted a faster de- velopment and increased the size of sole larvae by 30 days post hatch (dph) relatively to a lower temperature (15°C) [23]. Furthermore, muscle cellularity at equivalent devel- opmental stages was affected by temperature and a transi- ent differential gene expression associated with incubation temperature was also observed at several stages. For ex- ample, myf5, fst, myHC and mrf4 transcript levels were higher at 21°C compared to 15°C [23]. Background It is likely that this differential expression of myogenic genes is regulated by miRNAs to some extent but their role in thermal plasticity of development and growth remains to be investigated. Hence, we have used the SOLiD deep-sequencing plat- form to determine potential changes in the miRNA tran- scriptome in Senegalese sole embryos and larvae exposed to two different temperatures (15°C or 21°C) during em- bryonic development. Moreover, we have predicted targets of miRNAs that were differentially expressed with tem- perature, in order to understand their potential role in the phenotypic plasticity of growth in teleosts. In vertebrates, somatic and skeletal muscle growth is strongly stimulated by Igf-I (Insulin-like growth factor I) [3], whose functions are mediated by the Igf-I receptor (Igf1R) through activation of two major intracellular signalling pathways: mitogen-activated protein kinases (MAPKs) and mTOR (mammalian target of rapamycin) through phosphatidylinositol 3 kinase (PI3K)/Akt [4]. mTOR mediates signalling in response to nutrient avail- ability, cellular energy, mitogenic signals and various types of stressors. Phosphorylation of mTOR increases levels of protein synthesis by regulating essential proteins control- ling mRNA translation. The MAPK pathway is essential for muscle cell proliferation and differentiation [5,6], and the closely related hepatic insulin pathway acts upstream of Akt/mTOR. It recruits IRS proteins, which in turn re- cruit PI3K to phosphorylate Akt. Downstream of Akt, sub- sequent metabolic effects are largely mediated by mTOR and also FoxO1 [7]. mTOR then regulates S6K1 and 4- EBP1 to induce hepatic protein synthesis and to stimulate lipogenesis [7]. The physiological actions of these complex pathways are multiple and several miRNAs have already been identified as intervenients on their regulation. For in- stance, in human, miR-199a-3p and miR-101 can sup- press gene expression by directly binding to the 3’-UTR of mTOR [8,9]. Furthermore, miR-181 regulates PTEN, the principal negative regulator of PI3K [10]. MiRNAs that influence mTOR and MAPK pathways play crucial roles in myogenesis, since they are essential for muscle development and growth. For example, miR-181 is in- volved in myoblast differentiation and in establishing the muscle phenotype [11]. Additionally, some miRNAs (e.g., miR-1, miR-133 and miR-206) are specifically expressed in muscle and positively related with its growth [12]. Inter- estingly, in teleost like zebrafish (Danio rerio), fast muscle miRNA populations are differentially regulated during the transition from hyperplastic to hypertrophic muscle phe- notype in adult fish [13]. Also, the dynamic expression * Correspondence: jorge.fernandes@uin.no 2Faculty of Biosciences and Aquaculture, University of Nordland, Bodø 8049, Norway Full list of author information is available at the end of the article Campos et al. BMC Genomics 2014, 15:525 http://www.biomedcentral.com/1471-2164/15/525 Campos et al. BMC Genomics 2014, 15:525 http://www.biomedcentral.com/1471-2164/15/525 Campos et al. BMC Genomics 2014, 15:525 http://www.biomedcentral.com/1471-2164/15/525 Page 2 of 13 Differential expression of conserved miRNAs during Senegalese sole development and with embryonic temperature Hierarchical clustering of the 320 conserved miRNAs found with SOLiD sequencing in Senegalese sole revealed three main clusters: miRNAs with very low number of reads throughout development (Figure 2A), miRNAs generally highly expressed in all developmental stages (Figure 2B) and miRNAs with variable expression dur- ing development (Figure 2C). miRNA diversity and expression varied amongst de- velopmental stages (Figures 2, 3). Notably, several pas- senger strands showed very high expression values in one or more stages of development, such as miR-122-5p, miR-140-3p, miR-181a-3p, miR-199a-3p or miR-203b-5p (Figure 2B, C and Figure 3E). Generally, at earlier stages of development (i.e., 75Ep and 20S), there was less var- iety and lower level of miRNA transcripts than at later stages (Figure 2). The miRNA with highest number of reads during 75Ep in both temperature groups was miR- 430d (3.1 and 1.6% of the total number of reads at 15 and 21°C, respectively) (Figure 2B), followed by miR-430a and 430c. The miR-430 family was prominently expressed until hatching, but its expression decreased sharply after that and at metamorphosis hardly any expression could be The digital expression profiles obtained with SOLiD sequencing in Senegalese sole were validated by qPCR of selected miRNAs (r = 0.69, 0.76, 0.98, 0.91, 0.97, 0.98 and 0.81 for miR-17a-5p, mir-26a, miR-130c, miR-206-3p, miR-181a-5p, miR-181a-3p and miR-199a-5p, respect- ively). These miRNAs were selected based on: 1) previous knowledge of their functions in other species, namely in- volvement in development and growth, muscle differenti- ation or response to stress, and 2) differential expression during development and/or temperature, as evidenced by the above SOLiD sequencing results. Quantification of selected miRNAs was performed not only in the same samples used for SOLiD sequencing but also in pre- metamorphic and 30 dph larvae (Figure 3A-G). SOLiD sequencing of small RNAs To identify Senegalese sole miRNAs expressed during development, 8 small RNA libraries were prepared and sequenced using the SOLiD deep-sequencing platform. A total of 55,053,966 raw reads were obtained, resulting in 27,639,077 reads after sequence trimming. All small RNA-seq data have been deposited in Gene Expression Page 3 of 13 Campos et al. BMC Genomics 2014, 15:525 http://www.biomedcentral.com/1471-2164/15/525 Campos et al. BMC Genomics 2014, 15:525 http://www.biomedcentral.com/1471-2164/15/525 Campos et al. BMC Genomics 2014, 15:525 http://www.biomedcentral.com/1471-2164/15/525 detected (Figure 2B). At 75Ep, the passenger strands with the highest number of reads were miR-203b-5p, miR-202- 5p and miR-20a-3p (Figure 2B, C). During the 20S stage, miRNAs such as miR-203a, miR-17a-5p or miR-130c were found to be highly expressed (Figure 2B; Figure 3A, C). At hatching, miR-130c was the miRNA with the highest number of reads in both temperatures (2.4 and 6.3% of total miRNAs at 15 and 21°C, respectively). At this point, miR-203a, miR-204, miR-301c and miR-10d were also found in a large number of reads (Figure 2B). Other miRNAs were also mostly observed after hatching, such as miR-125, miR-199, miR-192, miR-214 and the let-7 family. During metamorphosis, miR-192 was the miRNA with the highest number of reads in the 15°C group (4.0%) and the second highest in the 21°C group (1.2%). miR- 130c, miR-125b-5p, miR-199a-5p and miR-181a-5p were also amongst the miRNAs with highest expression at this stage (Figure 2B, Figure 3C, D, F). Likewise, the passenger strands miR-122-5p, miR-199a-3p, miR-140-3p and miR- 181a-3p were expressed at high levels during metamor- phosis (Figure 2B, Figure 3E). Furthermore, expression of some passenger and guide strands of the same miRNA dif- fered during development. For example, miR-122-5p and miR-140-3p were generally expressed at higher levels throughout development than their guide strands, miR- 122-3p and miR-140-5p, respectively (Figure 2B andC). Omnibus under the accession GSE58297. After discard- ing rRNAs, tRNAs and snoRNAs, 320 conserved miR- NAs from 149 miRNA families were identified (Table 1). It is noteworthy that 48 of these miRNAs had not been previously described in fish (Additional file 1: Table S1). Read length distributions for each sample are shown in Figure 1. Two peaks in read length were found for all samples, one at 22–23 nt and another around 28–29 nt, which correspond to miRNAs/interfering miRNAs and piwi-interacting RNAs, respectively. The majority of reads were around the 22 nt peak, indicating miRNA enriched samples (Figure 1). SOLiD sequencing of small RNAs No novel miRNAs were identified amongst the publicly available nucleotide sequences from Senegalese sole. 75Ep: 75% epiboly, 20S: 20 somites, Hatch: hatching, Met: metamorphosis stage 3. Differential expression of conserved miRNAs during Senegalese sole development and with embryonic temperature After trimming, two peaks in read length were found for all samples: one at 22–23 nt and a smaller one at 28–29 nt, which correspond to miRNAs/short interfering miRNAs and piwi-interacting RNAs, respectively. The majority of reads were around the 22 nt peak, indicating miRNA enriched samples. Embryos were incubated at 15°C or 21°C and transferred to 21°C after hatching. The developmental stages analysed were 75% epiboly (75Ep), 20 somites (20S), hatching (hatch) and metamorphosis (met). Figure 1 Histogram of read lengths (nt, nucleotides) obtained by SOLiD sequencing of Senegalese sole RNA samples. After trimming, two peaks in read length were found for all samples: one at 22–23 nt and a smaller one at 28–29 nt, which correspond to miRNAs/short interfering miRNAs and piwi-interacting RNAs, respectively. The majority of reads were around the 22 nt peak, indicating miRNA enriched samples. Embryos were incubated at 15°C or 21°C and transferred to 21°C after hatching. The developmental stages analysed were 75% epiboly (75Ep), 20 somites (20S), hatching (hatch) and metamorphosis (met). their expression profiles analysed in Senegalese sole em- bryos and larvae subjected to two different incubation temperatures (15°C and 21°C). There was some tendency to observe a higher number of conserved miRBase reads at later developmental stages and at a lower water tem- perature. This is unlikely to be a technical bias, since all libraries were prepared simultaneously and run on the same slide. Instead, it reflects a dynamic miRNA transcriptome throughout early ontogeny, which is in- fluenced by environmental factors. From the 320 con- served miRNAs, 48 were not previously found in fish but were known in mammals, birds or nematodes. Up- or down-regulation of some of them is known to be involved in cancer-associated gene deregulation, namely miR-211 [24] and miR-509 [25]. miR-71 mediates the effects of germ cell loss on life span, and its over-expression was found to extend the life span of Caenorhabditis elegans lacking germ cells [26]. Most of these novel miRNAs were found in more than one small RNA library and were re- gulated, which indicates that they may play a key role in Senegalese sole development. Nevertheless, the specific functions of these miRNAs in embryogenesis remain to be ascertained. miRNAs analysed had the highest expression levels at the pre-metamorphosis stage (miR-26a, miR-17a-5p, miR-130c, miR-181a-5p and miR-181a-3p), whilst miR- 199-5p and miR-206-3p were preferentially expressed during metamorphosis (Figure 3). miRNA target prediction in Senegalese sole Several miRNAs had possible mRNAs targets belong- ing to or directly affecting MAPK and mTOR path- ways (Figure 4, Table 2). In particular, miR-130c was predicted to regulate eif4e (eukaryotic translation ini- tiation factor 4E) and mapk9, miR-17a-5p may bind to the 3’UTRs of sestrin3, mapk3 and mapk13, miR- 181a-5p could target ddit4 (DNA-damage-inducible tran- script 4) and mapk3, miR-181a-3p may regulate calpain1 and miR-206-3p was predicted to target sestrin1. There is increasing evidence that miRNAs that pos- sess both highly conserved -5p and -3p sequences, espe- cially within seed and anchor sequences, can originate two mature functional miRNAs, such as the case of miR- 18a, miR-140 or miR-17 [27]. In the present study, se- veral passenger strands were expressed at higher levels than the respective guide strands at one or more de- velopmental stages (Figure 2 and 3), which suggests some involvement of these strands in Senegalese sole development. Differential expression of conserved miRNAs during Senegalese sole development and with embryonic temperature Most he sequenced miRNA transcriptomes in Senegalese sole embryos and larvae incubated at 15°C Table 1 Summary of the sequenced miRNA transcriptomes in Senegalese sole embryos and larvae incubated at 15°C or 21°C Sample # Total reads # Trimmed (% Total) Average length (nt) # Annotated miRBase (% Total) # Conserved miRNAs 75Ep 15°C 6 642 089 2 802 986 (42.2) 23.5 200 224 (3.0) 232 75Ep 21°C 8 842 550 2 169 069 (24.5) 21.1 174 027 (2.0) 231 20S 15°C 6 566 636 3 320 266 (50.6) 22.4 993 981 (15.1) 265 20S 21°C 6 101 835 2 564 327 (42.0) 22.7 689 954 (11.3) 265 Hatch 15°C 7 268 950 4 312 425 (59.3) 23.0 1 467 039 (20.2) 285 Hatch 21°C 6 200 277 3 256 433 (52.5) 22.4 1 162 239 (18.7) 281 Met 15°C 7 105 697 4 965 849 (69.9) 23.0 2 391 674 (33.7) 288 Met 21°C 6 325 932 4 247 722 (67.2) 25.2 804 587 (12.7) 279 75Ep: 75% epiboly, 20S: 20 somites, Hatch: hatching, Met: metamorphosis stage 3. Campos et al. BMC Genomics 2014, 15:525 http://www.biomedcentral.com/1471-2164/15/525 Page 4 of 13 Page 4 of 13 2.0E+05 4.0E+05 6.0E+05 8.0E+05 1.0E+06 1.2E+06 1.4E+06 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 # reads Length (nt) 0 2.0E+05 4.0E+05 6.0E+05 8.0E+05 1.0E+06 1.2E+06 1.4E+06 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 # reads Length (nt) 0 Figure 1 Histogram of read lengths (nt, nucleotides) obtained by SOLiD sequencing of Senegalese sole RNA samples. After trimming, two peaks in read length were found for all samples: one at 22–23 nt and a smaller one at 28–29 nt, which correspond to miRNAs/short interfering miRNAs and piwi-interacting RNAs, respectively. The majority of reads were around the 22 nt peak, indicating miRNA enriched samples. Embryos were incubated at 15°C or 21°C and transferred to 21°C after hatching. The developmental stages analysed were 75% epiboly (75Ep), 20 somites (20S), hatching (hatch) and metamorphosis (met). Figure 1 Histogram of read lengths (nt, nucleotides) obtained by SOLiD sequencing of Senegalese sole RNA samples. Differential expression of conserved miRNAs during Senegalese sole development and with embryonic temperature There were several significant differences in miRNA expression between embryonic temperatures. miR-17a- 5p, miR-26a, miR-130c and miR-206-3p transcripts were significantly higher at 21°C than at 15°C during 75Ep (P < 0.05) (Figure 3A, B, C, G). At the 20S stage, miR- 130c and miR-181a-3p showed a 1.5- and 4.9-fold higher expression at 15°C than 21°C (P < 0.05), respect- ively (Figure 3C, E). At hatching, miR-17a-5p, miR-26a, miR-130c, miR-181a-5p, miR-199a-5p and miR-206- 3p were highly expressed at 21°C compared to 15°C (Figure 3A, B, C, D, F, G; P < 0.05). No significantly dif- ferences between temperatures were found after hatching. Characterisation of Senegalese sole miRNAs Campos et al. BMC Genomics 2014, 15:525 http://www.biomedcentral.com/1471-2164/15/525 Campos et al. BMC Genomics 2014, 15:525 Page 6 of 13 miR-130c 75Ep 20S Hatch PreMet Met 30dph Relative expression 0 20 40 60 80 100 * * * miR-26a 75Ep 20S Hatch PreMet Met 30dph Relative expression 0 1 2 3 4 5 6 * * miR-181a-5p 75Ep 20S Hatch PreMet Met 30dph Relative expression 0 10 20 30 40 50 60 miR-181a-3p 75Ep 20S Hatch PreMet Met 30dph Relative expression 0 5 10 15 20 25 30 35 * miR-17a-5p 75Ep 20S Hatch PreMet Met 30dph Relative expression 0 20 40 60 80 100 120 140 miR-199a-5p 75Ep 20S Hatch PreMet Met 30dph Relative expression 0 20 40 60 80 100 120 140 160 180 * A B C D E F * * * miR-17a-5p 75Ep 20S Hatch PreMet Met 30dph Relative expression 0 20 40 60 80 100 120 140 A * * miR-26a 75Ep 20S Hatch PreMet Met 30dph Relative expression 0 1 2 3 4 5 6 * * B B miR-130c 75Ep 20S Hatch PreMet Met 30dph Relative expression 0 20 40 60 80 100 * * * 75Ep 20S Hatch PreMet Met 30dph Relative expression 0 1 2 3 4 5 * * miR-181a-5p 75Ep 20S Hatch PreMet Met 30dph Relative expression 0 10 20 30 40 50 60 miR-181a-3p 75Ep 20S Hatch PreMet Met 30dph Relative expression 0 5 10 15 20 25 30 35 * 75Ep 20S Hatch PreMet Met 30dph Relative expression 0 20 40 60 80 100 120 miR-199a-5p 75Ep 20S Hatch PreMet Met 30dph Relative expression 0 20 40 60 80 100 120 140 160 180 * miR-206-3p 75Ep 20S Hatch PreMet Met 30dph Relative expression 0 5 10 15 20 25 30 35 * * C D E F G * * * Figure 3 Relative expressions of (A) miR-17a-5p, (B) miR-26a, (C) miR-130c, (D) miR-181a-5p, (E) miR-181a-3p, (F) miR-199a-5p and (G) miR-206-3p during Senegalese sole early development. Embryos were incubated at 15°C (blue bars) or 21°C (red bars) and transferred to 21°C after hatching. Six developmental stages were analysed (75% epiboly (75Ep), 20 somites (20S), hatching (hatch), pre-metamorphosis (pre-met), metamorphosis (met) and 30 days post-hatch (30 dph)) (N = 3 pools of embryos or larvae). Transcript levels were determined by qPCR and the control RNA Spike-in (Exiqon) was used to compare miRNA expression profiles. Characterisation of Senegalese sole miRNAs Characterisation of Senegalese sole miRNAs Characterisation of Senegalese sole miRNAs Using high-throughput SOLiD sequencing, 320 conserved miRNA guide and passenger strands were identified and Page 5 of 13 Page 5 of 13 Campos et al. BMC Genomics 2014, 15:525 http://www.biomedcentral.com/1471-2164/15/525 Campos et al. BMC Genomics 2014, 15:525 http://www.biomedcentral.com/1471-2164/15/525 p e early development. The complete e linkage) has 320 rows (miRNAs) and 8 o embryonic temperatures (15 and 21°C). hroughout development, B) miRNAs velopment. Yellow and bue indicate higher btained by SOLiD sequencing during Senegalese sole early development. The complete med reads, log2 transformed, Euclidean distance, complete linkage) has 320 rows (miRNAs) and 8 stages (75Ep, 20S, hatching and metamorphosis) and two embryonic temperatures (15 and 21°C). plit in 3 panels: A) miRNAs with a low number of reads throughout development, B) miRNAs ges, and C) miRNAs with variable expression through development. Yellow and bue indicate higher NAs frequently referred to in the main text are highlighted in larger font. Figure 2 Heatmap of conserved miRNAs obtained by SOLiD sequencing during Senegalese sole early development. The complete heatmap (normalised to the number of trimmed reads, log2 transformed, Euclidean distance, complete linkage) has 320 rows (miRNAs) and 8 columns that correspond to 4 developmental stages (75Ep, 20S, hatching and metamorphosis) and two embryonic temperatures (15 and 21°C). For visualisation purposes, the heatmap was split in 3 panels: A) miRNAs with a low number of reads throughout development, B) miRNAs consistently expressed at high levels at all stages, and C) miRNAs with variable expression through development. Yellow and bue indicate higher and lower expression levels, respectively. miRNAs frequently referred to in the main text are highlighted in larger font. e d 8 21°C). higher Figure 2 Heatmap of conserved miRNAs obtained by SOLiD sequencing during Senegalese sole early development. The complete heatmap (normalised to the number of trimmed reads, log2 transformed, Euclidean distance, complete linkage) has 320 rows (miRNAs) and 8 columns that correspond to 4 developmental stages (75Ep, 20S, hatching and metamorphosis) and two embryonic temperatures (15 and 21°C). For visualisation purposes, the heatmap was split in 3 panels: A) miRNAs with a low number of reads throughout development, B) miRNAs consistently expressed at high levels at all stages, and C) miRNAs with variable expression through development. Yellow and bue indicate higher and lower expression levels, respectively. miRNAs frequently referred to in the main text are highlighted in larger font. Characterisation of Senegalese sole miRNAs Significant differences between temperatures are indicated by asterisks (P < 0.05). miR-130c 75Ep 20S Hatch PreMet Met 30dph Relative expression 0 20 40 60 80 100 * * * 75Ep 20S Hatch PreMet Met 30dph R 0 1 * * miR-181a-5p 75Ep 20S Hatch PreMet Met 30dph Relative expression 0 10 20 30 40 50 60 miR-181a-3p 75Ep 20S Hatch PreMet Met 30dph Relative expression 0 5 10 15 20 25 30 35 * 75Ep 20S Hatch PreMet Met 30dph R 0 20 miR-199a-5p 75Ep 20S Hatch PreMet Met 30dph Relative expression 0 20 40 60 80 100 120 140 160 180 * C D E F * * miR-181a-5p 75Ep 20S Hatch PreMet Met 30dph Relative expression 0 10 20 30 40 50 60 D * miR-130c 75Ep 20S Hatch PreMet Met 30dph Relative expression 0 20 40 60 80 100 * * * C D miR-181a-3p 75Ep 20S Hatch PreMet Met 30dph Relative expression 0 5 10 15 20 25 30 35 * miR-199a-5p 75Ep 20S Hatch PreMet Met 30dph Relative expression 0 20 40 60 80 100 120 140 160 180 * E F miR-206-3p 75Ep 20S Hatch PreMet Met 30dph Relative expression 0 5 10 15 20 25 30 35 * * G G Figure 3 Relative expressions of (A) miR-17a-5p, (B) miR-26a, (C) miR-130c, (D) miR-181a-5p, (E) miR-181a-3p, (F) miR-199a-5p and (G) miR-206-3p during Senegalese sole early development. Embryos were incubated at 15°C (blue bars) or 21°C (red bars) and transferred to 21°C after hatching. Six developmental stages were analysed (75% epiboly (75Ep), 20 somites (20S), hatching (hatch), pre-metamorphosis (pre-met), metamorphosis (met) and 30 days post-hatch (30 dph)) (N = 3 pools of embryos or larvae). Transcript levels were determined by qPCR and the control RNA Spike-in (Exiqon) was used to compare miRNA expression profiles. Significant differences between temperatures are indicated by asterisks (P < 0.05). Campos et al. BMC Genomics 2014, 15:525 http://www.biomedcentral.com/1471-2164/15/525 Page 7 of 13 Page 7 of 13 Akt/ mTOR IGF MAPK/ ERK JNK/ p38 MAPK IGF-1R DDIT4 Sestrin1 Sestrin3 eIF4E Differentiation Proliferation Differentiation Proliferation Differentiation Proliferation MAPK 3 MAPK 13 MAPK 9 Calpain 1 Myoblast fusion miR-130c miR-206-3p miR-17a-5p miR-181a-5p miR-181a-3p Targets of Figure 4 Simplified model of predicted miRNA targets related to Calpain1, JNK/p38 MAPK, MAPK/ERK and Akt/mTOR pathways. Igf functions are mediated by the Igf-I receptor (Igf1R) through activation of two major intracellular signalling pathways: MAPK and mTOR. Characterisation of Senegalese sole miRNAs mTOR and p38 MAPK are required for myogenic differentiation and p38 MAPK also inhibits proliferation. miR-130c was predicted to regulate EIF4E and MAPK9, miR-17a-5p may bind to the 3’UTRs of Sestrin3, MAPK3 and MAPK13, miR-181a-5p could target DDIT4 and MAPK3, miR-181a-3p may regulate Calpain1 and miR-206-3p was predicted to target Sestrin1. In Senegalese sole, miR-17a, miR-181-5p and miR-206 levels were higher at 21°C than 15°C during somitogenesis, whereas miR-181a-3p was down-regulated at 21°C compared to 15°C. Arrows, circles and horizontal lines indicate activation, negligible effect and inhibition, respectively. Figure 4 Simplified model of predicted miRNA targets related to Calpain1, JNK/p38 MAPK, MAPK/ERK and Akt/mTOR pathways. Igf functions are mediated by the Igf-I receptor (Igf1R) through activation of two major intracellular signalling pathways: MAPK and mTOR. mTOR and p38 MAPK are required for myogenic differentiation and p38 MAPK also inhibits proliferation. miR-130c was predicted to regulate EIF4E and MAPK9, miR-17a-5p may bind to the 3’UTRs of Sestrin3, MAPK3 and MAPK13, miR-181a-5p could target DDIT4 and MAPK3, miR-181a-3p may regulate Calpain1 and miR-206-3p was predicted to target Sestrin1. In Senegalese sole, miR-17a, miR-181-5p and miR-206 levels were higher at 21°C than 15°C during somitogenesis, whereas miR-181a-3p was down-regulated at 21°C compared to 15°C. Arrows, circles and horizontal lines indicate activation, negligible effect and inhibition, respectively. sing miRanda and RNAhybrid. MiRanda score and minimum free energy (ΔG kcal · mol−1) calculated using miRanda and RNAhybrid are ’-UTR target prediction. Targets were predicted using miRanda and RNAhybrid. MiRanda score and minimum free energy (ΔG kcal · mol−1) calculated using miRanda and RNAhybrid are marked for each miRNA-3’-UTR target prediction. Targets were predicted using miRanda and RNAhybrid. MiRanda score and minimum free energy (ΔG kcal · mol−1) calculated using m marked for each miRNA-3’-UTR target prediction. Expression profile and thermal plasticity of conserved miRNAs during Senegalese sole development Senegalese sole miRNAs were expressed throughout early ontogeny, others were stage-specific. The miR-430 family is an example of the latter (highly expressed only in earlier stages) and it is well known to accelerate deadenylation and clearance of maternal transcripts during zygotic stages [34,35]. miRNAs mostly observed after hatching or during metamorphosis included miR-125, miR-199, miR-192, miR-214 and the let-7 family (Figure 2). The latter com- prises a family of miRNAs that are highly conserved from worms to humans [36] and are involved in various pro- cesses, such as regulating cell proliferation and differenti- ation during development. The muscle-specific miR-206, known to reinforce the myogenic program by inhibiting the expression of DNA polymerase α (responsible for cell proliferation) and in- directly down-regulating the MyoD inhibitors Id1-3 and MyoR [46], showed increased expression levels during embryogenesis at 21°C compared to 15°C, further sup- porting our observations that this temperature enhanced muscle growth [23,47]. Interestingly, MyoD was found to regulate the expression of miR-133 [48] and miR-206 [49]. Transcript levels of myod2 at the 20S stage in Sene- galese sole embryos incubated at 15°C or 21°C [23] are consistent with the expression of miR-206 observed in the present study, which is higher at 21°C than 15°C. A similar conclusion can be drawn for miR-133, based on the digital expression profiling from SOLiD sequencing. In zebrafish and medaka (Oryzias latipes), miR-17 is ubi- quitously expressed during embryonic development but becomes restricted to proliferative tissues later in deve- lopment [50]. In the present study, we observed increased expression of miR-17a-5p in the 21°C group at some de- velopmental stages (Figure 3A); whether this is related with increased cellular proliferation is however unknown. Activation of the miR-214/199 cluster coincides with the recruitment of myoD and myogenin [51]. Furthermore, miR-199, miR-206 and miR-17 are positively associated with muscle regeneration and remodelling [52]. This abil- ity to stimulate myogenesis may explain the differences in transcript levels of miR-199, miR-206 and miR-17 between the two temperature groups of Senegalese sole larvae at hatching. Higher levels of these miRNAs are consistent with the observed muscle phenotype in the 21°C group compared to larvae reared at 15°C [23]. g p Several miRNAs showed an expression peak during pre-metamorphosis or metamorphosis (Figures 2 and 3). Expression profile and thermal plasticity of conserved miRNAs during Senegalese sole development For instance, amongst the miRNAs validated by qPCR, miR-17a-5p, miR-26a, miR-130c,miR-181a-5p and miR- 181a-3p showed a peak of expression mainly during the pre-metamorphic stage (Figure 3A, B, C, D, E), whereas, miR-199a-5p and miR-206-3p had higher levels at me- tamorphosis or later (Figure 3F, G). Similar results were found for some miRNAs in the olive flounder (Paralichthys olivaceus) [37]. Flatfish larvae undergo a particularly im- pressive metamorphosis, resulting in an asymmetrical cra- niofacial remodelling and lateralized behaviour [38,39]. In olive flounder, an involvement of miR-206a has also been suggested in the metamorphic process, which is mediated by the thyroid hormone [40]. It is known that Senegalese sole larvae have a high growth rate and accumulate ener- getic compounds until the onset of metamorphosis [41] but growth rates decrease significantly once metamorpho- sis starts [38]. It is possible that up-regulation of some miRNAs at a pre-metamorphic/metamorphic stages is associated with changes in lipid metabolism and with the complex metamorphic process. Such alterations in lipid metabolism may also be correlated with the high expression of miR-122 during metamorphosis, as previ- ously observed in other flatfish like halibut [19] or flounder [42]. miR-122, which is a liver-specific miRNA, has a crucial role in lipid metabolism in teleosts [43]. It is therefore plausible that changes in this miRNA abun- dance might be related with physiological changes oc- curring during metamorphosis. The observed growth differences between temperature groups may also be associated with other metabolic func- tions (e.g., energy production), since the abundance of miRNAs related to lipid metabolism differed across temperature groups. For instance, the guide strand of the liver-specific miR-122 was expressed at higher levels at 15°C than at 21°C. Given the role of this miRNA in fatty-acid oxidation, and therefore in energy production, it is plausible that temperature affected lipid metabolism and energy utilisation of sole larvae, thus affecting growth. Furthermore, since the passenger strand was expressed at high levels, one can hypothesise that it may also play an active role in lipid metabolism. Some of the miRNAs identified in Senegalese sole are known to play a stimulatory role in somatic growth and specifically in skeletal muscle development. In mammals, miR-26a up-regulation is described to post- transcriptionally repress the histone methyltransferase Ezh2, which is a suppressor of skeletal muscle cell dif- ferentiation [44]. In fish, such as the common carp, this miRNA was also associated with myogenesis [14]. Expression profile and thermal plasticity of conserved miRNAs during Senegalese sole development allometric differences in growth of specific tissues may influence the observed miRNA expression profiles. Never- theless, substantial changes in the Senegalese sole miRNA transcriptome during development (Table 1) highlight the importance of miRNAs in tissue and organ differentiation. miRNA differential expression has been found during the development of other vertebrate species, and similarly to our work, generally an increasing number of miRNAs are observed from the earliest to the latest stages of develop- ment [19,33]. Furthermore, we found that whilst some Critical early ontogeny events like organogenesis, hatch- ing or metamorphosis in fish involve dramatic changes in signalling, physiology and morphology [28-31]. Well defined expression windows of myogenic and growth- related genes during Senegalese sole ontogeny and larval development have been previously characterised, since different genes are activated or down-regulated at diffe- rent stages of development [23,32]. It is plausible that argets of five differentially expressed miRNAs during early development of Senegalese sole Table 2 Predicted targets of five differentially expressed miRNAs during early development of Senegalese sole miRNA Ensembl Gene ID Gene name miRanda RNAhybrid Score Energy (kcal/mol) Energy (kcal/mol) miR-130c ENSGACG00000005536 EIF4E 156 −25.65 −28.7 miR-130c ENSGACG00000018076 MAPK9 142 −21.42 −24.4 miR-17a-5p ENSGACG00000017027 Sestrin3 143 −24.03 −26.8 miR-17a-5p ENSGACG00000013102 MAPK3 154 −21.58 −26.2 miR-17a-5p ENSGACG00000004911 MAPK13 140 −22.15 −24.1 miR-181a-5p ENSGACG00000019700 DDIT4 146 −20.26 −22.5 miR-181a-5p ENSGACG00000013102 MAPK3 161 −20.51 −26 miR-181a-3p ENSGACG00000018991 Calpain 1 156 −20.35 −31 miR-206-3p ENSGACG00000008895 Sestrin1 144 −20.56 −22.7 Targets were predicted using miRanda and RNAhybrid. MiRanda score and minimum free energy (ΔG kcal · mol−1) calculated using miRanda and RNAhybrid are marked for each miRNA-3’-UTR target prediction. Table 2 Predicted targets of five differentially expressed miRNAs during early development of Se Targets were predicted using miRanda and RNAhybrid. MiRanda score and minimum free energy (ΔG kcal · mol−1) calculated using miRanda and RNAhybrid are marked for each miRNA-3’-UTR target prediction. Page 8 of 13 Campos et al. BMC Genomics 2014, 15:525 http://www.biomedcentral.com/1471-2164/15/525 can be drawn from miR-181a-5p expression in Senegalese sole embryos. miR-181 is strongly up-regulated in regen- erating muscle from an in vivo mouse model of muscle injury [11] and endurance exercise was also found to significantly increase the expression of miR-181 in mice skeletal muscle [45]. These results are consistent with previous findings in Senegalese sole, where a lower in- cubation temperature (15°C) produced lighter larvae with smaller fast fibres [23]. Prediction of potential targets of the differentially regulated Senegalese sole miRNAs may have down-regulated this myogenic inhibitor. Fur- thermore, sestrin3 was predicted to be regulated by miR- 17a. Like Sestrin1 and Sestrin2, Sestrin3 promotes AMPK activity [60,61] and it has been shown that the induced ex- pression of Sestrin3 by FoxO1 transcription factor in- hibits mTOR Complex 1 (mTORC1), which regulates cell growth mostly through increased protein synthesis [61]. miR-17a up-regulation at hatching in larvae reared at 21°C is consistent with a possible down-regulation of ses- trin3 and a concomitant increase in growth. Several miRNAs were computationally predicted with high confidence to target mRNAs directly related to myo- genesis (Figure 4, Table 2). However, it should be noted that miRNAs can have multiple mRNAs targets and whole larvae were used to investigate miRNA expression in this study. Hence, it is likely that expression levels of some Senegalese sole miRNAs reflect a broader gene regulation, except for those that are muscle-specific, such as miR-206. mTOR and p38 MAPK pathways have overlapping yet distinct roles during myogenesis: both are required for myogenic differentiation and p38 MAPK, but not mTOR, can inhibit proliferation [6]. It was also suggested that the normoxia activation of MAPK/ERK signalling pathway not only stimulates myoblast proliferation [5] but also suppresses myogenic differentiation [6]. MAPKs can be activated by a wide variety of different stimuli, but in gen- eral MAPK1 and 3 (also called ERK2 and ERK1, respec- tively) are preferentially activated in response to growth factors whereas the JNK and p38 kinases (which include amongst others MAPK9 and MAPK13) are more respon- sive to stress stimuli like osmotic shock or ionizing radi- ation [53]. The major direct or indirect targets of mTOR appear to be mechanisms responsible for ribosome re- cruitment to mRNA, such as the eukaryotic translation initiation factor 4E (eIF4E), which is activated by the phos- phorylation of its repressors, the 4EBP proteins [54,55]. Phosphorylation of 4EBP1 by mTOR stimulates transla- tion initiation through the release of eIF4E from 4EBP1, allowing eIF4E to associate with eIF4G to enhance cap- dependent translation [55]. Calpains are known to play a role in myogenesis, namely in promoting myoblast fusion [62]. Senegalese sole miR- 181a-3p was predicted with high confidence to target the 3’ UTR of calpain 1. Expression profile and thermal plasticity of conserved miRNAs during Senegalese sole development In our study, the higher miR-26a expression observed in the 21°C group during hatching (and a similar trend during 75Ep and 20S) indicates a possible activation of the myogenic programme at a higher temperature. A similar conclusion Page 9 of 13 Campos et al. BMC Genomics 2014, 15:525 http://www.biomedcentral.com/1471-2164/15/525 Campos et al. BMC Genomics 2014, 15:525 http://www.biomedcentral.com/1471-2164/15/525 Prediction of potential targets of the differentially regulated Senegalese sole miRNAs Its 4.9-fold up-regulation at 15°C during somitogenesis indicates a possible repression of calpain 1, which would impair the fusion of myoblasts to multinucleated myotubes at this temperature and result in the observed muscle phenotype differences between the two temperature groups [23]. Conclusions We have identified 320 conserved miRNAs in Senegalese sole, most of them dynamically expressed during early development and 48 not previously described in teleosts. Embryonic temperature affected the expression of several miRNAs, and it seems that a higher incubation tempe- rature (21°C) promoted expression of miRNAs positively related with growth at specific developmental stages. In particular, higher expression levels of miR-17a, miR-181- 5p and miR-206 and down-regulation of miR-181a-3p at 21°C may promote myogenesis and are in agreement with previous studies in Senegalese sole, which reported en- hanced growth at higher embryonic temperatures com- pared to 15°C. Also, miRNAs related to lipid metabolism and energy production (e.g., miR-122) may also be in- volved in the differential growth of fish with temperature. Taken together, our data indicate that miRNAs may play a role in temperature-induced phenotypic plasti- city of growth in teleosts. The DNA-damage-inducible transcript 4 (REDD1) sup- presses mTOR activity through the TSC1/TSC2 heterodi- mer [56,57]. miR-181a-5p up-regulation in Senegalese sole hatchlings at 21°C may down-regulate this mTOR inhibi- tor. miR-181a-5p was also predicted to target the 3’ UTR of mapk3. Thus, the up-regulation of miR-181a-5p at 21°C during early development may have led to higher myogenic differentiation of this group. Mapk3 and mapk13 are potential targets of miR-17a-5p, which are involved in MAPK/ERK and JNK/p38 MAPK signalling pathways, respectively. In human cell lines, miR-17-5p targets the 3’UTR of Mapk9 [58]. MAPK9 is a negative regulator of cellular proliferation through the JNK/P38 MAPK path- way [59] and this interaction contributed to the prolifera- tive phenotype caused by miR-17-5p [58]. In the present study, miR-130c was predicted to target MAPK9 mRNA and may therefore have a role in regulating cell prolifera- tion at some developmental stages. Animal ethics All experiments were conducted by trained scientists fol- lowing FELASA category C recommendations and were carried out in accordance with the clear boundaries of EU legal frameworks, specifically those relating to the protection of animals used for scientific purposes (i.e. Directive 2010/63/EU) and under the Portuguese legisla- tion regarding the protection of animals used for scien- tific purposes (Decree-Law No. 113/2013). This study was performed at the research facilities of the CCMAR (Algarve, Portugal), which has been certified for animal experimentation by the competent authority (Direcção Geral de Alimentação e Veterinária, Portugal). miR-206 was predicted to target sestrin1, which codes for a negative regulator of mTOR signalling through acti- vation of AMPK and TSC2 phosphorylation [60]. The up- regulation of miR-206 at 21°C during early development Campos et al. BMC Genomics 2014, 15:525 http://www.biomedcentral.com/1471-2164/15/525 Campos et al. BMC Genomics 2014, 15:525 http://www.biomedcentral.com/1471-2164/15/525 Page 10 of 13 of less than 40 nucleotides (nt) was recovered by sodium acetate precipitation, assessed on Agilent 2100 Bioana- lyzer with small RNA chip (Agilent Technologies), and used for the amplified small RNA library template prep- aration using the SOLiD™total RNA-seq kit with the barcoded SOLiD™3’ primers (Life Sciences). The aver- age length of the amplified cDNA template was assessed in Agilent 2100 Bioanalyzer with the high sensitivity DNA chip kit (Agilent Technologies) to calculate the ratio of the desired miRNA ligation products (120– 130 bp). To quantify the precise cDNA yield of miRNA ligation products for template bead preparation, real-time quantitative PCR (qPCR) was carried out using KAPA SYBR FAST Light Cycler 480 qPCR kit with SOLiD™pri- mer mix and DNA standards (Kapa Biosystems). Each library template was clonally amplified on SOLiD™P1 beads by emulsion PCR and then sequenced on the SOLiD4 platform (Life Sciences) on a quadrant of a slide with 50 nt read length. Sequencing data analysis Sequencing data analysis The SOLiD raw colour space reads were pre-processed using the CLC Genomics Workbench 4.9 (CLCbio) to remove low quality reads and clip internal adaptor se- quence (CTGCTGTACGGCCAAGGCC), thus leaving reads ranging between 10 and 35 nucleotides (nt) in length. Reads corresponding to small RNAs other than miRNAs (such as tRNAS, rRNA, mtRNA or snRNAs) were discarded following BLAST searches against ncRNA databases. Unannotated clusters and singletons were mapped against miRNAs sequences deposited in miRBase 18.0 (http://www.mirbase.org/) in order to identify con- served miRNAs. The remaining reads were mapped against Pleurinectiformes and Solea EST databases (NCBI) to identify possible non-conserved miRNAs. For each mappable sequence between 16 and 28 nt and with 5 counts or above, hairpin folding was evaluated by se- quence analysis to identify the presence of a stem loop using the RNA secondary structure prediction tool in the CLC Genomics Workbench. The flanking EST sequence of each analysed read should be at least 60 nt, and the free energy of the fold-back structure (ΔG) should be ≤−18 kcal · mol−1. Sampling and RNA extraction Embryo and larvae samples were taken in triplicate according to the developmental stages described in Fernández-Díaz et al. [32]. The sampling points were: 75% epiboly (75Ep), 20 somites (20S), hatching (0 dph), pre- metamorphosis (Pre-Met, stage 0 according to the eye- translocation stage; 8 and 9 dph for larvae from 21°C and 15°C, respectively), metamorphosis climax (Met, stage 3 according to the eye-translocation stage; 14 dph and 15 dph in larvae incubated at 21°C and 15°C, respectively) and 30 dph. The embryos were snap-frozen in liquid ni- trogen, and larvae were sacrificed by terminal anaesthesia with MS-222 (400 mg · l−1) and snap-frozen in liquid nitrogen. All samples were stored at −80°C until further analysis. Total RNA was extracted using the mirVana miRNA isolation kit (Ambion) according to the manufac- turer’s instructions. Assessment of RNA quality was per- formed by agarose gel electrophoresis on a 1.2% (w/v) agarose gel containing SYBR safe DNA gel stain. RNA samples were then quantified with a Nanodrop spect- rophotometer (Nanodrop Technologies/Saven Werner). Absorbance ratios (260/280 nm) were greater than 1.9, in- dicating high purity RNA. The number of reads in each sample was normal- ised by dividing the number of reads of a given miRNA in a sample by the total number of trimmed miRNA reads in the same sample. To be able to differentiate the expression of each miRNA and categorise them according to their expression pattern, a heatmap chart was drawn and hierarchical clustering with Euclidean distance and complete linkage algorithms was per- formed using the MeV online software (http://www. tm4.org/mev.html). Fish husbandry Senegalese sole embryos were incubated at two tempera- tures (15.2 ± 0.3°C and 20.9 ± 0.3°C) in triplicate groups at the LEOA facility, CCMAR/University of Algarve, Portugal. Eggs obtained by natural spawning of captive broodstock fish were randomly distributed amongst 6 fi- breglass conical tanks (100 l) at a density of 100 eggs · l−1. After hatching all larvae were reared at the same tem- perature (20.8 ± 0.1°C) until the metamorphosis climax. Water temperature, O2, salinity, pH and nitrogenous com- pounds were monitored regularly during the entire trial, and larvae were exposed to an artificial photoperiod of 12 h:12 h light:dark. They were reared in 100 L conical tanks at a density of 40 larvae · L−1. At mouth opening lar- vae started to feed on rotifers (Brachionus sp.). Artemia nauplii were introduced at 5 days post-hatch (dph) and their density was gradually increased from 4 to 8 ml−1 until 8 dph. Live Artemia meta-nauplii were then intro- duced at this time point on increasing from 12 to 20 · ml−1 until 18 dph. Frozen meta-nauplii were offered from when larvae settled at the bottom of the tanks (around 18 dph) until 30 dph. SOLiD sequencing of Senegalese sole small RNAs SOLiD sequencing of Senegalese sole small RNAs Small RNA reads were generated from eight SOLiD (Life Technologies, Austin, Texas, USA) libraries prepared from pools of triplicate samples of embryos and larvae from the following stages and at both 15 and 21°C tem- perature groups: 75Ep, 20S, hatching and metamor- phosis climax stage 3. The enriched small RNA fraction Campos et al. BMC Genomics 2014, 15:525 http://www.biomedcentral.com/1471-2164/15/525 Page 11 of 13 Table 3 List of primers used in quantification of miRNAs by qPCR MicroRNA Mature sequence (5’ – 3’) ID qPCR primers (Exiqon) Amplicon (bp) Tm (°C) E (%) miR-26a UUCAAGUAAUCCAGGAUAGGCU 151790-1 49 69 98 miR-17a-5p CAAAGUGCUUACAGUGCAGGUA 151777-1 45 71 90 miR-130c CAGUGCAAUAUUAAAAGGGCAU 151782-1 48 69 94 miR-206-3p UGGAAUGUAAGGAAGUGUGUGG 151786-1 45 70 102 miR-199a-5p CCCAGUGUUCAGACUACCUGUUC 151813-1 46 70 95 miR-181a-5p AACAUUCAACGCUGUCGGUGAGU 151802-1 46 71 94 miR-181a-3p ACCAUCGACCGUUGAUUGUACC 151808-1 41 71 91 Amplicon sizes, melting temperatures (Tm) and PCR efficiencies (E) are indicated. Table 3 List of primers used in quantification of miRNAs by qPCR web server (bibiserv.techfak.uni-bielefeld.de/rnahybrid) [66]. MiRanda implements a dynamic search programme for maximal local complementarity alignments between the miRNA and the reference sequence and generate scores based on the position-specific empirical models [65]. The free energy of optimal strand-strand inter- action was calculated using the ViennaRNA package in- corporated within the program. Targets for Senegalese sole miRNAs were predicted using the following de- fault parameters: Gap open penalty, −9.0; gap extend penalty, −4.0; score threshold, 140.0; energy threshold, 1.0 kcal · mol−1; scaling parameter, 4.0. Targets predicted to have an energy threshold below −20 kcal · mol−1 were selected for further analysis. The RNAhybrid web server was further used to calculate the minimum free energy of the stable duplex between miRNA and target site within the 3’-UTR. Quantitative real-time PCR assay Real-time PCR (qPCR) was used to validate the SOLiD profile of 7 selected miRNAs (mir-17a-5p, mir-26a, mir- 130c, mir-206-3p, mir-181a-5p, mir-181a-3p and mir- 199a-5p) using LNA™-enhanced microRNA qPCR specific primers (Exiqon A/S) (Table 3). Expression analysis of these selected miRNAs was also performed in pre-metamorphic larvae (stage 0 according to the eye-translocation stage) and in 30 dph larvae, in order to have a more com- plete overview of these miRNA transcript levels dur- ing Senegalese sole development. g g p cDNA was synthesised from RNA diluted to 5 ng · μl−1 using the miRCURY LNA™Universal RT microRNA PCR Kit (Exiqon) and following the manufacturer’s in- structions. SOLiD sequencing of Senegalese sole small RNAs Reactions were performed using SYBR Green chemistry (Exiqon) on a Light Cycler 480® (Roche Applied Science) using white 96-well plates and the following run- ning conditions: denaturation at 95°C: 10 min followed by 45 amplification cycles at 95°C: 10 s and 60°C: 1 min. Spe- cificity of reaction was analysed by melting curve analysis [63]. Five-point standard curves of a 5-fold dilution series (1:1–1:625) of pooled RNA were used for PCR efficiency calculation [64]. All samples were run in duplicate. CT values were determined using the fit-point method using the LightCycler® 480 software with a fluorescence thresh- old arbitrarily set to 0.3. The provided control RNA Spike-in (Exiqon) was used as exogenous reference to examine the miRNAs expression profile during devel- opment and across temperatures, using the ΔΔCT method, according to Fernandes et al. [64]. Differences in the ex- pression of the target miRNAs between temperatures were evaluated by Student’s t-test using the SigmaPlot 11.0 stat- istical software (Systat Software, San Jose, CA, USA), since the normality assumption was met. Competing interests Th h d l h The authors declare that they have no competing interests. The authors declare that they have no competing interests. Authors’ contributions CC carried out the rearing experiment, performed the miRNAs sequence analysis and qPCR validation and drafted the manuscript. AYMS carried out the target prediction analysis. LMPV, LECC and SE participated in the design of the study. JMOF conceived the study, participated in its design and coordination, and helped to write the manuscript. All authors read and approved the final manuscript. Additional file Additional file 1: Table S1. Sequences of mature miRNAs identified in Senegalese sole.The 48 miRNAs that had not been previously described in fish are highlighted in yellow. Acknowledgements The authors acknowledge J. Sendão, M.F. Castanheira, P.Gavaia, E. Cabrita, H. Teixeira, F. Rocha and A. Santos (CCMAR, Portugal), for their invaluable help during the experimental setup and sampling of Senegalese sole embryos and larvae. This study was funded by Project EPISOLE - PTDC/MAR/110547/2009, granted by Fundação para a Ciência e a Tecnologia and Programa Operacional Temático Factores de Competitividade (COMPETE), FEDER (Portugal). J. Fernandes acknowledges the support provided by the Research Council of Norway (GrowCod project, ref. 190350/S40). C. Campos and S. Engrola were financially supported by Fundação para a Ciência e Tecnologia, Portugal, through grants SFRH/BD/43633/2008 and SFRH/BPD/49051/2008, respectively. Additional file 1: Table S1. Sequences of mature miRNAs identified in Senegalese sole.The 48 miRNAs that had not been previously described in fish are highlighted in yellow. 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BMC Genomics 2014, 15:525 http://www.biomedcentral.com/1471-2164/15/525 40. Fu YS, Shi ZY, Wang GY, Li WJ, Zhang JL, Jia L: Expression and regulation of miR-1, −133a, −206a, and MRFs by thyroid hormone during larval development in Paralichthys olivaceus. References Comp Biochem Physiol B 2012, 161(3):226–232. 62. Honda M, Masui F, Kanzawa N, Tsuchiya T, Toyo-oka T: Specific knockdown of m-calpain blocks myogenesis with cDNA deduced from the corresponding RNAi. Am J Physiol Cell Physiol 2008, 294(4):957–965. 63. Campos C, Valente LM, Borges P, Bizuayehu T, Fernandes JM: Dietary lipid levels have a remarkable impact on the expression of growth-related genes in Senegalese sole (Solea senegalensis Kaup). J Exp Biol 2010, 213(2):200–209. 41. Yufera M, Parra G, Santiago R, Carrascosa M: Growth, carbon, nitrogen and caloric content of Solea senegalensis (Pisces: Soleidae) from egg fertilization to metamorphosis. Marine Biol 1999, 134:43–49. 64. Fernandes JM, Mackenzie MG, Wright PA, Steele SL, Suzuki Y, Kinghorn JR, Johnston IA: Myogenin in model pufferfish species: Comparative genomic analysis and thermal plasticity of expression during early development. Comp Biochem Physiol Part D Genomics Proteomics 2006, 1(1):35–45. 42. Fu Y, Shi Z, Wu M, Zhang J, Jia L, Chen X: Identification and differential expression of microRNAs during metamorphosis of the Japanese flounder (Paralichthys olivaceus). PLoS One 2011, 6(7):e22957. 43. Mennigen JA, Martyniuk CJ, Seiliez I, Panserat S, Skiba-Cassy S: Metabolic consequences of microRNA-122 inhibition in rainbow trout: Oncorhynchus mykiss. BMC Genomics 2014, 15:70. 65. Enright AJ, John B, Gaul U, Tuschl T, Sander C, Marks DS: MicroRNA targets in Drosophila. Genome Biol 2003, 5(1):R1. 44. Wong CF, Tellam RL: MicroRNA-26a targets the histone methyltransferase Enhancer of Zeste homolog 2 during myogenesis. J Biol Chem 2008, 283(15):9836–9843. 66. 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References Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: 57. 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https://openalex.org/W3032129414
https://link.springer.com/content/pdf/10.1245/s10434-020-08653-w.pdf
English
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Patient Age and Survival After Surgery for Esophageal Cancer
Annals of surgical oncology
2,020
cc-by
5,753
ABSTRACT Background. Esophagectomy for esophageal cancer is associated with a substantial risk of life-threatening com- plications and a limited long-term survival. This study aimed to clarify the controversial questions of how age influences short-term and long-term survival. Methods. This population-based cohort study included almost all patients who underwent curatively intended esophagectomy for esophageal cancer in Sweden in 1987–2010, with follow-up through 2016. The exposure was age, analyzed both as a continuous and categorical variable. The probability of mortality was computed using a novel flexible parametric model approach. The reported probabilities are proper measures of the risk of dying, and the related odds ratios (OR) are therefore more suit- able measures of association than hazard ratios. The outcomes were 90-day all-cause mortality, 5-year all-cause mortality, and 5-year disease-specific mortality. A novel flexible parametric model was used to derive the instanta- neous probability of dying and the related OR along with 95% confidence intervals (CIs), adjusted for sex, education, comorbidity, tumor histology, pathological tumor stage, and resection margin status. Conclusions. Patient age 75 years or older at esophagec- tomy for esophageal cancer appears to be an independent risk factor for higher short-term mortality and lower long- term survival. The most effective treatment for locally advanced eso- phageal cancer is surgical resection (esophagectomy) combined with chemo(radio)therapy.1 Esophagectomy is an extensive procedure that carries a high risk (40–60%) of serious and sometimes lethal complications.1 The long- term (5-year) survival after surgery is limited—only 31% according to nationwide population-based studies from Sweden.2,3 The short-term safety and long-term benefits of esophagectomy in older people is controversial. Some studies have found no clear differences in short- or long- term mortality after esophagectomy when comparing patients older or younger than 70 years of age,4–10 but most of these studies were based on few and possibly selected patients from single centers. On the other hand, a number of other studies as well as a meta-analysis found an increased risk of short- and long-term mortality in elderly patients, although some of these studies also included patients who did not undergo curatively intended oesophagectomy.11–21 Thus, the role of age in relation to mortality after esophagectomy for esophageal cancer remains uncertain. If patient age independent of other Results. Among 1737 included patients, the median age was 65.6 years.  The Author(s) 2020 First Received: 9 March 2020; Published Online: 28 May 2020 G. Santoni, PhD e-mail: Giola.santoni@ki.se Patient Age and Survival After Surgery for Esophageal Cancer Jesper Lagergren, MD, PhD1,2, Matteo Bottai, ScD3, and Giola Santoni, PhD1 1Upper Gastrointestinal Surgery, Department of Molecular Medicine and Surgery, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden; 2School of Cancer and Pharmaceutical Sciences, King’s College London, London, UK; 3Division of Biostatistics, Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Swe 1Upper Gastrointestinal Surgery, Department of Molecular Medicine and Surgery, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden; 2School of Cancer and Pharmaceutical Sciences, King’s College London, London, UK; 3Division of Biostatistics, Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden 90-day all-cause mortality (OR 1.05, 95% CI 1.02–1.07), 5-year all-cause mortality (OR 1.02, 95% CI 1.01–1.03), and 5-year disease-specific mortality (OR 1.01, 95% CI 1.01–1.02). Compared with patients aged \ 70 years, those aged 70–74 years had no increased risk of any mortality outcome, while patients aged C 75 years had higher odds of 90-day mortality (OR 2.85, 95% CI 1.68–4.84), 5-year all-cause mortality (OR 1.56, 95% CI 1.27–1.92), and 5-year disease-specific mortality (OR 1.38, 95% CI 1.09–1.76). Ann Surg Oncol (2021) 28:159–166 https://doi.org/10.1245/s10434-020-08653-w Ann Surg Oncol (2021) 28:159–166 https://doi.org/10.1245/s10434-020-08653-w ORIGINAL ARTICLE – THORACIC ONCOLOGY ORIGINAL ARTICLE – THORACIC ONCOLOGY ABSTRACT When analyzed as a continuous variable, older age was associated with slightly higher odds of Electronic supplementary material The online version of this article (https://doi.org/10.1245/s10434-020-08653-w) contains supplementary material, which is available to authorized users. 160 J. Lagergren et al. Exposure factors influenced the survival after surgery it should be taken into account in the clinical decision making, i.e. when deciding whether esophagectomy should be recom- mended or not. The study exposure was the patient’s age at the date of surgery. This information was available in all registries and the medical records. We hypothesized that the short-term mortality was higher in older patients than in younger patients, while the long-term survival was independent of age. With the aim of testing these hypotheses, the present study took advantage of a well-established nationwide Swedish cohort with comprehensive data on key variables and complete follow- up for mortality. We availed ourselves of a novel statistical method that allowed estimation of mortality risk, properly defined as the probability of dying at any given time point. Design We used data from a Swedish, nationwide, population- based cohort study that has been previously validated and described in detail elsewhere.22,23 Briefly, this study included all patients with esophageal cancer treated with curative intent between 1987 and 2010, with follow-up until November 2014. From the Swedish Cancer Registry, patients with a diagnosis of esophageal cancer (150.0, 150.8, or 150.9) between 1987 and 2010 were identified according to the 7th edition of the International Classifi- cation of Diseases (ICD7). Patients with esophageal cancer who underwent esophagectomy were identified from the Swedish Patient Registry. The Swedish Causes of Death Registry was used to provide accurate data about the dates and causes of death. If the diagnosis of esophageal cancer was listed as a cause of death, then mortality was defined as disease-specific. The final source cohort consisted of 1820 patients who underwent curatively intended esophagec- tomy for esophageal adenocarcinoma or squamous cell carcinoma in Sweden between 1987 and 2010. They were followed up through 2016, thus allowing 5-year follow-up of all participants. After exclusion of patients with missing information on education (n = 69, 3.8%) or tumor stage (n = 14, 0.8%), the final sample included 1737 (95.4%) patients. The data were retrieved from medical records and five national Swedish health and population registries: Cancer Registry, Patient Registry, Cause of Death Reg- istry, Prescribed Drug Registry, and Longitudinal Integration Database for Health Insurance and Labor Market Studies (LISA). The linkages of individuals’ data were enabled by the personal identity numbers given to each Swedish resident upon birth or immigration.24 The study was approved by the Regional Ethical Review Board in Stockholm, Sweden. Covariates The confounders were seven variables known to be associated with both the exposure (age) and the outcome (mortality): (1) sex (male or female); (2) attained education level (B 9 years, 10–12 years, or [ 12 years of formal education); (3) comorbidity (Charlson comorbidity index score 0, 1, or C 2);27 (4) neoadjuvant therapy (typically chemoradiotherapy, no or yes); (5) tumor histology (ade- nocarcinoma or squamous cell carcinoma); (6) pathological tumor stage (0–II or III–IV); and (7) resection margin status (no tumor involved [R0] or tumor involved [R1 or R2]). Data on sex were retrieved from the medical records and all the registries used. Information on comorbidities was collected from the Patient Registry, which has a high completeness and accuracy of recording of diagnoses,28 while information on neoadjuvant therapy, tumor histol- ogy, pathological tumor stage, and resection margin status was obtained from review of all relevant surgery charts and histopathology records from the Swedish hospitals where the esophagectomy had been conducted.22,23 Outcomes The main outcome was 5-year all-cause mortality, which was selected because the 5-year cut-off reduces the influence of death due to factors unrelated to the esopha- geal cancer or its treatment, and the all-cause survival is the most complete and accurate assessment of survival. Sec- ondary outcomes were 90-day all-cause mortality and disease-specific 5-year mortality. The choice of 90 days as the short-term mortality cut-off was based on research showing a time shift of postoperative mortality from 30 days to 31–90 days with more recent calendar time.25 The definition of disease-specific mortality was mortality with an esophageal cancer diagnosis as a primary or con- tributing cause of death after excluding the first 90 days of surgery. The data on all mortality outcomes were retrieved from the Cause of Death Registry, which has over 99% completeness for date and cause of death.26 Statistical Analysis The probability of mortality was computed using a novel flexible parametric model approach29,30 and the results were displayed graphically. The probability of death was defined as the average probability of dying in an infinitely Age and Survival After Esophagectomy 161 TABLE 1 Sociodemographic and clinical characteristics of participants who have undergone esophagectomy for esophageal cancer in Sweden Characteristic N (%) Total 1737 (100.0) Age, years \60 505 (29.1) 60–69 655 (37.7) 70–74 340 (19.6) 75? 237 (13.6) Sex Males 1306 (75.2) Females 431 (24.8) Formal education, years B9 889 (51.2) 10–12 624 (35.9) [12 224 (12.9) Charlson comorbidity index 0 964 (55.5) 1 491 (28.3) 2? 282 (16.2) Neoadjuvant therapy No 1175 (67.6) Yes 562 (32.3) Tumor histology Adenocarcinoma 769 (44.3) Squamous cell carcinoma 968 (55.7) Pathological tumor stage 0–II 1042 (60.0) III–IV 695 (40.0) Resection margin status R0 1470 (84.6) R1-R2 267 (15.4) Deaths 90-day all-cause 195 (11.1) 5-year all-cause 1299 (74.8) 5-year disease-specific 1004 (65.1) short time interval. This quantity is related to the hazard function through the following mathematical expression: g(t) = 1 - exp[–h(t)], where g(t) is the probability func- tion, h(t) is the hazard function, and exp is the exponential function (see working paper at http://www.imm.ki.se/bios tatistics/eventprob/Working_paper_2020.pdf). This quan- tity can be readily estimated using general parametric survival models with a user-defined baseline hazard func- tion. Odds ratios (OR) of death as a function of both a linear age variable and a categorical age variable were reported. For comparison, flexible semi-parametric models were used to compute hazard ratios (HR) as a function of age at surgery. All analyses were conducted using the user- written Stata program stpreg available at http://www.imm. ki.se/biostatistics/eventprob. TABLE 1 Sociodemographic and clinical characteristics of participants who have undergone esophagectomy for esophageal cancer in Sweden We first analyzed age as a continuous variable in rela- tion to the mortality outcomes. Several models with different age and time functions, and age and time inter- actions, were tested and the final model was selected based on the Akaike’s information criterion. Age was introduced into the model as a linear predictor and through restricted cubic splines. Time since surgery was modeled with restricted cubic splines. The interaction between the exposure and time was also tested. Second, we analyzed age in three categories (\ 70, 70–74, and C 75 years) in relation to the probability of dying. RESULTS aged 75 years or older were alive 90 days after surgery (Fig. 1a). Figure 2 shows the changes in probability of dying within 90 days of surgery, depending on age or days after surgery. The probability of 90-day mortality increased from 10% at age 60 years to 60% or above at age 89 years (Fig. 2a). The odds of dying increased by 5% with each year of age (OR 1.05, 95% confidence interval [CI] 1.02–1.07) (Table 2). Patients aged 75 years or older had almost three times the odds of dying compared with patients younger than 70 years of age (OR 2.85, 95% CI 1.68–4.31). There was no statistically significant Patients Characteristics of the 1737 patients included in the study are described in Table 1. Median age at surgery was 65.6 years (interquartile range 5.6–71.9 years), and the age range was 18–89 years. Statistical Analysis The cut-off of 70 years of age was used because it is the most common cut-off used in the literature. The cut-off of 75 years of age was intro- duced to examine associations among older people. Patients entered the study at the date of surgery and remained in the cohort until the date of death or end of the study (31 December 2016), whichever occurred first. For each of the three mortality outcomes, we analyzed a crude model and an adjusted model. The adjusted model included the seven confounders presented above, using the same categorization. Stratified analyses were carried out for sex, tumor histology, pathological tumor stage, and resection margin status. 90-Day All-Cause Mortality The overall 90-day mortality rate was 11.2% (n = 195), and 90% of patients aged below 70 years and 85% of those 162 J. Lagergren et al. interaction between age and any of the seven covariates included in the model 5-Year Disease-Specific Mortality 0 0.00 0.25 0.50 0.75 1.00 15 30 45 60 75 90 y re g r u s retfa sra e Y y re g r u s retfa s y a D Kaplan-Meier survival estimates 0.00 0.25 0.50 0.75 1.00 Kaplan-Meier survival estimates Age <70 70-74 >75 Age <70 70-74 >75 0 1 2 3 4 5 B A FIG. 1 Survival estimates of age in relation to a 90-day all- cause mortality; and b 5-year all-cause mortality after esophagectomy for esophageal cancer 0 10 20 30 40 50 60 70 80 Probability (%) 20 30 40 50 60 70 80 90 Age at surgery 15 30 60 Days after surgery 0 10 20 30 40 50 60 70 80 Probability (%) 0 15 30 45 60 75 90 Days after surgery 50 60 70 Age at surgery B A FIG. 2 Probability of 90-day all-cause mortality after esophagectomy for esophageal cancer. a Probability versus age at surgery for three time points. 90-Day All-Cause Mortality b Probability versus years from surgery for three age groups y re g r u s retfa sra e Y 0.00 0.25 0.50 0.75 1.00 Kaplan-Meier survival estimates Age <70 70-74 >75 0 1 2 3 4 5 B 0 0.00 0.25 0.50 0.75 1.00 15 30 45 60 75 90 y re g r u s retfa s y a D Kaplan-Meier survival estimates Age <70 70-74 >75 A 0 0.00 0.25 0.50 0.75 1.00 15 30 45 60 75 90 y re g r u s retfa sra e Y y re g r u s retfa s y a D Kaplan-Meier survival estimates 0.00 0.25 0.50 0.75 1.00 Kaplan-Meier survival estimates Age <70 70-74 >75 Age <70 70-74 >75 0 1 2 3 4 5 B A B y re g r u s retfa s y a D 0 10 20 30 40 50 60 70 80 Probability (%) 20 30 40 50 60 70 80 90 Age at surgery 15 30 60 Days after surgery A 0 10 20 30 40 50 60 70 80 Probability (%) 0 15 30 45 60 75 90 Days after surgery 50 60 70 Age at surgery B B B A Age at surgery 5-Year Disease-Specific Mortality interaction between age and any of the seven covariates included in the model. The 5-year disease-specific mortality rate was 76.7% (n = 1183). The adjusted odds of disease-specific death increased by 1% for each year of age (OR 1.01, 95% CI 1.01–1.02) (Table 2). The association between age and probability of dying showed a pattern similar to that of the 5-year all-cause mortality (electronic supplementary Fig. 4). There was no difference between the 70–74 years age group and younger patients, while patients aged 75 years or older had 38% higher odds of dying than patients younger than 70 years of age (adjusted OR 1.38, 95% CI 1.09–1.76). There was no statistically significant interaction between age and any of the covariates in the model. In the stratified analyses, older age was associated with higher odds of 5-year disease-specific mortality in participants with clear resection margins (R0) and in patients with early-stage tumor (pathological tumor stage 0, I, or II) [electronic supplementary Table 6]. 5-Year All-Cause Mortality The 5-year all-cause mortality rate was 74.8% (n = 1299). At 5 years after surgery, 27% of patients aged below 70 years and 16% of those aged 75 years or older were alive (Fig. 1b). The association between age and probability of dying is depicted in Fig. 3. The probability of death increased with age (Fig. 3a), but the strength of the association decreased with time after surgery (Fig. 3b). The adjusted odds of 5-year mortality increased by 2% with each year of age (OR 1.02, 95% CI 1.01–1.03) (Table 2). Patients aged 75 years or older had 56% higher odds of dying than those younger than 70 years of age (OR 1.56, 95% CI 1.27–1.92) (Table 2). No interaction term between age and sex, tumor histology, tumor stage, or resection margin status was statistically significant. In the stratified analyses, the strength of the association between age and 5-year all-cause mortality was stronger in patients with clear resection margins (R0) (Table 3). The results remained similar after excluding the first 90 days after surgery (electronic supplementary Table 4 and electronic supplementary Table 5). Comparison Between Odds Ratios and Hazard Ratios Electronic supplementary Table 7 reports the powers of the probability of all-cause 90-day and 5-year mortality, which were equal to the ratios of the hazard of dying. Age and Survival After Esophagectomy 163 Deaths [N (%)] Unadjusted OR (95% CI) Adjusted OR (95% CI) 90-day all-cause mortalitya Age, linear 195 (11) 1.05 (1.02–1.07) 1.05 (1.02–1.07)b Age, years \ 70 113 (10) 1.00 (reference) 1.00 (reference)b 70–74 37 (11) 1.15 (0.72–1.83) 1.19 (0.72–1.96)b C 75 45 (19) 2.63 (1.60–4.31) 2.85 (1.68–4.84)b 5-year all-cause mortalityc Age, linear 1299 (75) 1.02 (1.01-1.03) 1.02 (1.01–1.03)b Age, years \ 70 849 (73) 1.00 (reference) 1.00 (reference)b 70–74 251 (74) 1.02 (0.86–1.21) 1.05 (0.88–1.26)b C 75 199 (84) 1.49 (1.22–1.81) 1.56 (1.27–1.92)b 5-year disease-specific mortalityd Age, linear 1004 (65) 1.01 (1.00–1.02) 1.01 (1.01–1.02)e Age, years \ 70 673 (64) 1.00 (reference) 1.00 (reference)e 70–74 193 (64) 0.98 (0.81–1.19) 1.06 (0.87–1.30)e C 75 138 (72) 1.28 (1.02–1.61) 1.38 (1.09–1.76)e OR odds ratio, CI confidence interval aLinear function of survival time bAdjusted for sex, education, Charlson comorbidity index, tumor histology, pathological tumor stage, and resection margin status cSpline function of time after surgery with 3 knots dSpline function of time after surgery with 4 knots eAdjusted for sex, education, neoadjuvant therapy, tumor histology, pathological tumor stage, and resection margin status Deaths [N (%)] Unadjusted OR (95% CI) Adjusted OR (95% CI) Deaths [N (%)] Unadjusted OR (95% CI) Adjusted OR (95% CI) Powers and ORs of dying were similar for values of the odds and of the hazards closer to one. DISCUSSION This study indicated that both short- and long-term mortality increased with older age of patients after esophagectomy for esophageal cancer, independent of other prognostic factors, including comorbidity and tumor stage. The probability of mortality was higher for patients aged 75 years or older at the time of surgery, particularly for short-term mortality. The association between older age at surgery and higher probability of dying was similar in the different strata of the cohort, but the 5-year mortality outcomes were more pronounced in patients with clear resection margins (R0) compared with those with tumor- involved margins (R1 or R2). Comparison Between Odds Ratios and Hazard Ratios 0 10 20 30 40 50 Probability (%) 20 30 40 50 60 70 80 90 Age at surgery Half Two Four Years after surgery 0 10 20 30 40 50 Probability (%) 0 1 2 3 4 5 Years from surgery Age 50 Age 60 Age 70 Age at surgery B A FIG. 3 Probability of 5-year all-cause mortality after esophagectomy for esophageal cancer. a Probability versus age at surgery for three time points after esophagectomy for esophageal cancer. b Probability versus years from surgery for three age groups 0 10 20 30 40 50 Probability (%) 20 30 40 50 60 70 80 90 Age at surgery Half Two Four Years after surgery A 0 10 20 30 40 50 Probability (%) 0 1 2 3 4 5 Years from surgery Age 50 Age 60 Age 70 Age at surgery B B B Probability (%) Probability ( stage. The probability of mortality was higher for patients aged 75 years or older at the time of surgery, particularly for short-term mortality. The association between older age at surgery and higher probability of dying was similar in the different strata of the cohort, but the 5-year mortality outcomes were more pronounced in patients with clear resection margins (R0) compared with those with tumor- involved margins (R1 or R2). Powers and ORs of dying were similar for values of the odds and of the hazards closer to one. DISCUSSION 90-day mortality 5-year all-cause mortality Deaths [N (%)] OR (95% CI)a Deaths [N (%)] OR (95% CI)a Sex Males 155 (12) 991 (76) Age \ 70 years 91 (10) 1.00 (reference) 661 (74) 1.00 (reference) Age 70–74 years 30 (12) 1.29 (0.73–2.28) 191 (77) 1.11 (0.90–1.36) Age C 75 years 34 (20) 2.71 (1.47–4.99) 139 (82) 1.40 (1.10–1.79) Females 40 (9) 431 (71) Age \ 70 years 22 (8) 1.00 (reference) 188 (69) 1.00 (reference) Age 70–74 years 7 (8) 0.90 (0.31–2.62) 60 (66) 0.92 (0.65–1.31) Age C 75 years 11 (16) 3.26 (1.17–9.09) 60 (88) 2.01 (1.37–2.94) Tumor histology Adenocarcinoma 69 (9) 523 (68) Age \ 70 years 35 (7) 1.00 (reference) 312 (65) 1.00 (reference) Age 70–74 years 16 (9) 1.31 (0.62–2.79) 121 (71) 1.19 (0.92–1.55) Age C 75 years 18 (15) 2.79 (1.27–6.11) 90 (76) 1.44 (1.08–1.94) Squamous cell carcinoma 126 (13) 776 (80) Age \ 70 years 78 (11) 1.00 (reference) 537 (79) 1.00 (reference) Age 70–74 years 21 (12) 1.09 (0.56–2.14) 130 (77) 0.93 (0.73–1.20) Age C 75 years 27 (23) 2.93 (1.43–6.00) 109 (92) 1.70 (1.27–2.28) Pathological tumor stage 0–II 108 (10) 671 (64) Age \ 70 years 65 (9) 1.00 (reference) 439 (63) 1.00 (reference) Age 70–74 years 19 (10) 0.98 (0.50–1.92) 122 (51) 0.96 (0.76–1.21) Age C 75 years 24 (17) 2.24 (1.13–4.45) 110 (78) 1.65 (1.28–2.14) III–IV 87 (12) 628 (90) Age \ 70 years 48 (10) 1.00 (reference) 410 (90) 1.00 (reference) Age 70–74 years 18 (13) 1.53 (0.71–3.29) 129 (91) 1.21 (0.91–1.62) Age C 75 years 21 (22) 4.04 (1.76–9.24) 89 (93) 1.40 (1.00–1.96) Resection margin status R0 135 (9) 1044 (71) Age \ 70 years 73 (8) 1.00 (reference) 664 (69) 1.00 (reference) Age 70–74 years 29 (10) 1.27 (0.74–2.20) 211 (71) 1.02 (0.84–1.23) Age C 75 years 33 (16) 2.75 (1.55–4.90) 169 (82) 1.61 (1.29–2.00) R1–R2 60 (22) 255 (95) Age \ 70 years 40 (21) 1.00 (reference) 185 (95) 1.00 (reference) Age 70–74 years 8 (20) 0.80 (0.24–2.67) 40 (98) 1.51 (0.83–2.77) Age C 75 years 12 (37) 3.68 (0.91–14.99) 30 (94) 1.18 (0.64–2.16) OR odds ratio, CI confidence interval a Adjusted for sex, education, Charlson comorbidity index, tumor histology, pathological tumor stage, and resection margin status TABLE 3 Odds ratios, with 95% confidence intervals, of age in relation to mortality after esophagectomy for esophageal cancer, stratified by sex or tumor characteristics OR odds ratio, CI confidence interval a Adjusted for sex, education, Charlson comorbidity index, tumor histology, pathological tumor stage, and resection margin status Among the methodological strengths of this study is the population-based design with inclusion of almost all patients in Sweden who underwent curatively intended esophagectomy for cancer. DISCUSSION This study indicated that both short- and long-term mortality increased with older age of patients after esophagectomy for esophageal cancer, independent of other prognostic factors, including comorbidity and tumor 164 J. Lagergren et al. TABLE 3 Odds ratios, with 95% confidence intervals, of age in relation to mortality after esophagectomy for esophageal cancer, stratified by sex or tumor characteristics DISCUSSION Data on the exposure (patient age) and outcomes (mortality) were complete and valid. The extensive clinical information from all hospitals in Sweden and nationwide registries allowed for adjustment for the main confounding factors and counteracted loss to follow-up. However, the study had no direct information about other potential confounders, such as smoking and obesity, which increase with age.31,32 Yet, these factors were partially controlled for by adjusting for comorbidities associated with smoking and obesity. A possible stricter selection of older patients considered fit enough to undergo esophagectomy or with tumors of less advanced stage might have introduced bias. However, current protocols for Age and Survival After Esophagectomy 165 suitable measures of association than HRs. The reported probabilities are not average probabilities of dying within a given interval of time, but rather the instantaneous proba- bilities of dying at any given time. Because obtaining instantaneous probabilities and their ORs is simple to interpret, we recommend calculating and reporting these instead of the traditional hazards.34 curative esophagectomy state that the selection for surgery should be independent of age, and the adjustment for comorbidity and tumor stage should have counteracted any such bias. The postoperative survival rates have improved during the long study period, but it is less likely that the study period influenced the association between age and survival. The main finding of this study was that older age increases the probability of dying not only in the short-term but also in the longer term. Only some of the association between older age and 5-year mortality was explained by the higher 90-day mortality. These results are in line with other studies that found increased in-hospital mortality and 5-year mortality in patients aged 70 years or older.15–17,19 The main finding of this study was that older age increases the probability of dying not only in the short-term but also in the longer term. Only some of the association between older age and 5-year mortality was explained by the higher 90-day mortality. These results are in line with other studies that found increased in-hospital mortality and 5-year mortality in patients aged 70 years or older.15–17,19 Older age was also associated with an increased disease- specific mortality in the present study. DISCUSSION A previous study suggested that older age does not increase the risk of 5-year disease-specific mortality, but increases the non-surgical complication rate and susceptibility to in-hospital mortality compared with younger counterparts.11 Other studies have found no influence of age on long- or short-term mortal- ity.4–10 A systematic review and meta-analysis found that age had little influence on mortality after oesophagec- tomy;33 however, some of the studies included in the meta- analysis contained few participants.5,7,9 DISCLOSURE The authors declare that they have 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. 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/. These findings may influence clinical decision making. The study suggests that age is an independent risk factor for worse survival, both in the short- and long-term, after esophageal cancer surgery. Thus, when patients are con- sidered for surgery or other treatment options, e.g. definite chemo(radio)therapy or a palliative route, age should be regarded as an independent prognostic factor in the clinical setting. This has not been made clear in the existing literature. The statistical method used in this paper allowed for obtaining curves for the probability of dying throughout the follow-up time. This method can be applied to any study where the aim is to analyze the time to an event of interest, such as diagnosis of cancer and onset of a disease. Tradi- tionally, in these types of studies, the analysis focuses on hazard ratios and hazard functions. The reported proba- bilities, unlike hazards, are proper measures of the risk of dying, and the related ORs are therefore more CONCLUSION This nationwide and population-based cohort study indicates that patient age 75 years or older should be taken into consideration as an independent poor prognostic factor when patients are considered for esophagectomy for eso- phageal cancer. Surgery also remains the best available treatment for older patients, but age should be part of the informed consent process. ACKNOWLEDGEMENTS Open access funding provided by Karolinska Institute. AUTHORS CONTRIBUTION All authors contributed to the study design. JL was responsible for the data collection. GS performed the data management and statistical analyses, and prepared the first draft of the paper. All authors contributed to the interpretation of the results, critically reviewed the manuscript, and had full access to all of the data (including statistical reports and tables) in the study. GS takes responsibility for the integrity of the data and the accuracy of the data analysis. The association between age and mortality in this study was not strongly affected by other factors such as comor- bidity or tumor characteristics. For the 5-year all-cause mortality, the association was stronger in patients with early-stage tumors and those who had surgery with tumor- free resection margins (R0), i.e. in those with more readily curable tumors. The lack of influence of age in patients with advanced tumor stage and non-radical resection could be explained by the fact that the vast majority of these patients died from tumor recurrence independent of age. FUNDING Swedish Research Council and Swedish Cancer Society. FUNDING Swedish Research Council and Swedish Cancer Society. DISCLOSURE The authors declare that they have no conflict of interest. DISCLOSURE The authors declare that they have no conflict of interest. REFERENCES 1. Lagergren J, Smyth E, Cunningham D, Lagergren P. Oesophageal cancer. Lancet. 2017;390(10110):2383–96. 2. Brusselaers N, Mattsson F, Lindblad M, Lagergren J. Association between education level and prognosis after esophageal cancer surgery: a Swedish population-based cohort study. PloS One. 2015;10(3):e0121928. J. Lagergren et al. 166 18. Ra J, Paulson EC, Kucharczuk J, et al. Postoperative mortality after esophagectomy for cancer: development of a preoperative risk prediction model. Ann Surg Oncol. 2008;15(6):1577–84. 3. Rutegard M, Charonis K, Lu Y, Lagergren P, Lagergren J, Rouvelas I. Population-based esophageal cancer survival after resection without neoadjuvant therapy: an update. Surgery. 2012;152(5):903–10. 19. Tapias LF, Muniappan A, Wright CD, et al. Short and long-term outcomes after esophagectomy for cancer in elderly patients. Ann Thorac Surg. 2013;95(5):1741–8. 4. Backemar L, Lagergren P, Johar A, Lagergren J. Impact of co- morbidity on mortality after oesophageal cancer surgery. Br J Surg. 2015;102(9):1097–105. 20. van Gestel YR, Lemmens VE, de Hingh IH, et al. Influence of comorbidity and age on 1-, 2-, and 3-month postoperative mor- tality rates in gastrointestinal cancer patients. Ann Surg Oncol. 2013;20(2):371–80. 5. Internullo E, Moons J, Nafteux P, et al. Outcome after esophagectomy for cancer of the esophagus and GEJ in patients aged over 75 years. Eur J Cardiothorac Surg. 2008;33(6):1096–104. 21. Yang HX, Ling L, Zhang X, Lin P, Rong TH, Fu JH. Outcome of elderly patients with oesophageal squamous cell carcinoma after surgery. Br J Surg. 2010;97(6):862–7. 6. Morita M, Egashira A, Yoshida R, et al. Esophagectomy in patients 80 years of age and older with carcinoma of the thoracic esophagus. J Gastroenterol. 2008;43(5):345–51. 22. Markar SR, Mackenzie H, Lagergren P, Hanna GB, Lagergren J. Surgical Proficiency Gain and Survival After Esophagectomy for Cancer. J Clin Oncol. 2016;34(13):1528–36. 7. Pultrum BB, Bosch DJ, Nijsten MW, et al. Extended esophagectomy in elderly patients with esophageal cancer: minor effect of age alone in determining the postoperative course and survival. Ann Surg Oncol. 2010;17(6):1572–80. 23. Wikman A, Ljung R, Johar A, Hellstadius Y, Lagergren J, Lagergren P. Psychiatric morbidity and survival after surgery for esophageal cancer: a population-based cohort study. J Clin Oncol. 2015;33(5):448–54. 8. Rahamim JS, Murphy GJ, Awan Y, Junemann-Ramirez M. The effect of age on the outcome of surgical treatment for carcinoma of the oesophagus and gastric cardia. Eur J Cardio-Thorac. 2003;23(5):805–10. 24. Ludvigsson JF, Otterblad-Olausson P, Pettersson BU, Ekbom A. REFERENCES The Swedish personal identity number: possibilities and pitfalls in healthcare and medical research. Eur J Epidemiol. 2009;24(11):659–67. 9. Ruol A, Portale G, Zaninotto G, et al. Results of esophagectomy for esophageal cancer in elderly patients: age has little influence on outcome and survival. J Thorac Cardiovasc Surg. 2007;133(5):1186–92. 25. Rutegard M, Lagergren P, Johar A, Lagergren J. Time shift in early postoperative mortality after oesophagectomy for cancer. Ann Surg Oncol. 2015;22(9):3144–9. 10. Zehetner J, Lipham JC, Ayazi S, et al. Esophagectomy for cancer in octogenarians. Dis Esophagus. 2010;23(8):666–9. 26. Brooke HL, Talback M, Hornblad J, et al. The Swedish cause of death register. Eur J Epidemiol. 2017;32(9):765–73. 11. Cijs TM, Verhoef C, Steyerberg EW, et al. Outcome of esophagectomy for cancer in elderly patients. Ann Thorac Surg. 2010;90(3):900–7. 27. Brusselaers N, Lagergren J. The Charlson comorbidity index in registry-based research. Methods Inf Med. 2017;56(5):401–6. 12. Dhungel B, Diggs BS, Hunter JG, Sheppard BC, Dolan JP. Patient and perioperative predictors of morbidity & mortality after esophagectomy: American College of Surgeons National Surgical Quality Improvement Program (ACS-NSQIP), 2005–2007. Gastroenterology. 2010;138(5):S867. 28. Ludvigsson JF, Andersson E, Ekbom A, et al. External review and validation of the Swedish national inpatient register. BMC Public Health. 2011;11:450. 29. Bottai M. A regression method for modelling geometric rates. Stat Methods Med Res. 2017;26(6):2700–7. 13. Ferguson MK, Martin TR, Reeder LB, Olak J. Mortality after esophagectomy: risk factor analysis. World J Surg. 1997;21(6):599–604. 30. Discacciati A, Bottai M. Instantaneous geometric rates via gen- eralized linear models. Stata J. 2017;17(2):358–71. 31. Peeters A, Barendregt JJ, Willekens F, et al. Obesity in adulthood and its consequences for, life expectancy: a life-table analysis. Ann Intern Med. 2003;138(1):24–32. 14. Hodari A, Hammoud ZT, Borgi JF, Tsiouris A, Rubinfeld IS. Assessment of morbidity and mortality after esophagectomy using a modified frailty index. Ann Thorac Surg. 2013;96(4):1240–5. 32. Stegmayr B, Eliasson M, Rodu B. The decline of smoking in northern Sweden. Scand J Public Health. 2005;33(4):321–4. 15. Markar SR, Karthikesalingam A, Thrumurthy S, Ho A, Muallem G, Low DE. Systematic review and pooled analysis assessing the association between elderly age and outcome following surgical resection of esophageal malignancy. Dis Esophagus. 2013;26(3):250–62. 33. Skorus UA, Kenig J. Outcome of esophageal cancer in the elderly—systematic review of the literature. Videosurgery Mini- inv. 2017;12(4):341–9. 34. Sutradhar R, Austin PC. Relative rates not relative risks: addressing a widespread misinterpretation of hazard ratios. Ann Epidemiol. 2018;28(1):54–7. 16. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. REFERENCES Moskovitz AH, Rizk NP, Venkatraman E, et al. Mortality increases for octogenarians undergoing esophagogastrectomy for esophageal cancer. Ann Thorac Surg. 2006;82(6):2031–6. (dis- cussion 2036). Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 17. Poon RTP, Law SYK, Chu KM, Branicki FJ, Wong J. Esophagectomy for carcinoma of the esophagus in the elderly: results of current surgical management. Ann Surg. 1998;227(3):357–64.
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Peer Review #3 of "RNA interference-mediated silencing of genes involved in the immune responses of the soybean pod borer Leguminivora glycinivorella (Lepidoptera: Olethreutidae) (v0.1)"
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Manuscript to be reviewed RNA interference-mediated silencing of genes involved in the immune responses of the soybean pod borer Leguminivora glycinivorella (Lepidoptera: Olethreutidae) 1 Key Laboratory of Soybean Biology in the Chinese Ministry of Education, Northeast Agricultural University, Harbin, Heilongjiang, China 2 Colleges of Life Science, Northeast Agricultural University, Harbin, Heilongjiang, China 3 Key Laboratory of Biology and Genetics & Breeding for Soybean in Northeast China, Ministry of Agriculture, Northeast Agricultural University, Harbin, Heilongjiang, China Corresponding Author: Fanli Meng Email address: mengfanli@neau.edu.cn RNA interference (RNAi) technology may be useful for developing new crop protection strategies against the soybean pod borer (SPB; Leguminivora glycinivorella), which is a critical soybean pest in northeastern Asia. Immune-related genes have been recently identified as potential RNAi targets for controlling insects. However, little is known about these genes or mechanisms underlying their expression in the SPB. In this study, we completed a transcriptome-wide analysis of SPB immune-related genes. We identified 41 genes associated with SPB microbial recognition proteins, immune-related effectors or signalling molecules in immune response pathways (e.g., Toll and immune deficiency pathways). Eleven of these genes were selected for a double-stranded RNA artificial feeding assay. The down-regulated expression levels of LgToll-5-1a and LgPGRP-LB2a resulted in relatively high larval mortality rates and abnormal development. Our data represent a comprehensive genetic resource for immune-related SPB genes, and may contribute to the elucidation of the mechanism regulating innate immunity in Lepidoptera species. Furthermore, two immune-related SPB genes were identified as potential RNAi targets, which may be used in the development of RNAi-mediated SPB control methods. PeerJ reviewing PDF | (2018:02:24130:2:0:NEW 14 May 2018) 1 Running title: Immune -related genes of Leguminivora glycinivorella 2 RNA interference-mediated silencing of genes involved in the immune responses of the 3 soybean pod borer Leguminivora glycinivorella (Lepidoptera: Olethreutidae) 4 Ruixue Ran 1, Tianyu Li 1, Xinxin Liu 1, HejiaNi2, Wenbin Li 1,3, Fanli Meng 1,3 * 5 1 Key Laboratory of Soybean Biology in the Chinese Ministry of Education, Northeast 6 Agricultural University, Harbin 150030, China 7 2 Colleges of Life Science, Northeast Agricultural University, Harbin, China, 150030 8 3 Division of Soybean Breeding and Seeds, Soybean Research & Development Center, CARS 9 (Key Laboratory of Biology and Genetics & Breeding for Soybean in Northeast China, Ministry 10 of Agriculture), Northeast Agricultural University, Harbin 150030, China 11 12 Ruixue Ran: ranruixue@outlook.com 13 Tianyu Li: litianyu151243@outlook.com 14 Xinxin Liu: Liu_xinxin6@163.com 15 HejiaNi: nhjwinner@163.com 16 Wenbin Li: wenbinli@neau.edu.cn 17 Fanli Meng: mengfanli@neau.edu Manuscript to be reviewe Manuscript to be reviewed 1 Running title: Immune -related genes of Leguminivora glycinivorella 18 Ruixue Ran and Tianyu Li contributed equally to this work 19 *Corresponding author: 20 Fanli Meng 21 Key Laboratory of Soybean Biology in the Chinese Ministry of Education, Northeast Agricultural 22 University, Harbin, China, 150030 23 Email: mengfanli@neau.edu 24 Phone: (+86) 451-55191413 25 Fax: (+86) 451-55103336 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 Manuscript to be reviewed Manuscript to be reviewed 18 Ruixue Ran and Tianyu Li contributed equally to this work 19 *Corresponding author: 20 Fanli Meng 21 Key Laboratory of Soybean Biology in the Chinese Ministry of Education, Northeast Agricultural 22 University, Harbin, China, 150030 23 Email: mengfanli@neau.edu 24 Phone: (+86) 451-55191413 25 Fax: (+86) 451-55103336 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 18 Ruixue Ran and Tianyu Li contr 19 *Corresponding author: 20 Fanli Meng 21 Key Laboratory of Soybean Bio 22 University, Harbin, China, 1500 23 Email: mengfanli@neau.edu 24 Phone: (+86) 451-55191413 25 Fax: (+86) 451-55103336 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 PeerJ reviewing PDF | (2018:02:24130:2:0:NEW 14 May 2018) 44 45 46 47 48 49 50 Abstract 51 RNA interference (RNAi) technology may be useful for developing new crop protection strategies 52 against the soybean pod borer (SPB; Leguminivora glycinivorella), which is a critical soybean pest 53 in northeastern Asia. Immune-related genes have been recently identified as potential RNAi targets 54 for controlling insects. However, little is known about these genes or mechanisms underlying their 55 expression in the SPB. In this study, we completed a transcriptome-wide analysis of SPB immune- 56 related genes. We identified 41 genes associated with SPB microbial recognition proteins, 57 immune-related effectors or signalling molecules in immune response pathways (e.g., Toll and 58 immune deficiency pathways). Eleven of these genes were selected for a double-stranded RNA 59 artificial feeding assay. The down-regulated expression levels of LgToll-5-1a and LgPGRP-LB2a 60 resulted in relatively high larval mortality rates and abnormal development. Our data represent a 61 comprehensive genetic resource for immune-related SPB genes, and may contribute to the 62 elucidation of the mechanism regulating innate immunity in Lepidoptera species. Furthermore, 63 two immune-related SPB genes were identified as potential RNAi targets, which may be used in Manuscript to be review Manuscript to be reviewed PeerJ reviewing PDF | (2018:02:24130:2:0:NEW 14 May 2018) Manuscript to be reviewed 64 the development of RNAi-mediated SPB control methods. 64 the development of RNAi-mediated SPB control methods. 65 INTRODUCTION 66 Leguminivora glycinivorella (Mats.) obraztsov [soybean pod borer (SPB)] belongs to the 67 order Lepidoptera and family Olethreutidae. The SPB is the major pest of soybean in northeastern 68 Asia (Zhao et al., 2008; Meng et al., 2017a). The larvae use the immature beans as a food source 69 until they reach maturity, resulting in soybean yield losses of up to 40% (Meng et al., 2017b). 70 Insecticides have been used to control SPB infestations over the past three decades. However, 71 larvae within soybean pods that are under a closed canopy are often not exposed to the applied 72 insecticides. Because of the lack of effective SPB-resistant germplasm, conventional breeding has 73 not resulted in the production of new SPB-resistant cultivars. Therefore, the SPB remains a major 74 pest and is responsible for substantial soybean yield losses (Wang et al., 2014; Song et al., 2015). 75 Consequently, soybean breeders and growers are interested in developing new strategies for 76 controlling SPB infestations, with RNA interference (RNAi) representing a promising option 77 (Khajuria et al., 2015; Fishilevich et al., 2016). 78 RNAi involves the degradation of specific endogenous mRNAs by homologous double-stranded 78 RNAi involves the degradation of specific endogenous mRNAs by homologous double-stranded 79 RNAs (dsRNAs) (Fire et al., 1998). Depending on the function of the targeted gene, RNAi can 80 inhibit insect growth or result in death (Joga et al., 2016; Christiaens et al., 2014). RNAi is 78 RNAi involves the degradation of specific endogenous mRNAs by homologous double-stranded 79 RNAs (dsRNAs) (Fire et al., 1998). Depending on the function of the targeted gene, RNAi can 80 inhibit insect growth or result in death (Joga et al., 2016; Christiaens et al., 2014). RNAi is PeerJ reviewing PDF | (2018:02:24130:2:0:NEW 14 May 2018) Manuscript to be reviewed Manuscript to be reviewed 98 SPB immune-related genes or the associated immune responses. 99 To identify the immune-related genes of SPB, we generated SPB transcriptome datasets 100 based on Illumina sequencing. These datasets were used to identify many genes associated with 101 microbe recognition, immune-related signalling, and defence effectors. Furthermore, RNAi was 102 applied to study the effects of silencing immune-related genes on first instar larvae. A feeding 103 assay involving an artificial diet supplemented with dsRNA was used to identify candidate target 104 genes for controlling the SPB by RNAi. 105 MATERIALS& METHODS 106 Insect rearing 107 L. glycinivorella eggs collected from a naturally infested soybean field at the experimental 108 station of Northeast Agricultural University in Harbin, China were hatched at 26 °C. The resulting 109 larvae were reared on an artificial diet prepared in our laboratory (Meng et al., 2017a). Adult moths 110 were fed a 5% honey solution, and were allowed to oviposit on young bean pods. The first instar 111 larvae were selected and subjected to artificial diet feeding experiments. 112 Illumina sequencing 113 The T3 dsSpbP0 (double-stranded SBP ribosomal protein P0 RNAi transgenic soybean line) 114 and wild-type ‘DN50’ plants, provided by the Key Laboratory of Soybean Biology of the Chinese Manuscript to be reviewe 98 SPB immune-related genes or the associated immune responses. 98 SPB immune-related genes or the associated immune responses. 98 SPB immune-related genes or the associated immune responses. 99 To identify the immune-related genes of SPB, we generated SPB transcriptome datasets 100 based on Illumina sequencing. These datasets were used to identify many genes associated with 101 microbe recognition, immune-related signalling, and defence effectors. Furthermore, RNAi was 102 applied to study the effects of silencing immune-related genes on first instar larvae. A feeding 103 assay involving an artificial diet supplemented with dsRNA was used to identify candidate target 104 genes for controlling the SPB by RNAi. g 107 L. glycinivorella eggs collected from a naturally infested soybean field at the experimental 108 station of Northeast Agricultural University in Harbin, China were hatched at 26 °C. The resulting 109 larvae were reared on an artificial diet prepared in our laboratory (Meng et al., 2017a). Adult moths 110 were fed a 5% honey solution, and were allowed to oviposit on young bean pods. The first instar 111 larvae were selected and subjected to artificial diet feeding experiments. Manuscript to be reviewed 81 conserved in nearly all eukaryotic organisms, and feeding insect pests dsRNA molecules may be 82 useful in protecting agriculturally important crops (feeding RNAi or plant-mediated RNAi) (Mao 83 & Zeng, 2014; Ulrich et al., 2015). However, microinjection RNAi is particularly successful when 84 targeting genes involved in immune responses (Terenius et al., 2011). The effectiveness of the 85 RNAi technique for controlling pests depends on whether appropriate candidate genes are targeted 86 because RNAi efficacy and the RNAi signal transmission vary among genes (Huvenne & 87 Smagghe, 2010). Whileapplying RNAi technology to control Lepidoptera insects (i.e., moths 88 and butterflies) has been problematic (Shukla et al., 2016), the information available regarding its 89 efficiency has recently increased. 90 Insects such as Drosophila melanogaster and Bombyx mori have a vigorous innate immune 91 systems with which to defend against microbial infections (Myllymäki et al., 2014; Parsons et al., 92 2016; Yang et al., 2017; Chen et al., 2018). Peptidoglycan recognition proteins (PGRPs) are 93 important pattern recognition receptors that detect peptidoglycan (PGN) in the cell walls of gram- 94 negative and gram-positive bacteria. PGRPs activate the Toll or immune deficiency (IMD)/JNK 95 pathways or induce proteolytic cascades that generate antimicrobial peptides (Gao et al., 2015; 96 Chen et al., 2014). Antimicrobial peptides are critical for defending against invading pathogens 97 and for protecting insects against infections (Gegner et al., 2018). However, little is known about Manuscript to be reviewe 90 Insects such as Drosophila melanogaster and Bombyx mori have a vigorous innate immune 91 systems with which to defend against microbial infections (Myllymäki et al., 2014; Parsons et al., 92 2016; Yang et al., 2017; Chen et al., 2018). Peptidoglycan recognition proteins (PGRPs) are 93 important pattern recognition receptors that detect peptidoglycan (PGN) in the cell walls of gram- 94 negative and gram-positive bacteria. PGRPs activate the Toll or immune deficiency (IMD)/JNK 95 pathways or induce proteolytic cascades that generate antimicrobial peptides (Gao et al., 2015; 96 Chen et al., 2014). Antimicrobial peptides are critical for defending against invading pathogens 97 and for protecting insects against infections (Gegner et al., 2018). However, little is known about PeerJ reviewing PDF | (2018:02:24130:2:0:NEW 14 May 2018) Manuscript to be reviewed 112 Illumina sequencing 113 The T3 dsSpbP0 (double-stranded SBP ribosomal protein P0 RNAi transgenic soybean line) 114 and wild-type ‘DN50’ plants, provided by the Key Laboratory of Soybean Biology of the Chinese P J i i PDF | (2018 02 24130 2 0 NEW 14 M 2018) 113 The T3 dsSpbP0 (double-stranded SBP ribosomal protein P0 RNAi transgenic soybean line) 114 and wild-type ‘DN50’ plants, provided by the Key Laboratory of Soybean Biology of the Chinese PeerJ reviewing PDF | (2018:02:24130:2:0:NEW 14 May 2018) Manuscript to be reviewed 115 Education Ministry, Harbin, China, were grown in a greenhouse at 24 ± 1°C with 60% relative 116 humidity under a 16-h light/8-h dark cycle (Meng et al., 2017b). At the R5 soybean stage (fully 117 developed pods), three replicates of 50 first-instar larvae were reared on soybean pods of DN50 118 and T3 dsSpbP0 plants. All larvae were collected after 3 d and used to construct cDNA library. 119 cDNA library preparation and Illumina sequencing were conducted by the Biomarker Technology 120 Company (Beijing, China). Briefly, total RNA was extracted from six pooled larvae using TRIzol 121 reagent (Invitrogen, Carlsbad, CA, USA). The first-strand cDNA was synthesised using random 122 hexamer-primers from purified Poly (A) mRNA. Second-strand cDNA was synthesised using 123 DNA polymerase I and RNaseH, and then purified using a QiaQuick PCR extraction kit (Qiagen, 124 Hilden, Germany). cDNA fragments of a suitable length (300–500 bp) were obtained by agarose 125 gel electrophoresis and amplified by PCR to construct the final cDNA libraries using NEB Next 126 Ultra RNA Library Prep Kit for Illumina (NEB, Ipswich, MA,USA). The cDNA library was 127 sequenced on Illumina HiSeq 2000 system. The unigenes from six samples were combined to 128 create the SPB unigene database (Chen et al., 2014; Meng et al., 2017c). All raw transcriptome 129 data have been deposited in the NIH Short Read Archive (accession numbers SRR5985984– 130 SRR5985989). 131 Identification of immune-related genes Manuscript to be reviewed 132 A list of immune-related genes was compiled based on the available relevant literature (Table 133 S1), and homologous B. mori, D. melanogaster, Danaus plexippus and Papilio polytes genes in 134 the GenBank database were identified (Guan & Mariuzza, 2007; Xu et al., 2012). The tBLASTn 135 algorithm-based tool was used to complete sequence similarity searches of the SPB transcriptome 136 database (Boratyn et al., 2013). The 41 SPB immune-related genes were submitted 137 to NCBI GenBank and their Accession numbers are shown in TableS1. 138 Phylogenetic and domain analyses 139 Amino acid sequences were aligned with the Multiple Alignment program clustal omega 140 (https://www.ebi.ac.uk/Tools/msa/clustalo/), and the phylogenetic tree was constructed in MEGA 141 5 based on the neighbour-joining method with 1000 bootstrap replicates (Tamura et al., 2011; 142 Sievers et al., 2011). The architecture of the protein domains was analysed using the SMART 143 program (http://smart.embl-heidelberg.de/). 144 dsRNA synthesis 145 We synthesised dsRNAs using the T7 RiboMAX Express Large Scale RNA Production 146 System (Promega, Madison, WI, USA). The T7 RNA polymerase promoter sequence was added 147 to each end of the DNA templates during PCR amplifications. The primers containing the T7 RNA 132 A list of immune-related genes was compiled based on the available relevant literature (Table 133 S1), and homologous B. mori, D. melanogaster, Danaus plexippus and Papilio polytes genes in 134 the GenBank database were identified (Guan & Mariuzza, 2007; Xu et al., 2012). The tBLASTn 135 algorithm-based tool was used to complete sequence similarity searches of the SPB transcriptome 136 database (Boratyn et al., 2013). The 41 SPB immune-related genes were submitted 137 to NCBI GenBank and their Accession numbers are shown in TableS1. 134 the GenBank database were identified (Guan & Mariuzza, 2007; Xu et al., 2012). The tBLASTn 135 algorithm-based tool was used to complete sequence similarity searches of the SPB transcriptome 136 database (Boratyn et al., 2013). The 41 SPB immune-related genes were submitted 137 to NCBI GenBank and their Accession numbers are shown in TableS1. 138 Phylogenetic and domain analyses 139 Amino acid sequences were aligned with the Multiple Alignment program clustal omega 140 (https://www.ebi.ac.uk/Tools/msa/clustalo/), and the phylogenetic tree was constructed in MEGA 141 5 based on the neighbour-joining method with 1000 bootstrap replicates (Tamura et al., 2011; 142 Sievers et al., 2011). The architecture of the protein domains was analysed using the SMART 143 program (http://smart.embl-heidelberg.de/). PeerJ reviewing PDF | (2018:02:24130:2:0:NEW 14 May 2018) Manuscript to be reviewed Manuscript to be reviewed 144 dsRNA synthesis 145 We synthesised dsRNAs using the T7 RiboMAX Express Large Scale RNA Production 146 System (Promega, Madison, WI, USA). The T7 RNA polymerase promoter sequence was added 147 to each end of the DNA templates during PCR amplifications. The primers containing the T7 RNA 148 polymerase promoter were designed using Primer-BLAST PeerJ reviewing PDF | (2018:02:24130:2:0:NEW 14 May 2018) 137 to NCBI GenBank and their Accession numbers are shown in TableS1. 137 to NCBI GenBank and their Accession numbers are shown in TableS1. 144 dsRNA synthesis 145 We synthesised dsRNAs using the T7 RiboMAX Express Large Scale RNA Production 146 System (Promega, Madison, WI, USA). The T7 RNA polymerase promoter sequence was added 147 to each end of the DNA templates during PCR amplifications. The primers containing the T7 RNA 148 polymerase promoter were designed using Primer-BLAST PeerJ reviewing PDF | (2018:02:24130:2:0:NEW 14 May 2018) Manuscript to be reviewed Manuscript to be reviewed 149 (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) (Table S2). For the negative control, the green 150 fluorescent protein (GFP) gene was amplified from the PCAMBIA1302 vector as a template for 151 GFP dsRNA synthesis. The template DNA and single-stranded RNA were eliminated from the 152 transcription reaction by DNase I and RNase A, respectively. The prepared dsRNAs were purified 153 by a phenol/chloroform extraction followed by an ammonium acetate precipitation. The dsRNAs 154 were ultimately suspended in ultrapure water and quantified using a Nano Drop 2000 155 spectrophotometer (Thermo Scientific, Waltham, MA, USA). 156 SPB feeding bioassay 157 The first-instar SPB larvae were fed an artificial diet containing dsRNA (10 μg/g) for specific 158 target genes as described by Meng et al. (2017a). Control larvae were treated with the same 159 concentration of GFP dsRNA. The feeding bioassay was completed in triplicate with 50 larvae per 160 treatment or control. Three biological replicates were used for each treatment. The larvae were 161 reared for 15 d at 26 °C under a 16-h light/8-h dark cycle with 65% relative humidity. The dsRNA- 162 supplemented artificial diet was refreshed every 3 d. Body weight, mortality, and phenotypic 163 abnormalities were recorded every 3 d. 164 Quantitative real-time PCR (qRT-PCR) 165 For every treatment, two surviving larvae were randomly collected at each time point from 149 (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) (Table S2). For the negative control, the green 150 fluorescent protein (GFP) gene was amplified from the PCAMBIA1302 vector as a template for 151 GFP dsRNA synthesis. The template DNA and single-stranded RNA were eliminated from the 152 transcription reaction by DNase I and RNase A, respectively. The prepared dsRNAs were purified 153 by a phenol/chloroform extraction followed by an ammonium acetate precipitation. The dsRNAs 154 were ultimately suspended in ultrapure water and quantified using a Nano Drop 2000 155 spectrophotometer (Thermo Scientific, Waltham, MA, USA). 165 For every treatment, two surviving larvae were randomly collected at each time point from 165 For every treatment, two surviving larvae were randomly collected at each time point from PeerJ reviewing PDF | (2018:02:24130:2:0:NEW 14 May 2018) Manuscript to be reviewed Manuscript to be reviewed 166 all of the biological replicates from 0 to 15 d after larvae were fed the artificial diet containing the 167 target gene’s dsRNA, frozen in liquid nitrogen and stored at −80°C. Total RNA was extracted from 168 two pooled larvae using the RNApure Tissue Kit (DNase I) (CWBIO, Beijing, China). 169 Additionally, primer sets were synthesised (Table S2). The extracted RNA samples were treated 170 with DNase I (Invitrogen) to remove any contaminating genomic DNA. They were then used as 171 the template for first-strand cDNA synthesis with the TIANScript RT Kit (Tiangen, Beijing, 172 China). The qRT-PCR analysis was completed using the SYBR Green kit (Bio-Rad, Hercules, CA, 173 USA) and a Roche LightCycler® 480 real-time PCR system (Roche, Basel, Switzerland). Each 174 reaction used cDNA corresponding to 50 ng of total RNA and each primer at a final concentration 175 of 100 nm in 20 µl reaction. Controls included non-RT controls (50ng total RNA without reverse 176 transcription was used to detect genomic DNA contamination) and non-template controls (water 177 template).The qRT-PCR conditions were as follows: 95°C for 5 min; 40 cycles at 95°C for 30 s, 178 60 °C for 15 s, and 72°C for 45 s; 95°C for 1 min and 55°C for 1 min. At the end of each qRT- 179 PCR experiment, a melt curve was generated to check for primer-dimer formation. The efficiencies 180 of the qRT-PCR primer pairs were greater than 90% (Table S2).The qRT-PCR analysis contained 181 three biological replicates, each having three technical replicates (Meng et al., 2017c). Each target 182 gene’s qRT-PCR products were sequenced to confirm their identities. PeerJ reviewing PDF | (2018:02:24130:2:0:NEW 14 May 2018) Manuscript to be reviewed Manuscript to be reviewed 183 Relative expression levels were calculated by the following formula using LightCycler 480 184 Software v1.5.0: R = 2−(ΔCt sample-ΔCt calibrator), where Rrepresents the relative expression level, ΔCt 185 sample is the average difference between the Ct of the gene and that of SBPß-actin in the 186 expreimental sample (Meng et al., 2017b), and ΔCt calibrator is the average difference between 187 the Ct of the gene and the e SBPß-actin in the calibrator. A representative sample was set as the 188 calibrator (Bustin et al., 2009). The differences in Ct values between technical replicates was less 189 than 0.5. The relative expression level of 0d for each target gene was set as a benchmark. The 190 relative expression level of other time point for each target gene was used to compare with 0d 191 value. 192 Statistical analyses 193 All of the data in this study are presented as mean ± SE. Significant differences were 194 determined by one-way analysis of variance followed by least significant difference tests for mean 195 comparisons. The statistical analysis was performed with SAS 9.21 software (SAS Institute, Cary, 196 NC, USA). P-values were corrected by Bonferroni. 197 RESULTS 198 Identification of Leguminivora glycinivorella immune-related genes 199 Amino acid sequences encoded by D. melanogaster, B. mori, and Manduca sexta 200 (Lepidoptera) immune-related genes were used to search SPB transcriptome sequences. The 41 p Manuscript to be reviewed 201 identified putative SPB immune-related genes were functionally classified into three groups, 202 microbial recognition, immune signalling, and immune effector molecules (Table S1). 201 identified putative SPB immune-related genes were functionally classified into three groups, 202 microbial recognition, immune signalling, and immune effector molecules (Table S1). 201 identified putative SPB immune-related genes were functionally classified into three groups, 202 microbial recognition, immune signalling, and immune effector molecules (Table S1). 201 identified putative SPB immune-related genes were functionally classified into three groups, 202 microbial recognition, immune signalling, and immune effector molecules (Table S1). 203 Microbial recognition molecules 204 The PGRPs recognize conserved molecular patterns present in pathogens, but absent in the 205 host, including PGNs, which are essential cell wall components of almost all bacteria. The PGRPs 206 are encoded by a highly conserved gene family in insects, and are generally classified into two 207 types (short and long) (Dziarski & Gupta, 2006; Yang et al., 2017). We identified eight SPB 208 PGRPs, four short and four long types, with similarities to D. melanogaster PGRP-SC, PGRP-SD, 209 and PGRP-LB (Fig. 1). Five (LgPGRP-SC1a, LgPGRP-SC1b, LgPGRP-SD1b, LgPGRP-LB1, 210 and LgPGRP-LB2b) of the eight identified PGRPs were predicted to be secreted proteins, based 211 on the presence of putative signal peptides, that function as amidases. LgPGRP-LB lacks a putative 212 signal peptide, but contains a transmembrane region and amidase domain, suggesting that it serves 213 as a transmembrane PGN receptor. In contrast, LgPGRP-SD1a and LgPGRP-LB2a carry only the 214 PGRP domain, implying that they are intracellular proteins (Table S1). 215 The gram-negative bacteria-binding proteins (GNBPs) and β-1,3-glucan recognition proteins 216 (βGRPs) belong to a subfamily of pattern recognition receptors and have strong affinities for the Manuscript to be reviewed 218 of gram-positive bacteria. Of the three GNBPs produced by D. melanogaster (GNBP1, GNBP2, 219 and GNBP3), GNBP1 interacts with PGRP-SA to form a hydrolytic complex that activates the 220 Toll pathway in response to gram-positive bacteria, while GNBP3 is required for detecting fungi 221 and activating the Toll pathway (Hughes, 2012; Rao et al., 2017). We identified one GNBP gene 222 in the SPB transcriptome datasets. The neighbour-joining phylogenetic analysis indicated that 223 LgGNBP3 is an ortholog of DmGNBP3 (Fig. S1). A comparison between the deduced amino acid 224 sequences and the D. melanogaster GNBP sequences indicated that LgGNBP3 contains a putative 225 N-terminal β-1,3-glucan-recognition domain (CBM39) and a C-terminal glucanase-like domain 226 (glycosyl hydrolase family 16), suggesting that LgGNBP3 may bind to fungal β-1,3-glucan. 227 Immune signalling molecules 228 After specific ligands are detected, microbial recognition molecules activate or modulate 229 various immune response pathways. Here, we identified genes associated with the Toll and IMD 230 pathways, which are the major signalling pathways that mediate the innate immunity of insects. 231 The Toll pathway regulates the production of antimicrobial peptides in response to infections by 232 fungi or gram-positive bacteria with lysine-type PGNs in their cell walls (Roh et al., 2009). The 233 Toll receptor, which is responsible for the signal transduction associated with the Toll pathway, is 234 vital for insect innate immune responses and embryo development (Takeda & Akira, 2004; Benton PeerJ reviewing PDF | (2018:02:24130:2:0:NEW 14 May 2018) Manuscript to be reviewed 235 et al., 2016). In this study, we identified 10 genes encoding Toll receptors in the SPB transcriptome 236 datasets. The TIR domain is highly conserved in insect Toll families. To investigate the 237 orthologous relationships among these genes, we constructed a phylogenetic tree based on an 238 alignment of the TIR domains from all SPB and D. melanogaster Toll proteins. The Toll receptors 239 analysed in this study formed six major clusters, namely Toll-3, Toll-4, Toll-5, Toll-6, Toll-7 and 240 Toll-8 (Fig. 2A). Based on the phylogenetic tree, the L. glycinivorella Toll genes were designated 241 as LgTLR-3, LgTLR-4, LgToll-5-1a, LgToll-5-1b, LgToll-6, TLR-6-1, TLR-6-2, LgToll-7, LgTLR- 242 7 and LgToll-8. All 10 predicted proteins contain an extracellular LRR domain as well as 243 transmembrane and cytoplasmic TIR domains (Fig. 2B). We also identified sequences matching 244 the intracellular components, ECSIT and Tollip, which affect the Toll signalling pathway (Table 245 S1). 246 The IMD pathway is mainly activated by gram-negative bacterial infections. Additionally, 247 IMD signal transduction is reportedly mediated by IMD, fas-associated death domain protein 248 (FADD), death-related ced-3/Nedd2-like caspase (Dredd), inhibitor of apoptosis protein 2 (IAP2), 249 transforming growth factor β-activated kinase (TAK1), TAK1-binding 2 (Tab2), ubiquitin 246 The IMD pathway is mainly activated by gram-negative bacterial infections. Additionally, 247 IMD signal transduction is reportedly mediated by IMD, fas-associated death domain protein 248 (FADD), death-related ced-3/Nedd2-like caspase (Dredd), inhibitor of apoptosis protein 2 (IAP2), 249 transforming growth factor β-activated kinase (TAK1), TAK1-binding 2 (Tab2), ubiquitin 250 conjugating 13 (Ubc13), and an inhibitor of nuclease factor B kinase subunits b and g (IKKb and 251 IKKg)( Bao et al., 2013; Myllymäki et al., 2014). Of these,we only identified sequences that were PeerJ reviewing PDF | (2018:02:24130:2:0:NEW 14 May 2018) Manuscript to be reviewed 252 homologous to FADD, Dredd, and IAP2 (Table S1). 253 Immune-related effector genes 254 The PGRPs and βGRPs detect PGNs and β-1,3-glucans, which activates a clip-domain 255 serine protease (CLIP) cascade that converts prophenoloxidase to active phenoloxidase, leading 256 to the melanisation responses involved in eliminating pathogens(Monwan, Amparyup & 257 Tassanakajon, 2017; Li et al., 2016). We identified two CLIP genes (LgSnake-1 and LgSnake-2) 258 in the SPB transcriptome datasets. The deduced amino acid sequences each contain a clip 259 domain at the N-terminus and a serine protease domain at the C-terminus (Fig. S2). Serine 260 protease inhibitors (i.e., serpins) negatively regulate prophenoloxidase activation, which prevents 261 the excessive activation of the CLIP cascade. In this study, we identified three serpin genes, 262 Lgserpin1, Lgserpin2, and Lgserpin3, in the SPB transcriptome datasets. Their deduced amino 263 acid sequences each contained a putative signal peptide sequence and a core serpin domain, 264 suggesting that they are secreted proteins (Table S1). 265 Immune response effector genes 266 Antibacterial peptides are immune response effectors that are induced by immune 267 challenges and are important for defence responses against insects Diverse antibacterial peptide 252 homologous to FADD, Dredd, and IAP2 (Table S1). 268 genes have been identified in many insect species, including genes encoding defensins, reeler, Manuscript to be reviewed 269 and lysozyme (Imler & Bulet, 2005; Bao et al., 2011). In this study, we identified two defensin 270 genes, Lgdefensin1 and Lgdefensin2, in the SPB transcriptome datasets. The encoded amino acid 271 sequences each consisted of a putative signal peptide sequence and a core Knot1 domain (Table 272 S1). We also identified seven chicken-type (C-type) lysozymes and two invertebrate-type (I- 273 type) lysozymes in the L SPB transcriptome. The C-type lysozymes are bacteriolytic enzymes 274 that hydrolyse the β (1- 4) bonds between N-acetylglucosamine and N-acetylmuramic acid in the 275 PGN of prokaryotic cell walls. The predicted SPB C-type proteins,with the exception of the C- 276 type 3 protein, each include an N-terminal signal peptide sequence (Table S1). Additionally, we 277 detected eight conserved cysteine residues in the L. glycinivorella C-type lysozymes (Fig. S3A) 278 as well as 12 conserved cysteine residues in the deduced SPB I-type lysozyme sequences (Fig. 279 S3B). These cysteine residues may form intramolecular disulphide bonds to enhance stability 280 and resistance against proteolytic degradation. 281 Potential RNAi targets identified in an artificial feeding assay and effects of double-stranded RNA 282 on soybean pod borer development and mortality 283 In total, 11genes representing the immune-related SPB genes were selected and analysed to 281 Potential RNAi targets identified in an artificial feeding assay and effects of double-stranded RNA 282 on soybean pod borer development and mortality 283 In total, 11genes representing the immune-related SPB genes were selected and analysed to 284 identify potential new RNAi targets useful for controlling the SPB. We synthesised the 285 corresponding dsRNAs in vitro and mixed them in an artificial diet. The mortality rates 3d after 283 In total, 11genes representing the immune-related SPB genes were selected and analysed to 284 identify potential new RNAi targets useful for controlling the SPB. We synthesised the 285 corresponding dsRNAs in vitro and mixed them in an artificial diet. The mortality rates 3d after PeerJ reviewing PDF | (2018:02:24130:2:0:NEW 14 May 2018) Manuscript to be reviewed Manuscript to be reviewed 286 larvae were fed artificial diets, which contained containing 10 µg/g dsRNA for LgPGRP-LB, 287 LgPGRP-LB2b, LgToll-5-1a, LgToll-5-1b, LgTLR-7, LgSerpin2 or LgChaoptin were 37%–92%. 288 These mortality rates were significantly greater than those of control larvae treated with GFP 289 dsRNA. Additionally, the final mortality rates of the larvae fed LgPGRP-LB, LgPGRP-LB2b, 290 LgToll-5-1a, LgSerpin2 and LgChaoptin dsRNA were even higher at 15 d. In contrast, the artificial 291 diets containing dsRNA independently targeting LgPGRP-LB2a, LgTLR-3 and Lgitype-1 did not 292 have any statistically significant effects on larval mortality (Fig. 3). Moreover, two main 293 phenotypic differences were observed among the surviving larvae after 15 d of feeding. First, the 294 weights of the larvae fed LgToll-5-1b and LgItype-1 dsRNA increased more gradually than those 295 of larvae fed GFP dsRNA, and was ultimately lower after 15 d of feeding (Fig. 4). Additionally, 296 the LgToll-5-1b RNAi treatment resulted partly black cuticles. Second, larvae fed dsRNA targeting 297 LgPGRP-LB or LgPGRP-LB2a underwent early pupation, with pupation rates of 25 and 50%, 298 respectively. The remaining larvae developed abnormally with stunted and twisted bodies (Fig. 5). 299 To investigate how larval mortality and abnormal development are correlated with the 300 relative expression levels of specific target genes, we performed qRT-PCR using total RNA 301 extracted from the surviving larvae at different time points after feeding on artificial diets. The Manuscript to be reviewed 303 decreased in larvae 3 d after being treated with the respective dsRNAs, while the expression levels 304 of all of the genes decreased significantly after 6 or 9 d (Fig. 6). Thus, the increased mortality rates 305 and abnormal development of larvae fed dsRNA may result from the down-regulated expression 306 of specific target genes. Moreover, unigenes LgToll-5a and LgPGRP-LB2a may represent good 307 RNAi targets for controlling the SPB. 308 DISCUSSION 309 Insects possess efficient innate immune system that protect them from microorganisms and 310 aid in abiotic stresses (Hillyer, 2015; Parsons& Foley, 2016). In this study, we identified 41 genes 311 in the SPB transcriptome that encode components of conserved immune signalling pathways (Toll 312 and IMD pathways) as well as pathogen recognition and immune response effectors. Most of these 313 genes contained conserved sequences that exist in orthologous D. melanogaster and B. mori genes 314 (Table S1). However, immune-related gene families have expanded or contracted in different taxa. 315 For example, the PGRP gene families in D. melanogaster, B. mori, SPB have 13, 12, and 8 316 members, respectively (Hillyer, 2015; Yang et al., 2015). In addition to sequence differences 317 among the immune-related genes, the encoded proteins exhibited diverse activities. For example, 318 four of the SPB’s PGRPs are closely related to each other and form an independent cluster with 319 D. melanogaster PGRP LB (Fig. 1). Two of them contain a putative signal peptide and a conserved 307 RNAi targets for controlling the SPB. 308 DISCUSSION 309 Insects possess efficient innate immune system that protect them from microorganisms and 310 aid in abiotic stresses (Hillyer, 2015; Parsons& Foley, 2016). In this study, we identified 41 genes 311 in the SPB transcriptome that encode components of conserved immune signalling pathways (Toll 312 and IMD pathways) as well as pathogen recognition and immune response effectors. Most of these 313 genes contained conserved sequences that exist in orthologous D. melanogaster and B. mori genes 314 (Table S1). However, immune-related gene families have expanded or contracted in different taxa. 315 For example, the PGRP gene families in D. melanogaster, B. mori, SPB have 13, 12, and 8 316 members, respectively (Hillyer, 2015; Yang et al., 2015). In addition to sequence differences 317 among the immune-related genes, the encoded proteins exhibited diverse activities. PeerJ reviewing PDF | (2018:02:24130:2:0:NEW 14 May 2018) Manuscript to be reviewed Manuscript to be reviewed 320 Ami_2 domain, while the others lack a signal peptide (Table S1). Furthermore, silencing LgPGRP- 321 LB and LgPGRP-LB2a induced early pupation and abnormal larval development, while silencing 322 LgPGRP-LB2b lead to significantly higher mortality rates at 3d and 6 d, indicating that LgPGRP- 323 LB2b may be essential for early larval development (Fig. 5). PGRP-LB is a catalytic amidase that 324 can degrade PGN and regulate host immune responses to infectious microorganisms by down- 325 regulating the IMD pathway (Zaidman-Rémy et al., 2006; Troll et al., 2009;Gendrin et al., 2017), 326 which protects the beneficial microbes in insects and prevents host-inflicted damage during 327 development (Hashimoto et al., 2007). In the Tsetse fly (Diptera: Glossinidae), silencing PGRP- 328 LB by RNAi decreases host fecundity because of the associated cost of activating the host immune 329 response (Wang & Aksoy, 2012). 330 The SPB is a univoltine insect. The mature larvae make cocoons in the soil, enter diapause 331 during the winter and pupate in mid-July, resulting in a diapause period of 10 months (Meng et 332 al., 2017b). In our study, LgPGRP-LB and LgPGRP-LB2a were silenced by RNAi, which broke 333 diapause and caused mature larvae to pupate. This termination of diapause result from an immune 334 response that was initiated to prevent host-inflicted damage. Further research is needed to confirm 335 that LgPGRP-LB influences the host immune responses’ activation.. 336 The Toll pathway is critical for innate immunity against bacteria and also affects embryonic 320 Ami_2 domain, while the others lack a signal peptide (Table S1). Furthermore, silencing LgPGRP- 321 LB and LgPGRP-LB2a induced early pupation and abnormal larval development, while silencing 322 LgPGRP-LB2b lead to significantly higher mortality rates at 3d and 6 d, indicating that LgPGRP- 323 LB2b may be essential for early larval development (Fig. 5). PGRP-LB is a catalytic amidase that 324 can degrade PGN and regulate host immune responses to infectious microorganisms by down- 325 regulating the IMD pathway (Zaidman-Rémy et al., 2006; Troll et al., 2009;Gendrin et al., 2017), 326 which protects the beneficial microbes in insects and prevents host-inflicted damage during 327 development (Hashimoto et al., 2007). In the Tsetse fly (Diptera: Glossinidae), silencing PGRP- 328 LB by RNAi decreases host fecundity because of the associated cost of activating the host immune 329 response (Wang & Aksoy, 2012). 330 The SPB is a univoltine insect. Manuscript to be reviewed For example, 318 four of the SPB’s PGRPs are closely related to each other and form an independent cluster with 319 D. melanogaster PGRP LB (Fig. 1). Two of them contain a putative signal peptide and a conserved PeerJ reviewing PDF | (2018:02:24130:2:0:NEW 14 May 2018) Manuscript to be reviewed Manuscript to be reviewed The mature larvae make cocoons in the soil, enter diapause 331 during the winter and pupate in mid-July, resulting in a diapause period of 10 months (Meng et 332 al., 2017b). In our study, LgPGRP-LB and LgPGRP-LB2a were silenced by RNAi, which broke 330 The SPB is a univoltine insect. The mature larvae make cocoons in the soil, enter diapause 331 during the winter and pupate in mid-July, resulting in a diapause period of 10 months (Meng et 332 al., 2017b). In our study, LgPGRP-LB and LgPGRP-LB2a were silenced by RNAi, which broke 333 diapause and caused mature larvae to pupate. This termination of diapause result from an immune 334 response that was initiated to prevent host-inflicted damage. Further research is needed to confirm 335 that LgPGRP-LB influences the host immune responses’ activation.. 336 The Toll pathway is critical for innate immunity against bacteria and also affects embryonic 336 The Toll pathway is critical for innate immunity against bacteria and also affects embryonic Manuscript to be reviewed 337 development, olfactory neuron processes, and TNF-induced JNK-dependent cell death in D. 338 melanogaster (Yang et al., 2015; Valanne et al., 2011; Wu et al., 2015). Knocking down the fusilli 339 and cactin genes, which are part of the Toll pathway, is lethal for the red flour beetle (Tribolium 340 castaneum), and the silencing cactin is 100% lethal at all developmental stages (from larva to 341 adult). Additionally, the knockdown of pelle and dorsal prevents eggs from hatching in the next 342 generation (Bingsohn et al., 2017). In our study, a 10% knocked down of LgToll-5-1a lead to 343 92%larval mortality rate 3 d after feeding. While LgToll-5-1a is more critical for the first instar 344 larvae development. A 56% knocked down of LgToll-5-1b did not impact on the survival rate of 345 larvae 3 d after feeding. Knocking down LgToll-5b took considerably longer (15 d) to have an 346 impact on body weights, and it prevented old cuticles from separating from larval bodies. LgToll- 347 5-1b may influence mid-to-late larval development. Thus, LgToll-5 may be play critical roles on 348 larval development, and it may function in immunity in adults. A future study will challenge Toll- 349 5-1a or LgToll-5-1b RNAi-treated insects with pathogen infections to determine their roles, if any, 350 in immunity. 351 Lysozymes are widely distributed immune effectors that exhibit muramidase activities against 352 the PGNs in bacterial cell wall to induce cell lysis (Zhou et al., 2017). In our study, the LgI-type- PeerJ reviewing PDF | (2018:02:24130:2:0:NEW 14 May 2018) Manuscript to be reviewed Manuscript to be reviewed 354 activities. The pI of LgI-type-1 is 7.93 (Table S1). Researchers have proposed that I-type 355 lysozymes with high pI values influence immunity (Kurdyumov et al., 2015; Xue et al., 2004). 356 Thus, LgI-type-1 may have isopeptidase activity and play a role in the SPB’s immune system 357 CONCLUSION 358 We identified 41 genes associated with SPB microbial recognition proteins, immune-related 359 effectors, or signalling molecules of immune response pathways (e.g., Toll and immune deficiency 360 pathways). This will be useful as a comprehensive genetic resource for immune-related SPB genes 361 and may help elucidate the mechanism regulating innate immunity in Lepidoptera species. In 362 addition, the in vivo functions of 11 genes were analysed in RNAi experiments, which indicated 363 that three genes may be appropriate RNAi targets for controlling the SPB. The observations 364 described herein may be useful for future analyses of the mechanisms underlying the SPB mmune 365 response pathways and for developing RNAi-mediated methods to control SPB infestations. 366 ACKNOWLEDGEMENTS 367 We thank Professor Xiaoyun Wang (Northeast Agricultural University, China) for her technical 368 support in culturing the SPB. 369 REFERENCES 370 Bao YY, Qu LY, Zhao D, Chen LB, Jin HY, Xu LM, Cheng JA, Zhang CX. 2013. The genome- PeerJ reviewing PDF | (2018:02:24130:2:0:NEW 14 May 2018) Manuscript to be reviewed 373 Bao YY, Xue J, Wu WJ, Wang Y, Lv ZY, Zhang CX. 2011. An immune-induced reeler protein is 374 involved in the Bombyx mori melanization cascade. 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Insect Mol Biol. 26(6):665- 530 676 DOI 10.1111/imb.12330 531 Zaidman-Rémy A1, Hervé M, Poidevin M, Pili-Floury S, Kim MS, Blanot D, Oh BH, Ueda R, 532 Mengin-Lecreulx D, Lemaitre B. 2006. The Drosophila amidase PGRP-LB modulates the immune 533 response to bacterial infection. Immunity 24(4):463-73 DOI 10.1016/j.immuni.2006.02.012 531 Zaidman-Rémy A1, Hervé M, Poidevin M, Pili-Floury S, Kim MS, Blanot D, Oh BH, Ueda R, 532 Mengin-Lecreulx D, Lemaitre B. 2006. The Drosophila amidase PGRP-LB modulates the immune 533 response to bacterial infection. Immunity 24(4):463-73 DOI 10.1016/j.immuni.2006.02.012 Manuscript to be reviewed PeerJ reviewing PDF | (2018:02:24130:2:0:NEW 14 May 2018) PeerJ reviewing PDF | (2018:02:24130:2:0:NEW 14 May 2018) Manuscript to be reviewed Figure 2 The analysis of Toll receptors of theL. glycinivorella Phylogenetic relationships among PGRPs from Leguminivora glycinivorella and Drosophila melanogaster Phylogenetic relationships among PGRPs from Leguminivora glycinivorella and Drosophila melanogaster The phylogenetic tree was constructed using MEGA5.0 with a neighbour-joining approach. The bootstrap percentages (1,000 replicates) are provided next to the branches. The first two letters of each PGRP name indicate the species (Dm, D. melanogaster; Lg, L. glycinivorella). PeerJ reviewing PDF | (2018:02:24130:2:0:NEW 14 May 2018) Manuscript to be reviewed PeerJ reviewing PDF | (2018:02:24130:2:0:NEW 14 May 2018) Manuscript to be reviewed The analysis of Toll receptors of theL. glycinivorella A) Phylogenetic relationships among Toll receptors from Leguminivora glycinivorella,Bombyx mori and Drosophila melanogaster. The phylogenetic tree was constructed using MEGA5.0 with a neighbour-joining approach. Lg,L. glycinivorella; Bm,Bombyx mori; Dm,D. melanogaster; TLR, Toll like receptor. (B) Predicted domains of the L.glycinivorellaToll receptors. The domain organization were predicted using the SMART program (http://smart.embl.de/). The extracellular leucine-rich repeats (LRRs), rectangles; LRR C- terminal domain, small ellipses; intracytoplasmic TIR domains, big ellipses; signal peptides, red rectangles; transmembrane domain, blue bar. PeerJ reviewing PDF | (2018:02:24130:2:0:NEW 14 May 2018) Manuscript to be reviewed Figure 3 Mortality rates of soybean pod borer larvae fed an artificial diets supplemented separately with dsRNA (10 μg/g) for 11 candidate RNA interference target genes. Columns represent mean±SE. Different letters above the column indicate significant difference (p < 0.0009). Mortality rates of soybean pod borer larvae fed an artificial diets supplemented separately with dsRNA (10 μg/g) for 11 candidate RNA interference target genes. Columns represent mean±SE. Different letters above the column indicate significant difference (p < 0.0009). difference (p < 0.0009). PeerJ reviewing PDF | (2018:02:24130:2:0:NEW 14 May 2018) PeerJ reviewing PDF | (2018:02:24130:2:0:NEW 14 May 2018) Manuscript to be reviewed Figure 4 Body weights of soybean pod borer larvae fed artificial diets supplemented separately with dsRNA (10 μg/g) for 11 candidate RNA interference target genes. Columns represent mean±SE. Different letters above the column indicate significant difference (p < 0.0009). PeerJ reviewing PDF | (2018:02:24130:2:0:NEW 14 May 2018) Manuscript to be reviewed Figure 5 Images of soybean pod borer larvae fed an artificial diet supplemented separately with dsRNA (10 μg/g) for 11 candidate RNA interference target genes for 15 d. A) The larvae fed an artificial diet containing dsRNA for LgPGRP-LB. (B) The larvae fed an artificial diet containing dsRNA for LgPGRP-LB2a. (C) The larvae fed an artificial diet containing dsRNA for LgPGRP-LB2b. (D) The larvae fed an artificial diet containing dsRNA for LgToll-5-1a, only one survived after 15 d. (E) The larvae fed an artificial diet containing dsRNA for LgToll-5-1b. (F) The larvae fed an artificial diet containing dsRNA for LgTLR-6-1b. (G) The larvae fed an artificial diet containing dsRNA for LgTLR-7. (H) The larvae fed an artificial diet containing dsRNA for LgTLR-3. (I)The larvae fed an artificial diet containing dsRNA for LgChaoptin. (J) The larvae fed an artificial diet containing dsRNA for LgSerpin-2. (K) The larvae fed an artificial diet containing dsRNA for LgItype-1. (L) The larvae fed an artificial diet containing dsRNA for GFP. PeerJ reviewing PDF | (2018:02:24130:2:0:NEW 14 May 2018) PeerJ reviewing PDF | (2018:02:24130:2:0:NEW 14 May 2018) Figure 6 Relative expression levels of 11 candidate RNA interference target genes at different time points after soybean pod borer larvae were fed separate artificial diets containing the respective dsRNA (10 μg/g). Quantitative real-time polymerase chain reactions were completed using total RNA extracted from surviving larvae. Columns represent mean±SE. Different letters above the column indicate significant difference (p < 0.002) PeerJ reviewing PDF | (2018:02:24130:2:0:NEW 14 May 2018)
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Martin Wagenschein (1896-1988) und seine Lehrkunst - heute
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Christoph Berg, Hans Martin Wagenschein (1896-1988) und seine Lehrkunst - heute Beiträge zur Lehrerbildung 7 (1989) 1, S. 11-18 Quellenangabe/ Reference: Christoph Berg, Hans: Martin Wagenschein (1896-1988) und seine Lehrkunst - heute - In: Beiträge zur Lehrerbildung 7 (1989) 1, S. 11-18 - URN: urn:nbn:de:0111-pedocs-131436 - DOI: 10.25656/01:13143 https://nbn-resolving.org/urn:nbn:de:0111-pedocs-131436 https://doi.org/10.25656/01:13143 in Kooperation mit / in cooperation with: http://www.bzl-online.ch Nutzungsbedingungen Terms of use Gewährt wird ein nicht exklusives, nicht übertragbares, persönliches und beschränktes Recht auf Nutzung dieses Dokuments. Dieses Dokument ist ausschließlich für den persönlichen, nicht-kommerziellen Gebrauch bestimmt. 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You are not allowed to alter this document in any way, to copy it for public or commercial purposes, to exhibit the document in public, to perform, distribute or otherwise use the document in public. By using this particular document, you accept the above-stated conditions of use. Kontakt / Contact: peDOCS DIPF | Leibniz-Institut für Bildungsforschung und Bildungsinformation Informationszentrum (IZ) Bildung E-Mail: pedocs@dipf.de Internet: www.pedocs.de http://www.bzl-online.ch Würdigung MARTIN WAGENSCHEIN (1896-1988) UND SEINE LEHRKUNST - HEUTE GENETISCH-SOKRATISCH-EXEMPLARISCH VERSTEHEN LERNEN UND LEHREN Hans Christoph Berg . Martin Wagenschein, geboren 1896, Professor Dr., Dr.h.c., studierte Physik und Mathematik. Promotion i n Physik. I n den Zwanziger Jahren e r h i e l t e r entscheidende Anregungen und Erfahrungen i n Paul Geheebs F r e i e r Schulgemeinde "Odenwaldschule", war danach Lehrer an s t a a t 1ichen Gymnasien und nach 1945 M i t a r b e i t e r an Schul versuchen und Bi1 dungsplänen; von 1960-65 a r b e i t e t e e r i m Ausschuss "Höhere Schule'' d e s "Deutschen Ausschusses"; s e i t 1965 Honorarprofessor an d e r U n i v e r s i t ä t Tübingen, wo e r über 20 Jahre lang s e i n Seminar h i e l t . 1978 wurde ihm d i e Ehrendoktorwiirde d e r Technischen Hochschule Darmstadt verliehen. Dort h a t t e e r einen Lehrauftrag s e i t 1950. Martin Wagenschein s t a r b Ostern 1988. 1. WAGENSCHEIN IN KLASSISCHER PADAGOGIK-TRADITION I n d e r pädagogischen Diskussion i s t Wagenschein mehr m i t s e i n e r Konzeption und weniger m i t seinen Beispielen d e s Genetisch-sokratischExemplarischen Lehrens bekannt und berühmt geworden. Und s o werden d i e meisten Kollegen Hentigs U r t e i l : "Wir brauchen i n Deutschlands Schulen n i c h t s dringender a l s Wagenscheins Lehrkunst" s o zurecht l e sen, a l s h ä t t e Hentig bloß geschrieben: ' w i r brauchen i n d e r Lehrerbildung n i c h t s dringender a l s Wagenscheins Lehrmethode'. Aber Wagenschein war z u e r s t und z u l e t z t e i n p a s s i o n i e r t e r und verantwortl i c h e r Physik- und Mathematiklehrer, d e r aus w e i t e r und t i e f e r Menschl i c h k e i t d i e Naturforschung l i e b t e , und d i e Kinder, und vor allem d i e Natur: " W i r kennen d a s , wovon d i e Mondgedichte reden a l l e , u n t e r ganz a l l t ä g l i c h e n Umständen: Ist n i c h t d e r Mond d e r große Stimmungsmeister? Niemals d e r Gleiche, wie auch man s e l b s t . Unberechenbar wie d a s Geschick d e s Tages: wenn w i r frühmorgens, noch i m Dunkeln, v o r ' s Haus t r e t e n , sehen w i r uns nach ihm um, ob e r Wache h ä l t , wie e s ihm heute geht und wie e r uns f i n d e t : e i n e r n s t e r Grußwechsel m i t dem Uberirdischen. E r e r w i d e r t unseren fragenden Blick, prüfend, m i t Ermutigung oder Einwand. Er i s t noch da. Er h a t uns bemerkt, - doch s c h n e l l i s t e r wieder f ü r s i c h , e n t r ü c k t , t i e f b e s c h ä f t i g t i m Wolkenland und i m Meer d e r Sterne." (WAGENSCHEIN: Erinnerungen S. 155 f = E 1 5 5 ) . In s o l c h e r Haltung fand e r den D u r c h s c h n i t t s u n t e r r i c h t zu l i e b l o s infolgedessen auch zu unwissenschaftlich und zu u n w i r t s c h a f t l i c h und so suchte und fand e r i n Verantwortung vor Kepler und G a l i l e i , vor den Kindern und Laien, und vor dem Tautropfen und dem Mond einen s a c h l i c h und menschlich angemesseneren Lehrweg: d a s Genetisch-Sokratisch-Exemplarische Lehren. Und diesen Weg i s t e r z e i t l e b e n s i m Unterr i c h t gegangen, m i t Kindern, Jugendlichen, Studenten, Lehrern, Laien b i s i n s e i n neunzigstes J a h r u r g r o ß v ä t e r l i c h Seminar haltend. Zeitlebens b l i e b e r z u e r s t Physik- und Mathematiklehrer und wurde e r s t dabei und danach Methodiker: e r wurde a l s Wegwanderer zum Wegweiser. In d i e s e r Ganzheitlichkeit wurde Wagenschein zu e i n e r s e l t e n e n Ge- ... ll Würdigung Würdigung stalt in unserer leider doppelt halbierten Pädagogik - schon die Schule lebensfern und obendrein noch die Erziehungswissenschaft schulfern zu einer seltenen Gestalt, wie sie allerdings immer wieder von den "Klassikern der Pädagogik'' (SCHEUERL 1979) verwirklicht wurde, besonders deutlich von Diesterweg: erstens erfolgreicher Autor von fünf Schulbüchern - Lesebuch, Sprachlehre, Rechnen, Geometrie, Himmelskunde ("populäre Himmelskunde und astronomische Geographie"; hatte mit 26 Auflagen 100 Jahre Bestand!), zweitens daraufhin sein "Wegweiser zur Bildung", eine gründliche und handliche Methodik, zugespitzt zu drei Dutzend Lehrregeln, orientiert auf die "organischgenetisch-dialogische" Lehrmethode, die er Schleiermacher abgelauscht hatte (DIESTERWEG 1834). Diese ganzheitliche Tradition der Pädagogik blieb glücklicherweise klassische Norm, blieb aber leider seltene Wirklichkeit. Und so wurde in der mehrheitlich einseitigen Pädagogik Wagenscheins beidseitiger Ansatz meist vereinseitigt: die Physik- und Mathematiklehrer studierten seine Unterrichtsexempel, die (meist mathematikgeschädigten) Allgemeindidaktiker studierten seine Konzeptartikel. In diesem Aufsatz will ich mich an den ganzen Wagenschein halten und seine Leitbegriffe Genetisch-Sokratisch-Exemplarisch entwickeln an zwei seiner Lehrgangsskizzen - erstens zur Kreisformel, zweitens zur Erdkugel (in WAGENSCHEIN: Naturphänomene S. 278 ff bzw. S. 298 ff). - ... 2. SOKRATISCH - "Als er Kind war, Schüler, Jüngling, Student, da sagte man ihm: 4 pi r2, so rechnet man die Fläche der Kugel aus. 4 mal diese Zahl (die etwa 3,14 ist) mal r, der Länge des Halbmessers, und dann mal r noch einmal. Auch bewiesen hatte man es; es ging auf die Griechen zurück. Erst als er dann später, dreißig Jahre alt, begann, eben dies die Kinder zu lehren, bemerkte er - nun, er bemerkte nichts ande. res, als was wir auch heute, sechzig Jahre später, genauso bemerken können, wenn wir uns trauen: da0 wir (vielleicht) diese Formel können - Wagenschein sagt: "bedienen können" - da8 wir diese Formel aber zumeist nicht anschauen, geschweige durchschauen können. Fast alle Wagenscheinstücke beginnen so ähnlich: zuerst repetiert er das konventionell Gelernte - "apportieren" nennt er das - und dann wendet er sich davon ab: solches Scheinwissen als Geistesnahrung - nein danke. "Wie tödlich langweilig ist es und allzuleicht zu verstehen, daß an zwei parallelen geraden Linien, die von einer dritten gekreuzt werden, Winkel sich finden lassen (mit schwer zu behaltenden Namen), die gleich groß sind." (N 298) Ob das angelernte Schulwissen richtig oder falsch ist, ist minderwichtig, denn sowohl Richtiges wie Falsches ist minderwertig, weil und sofern bloß oberflächlich angelernt. Wagenschein prüft in sokratischer Unerbittlichkeit sich und jedein) andere(n1: Hast du auch wirklich selber gesehen und gedacht, und hast auch wirklich Du selber gesehen und gedacht, oder wo läßt Du sehen und denken? Lieber vorwissenschaftlich naiv aber in eigener ..." Denkanstrengung darüber rätseln "'Willi der Stein oder 'muß' er fallen?" (N 1971, als ein erstaunliches Alltagsphänomen pseudowissenschaftlich superklug mit den unverstanden hergeholten Molekülen zu erklären (N 32): fremde Federn mogen schmücken aber helfen nicht fliegen; sie machen uns bloß zu "Prothesengöttern" (FREUD). Und so prüft Wagenschein unser aller Schulwissen auf Scheinwissen: "Gemeinsam ist ihnen, Wagenschein und Sokrates, da0 sie eine geistige Bewegung, einen Frage-Prozeß und Frage-Progreß auslösen, der dazu führt, da6 man 'versteht', nicht nur auswendig weiß" (HENTIG 1986, S. 41). Und wie Sokrates, so ist auch Wagenschein beides, "Stechfliege" und "Hebamme". Und wie Sokrates zwischen die Fronten geriet zwischen staatserhaltenden Traditionalisten einerseits und selbstherrlich progressiven Sophisten andererseits, so ging auch Wagenschein einen dritten Weg zwischen Restauration und Reformismus - daher seine Aktualität und Attraktivität in der jetzigen Phase der ''Reform der ~eform".Mit Sokrates also nimmt Wagenschein Abschied von leerem konventionellem Schulwissen. Aber Wagenschein wird kein "~okratist",er versteift sich nicht zu einer kritizistischen Prinzipienreiterei - suchte doch selbst Kafkas Hungerkünstler eine Speise, die ihm schmeckt - Wagenschein geht, auch lebensgeschichtlich, auf die Suche nach besserem Wissen. Nach zweijährigem Referendariat Überlegt er: "ES gab also zwar Lichtblicke in dieser Staatsschule, vereinzelte. Aber im ganzen: das war es nicht! Aber wie und wo ? Gab es da nicht diese 'Odenwaldschule'?" (E 24) Und dort, in dieser kühnen freien Schult? Geheebs (vgl. GEHEEB~LGNER1924 - nach der Emigration 1934 seit 1946 in Goldern im Berner Oberland, vgl. NÄF 1986), dort in der alten Odenwaldschule haben wir ihn bei seiner Ich-weiß-daß-ich-nichts-weiß-Bemerkung getroffen, zu der wir gleich zurückkommen werden. ... - - 3. GENETISCH "Der Begriff soll ja nicht nur den Tatbestand klären, wie es freilich die meisten Begriffsbestimmungen tun, sondern auch den Grund enthalten und hervorleuchten lassen. Heutzutage sind aber die Ausdrücke für einen Begriff nur die Schlußsätze" - "heutzutage", sagt Aristoteles vor 2500 Jahren (Von der Seele, 41Zb). Also den Dingen auf den Grund gehen: "Mut zur Gründlichkeit" in Wagenscheins Worten - eine Variation der Aufklärungsparole vom "Sapere aude" - woraus dann auch der " ~ u tzur Lücke" folgt. Zurück zur Ausgangsszene in die Odenwaldbemerkte er, da0 sich diese Formel auch anschauen schule 1926: ließ, ja ursprünglich von den Griechen angeschaut worden war und auch nur angeschaut werden konnte. Da0 dieses 4 pi r2 nur eine späte Kurzschrift ist, gemacht für die Berechnenden, nicht mehr für die Schauenden; ein Automat, zu bedienen von jedermann. Daß es ja als "4 mal pi r2 " nichts anderes saqt, al6 daß die Kugeloberfläche genau viermal so groß sei als pi r2 Dies pi r2 aber war die Rechenvorschrift für den "größten Kreis" der Kugel, den sie, durch ihre "... . Würdigung Würdigung Mitte aufgeschnitten, sehen l ä ß t . So ist a l s o , was uns d e r Apfel i n d e r greifenden Hand a l s Schale b i e t e t , v i e r f a c h d a s , was e r von seinem Innern p r e i s g i b t , wenn w i r ihn geradewegs mitten durchschneiden und auf d i e f r i s c h e Innenfläche blicken." (N 278) - Sind w i r zuf r i e d e n m i t diesem Werden und Wachsen des Wissens? Wohl kaum. Wagenschein auch noch n i c h t . Immerhin h a t d a s Verstehen Wurzel g e f a ß t , i s t angekeimt, i s t aber noch n i c h t r e i f ; Z e i t und Geduld s i n d e r f o r d e r l i c h , besser gesagt " i n s t ä n d i g e s Harren". Der Bericht g e h t weiter: " I n seinem einundfünfzigsten J a h r schaute e r e i n e s Abends d i e goldene Mondscheibe an, wie s i e über dem Wald a u f s t i e g : da sah e r e s noch einmal neu! Er begann zu grübeln und zu s c h ä t i e n , wievielmal nun wohl d i e s e von ihm gesehene, wirklich gewölbte Mondfläche größer s e i a l s s i e , f ä l s c h l i c h f l a c h gesehen, e r s c h e i n t ? - Bis e s ihn durchzuckte, daß e r d a s s e i t langem wußte (und wohl doch n i c h t wußte?): daß s i e j a eben genau doppelt s o groß s e i n müsse: Denn wenn d i e ganze Kugel i h r e n größten Kreis viermal f a ß t , so muß i h n d i e halbe Kugel, d i e a l l e i n ich j a übersehen kann, zweimal e n t h a l t e n . Dieser größte Kreis i s t aber gerade das, was i c h vom Mond zu sehen glaube, indem i c h ihn a l s flachen T e l l e r nehme. Zweimal mehr Fläche b i e t e t m i r d e r Mond, a l s i c h zu sehen meine. Und s o i s t e s b e i j e d e r Kugel, wenn i c h s i e weit genug von m i r h a l t e . Aber d e r Mond h a t t e e s i h n g e l e h r t , was d i e Formel 4 p i r 2 oder 2 p i r2 i m Grunde sagen w i l l . " (N 278 f ) Hier i s t v i e l e s vom Ansatz des genetischen Lehrens - "der Werdegang a l s Lehrgang" (BERG 1985) bei.Wagenschein zu sehen: Es geht ihm zue r s t um d a s Regenerieren d e s eigenen degenerierten Wissens. Und w i r lernen i n d i e s e r Szene einen s e i n e r d r e i Haupthelfer genauer kennen: den u n v e r s t e l l t e n wirklichen Gegenstand. Auch d e r zweite Helfer wird benannt: u n v e r s t ö r t e , unverschulte Kinder: .. e s bedarf v i e l l e i c h t d e s Mondes a l s e i n e s immer neu an den Himmel gesetzten Fragezeichens, um wieder a k t i v i e r t und wieder naiv gemacht zu werden. Der andere, d e r h i l f r e i c h s t e Weg, i s t d e r Umgang m i t Kindern. S i e h e l f e n uns zu dem, was w i r ihnen h ä t t e n bringen s o l l e n . " (N 280). Wagenscheins d r i t t e r Helfer sind d i e a l t e n Forscher, e i g e n t l i c h b e s s e r d i e jungen Forscher, d i e o r i g i n ä r e n , ursprünglichen Forscher; s i e zeigen s i c h i m Erdkugelbeispiel: "Wie anders s i e h t d i e s e langweilige Winkelfigur aus, wenn s i e aus e i n e r unser Denken herausfordernden Wirklichkeit herausgelesen wird, herausspringt: 'ursprünglich', indem s i e uns d i e Antwort auf e i n e Frage z u s p i e l t : Eratosthenes (275 b i s 195 V. Chr.) w o l l t e herausbekommen, wie groß d i e Erdkugel ist. Er wußte schon, da5 s i e e i n e schwebende Kugel i s t , (N 298). Und nun v o l l z i e h t Wagenschein d i e damalige Erdkugelberechnung s e l b s t nach, e r b e r i c h t e t s i e f ü r uns mitvollziehbar, und e r g i b t (inzwischen r e a l i s i e r t e ) Hinweise auf i h r e n heutigen schulischen Nachvollzug. ... - ". ..." 4. - EXEMPLARISCH Exemplarisch Lehren h e i ß t zugleich am B e i s p i e l und verallgemeinbar Lehren - und zwar beides gründlich, i n jener besonderen "Gründlich- k e i t , d i e i m Einzelnen a u f s Ganze geht": "Die Welt i m Wassertropfen", mundus i n g u t t a , h i e ß e s b e i den Römern; b e i den Engländern " a l l i n a n u t s h e l l " . Es g i l t , beim Lehrgang d i e E i n s t i e g e zu suchen, möglichst a l l t ä g l i c h e E i n s t i e g e , d i e i n d i e T i e f e und Weite d e r Welt führen, E i n s t i e g e , b e i denen man m i t Leib und S e e l e und G e i s t i n d i e Wirklichk e i t d e r Welt e i n t a u c h t . Exemplarisch Lehren h e i ß t darum auch philosophisch u n t e r r i c h t e n : Physik ohne Metaphysik b l i e b e o b e r f l ä c h l i c h , würde e r s t e n s d i e Geistesgeschichte d e r Physik v e r r a t e n und wäre bloß p h y s i k a l i s t i s c h s t a t t physikalisch, würde zweitens auch den Bildungshunger d e r Kinder mißachten und gäbe ihnen bloß S t e i n e s t a t t Brot, wäre bloß pädokratisch aber n i c h t pädagogisch: "Kein von seinem Fach benommener, kein philosophisch n i c h t angeriihrter Lehrer ist imstande, allgemeinbildend zu u n t e r r i c h t e n " . Wagenschein h a t d i e s e n philosophischen S c h r i t t , diesen S c h r i t t aus dem sauber erschlossenen Fachwissen i n d i e fundarnontale und überfachliche Wirklichkeit immer wieder markiert. Eine d i e s e r Fundamentalerfahrungen i s t i h m d i e Mathematisierb a r k e i t d e r Natur, s e i n Kronzeuge Heisenberg: " daß d i e Mathematik i n irgendeiner Weise auf d i e Gebilde unserer Erfahrung p a ß t , empfand i c h a l s außerordentlich merkwürdig und aufregend Gewöhnlich l ä ß t der S c h u l u n t e r r i c h t d i e verschiedenen Landschaften d e r g e i s t i g e n Welt vorbeiziehen, ohne da6 w i r i n ihnen r e c h t heimisch werden. Er bej e nach den Fähigkeiten d e s Lehrers m i t einem mehr leuchtet s i e oder weniger h e l l e n L i c h t , und d i e Bilder h a f t e n längere oder kürzere Z e i t i n unserer Erinnerung. Aber i n einigen s e l t e n e n Fällen f ä n g t e i n Gegenstand, d e r so i n s B l i c k f e l d g e t r e t e n i s t , p l ö t z l i c h an, i m eigeund s c h l i e ß l i c h f ü l l t d a s von ihm ausgenen Lichte zu leuchten s t r a h l t e L i c h t einen immer größeren Raum i n unserem Denken, g r e i f t auf andere Gegenstände über und wird s c h l i e ß l i c h zu einem wichtigen T e i l unseres eigenen Lebens. - So ging e s m i r damals m i t d e r Erkenntn i s , daß d i e Mathematik auf d i e Dinge unserer Erfahrung paßt (V 21). In d i e s e r Grunderfahrung über d i e Mathematisierbarkeit d e r Natur, i m Staunen Über d i e s o unerwartbar g l a t t e Passung mündet Wagenschein auch i n s e i n e r Betrachtung über d a s Zahlenverhältnis von Umriß und (Mond)Halbkugel: "Zwei und n i c h t mehr und n i c h t minder, d i e g o t t geschaffene zweite Zahl, d i e verdoppelte Einheit. Damals b e i den Griechen erwuchs d i e s e Blüte. Und n i c h t nur, daß s i e e s wußten: s i e erwiesen e s auch, da6 e s s o und n i c h t anders s e i n müsse. So rund und g l a t t das Ergebnis, so wußten s i e doch, e s war e i n f e i n e r , e i n unendlicher Prozeß zu überstehen, ehe man e s erkennen konnte. S i e dachten i h n zu Ende, und i h r Scharfsinn l i e ß s i e einen Fund machen von e i n f a c h s t e r Anschaulichkeit, d e r golden an ihrem Himmel stand." (N 279). Ähnlich weitreichend und t i e f g r ü n d i g i s t d e r Bildungsertrag i n unserem zweiten B e i s p i e l , d e r Erdkugelberechnung nach Eratosthenes: "Lernt man den S a t z , daß jene zwei Winkel an P a r a l l e l e n einander g l e i c h s i n d , an d e r s t e r i l e n Figur, s o l e r n t man f a s t g a r n i c h t s . Lernt man i h n aber g l e i c h b e i s e i n e r Entdeckung aus e i n e r Wirklichkeit herauslesen s o s p ü r t man s o f o r t etwas davon, was Mathematik i s t und kann. (Nur etwas. Vom ' ~ e w e i s e n ' i s t noch g a r keine Rede. Dazu i s t d e r ' S a t z ' ZU s e l b s t v e r s t ä n d l i c h . Man braucht d i e G l e i c h h e i t d e r beiden Winkel nur ... ... ... ... ... ..." 15 Würdigung Würdigung zu vermuten und sagt auch schon ja dazu.) Man lernt das Abstrahieren, das Herausholen der Figur aus der Wirklichkeit. Die Wirklichkeit, das ist hier: die Weltinsel der Antike; die agyptische hohe Sonne; die stillen Schatten im Mittagsglanz; das Tappen der Sohlen der Läufer, die damals die Strecke durchmessen mußten; der mitlaufende, also ferne Mond; die noch fernere, weil sichtlich hinter der Mondsichel sitzende Sonne. Aus dieser Wirklichkeit ziehen wir heraus, was damals wie heute gilt. Die unvergänglichen Denklinien der Geometrie, wn eine Figur zu gewinnen, die zu einem abstrakten Reich gehört und uns doch die Macht gibt, die ganze Erde zu umspannen, ohne ein kleines Land zu verlassen." (N 301) Klafki hat seinerseit den Ausdruck "didaktische Analyse" eingebürgert; Heimann verwendet daneben auch den Ausdruck "didaktische Exerzitien", und sogar "didaktische Meditation" (HEIMANN 1962, S. 417). Bei Heimann hatte dieser Begriff noch nicht die heutigen Obertöne; Wagenschein dagegen in seinen didaktischen Betrachtungen undSBesinnungen,insbesondere in seinem Aufsatz "Über die Aufmerksamkeit" von 1959 (U I S. 351 ff) präludiert der heutigen Begriffsbedeutung. In seiner Autobiografie schreibt er: "Und als 1953 der Hessische Kultus-Minister die Leitung des entstehenden großen 'Schuldorfes Bergstraße' mir hatte zutrauen wollen, schrieb ich ihm: 'Ich sehe - und mehr noch sehen es andere, die es mir sagen -, da% ich einen ganz bestimmten Auftrag habe. Er kommt unmittelbar aus dem praktischen Unterricht, und zwar aus dem physikalischen. Sein ziel ist aber nicht fachlich im engeren Sinne. Er gilt der Humanisiening der mathematischen Naturwissenschaften, ihrer In-Eins-Setzung mit den küristlerisch undreligiösen Grundkräften des Menschen'." (E 74) 5. VERSTEHEN LERNEN VOR VERSTEHEN LEHREN "Zweierlei Wissen" war der Titel von Wagenscheins "didaktischer Betrachtung" (E 80) oder "didaktischer Analyse" (nach Klafki) oder "didaktischer Exerzitie und Meditation" (nach Heimann). Bei einer ähnlichen Unterrichtsvorbereitung sehen wir ihn beim Spüreisen (N 15 f, kommentiert durch Wagenschein selbst; E 78 821, oder in der vielleicht schönsten Miniatur "Das Licht und die Dinge" (N 113 £1. Wagenschein ist hier Physiker und wird Pädagoge; er vollzieht hier seine eigene Lehrerbildung. Hierbei orientiert er sich nicht an der Forschungsspitze der Physik "Hochschulphysiker leben heute fast alle oberhalb der Baumgrenze. Man sollte sie nicht nach Waldwegen fragen" (E 83) - sondern er geht zurück ins Ursprüngliche: "Der künftige Lehrer der Physik aber braucnt nichts dringender als, ungeachtet seiner präzisen Fachkenntnisse, fähig zu bleiben oder wieder zu werden, auf diese Vorstufe sich zurücksinken zu lassen" (N 114). In den vielen bitteren Auseinaw3ersetzungen mit dem mittleren Wissenschaftsmanagement in der Physik war für Wagenschein der Zuspruch der Spitzenforscher ermutigend: "Nichts ist geeigneter, das Interesse für die . Probleme der Physik in weitere Kreise zu tragen, als eine Vorstellung nach Ihrer Art", schrieb ihm Max Planck. - - 6. WAGENSCHEINS LEHRKUNST - HEUTE? Im Jahr 1986 waren zu Wagenscheins 90. Geburtstag über 50 Gedenkaufsätze publiziert worden, woraus sich unter der Obhut der deutschsprachigen Sektion des Weltbundes f S Erneuerung der Erziehung die Einladung an alle Autoren zur Wagenscheintagung 1987 entwickelte (~er~/Buck/Lüthi: "Die Pädagogik Martin ~agenscheins") Glücklicherweise konnten wir in der Ecole d'Humanit6 tagen, wo Martin und Vera Wagenschein mehrfach Gastlehrer gewesen waren, und wo Armin und Nathalie Lüthi als Nachfolger Paul Geheebs schon lange ein Zeichen gesetzt hatten mit dem Martin-Wagenschein-Haus; inzwischen ist dort 1988 auch der Wagenschein-Nachlaß aufgenommen. Mit der zweiten Wagenscheintagung im Evangelischen Trifelsgymnasium in Annweiler/Pfalz (Berg/Buck/Gerth: "Von Wagenscheins Lehrkunst Lernen") erweiterten sich Gesprächsthemen und Gesprächskreis durch Vertreter anderer Schulen, die sich um den Einbau von Wagenscheins Lehrkunst im heutigen Unterricht bemühen (vgl. O?T 1987; 1988). Die dritte Wagenscheintagung in Goldern vom 2. - 5. Juli 1989 wird sich~ausdrücklichdiesem Thema widmen: ~erg/~uck/~üthi: "Wagenscheins Lehrkunst in Schulvielfalt". Voraussichtlich wird sich dieser Wagenscheinkreis rondoartig-alle drei Jahre in Goldern treffen und dazwischen in anderen "wagenscheinfreundlichen'' Schulen: für 1990 liegt eine Einladung aus dem Landerziehungsheim Marienau/~üneburger Heide vor (Hasenclever), f S 1991 aus der ~aborschule/~ielefeld(~offmann/~Ütgert/Schulze)und aus der katholischen Stiftsschule St. Johann/Amöneburg (Hildebrandt-Günther). In einigen Jahren werden wir hoffentlich berichten können, wie wir Wagenscheins Lehrstücke studiert, novelliert, weiterkomponiert und didaktisch inszeniert haben, sein "Fallgesetz im Brunnenstrahl", seine "Erde unter den Sternen", seine legendären "primzahlen" (nicht nur ein einziges Mal in Goldern), und eben auch seine Skizzen von "Kern und Schale runder Dinge" und "Eratosthenes' Erdkugelmessung"; des weiteren Lehrstücke aus der Tradition, in der er steht, wie Faradays "Naturgeschichte einer Kerze", Keplers "Vom sechseckigen Schnee", Lessings ''Fabeln samt Abhandlungen Über die Fabel"; und natürlich neue Lehrstücke im Geiste Wagenscheins, wie Raebigers "Schwebestab" und Stettlers "Mit der Lupe in die Ferne sehen". Denn auch für Martin und Vera Wagenscheins still leuchtendes Lebenswerk danken wir am besten gemäß dem Spruch: "Tradition heißt nicht Asche aufheben, sondern ein Feuer am Brennen halten". . LITERATUR BERG, H. Chr. (1985): Methoden, Genetische. In: ~nzyklopädieErziehungswissenschaft, Bd. 4, Stuttgart: Klett-Cotta. / DIESTERWEG, A. (1834): tiber die Lehrmethode Schleiermachers: In: A. D.: Sämtliche Werke, Bd. 111. / GEHEEB, P./ILGNER, A. (1924): Odenwaldschule. In: HLLKER, F. (1924): Deutsche Schulversuche. Berlin: Schwetschke. / Würdigung HEIMANN, P. (1962): Didaktik als Theorie und Lehre. Die Deutsche Schule. / HENTIG, H. V. (1987): "Humanisierung". Eine verschämte Rückkehr zur Pädagogik? Andere Wege zur Veränderung der Schule. Stuttgart: Klett-Cotta. / NÄF, M. (1986): Die Ecole dtHumanit& in Goldern - der Neubeginn Geheebs. In: RÖHRS, H. (1986): Die Schulen der Reformpädagogik heute. Handbuch reformpädagogischer Schulideen und Schulwirklichkeit. Düsseldorf: Schwann. / OTT, E. H. (1987,1988): Berichte über die Wagenschein-Symposien Goldern 1987 und Annweiler 1988. Erziehungswissenschaft - Erziehungspraxis 31987; Forum Pädagogik 4/1988. / SCHEUERL, H. (Hg.) (1979): Klassiker der Pädagogik. München: Beck. / WAGENSCHEIN, M. (1983): Erinnerung für morgen. Eine pädagogische Autobiografie. ( = E) Weinheim: Beltz. / WAGENSCHEIN, M. (2/1988): Eaturphänomene sehen und verstehen - genetische Lehrgänge. (= N) Herausgegeben von H. Chr. Berg. Stuttgart: Klett. / WAGENSCHEIN, M. (1965): xrsprüngliches Verstehen und exaktes Denken I. (=U I) Stuttgart: Klett. / WAGENSCHEIN, M. (7/1982) : yerstehen lehren. Genetisch-Sokratisch-Exemplarisch. (= V ) Weinheim: Beltz ANMERKUNG: Die drei länger vergriffenen Schriften von Wagenschein - Verstehen Lehren; Kinder auf dem Weg zur Physik; Erinnerungen für morgen - erscheinen demnächst wieder unter dem Lektorat von Peter Kalb in der Pädagogischen Bibliothek Beltz V o r t r a g i n München: 'IRettet die Phänomene!" 18 (1975) Inhaltsverzeichnis Editorial Fritz Schoch, Peter Füglister Kurt Reusser - Klassenlehrer Würdigung Iwan Rickenbacher Der Klassenlehrer - pädagogische Möglichkeiten und Grenzen Hans Christoph Berg Martin Wagenschein (1896-1988) und seine Lehrkunst - heute , Gita Steiner-Khamsi Migrationsgeschichten: Ein didaktischer Ansatz in der interkulturellen Pädagogik Angela Frey-Eiling, Kar1 Frey und Alfons Frei Anforderungen an die Allgemeine Didaktik Herman.n Landolt Ausbildung und Reflexion 5 11 , Interkulture,lle Erziehung Allgemeine Didaktik an der ETH Didaktik der Lehrerbildung Kurzportrait Verbandstei 1 Lehrerbildung und Berufspraxis im Rückblick Lehrerfortbildung von morgen Peter Kradolfer Bericht über einen Besuch beim "Deutschen Institut for Fernstudien an der Universität Tübingen" (DIFF) Heinz Mandl Aufgaben und Ziele des DIFF Verzeichnis der lieferbaren Studienbriefe und Lehrmaterial ien des DIFF 19 30 40 46 51 53 Protokoll.der Jahresversammlung des SPV vom 4. November 1988 in Chur Jahresbericht 1988 des Präsidenten Rechnung und Budget SPV Rechnung und Budget BzL 57 Gertrude Hirsch, Gilbert Ganguillet und Uri P. Trier Welche Bedeutung messen Oberstufenlehrer der Lehrerbildung im Rückblick auf ihre Berufserf ahrungen zu? Hans Rudolf Lanker . Lehrerfortbi1dung:mehralsKurse 65 59 61 63
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Influence of habitat, density, lignin structure, and extraction treatment on thermal-softening properties of water-swollen wood: a study of 87 wood specimens
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Yuka Miyoshi , Hisashi Abe, Hiroaki Horiyama, Keisuke Kojiro and Yuzo Furuta Influence of habitat, density, lignin structure, and extraction treatment on thermal-softening properties of water-swollen wood: a study of 87 wood specimens https://doi.org/10.1515/hf-2023-0083 Received August 9, 2023; accepted December 12, 2023; published online January 15, 2024 the prior research showed correlation between therm softening temperature and methoxyl group content of w Keywords: viscoelasticity; lignin structure; density; ext the prior research showed correlation between thermal- softening temperature and methoxyl group content of wood. https://doi.org/10.1515/hf-2023-0083 Received August 9, 2023; accepted December 12, 2023; published online January 15, 2024 Keywords: viscoelasticity; lignin structure; density; extrac- tion; habitat Abstract: This study aims to reveal the diversity of thermal- softening temperatures and identify the factors that deter- mine this temperature. To achieve this, the thermal- softening properties of the radial direction of wood were measured under water-saturated conditions for 15 softwood and 72 hardwood specimens. Wood samples were obtained from the xylarium of the Forestry and Forest Products Research Institute, Japan. A dynamic viscoelastic measure- ment was performed on samples with uniform heating and cooling history because the difference in cooling rate can alter in the mechanical properties of wood. The storage and loss elastic moduli increased linearly as wood density increased, regardless of the wood species. However, the thermal-softening temperature (defined in this study as the peak temperature of loss tangent) was unrelated to the density, anatomical features, species, latitude, and annual rainfall in the habitat. When the relationship between thermal-softening temperature and lignin structure was investigated, a negative correlation was observed between the thermal-softening temperature and the syringyl ratio (syringyl/(syringyl+guaiacyl)) of lignin aromatics. This indicates that the thermal-softening temperature is higher for wood species with denser lignin structures, supporting Holzforschung 2024; 78(2): 109–120 Wood Physics/Mechanical Properties *Corresponding author: Yuka Miyoshi, Department of Wood Properties and Processing, Forestry and Forest Products Research Institute, Matsunosato 1, Tsukuba, Ibaraki 305-8687, Japan, E-mail: ymiyoshi@ffpri.affrc.go.jp Hisashi Abe, Department of Wood Properties and Processing, Forestry and Forest Products Research Institute, Matsunosato 1, Tsukuba, Ibaraki 305- 8687, Japan, E-mail: abeq@ffpri.affrc.go.jp Hiroaki Horiyama, Keisuke Kojiro and Yuzo Furuta, Division of Environmental Sciences, Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, Hangi-cho, Shimogamo, Sakyo-ku, Kyoto 606-8522, Japan, E-mail: s821732002@kpu.ac.jp (H. Horiyama), kojiro@kpu.ac.jp (K. Kojiro), furuta@kpu.ac.jp (Y. Furuta) uthor(s), published by De Gruyter. This work is licensed under the Creative Commons Attribution 4.0 International License. 2.1 Pretreatment of samples A total of 15 softwood and 72 hardwood specimens were obtained from the xylarium of the Forestry and Forest Products Research Institute (TWTw). The annual rainfall data were obtained from the Japan Mete- orological Agency (https://www.data.jma.go.jp/gmd/risk/obsdl/) and the World Weather Online (https://www.worldweatheronline.com/), based on the registered latitude and longitude information. For specimens without latitude information, the latitude data was estimated from the name of the registered region or representative point in the country (Table 1). For DMA measurement, each wood sample was cut in the longi- tudinal direction into an end-grain plate (1 mm thickness). Subse- quently, the strip-shaped DMA samples (3.2 mm (tangential) × 30 mm (radial)) and square-shaped weight measurement samples (15 mm (tangential) × 15 mm (radial)) were cut from the plate (Figure 1). Half of the DMA samples were extracted for 6 h with ethanol:benzene (1:2, v/v) in a Soxhlet extractor to remove the resin, fat, wax, and soluble tannin. The extracted DMA samples were air-dried at room temperature and freeze-dried for 6 h to completely volatilize the liquid. The weight measurement samples were first treated in the same manner as the extracted DMA samples. Next, they were boiled for 10 min and cooled naturally to room temperature in water. This process was performed two times according to the temperature rise and fall program of the DMA samples. The weight loss rate due to the extraction treatment was calculated by weighing the oven-dried (105 °C) samples (Figure 2). The density of all the samples was obtained from the dimensions and weight of the oven-dried weight measurement samples before extraction. The weight loss due to extraction and density were calculated from the average value measured from two or three weight measurement sam- ples that could be taken from each specimen. In addition, the thermal-softening properties of water- swollen wood are affected by the drying history and cooling rate before measurement, even in the same sample (Furuta et al. 2001, 2008a, b; Kojiro et al. 2008). Moreover, the thermal- softening properties of samples collected from a certain part of a tree with large amounts of extractives, such as the base of the branch can be altered by removing the extractives (Furuta et al. 2014). Therefore, to comprehensively discuss the factors affecting the thermal-softening temperatures of various wood species, it is necessary to conduct experiments that consider the thermal history and extraction treatment of the samples before measurement. did not provide any basis for this in their results. Olsson and Salmén (1997) investigated the relationship between the thermal-softening temperature of wood and the lignin con- tent or methoxyl group content, which was calculated as a molar percentage of the methoxyl groups per phenylpropane unit of lignin. They showed a correlation between thermal- softening temperature and the methoxyl group content. Furuta et al. (2010) measured the thermal-softening proper- ties of delignified wood. They clarified that the thermal- softening temperature of water-saturated wood in the lateral direction varied widely under the influence of a slight change in the lignin structure, rather than a reduction in the amount of lignin. The above researches indicate that there is a main relation between the diversity in thermal-softening proper- ties and lignin structure. However, the reasonable grounds for wood why the diverse thermal-softening properties are expressed cannot be discussed. to obtain the precise thermal-softening properties in water- saturated conditions. Since the thermal-softening properties in lateral direction are affected by the density and anatomic features of the wood, the correlation between the thermal- softening property and the density and vessel arrangement type of the wood was investigated. In addition, to clarify the factors that determine the diversity in thermal-softening temperature, the syringyl ratio of lignin (syringyl/(syrin- gyl+guaiacyl)) was measured in the samples used for DMA measurement, and the relationship between thermal- softening temperature and lignin structure was investi- gated. Elucidating the factors contributing to the diversity of the thermal-softening properties of wood is valuable for basic research and for facilitating advanced discussions on the mechanisms of thermal-softening properties. 2.1 Pretreatment of samples In this study, the thermal-softening properties of various wood specimens, registered in the xylarium of the Forestry and Forest Products Research Institute (TWTw), were measured to reveal the diversity of thermal-softening prop- erties and identify the factors that determine the thermal- softening temperature. Dynamic viscoelastic analysis (DMA) was conducted on specimens subjected to the same heating- cooling history, both with and without extraction treatment, 2 Materials and methods Wu et al. (1992) investigated the lignin structure of hardwood using ultraviolet and visible microspectropho- tometry, and clarified that the distributions of the syringyl and guaiacyl units of lignin in the vessel and fiber differed according to the habitat and porosity of the wood. According to the classification by Wu et al. (1992), the wood species in sub-frigid zones are rich in syringyl units, while those in tropical zones are rich in guaiacyl units. Therefore, the diversity of thermal-softening temperatures may be related to the lignin structure classified according to the climatic zone of the trees. If such a relationship exists between the physical properties and chemical structure of wood and its growing environment, an understanding of tree evolution and growth strategies can be developed. 1 Introduction Many studies have been conducted on the thermal- softening properties of wood in the lateral direction. One study found that the peak temperature of the logarithmic decrement, measured by the torsional dynamic viscoelas- ticity of the radial sample, becomes lower at a higher moisture content and reaches around 80 °C when the moisture content is 20 % or more (Becker and Noack 1968). Although the thermal-softening temperature varies with the frequency of measurement and the loading direction of the sample, it is recognized that the softening behavior observed around 80 °C for the water-saturated wood is based on the glass transition of lignin (Salmén 1984). It is known that the thermal-softening temperature of water- saturated wood is higher in softwood than in hardwood (Placet et al. 2007). The lignin structure differs between softwood and hardwood because softwood contains only the guaiacyl nucleus and hardwood contains both guaiacyl and syringyl nuclei. To investigate the effect of lignin on the thermal-softening properties of various species of wood in water-saturated condition, Furuta et al. (2001, 2008a, b) measured the peak temperatures of the loss tangent (tan δ), as the thermal-softening temperature, by dynamic mechanical analysis, and compared this temperature for water-saturated wood samples (10 softwood and 12 hardwood) in the radial direction. The results showed that the thermal-softening temperature is higher for softwood species, with a difference of up to 30 °C. The authors proposed that the difference in thermal-softening temperature between tropical hardwood and Japanese hardwood is caused by a variation in the lignin structure owing to the aromatic nuclei of lignin; however, it Y. Miyoshi et al.: Thermal-softening properties of water-swollen wood of various species 110 2.2 Dynamic viscoelasticity measurement A forced-vibration dynamic viscoelastometer (DMA 42E-SF 28 Artemis, NETZSCH Japan K.K., Kanagawa, Japan) was used to measure the Y. Miyoshi et al.: Thermal-softening properties of water-swollen wood of various species 111 111 Table : List of examined wood species. The syringyl ratio and peak temperature of tan δ were measured for water-saturated, untreated specimens in radial direction. Codes in Köppen climate classification (Kottek et al. ); tropical rainforest climate (Af); tropical savanna, wet (Aw); humid subtropical climate (Cfa); hot-summer humid continental climate (Dfa); and warm-summer humid continental climate (Dfb). The specimen with TWTw ID was classified as Af because it is a tropical wood species lacking provenance. 2.2 Dynamic viscoelasticity measurement Scientific name TWTw sample ID Vessel arrangement type Provenance Köppen climate classification Latitude (°) Annual rainfall (mm) Density (kg/m) Total extraction (%) Syringyl ratio Untreated sample Extracted sample Peak temperature of tan δ (°C) tan δ value of peak temperature (°C) Peak temperature of tan δ (°C) tan δ value of peak temperature (°C) Quercus gilva Radial-porous Taiwan Cfa .   . .  .  . Quercus gilva Radial-porous Taiwan Cfa .   . .  .  . Quercus gilva Radial-porous Taiwan Cfa .   . .  .  . Quercus gilva Radial-porous Chiba, Japan Cfa .   . .  .  . Quercus gilva Radial-porous Miyazaki, Japan Cfa .   . .  .  . Quercus gilva Radial-porous Miyazaki, Japan Cfa .   . .  .  . Quercus morii Radial-porous Taiwan Cfa .   . .  .  . Quercus morii Radial-porous Taiwan Cfa .   . .  .  . Quercus morii Radial-porous Taiwan Cfa .   . .  .  . Quercus longinux Radial-porous Taiwan Cfa .   . .  .  . Quercus pseudomolucea Radial-porous Indonesia Af −.   . .  .  . Quercus gamelliflora Radial-porous Malaysia Af .   . .  .  . Quercus acuta Radial-porous Gifu, Japan Cfa .   . .  .  . Quercus glaber Radial-porous Miyazaki, Japan Cfa .   . .  .  . Quercus crispula Ring-porous Mie, Japan Cfa .   . .  .  . Quercus crispula Ring-porous Russia Dfa .   . .  .  . Quercus crispula Ring-porous Oita, Japan Cfa .   . .  .  . Quercus crispula Ring-porous Hokkaido, Japan Dfa .   . .  .  . Quercus crispula Ring-porous Hokkaido, Japan Dfa .   . .  .  . Quercus crispula Ring-porous Hokkaido, Japan Dfa .   . .  .  . Lithocarpus soleriana Radial-porous Philippines Af .   . .  .  . Lithocarpus edulis Radial-porous Okinawa, Japan Cfa .   . .  .  . Table : List of examined wood species. The syringyl ratio and peak temperature of tan δ were measured for water-saturated, untreated specimens in radial direction. 2.2 Dynamic viscoelasticity measurement Codes in Köppen climate classification (Kottek et al. ); tropical rainforest climate (Af); tropical savanna, wet (Aw); humid subtropical climate (Cfa); hot-summer humid continental climate (Dfa); and warm-summer humid continental climate (Dfb). The specimen with TWTw ID was classified as Af because it is a tropical wood species lacking provenance. Scientific name TWTw sample ID Vessel arrangement type Provenance Köppen climate classification Latitude (°) Annual rainfall (mm) Density (kg/m) Total extraction (%) Syringyl ratio Untreated sample Extracted sample Peak temperature of tan δ (°C) tan δ value of peak temperature (°C) Peak temperature of tan δ (°C) tan δ value of peak temperature (°C) Table : List of examined wood species. The syringyl ratio and peak temperature of tan δ were measured for water-saturated, untreated specimens in radial direction. Codes in Köppen climate classification (Kottek et al. ); tropical rainforest climate (Af); tropical savanna, wet (Aw); humid subtropical climate (Cfa); hot-summer humid continental climate (Dfa); and warm-summer humid continental climate (Dfb). The specimen with TWTw ID was classified as Af because it is a tropical wood species lacking provenance. Scientific name TWTw sample ID Vessel arrangement type Provenance Köppen climate classification Latitude (°) Annual rainfall (mm) Density (kg/m) Total extraction (%) Syringyl ratio Untreated sample Extracted sample Peak temperature of tan δ (°C) tan δ value of peak temperature (°C) Peak temperature of tan δ (°C) tan δ value of peak temperature (°C) 112 Y. Miyoshi et al.: Thermal-softening properties of water-swollen wood of various species 112 Y. 2.2 Dynamic viscoelasticity measurement Miyoshi et al.: Thermal-softening properties of water-swollen wood of various species 112 Scientific name TWTw sample ID Vessel arrangement type Provenance Köppen climate classification Latitude (°) Annual rainfall (mm) Density (kg/m) Total extraction (%) Syringyl ratio Untreated sample Extracted sample Peak temperature of tan δ (°C) tan δ value of peak temperature (°C) Peak temperature of tan δ (°C) tan δ value of peak temperature (°C) Lithocarpus edulis Radial-porous Kagoshima, Japan Cfa .   . .  .  . Lithocarpus edulis Radial-porous Ibaraki, Japan Cfa    . .  .  . Lithocarpus clementiana Radial-porous Malaysia Af .   . .  .  . Lithocarpus glaber Radial-porous Kumamoto, Japan Cfa .   . .  .  . Acer carpinifolium Diffuse-porous Gifu, Japan Cfa .   . .  .  . Acer carpinifolium Diffuse-porous Yamanashi, Japan Cfa .   . .  .  . Acer carpinifolium Diffuse-porous Nagano, Japan Cfa .   . .  .  . Acer mono Diffuse-porous Hokkaido, Japan Dfa .   . .  .  . Acer mono Diffuse-porous Tokyo, Japan Cfa .   . .  .  . Acer mono Diffuse-porous China Dfa .   . .  .  . Gardenia jasminoides Diffuse-porous Okinawa, Japan Cfa .   . .  .  . Gardenia jasminoides Diffuse-porous Okinawa, Japan Cfa .   . .  .  . Gardenia jasminoides Diffuse-porous Kagoshima, Japan Cfa .   . .  .  . Distylium racemosum Diffuse-porous Fukuoka, Japan Cfa .   . .  .  . Distylium racemosum Diffuse-porous Tokyo, Japan Cfa .   . .  .  . Distylium racemosum Diffuse-porous Nagasaki, Japan Cfa .   . .  .  . Cercidiphyllum japonicum Diffuse-porous Saitama, Japan Cfa .   . .  .  . Cercidiphyllum japonicum Diffuse-porous Hokkaido, Japan Dfa .   . .  .  . Cercidiphyllum japonicum Diffuse-porous Ibaraki, Japan Cfa .   . .  .  . Y. 2.2 Dynamic viscoelasticity measurement Miyoshi et al.: Thermal-softening properties of water-swollen wood of various species 113 Scientific name TWTw sample ID Vessel arrangement type Provenance Köppen climate classification Latitude (°) Annual rainfall (mm) Density (kg/m) Total extraction (%) Syringyl ratio Untreated sample Extracted sample Peak temperature of tan δ (°C) tan δ value of peak temperature (°C) Peak temperature of tan δ (°C) tan δ value of peak temperature (°C) Populus maximowiczii Diffuse-porous Kyoto, Japan Cfa .   . .  .  . Populus maximowiczii Diffuse-porous Hokkaido, Japan Dfb .   . .  .  . Populus maximowiczii Diffuse-porous Hokkaido, Japan Dfb .   . .  .  . Hebe salicifolia – Diffuse-porous New Zealand Cfa −.   . .  .  . Ceiba pentandra Diffuse-porous Mexico Cfa .   . .  .  . Ceiba pentandra Diffuse-porous Mexico Cfa .   . .  .  . Ceiba pentandra Diffuse-porous Nigeria Cfa .   . .  .  . Falcataria moluccana Diffuse-porous – Af – –  . .  .  . Falcataria moluccana Diffuse-porous New Britain, Papua New Guinea Af −.   . .  .  . Falcataria moluccana Diffuse-porous Indonesia Af −.   . .  .  . Shorea parvistipulata Diffuse-porous Malaysia Af .   . .  .  . Shorea leprosula Diffuse-porous Borneo, Indonesia Af −.   . .  .  . Shorea pauciflora Diffuse-porous Malaysia Af .   . .  .  . Eusideroxylon zwageri Diffuse-porous Indonesia Af −.   . .  .  . Eusideroxylon zwageri Diffuse-porous Borneo, Indonesia Af −.   . .  .  . Eusideroxylon zwageri Diffuse-porous Borneo, Indonesia Af −.   . −.  .  . Fraxinus mandshurica Ring-porous Hokkaido, Japan Dfb .   . .  .  . Fraxinus mandshurica Ring-porous Hokkaido, Japan Dfb .   . .  .  . Fraxinus mandshurica Ring-porous China Dfb .   . .  .  . Zelkova serrata Ring-porous Ibaraki, Japan Cfa .   . .  .  . Table : (continued) 114 Y. Miyoshi et al.: Thermal-softening properties of water-swollen wood of various species 114 Y. Miyoshi et al.: Thermal-softening properties of water-swollen wood of various species 114 Scientific name TWTw sample ID Vessel arrangement type Provenance Köppen climate classification Latitude (°) Annual rainfall (mm) Density (kg/m) Total extraction (%) Syringyl ratio Untreated sample Extracted sample Peak temperature of tan δ (°C) tan δ value of peak temperature (°C) Peak temperature of tan δ (°C) tan δ value of peak temperature (°C) Zelkova serrata Ring-porous Tokyo, Japan Cfa .   . .  .  . Zelkova serrata Ring-porous Gunma, Japan Cfa .   . .  .  . Tetracentron sinense Non-porous China Aw .   . .  .  . Tetracentron sinense Non-porous China Aw .   . .  .  . Tetracentron sinense Non-porous British Aw .   . .  .  . Trochodendron aralioides Non-porous Kyoto, Japan Cfa .   . .  .  . Trochodendron aralioides Non-porous Kumamoto, Japan Cfa .   . .  .  . Trochodendron aralioides Non-porous Tokyo, Japan Cfa .   . .  .  . Gnetum gnemon Diffuse-porous Philippines Af .   . .  .  . Gnetum gnemon Diffuse-porous Java, Indonesia Af −.   . .  .  . Gnetum gnemon Diffuse-porous Malaysia Af .   . .  .  . Ginkgo biloba Softwood Ibaraki, Japan Cfa .   . −.  .  . Ginkgo biloba Softwood Tokyo, Japan Cfa .   . .  .  . Ginkgo biloba Softwood Hokkaido, Japan Dfa .   . .  .  . Cryptomeria japonica Softwood Akita, Japan Cfa .   . .  . + .+ Cryptomeria japonica Softwood Tokyo, Japan Cfa .   . −. + .+ + .+ Cryptomeria japonica Softwood Okinawa, Japan Cfa .   . −.  . + .+ Chamaecyparis obtusa Softwood Ibaraki, Japan Cfa .   . −.  . + .+ Chamaecyparis obtusa Softwood Nagano, Japan Cfa .   . .  . + .+ Chamaecyparis obtusa Softwood Tokyo, Japan Cfa .   . −.  . + .+ obtusa Y. Miyoshi et al.: Thermal-softening properties of water-swollen wood of various species 115 Figure 1: Sample dimensions used for measurements. As an example of the measured DMA data, the results of the temperature dependence of tan δ before and after extraction for soft- wood and hardwood samples are shown in Figure 3. The peak top of tan δ was read as the peak temperature. temperature-dependent, dynamic viscoelastic properties of the unex- tracted and extracted water-swollen samples. The dynamic viscoelastic properties of wood changed due to the drying history or cooling rate, even for the same samples (Furuta et al. 2001, 2008a, b; Kojiro et al. 2008). Therefore, to unify the heating history immediately before viscoelastic measurement for all the specimens, this study recorded the data in the second temperature rise after cooling them from 95 °C to 5 °C at 1 °C/min (Figure 2). The data for temperatures up to 98 °C were obtained origi- nally, which is very close to the boiling point of water. Since some samples contained noise in the temperature range higher than 95 °C, DMA data up to 95 °C, which could be stably measured, was analyzed to compare the results. During the viscoelastic measurement, the span was 10 mm in radial direction (Figure 1) and a 0.1 Hz sine wave with a tension displacement amplitude of 5 µm was applied to the sample. In the pre- liminary experiments conducted on several wood samples, the offset loads in the measured temperature range, when the displacement amplitude was controlled at 5 µm, were within the linear region of the stress-strain line. DMA measurement was performed one time for each sample because the preliminary experiments confirmed that the same temperature program would produce nearly identical measurement results. As an example of the measured DMA data, the results of the temperature dependence of tan δ before and after extraction for soft- wood and hardwood samples are shown in Figure 3. The peak top of tan δ was read as the peak temperature. Scientific name TWTw sample ID Vessel arrangement type Provenance Köppen climate classification Latitude (°) Annual rainfall (mm) Density (kg/m) Total extraction (%) Syringyl ratio Untreated sample Extracted sample Peak temperature of tan δ (°C) tan δ value of peak temperature (°C) Peak temperature of tan δ (°C) tan δ value of peak temperature (°C) Larix kaempferi Softwood Ibaraki, Japan Cfa .   . −.  .  . Larix kaempferi Softwood Nagano, Japan Cfa .   . −. + .+ + .+ Larix kaempferi Softwood Netherlands Dfb .   . .  .  . Agathis borneensis Softwood Malaysia Af .   . −.  .  . Agathis borneensis Softwood Malaysia Af .   . .  .  . Agathis borneensis Softwood Indonesia Af −.   . .  .  . Figure 1: Sample dimensions used for measurements. temperature-dependent, dynamic viscoelastic properties of the unex- tracted and extracted water-swollen samples. The dynamic viscoelastic properties of wood changed due to the drying history or cooling rate, even for the same samples (Furuta et al. 2001, 2008a, b; Kojiro et al. 2008). Therefore, to unify the heating history immediately before viscoelastic measurement for all the specimens, this study recorded the data in the second temperature rise after cooling them from 95 °C to 5 °C at 1 °C/min (Figure 2). The data for temperatures up to 98 °C were obtained origi- nally, which is very close to the boiling point of water. Since some samples contained noise in the temperature range higher than 95 °C, DMA data up to 95 °C, which could be stably measured, was analyzed to compare the results. During the viscoelastic measurement, the span was 10 mm in radial direction (Figure 1) and a 0.1 Hz sine wave with a tension displacement amplitude of 5 µm was applied to the sample. In the pre- liminary experiments conducted on several wood samples, the offset loads in the measured temperature range, when the displacement amplitude was controlled at 5 µm, were within the linear region of the stress-strain line. DMA measurement was performed one time for each sample because the preliminary experiments confirmed that the same temperature program would produce nearly identical measurement results. 2.3 Estimation of the syringyl ratio No uniform trend was observed among the wood species and no correlation was observed between the peak temperature and the value of tan δ in Figure 4. The peak temperature of tan δ is generally lower for hardwood and higher for softwood, as reported in previous studies (Furuta et al 2001 2008a b) In hardwood species the peak tem Figure 2: Sample preparation procedures and temperature program for dynamic viscoelastic measurement. Figure 3: Examples of DMA data for untreated and extracted samples. Figure 2: Sample preparation procedures and temperature program for dynamic viscoelastic measurement. Figure 2: Sample preparation procedures and temperature program for dynamic viscoelastic measurement. Figure 2: Sample preparation procedures and temperature program for dynamic viscoelastic measurement. the peak temperatures of tan δ were read for samples for which the peak was observed in the range of 95 °C. Samples for which no peak was observed at 95 °C are indicated as 95+ in Table 1, and the legend is marked in red in the figure. The tan δ values for the 95+ samples were read at 95 °C and indicated in Table 1. Figure 4 shows the relationship between the peak temperature of and the value of tan δ. The peak temperatures of tan δ were slightly different between the extracted and unextracted samples (Figure 4a and b, respectively). Six softwood samples were identified in which the extraction process changed the peak temperature to 95 ° C or higher. Table 1 shows that the peak temperature of tan δ in softwoods changed to higher temperatures after extrac- tion. However, some hardwood samples showed a decrease in the peak temperature of tan δ due to extraction. Although the thermal-softening temperatures changed by a maximum of 6 °C due to extraction, the temperature range and the direction of increase or decrease varied among individual samples. No uniform trend was observed among the wood species and no correlation was observed between the peak temperature and the value of tan δ in Figure 4. The peak temperature of tan δ is generally lower for hardwood and higher for softwood, as reported in previous studies (Furuta et al. 2001, 2008a, b). In hardwood species, the peak tem- perature of tan δ in the radial-, ring-, and non-porous sam- ples was in a temperature range from 70 to 80 °C; however, the temperature range was wider for diffuse-porous sam- ples. 2.3 Estimation of the syringyl ratio The two diffuse-porous samples (both were Ulin (Eusi- deroxylon zwageri), which is a tropical hardwood) showed higher tan δ values than the others. Table 1 lists the peak temperature of tan δ for unextracted specimens, showing some variation within the same wood species. Figure 3: Examples of DMA data for untreated and extracted samples. Figure 3: Examples of DMA data for untreated and extracted samples. specimens from their IR spectra. The peak areas were measured three times for each sample, and after confirming that there were no outliers, area ratios were calculated from the data that showed the clearest spectrum with the least noise. 2.3 Estimation of the syringyl ratio The extracted DMA samples, which were used for the dynamic visco- elastic measurements, were analyzed using an IR spectrometer (FT/ IR-4700, JASCO Corporation, Tokyo, Japan). Fine wood meal was pre- pared from the middle length portion of dried DMA samples, using a glass file, mixed at 1 % with KBr and pressed into a disc. Each spectrum was based on 64 scans with a wavenumber range from 4000 to 500 cm−1. Following Huang et al. (2012), the areas under the IR peak at 1595 cm−1 (range: 1605–1574 cm−1, A1595) and 1509 cm−1 (range: 1535–1492 cm−1, A1509) were measured. Huang et al. (2012) presented a linear regression equation with a correlation coefficient of 0.98 to describe the relation- ship between the syringyl ratio (syringyl/(syringyl+guaiacyl)) of lignin aromatics, measured by alkaline nitrobenzene oxidation analysis, and log (peak area ratio of A1595/A1509). In the present study, the same regression equation was used to determine the syringyl ratio of all the Table : (continued) 116 Y. Miyoshi et al.: Thermal-softening properties of water-swollen wood of various species 116 specimens from their IR spectra. The peak areas were measured three times for each sample, and after confirming that there were no outliers, area ratios were calculated from the data that showed the clearest spectrum with the least noise. the peak temperatures of tan δ were read for samples for which the peak was observed in the range of 95 °C. Samples for which no peak was observed at 95 °C are indicated as 95+ in Table 1, and the legend is marked in red in the figure. The tan δ values for the 95+ samples were read at 95 °C and indicated in Table 1. Figure 4 shows the relationship between the peak temperature of and the value of tan δ. The peak temperatures of tan δ were slightly different between the extracted and unextracted samples (Figure 4a and b, respectively). Six softwood samples were identified in which the extraction process changed the peak temperature to 95 ° C or higher. Table 1 shows that the peak temperature of tan δ in softwoods changed to higher temperatures after extrac- tion. However, some hardwood samples showed a decrease in the peak temperature of tan δ due to extraction. Although the thermal-softening temperatures changed by a maximum of 6 °C due to extraction, the temperature range and the direction of increase or decrease varied among individual samples. However, this study suggests that the latitude does not necessarily correspond to the thermal-softening temperature among various wood species. Previous studies have reported that among hardwood species, tropical species show higher thermal-softening temperatures (Furuta et al. 2010) and are rich in guaiacyl units (Wu et al. 1992). If both the physical properties and chemical structures observed in tropical wood species are determined by their habitat, the regional climate may exert an influence on the thermal-softening temperature. To confirm this possibility, the relationship between the peak temperature of tan δ in untreated specimens and the latitude and annual rainfall at the collection site is plotted in Figure 5. annual rainfall and peak temperature of tan δ (Figure 5b). Among the diffuse-porous specimens, there was only a weak positive correlation between the two parameters. However, this correlation could be enhanced in certain tropical diffuse- porous wood, because many areas with high rainfall are located near the equator (see the clustering of diffuse-porous specimens near 0° latitude in Figure 5a). Overall, neither the latitude nor annual precipitation were related to the thermal- softening temperatures of wood in this study. The softwood specimens showed high peak tempera- tures of tan δ, regardless of latitude. Among the hardwood specimens, the radial-porous and diffuse-porous wood cover a wide latitude range. Certain diffuse-porous speci- mens collected from near the equator exhibited high peak temperatures compared to softwood. This distribution of the peak temperature of tan δ was similar to the high peak temperatures of tan δ previously observed in tropical hardwood species (Furuta et al. 2001, 2008a, b). However, this study suggests that the latitude does not necessarily correspond to the thermal-softening temperature among various wood species. Figure 5: Relationship between habitat and peak temperature of tan δ of water-saturated, untreated radial specimens measured at 0.1 Hz. The symbol for hardwood species in- dicates the vessel arrangement type. “Non” = vesselless. Samples collected from tropical climates are presented in green. Samples for which the peak temperature of tan δ could not be observed by 95 °C are presented in red. Figure 4: Relationship between loss tangent (tan δ) and peak temperature of tan δ in water- saturated radial samples measured at 0.1 Hz. (a) Untreated samples and (b) extracted sam- ples. The symbol for hardwood species in- dicates the vessel arrangement type. “Non” = vesselless. Samples collected from tropical climates are presented in green. Samples for which the peak temperature of tan δ could not be observed by 95 °C are presented in red. Figure 4: Relationship between loss tangent (tan δ) and peak temperature of tan δ in water- saturated radial samples measured at 0.1 Hz. (a) Untreated samples and (b) extracted sam- ples. The symbol for hardwood species in- dicates the vessel arrangement type. “Non” = vesselless. Samples collected from tropical climates are presented in green. Samples for which the peak temperature of tan δ could not be observed by 95 °C are presented in red. Figure 4: Relationship between loss tangent (tan δ) and peak temperature of tan δ in water- saturated radial samples measured at 0.1 Hz. (a) Untreated samples and (b) extracted sam- ples. The symbol for hardwood species in- dicates the vessel arrangement type. “Non” = vesselless. Samples collected from tropical climates are presented in green. Samples for which the peak temperature of tan δ could not be observed by 95 °C are presented in red. Figure 4: Relationship between loss tangent (tan δ) and peak temperature of tan δ in water- saturated radial samples measured at 0.1 Hz. (a) Untreated samples and (b) extracted sam- ples. The symbol for hardwood species in- dicates the vessel arrangement type. “Non” = vesselless. Samples collected from tropical climates are presented in green. Samples for which the peak temperature of tan δ could not be observed by 95 °C are presented in red. Previous studies have reported that among hardwood species, tropical species show higher thermal-softening temperatures (Furuta et al. 2010) and are rich in guaiacyl units (Wu et al. 1992). If both the physical properties and chemical structures observed in tropical wood species are determined by their habitat, the regional climate may exert an influence on the thermal-softening temperature. To confirm this possibility, the relationship between the peak temperature of tan δ in untreated specimens and the latitude and annual rainfall at the collection site is plotted in Figure 5. The softwood specimens showed high peak tempera- tures of tan δ, regardless of latitude. Among the hardwood specimens, the radial-porous and diffuse-porous wood cover a wide latitude range. Certain diffuse-porous speci- mens collected from near the equator exhibited high peak temperatures compared to softwood. This distribution of the peak temperature of tan δ was similar to the high peak temperatures of tan δ previously observed in tropical hardwood species (Furuta et al. 2001, 2008a, b). 3.1 Relationship between thermal-softening temperature and the growing environment of the wood In this study, the peak temperature of tan δ is defined as the thermal-softening temperature of the sample. In this paper, Y. Miyoshi et al.: Thermal-softening properties of water-swollen wood of various species 117 (2005) investigated the relationship between the partial structure of lignin and the syringyl ratio of lignin aromatics in various wood species and found that the biphenyl structure content decreased quadratically with an increase in the syringyl ratio. Based on previous research and this study’s results, it can be concluded that woodspecies witha lowsyringyl ratiohave a dense lignin structure, withseveralcondensed structures derivedfrom the guaiacyl unit, whereas wood species with a high syringyl ratio have a sparse lignin structure, with several linear partial structures. responsible for its branch chain. The composition ratio of the partial structures changes according to the aromatic nucleus during lignin production. The main reason for this is that the aromatic nuclei of guaiacyl can be coupled at the 5-position to form fused structures, such as β-5, 5-5, and 4-O-5, whereas the aromatic nuclei of syringyl cannot be coupled alone at the 5-position due to the methoxy group, which acts as the sub- stitute (Stewart et al. 2009). Akiyama et al. (2005) investigated the relationship between the partial structure of lignin and the syringyl ratio of lignin aromatics in various wood species and found that the biphenyl structure content decreased quadratically with an increase in the syringyl ratio. Based on previous research and this study’s results, it can be concluded that woodspecies witha lowsyringyl ratiohave a dense lignin structure, withseveralcondensed structures derivedfrom the guaiacyl unit, whereas wood species with a high syringyl ratio have a sparse lignin structure, with several linear partial structures. 118 Figure 6: Relationship between data from dynamic viscoelastic measurements at 0.1 Hz and the density of extracted radial specimens in water-saturated conditions. (a) Storage elastic modulus (E′) at 30 °C, (b) loss elastic modulus (E″) at 30 °C, and (c) peak tempera- tures of tan δ. The symbol for hardwood spe- cies indicates the vessel arrangement type. “Non” = vesselless. Samples collected from tropical climates are presented in green. Samples for which the peak temperature of tan δ could not be observed by 95 °C are pre- sented in red. Figure 6: Relationship between data from dynamic viscoelastic measurements at 0.1 Hz and the density of extracted radial specimens in water-saturated conditions. (a) Storage elastic modulus (E′) at 30 °C, (b) loss elastic modulus (E″) at 30 °C, and (c) peak tempera- tures of tan δ. The symbol for hardwood spe- cies indicates the vessel arrangement type. “Non” = vesselless. Samples collected from tropical climates are presented in green. Samples for which the peak temperature of tan δ could not be observed by 95 °C are pre- sented in red. at slightly lower locations and did not display a clear pos- itive correlation with density. The relationship between E″ and density showed a positive correlation when the density was 1000 kg/m3 or less. The distribution patterns of E″ and E′ were nearly the same. Figure 6a and b presents three Ulin specimens with the highest densities (1000 kg/m3 or more). Their E′ and E″ values deviate from the linear distribution of the other specimens. While E′ and E″ show a positive correlation with wood density, the peak temperature of tan δ shows no such relationship (Figure 6c). Thus, the wood species exhibited a wide range of thermal-softening temperatures, regardless of their density. responsible for its branch chain. The composition ratio of the partial structures changes according to the aromatic nucleus during lignin production. The main reason for this is that the aromatic nuclei of guaiacyl can be coupled at the 5-position to form fused structures, such as β-5, 5-5, and 4-O-5, whereas the aromatic nuclei of syringyl cannot be coupled alone at the 5-position due to the methoxy group, which acts as the sub- stitute (Stewart et al. 2009). Akiyama et al. 3.2 Relationship between thermal-softening property and density In this section, the thermal-softening properties of extracted samples are presented to discuss the relationship between the wood cell wall constituents and the thermal-softening properties. Figure 6a–c plot the storage elastic modulus (E′) at 30 °C, loss elastic modulus (E″) at 30 °C, and peak tem- peratures of tan δ versus the wood density, respectively. A positive correlation was observed between E′ and density in softwood, ring-porous hardwood, non-porous hardwood, and diffuse-porous hardwood, all following the same linear relationship. However, radial-porous wood was distributed Specifically, in softwood and hardwood except diffuse- porous wood, no relationship was observed between the Figure 5: Relationship between habitat and peak temperature of tan δ of water-saturated, untreated radial specimens measured at 0.1 Hz. The symbol for hardwood species in- dicates the vessel arrangement type. “Non” = vesselless. Samples collected from tropical climates are presented in green. Samples for which the peak temperature of tan δ could not be observed by 95 °C are presented in red. Figure 5: Relationship between habitat and peak temperature of tan δ of water-saturated, untreated radial specimens measured at 0.1 Hz. The symbol for hardwood species in- dicates the vessel arrangement type. “Non” = vesselless. Samples collected from tropical climates are presented in green. Samples for which the peak temperature of tan δ could not be observed by 95 °C are presented in red. Figure 5: Relationship between habitat and peak temperature of tan δ of water-saturated, untreated radial specimens measured at 0.1 Hz. The symbol for hardwood species in- dicates the vessel arrangement type. “Non” = vesselless. Samples collected from tropical climates are presented in green. Samples for which the peak temperature of tan δ could not be observed by 95 °C are presented in red. 118 Y. Miyoshi et al.: Thermal-softening properties of water-swollen wood of various species 3.3 Relationship between thermal-softening property and lignin structure To investigate the relationship between thermal-softening temperature and lignin structure, the syringyl ratio of lignin aromatics was estimated from the peak area ratio of the IR spectrum. Since the sample used for the IR measurement had undergone a heating history during DMA measure- ment, there was a possibility that the syringyl ratio would be slightly different from that of the untreated sample. However, this experiment prioritized the comparison of the results with the same sample. In Figure 7, the syringyl ratio is distributed in a wide range from 0 to 0.8 (approximately), and almost 0 in soft- wood and tropical hardwood species. Since softwood has no syringyl aromatic nuclei, the syringyl ratio should be exactly 0. However, the syringyl ratio was estimated using a calibration curve with an error of about ±0.2. Tropical hardwood samples showed a wide range of syringyl ratios, indicating that these samples are not necessarily rich in the guaiacyl unit. Nevertheless, several samples showed particularly low syringyl ratios. Hardwoods with syringyl ratios near 0 possessed a high peak temperature of tan δ compared to softwoods. Thus, the syringyl ratio and peak temperature of tan δ display a negative correlation. Based Understanding the partial structure of lignin is useful for inferring its structural characteristics. The β-O-4 and phenyl- coumarin structures are the specific partial structures responsible for lignin’s linear chain, while the biphenyl (5-5 bond) and diaryl ether structures (4-O-5 structure) are Y. Miyoshi et al.: Thermal-softening properties of water-swollen wood of various species 119 Figure 7: Relationship between peak temperature of tan δ measured at 0.1 Hz and syringyl ratio of extracted radial specimens. The symbol for hardwood species indicates the vessel arrangement type. “Non” = vesselless. Samples collected from tropical climates are presented in green. Samples for which the peak temperature of tan δ could not be observed by 95 °C are presented in red. peak temperature of tan δ and the latitude and annual rainfall of the habitat. Furthermore, the peak temperature of tan δ was not uniquely determined by anatomical fea- tures and wood species. E′ and E″ showed strong correla- tions with density, regardless of species; however, there was no relationship between the peak temperature of tan δ and density. Furthermore, a positive correlation was observed between the peak temperature of tan δ and the syringyl ratio, which depends on the amounts of different partial structures in lignin. References Akiyama, T., Goto, H., Nawai, D.S., Syafii, W., Matsumoto, Y., and Meshitsuka, G. (2005). Erythro/threo ratio of β-O-4-5 structures as an important structural characteristic of lignin. Part 4: variation in the erythro/threo ratio in softwood and hardwood lignins and its relation to syringyl/guaiacyl ratio. Holzforschung 59: 276–281. Becker, H. and Noack, D. (1968). Studies on dynamic torsional viscoelasticity of wood. Wood Sci. Technol. 2: 213–230. 3.3 Relationship between thermal-softening property and lignin structure This result indicates that the thermal-softening temperature is higher for wood species with a denser lignin structure (low syringyl ratio) and lower for species with a sparse lignin structure (high syringyl ratio). Acknowledgments: The authors express their appreciation of Dr. Takami Saito (Forestry and Forest Product Research Institute, Japan) and Dr. TakuyaAkiyama (University of Tokyo, Japan) for providing helpful literature and suggestions. Figure 7: Relationship between peak temperature of tan δ measured at 0.1 Hz and syringyl ratio of extracted radial specimens. The symbol for hardwood species indicates the vessel arrangement type. “Non” = vesselless. Samples collected from tropical climates are presented in green. Samples for which the peak temperature of tan δ could not be observed by 95 °C are presented in red. Figure 7: Relationship between peak temperature of tan δ measured at 0.1 Hz and syringyl ratio of extracted radial specimens. The symbol for hardwood species indicates the vessel arrangement type. “Non” = vesselless. Samples collected from tropical climates are presented in green. Samples for which the peak temperature of tan δ could not be observed by 95 °C are presented in red. Author contributions: YM conducted the experiments and wrote the manuscript, HA selected and provided the wood specimens from the wood library of the Forestry and Forest Products Research Institute, HH and KK participated in result discussions, and YF supervised the work. All the authors approved the final version of the manuscript. on the interpretation of the partial structure of lignin, the peak temperature of tan δ is considered to be higher for wood species with denser lignin structures and lower for those with sparser lignin structures. It is known that in polymers, the glass transition temperature (Tg) shifts to higher values as the crosslink density increases (Nielsen 1962). In wood, the peak temperature of tan δ, derived from lignin, could similarly depend on the lignin’s condensation structure in each wood species. Although this study’s results were obtained by using the calibration curves of IR spectra, they supported the correlation shown by Olson and Salmén (1997) between thermal-softening temperature and methoxyl group content. Competing interests: The authors states no conflict of interest. Research funding: This study was supported by the JSPS KAKENHI (grant number: JP20K15570). Data availability: The raw data can be obtained on request from the corresponding author. 4 Conclusions Furuta, Y., Soma N., Obata, Y., and Kanayama, K. (2001). Research to make better use of wood as sustainable resource – physical property change of wood to heating and drying histories. In: Proceedings of the fourth international conference on materials for resources 2001 Akita (ICMR 2001), October 11-13, 2001. SMERJ, Akita, pp. 260–265. To elucidate the diversity of the thermal-softening prop- erties of wood, the peak temperature of tan δ was measured for 87 wood specimens from the global collection of the xylarium of Forestry and Forest Products Research Insti- tute. The peak temperature of tan δ was found to be higher for softwood and tropical hardwood species as compared to other hardwood species, regardless of the extraction treatment. There was no clear relationship between the Furuta, Y., Kojiro, J., Nakatani,T., Nakajima, M., and Ishimaru, Y. (2008a). The dynamic viscoelastic properties of wood in nonequilibrium states. J. Soc. Mater. Sci. Jpn. 57: 338–343. Furuta, Y., Nakajima, M., Nakatani, T., Kojiro, K., and Ishimaru, Y. (2008b). Effects of the lignin on the thermal-softening properties of the water- swollen wood. J. Soc. Mater. Sci. Jpn. 57: 344–349. 120 Y. Miyoshi et al.: Thermal-softening properties of water-swollen wood of various species 120 Nielsen, L.E. (1962). Mechanical properties of polymers. Reinhold Publishing Corp., New York. Nielsen, L.E. (1962). Mechanical properties of polymers. Reinhold Publishing Corp., New York. Furuta, Y., Nakajima, M., Nakanii, E., and Ohkoshi, M. (2010). The effects of lignin and hemicellulose on thermal-softening properties of water- swollen wood. Mokuzai Gakkaishi 56: 132–138. Olsson, A.M. and Salmén, L. (1997). The effect of lignin composition on the viscoelastic properties of wood. Nord. Pulp Paper Res. J. 12: 140–144. Furuta, Y., Okuyama, T., Kojiro, K., Miyoshi, Y., and Kiryu, T. (2014). Temperature dependence of the dynamic viscoelasticity of bases of Japanese cypress branches and the trunk close to the branches saturated with water. J. Wood Sci. 60: 249–254. Placet, V., Passard, J., and Perré, P. (2007). Viscoelastic properties of green wood across the grain measured by harmonic tests in the range 0–95 °C: hardwood vs. softwood and normal wood vs. reaction wood. Holzforschung 61: 548–557. Huang, Y., Wang, L., Chao, Y., Nawai, D.S., Akiyama, T., Yokoyama, T., and Matsumoto, Y. (2012). Analysis of lignin aromatic structure in wood based on the IR spectrum. J. Wood Chem. Technol. 32: 294–303. Salmén, L. (1984). Viscoelastic properties of in situ lignin under water- saturated conditions. J. Mater. Sci. 19: 3090–3096. Salmén, L. (1984). Viscoelastic properties of in situ lignin under water- saturated conditions. J. Mater. Sci. 19: 3090–3096. Stewart, J.J., Akiyama, T., Chapple, C., Ralph, J., and Mansfield, S.D. (2009). The effects on lignin structure of overexpression of ferulate 5-hydroxylase in hybrid poplar. Plant Physiol. 150: 621–635. Kojiro, K., Furuta, Y., and Ishimaru, Y. (2008). Influence of histories on dynamic viscoelastic properties and dimensions of water-swollen wood. J. Wood Sci. 54: 95–99. Stewart, J.J., Akiyama, T., Chapple, C., Ralph, J., and Mansfield, S.D. (2009). The effects on lignin structure of overexpression of ferulate 5-hydroxylase in hybrid poplar. Plant Physiol. 150: 621–635. Kottek, M., Griester, J., Beck, C., Rudolf, B., and Rubel, F. (2006). World map of the Köppen-Geiger climate classification updated. Meteorol. Zeitschrift 15: 259–263. Wu, J., Fukazawa, K., and Ohtani, J. (1992). Distribution of syringyl and guaiacyl lignins in hardwoods in relation to habitat and porosity form in wood. Holzforschung 46: 181–185.
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Identification of risk areas and practices for Taenia saginata taeniosis/cysticercosis in Ethiopia: a systematic review and meta-analysis
Parasites & vectors
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Identification of risk areas and practices for Taenia saginata taeniosis/cysticercosis in Ethiopia: a systematic review and meta‑analysis Edilu Jorga1, Inge Van Damme2, Bizunesh Mideksa1 and Sarah Gabriël2* © The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat​iveco​ mmons​.org/licen​ses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creat​iveco​mmons​.org/publi​cdoma​in/ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Abstract Background:  Bovine cysticercosis (BCC) is an infection of cattle with the metacestode stage of Taenia saginata, the beef tapeworm, which causes taeniosis in humans. BCC is responsible for considerable economic losses in the meat sector worldwide. This systematic review and meta-analysis summarizes the prevalence, risk factors and treatment efforts made so far on T. saginata infections in Ethiopia, providing a detailed analysis of different factors influencing the varying prevalence estimates in Ethiopia to gain more insight into the occurrence and risk factors of T. saginata taeniosis and cysticercosis to date. Methods:  A systematic review and meta-analysis was conducted on data collected from published and grey litera- ture accessed through an electronic database and manual search. Results:  The literature search resulted in 776 outputs of which 132 conformed to the predefined criteria. The average zonal prevalence of meat inspection-based BCC ranged from 2% in Buno-Bedele to 24.6% in Sidama zone. The pooled prevalence of BCC was influenced by the number of muscle/organs inspected, ranging from 3.4% (95% CI: 1.7–5.1%) using fewer predilection sites to 19.4% (95% CI: 13.3–25.4%) using inspection of a maximum number of predilec- tion sites. None of the tested variables were significantly associated with BCC. Questionnaire-based taeniosis ranged between 19.0% in Halaba special woreda to 70.0% in Gedeo zone and stool test-based taeniosis varied from 0.6% in central Tigray to 10.7% in Gurage zone. Questionnaire-based prevalence of taeniosis was higher in people with a frequent raw beef consumption habit (pooled OR, pOR: 10.5, 95% CI: 6.0–17.9), adults (pOR: 2.5, 95% CI: 1.7–3.6), men (pOR: 2.8, 95% CI: 2.1–3.6), and Christians (pOR: 2.0, 95% CI: 1.4–2.8) compared to less frequent raw beef consumers, younger people, women and Muslims, respectively. Conclusions:  This review revealed a widespread but variable occurrence of BCC and taeniosis in Ethiopian regions and zones, urging for harmonized and enhanced detection for improved control of the parasite. Accurate prevalence estimates using more sensitive tests, detailed risk factor analysis, as well as data on financial losses are needed to develop effective control strategies for the Ethiopian epidemiologic condition. Abstract Keywords:  Bovine cysticercosis, Ethiopia, Prevalence, Review, Risk factors, Taeniosis, Taenia saginata *Correspondence: sarah.gabriel@ugent.be *Correspondence: sarah.gabriel@ugent.be 2 Department of Veterinary Public Health, Laboratory of Foodborne Parasitic Zoonoses, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium Full list of author information is available at the end of the article *Correspondence: sarah.gabriel@ugent.be 2 Department of Veterinary Public Health, Laboratory of Foodborne Parasitic Zoonoses, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium Full list of author information is available at the end of the article Parasites & Vectors Parasites & Vectors Jorga et al. Parasites Vectors (2020) 13:375 https://doi.org/10.1186/s13071-020-04222-y *Correspondence: sarah.gabriel@ugent.be 2 Department of Veterinary Public Health, Laboratory of Foodborne Parasitic Zoonoses, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium Full list of author information is available at the end of the article Background Bovine cysticercosis (BCC) is an infection of cattle with the metacestode stage of the tapeworm Taenia saginata [1]. Despite its global distribution, the highest numbers of tapeworm carriers are observed within communities in developing countries. However, due to the limited public health impact of taeniosis, lack of data on the economic impact of BCC and taeniosis, the existence of other pri- ority diseases and limited resources, T. saginata taenio- sis/cysticercosis remains a neglected zoonosis [2, 3]. The definitive host (human) becomes infected with T. saginata by ingestion of viable cysticerci in raw or under- cooked beef. In the intestine, the adult worm stage meas- ures 4–12 meters in length and individuals may remain infected for several years. About 6–9 proglottids are shed daily, either on defecation or by active migration. Each proglottid contains 50,000–80,000 eggs [4], and up to 720,000 T. saginata eggs can be released daily into the environment by a single infected human. The life-cycle is maintained when infected people contaminate the envi- ronment/animal directly (as a result of open defecation, active migration of the proglottids into the environment (including feed) or unhygienic practices leading to con- tamination/infection via hands), or indirectly via urban sewage effluent [5]. fl Following ingestion of the eggs with contaminated feed, fodder or water by the intermediate host (cattle), the oncosphere penetrates the intestinal wall to reach the skeletal and cardiac muscles and other tissues, where they develop into cysticerci (BCC) and become infective to humans after 10 weeks [4]. The cysticerci in the stri- ated muscles start to degenerate and calcify within a few months following infection, and after 9 months the num- ber of viable cysticerci is reduced substantially [3]. Over the last decades, several studies on T. saginata cysticercosis and taeniosis have been conducted in differ- ent regions in Ethiopia, applying different sampling and often poor diagnostic methodologies, resulting in varying outcomes. Prevalence estimates based on meat inspec- tion have been reported between 1.2% [20] and 32.2% [21] and prevalence estimates of questionnaire-based human taeniosis have been described between 19.0% [22] and 82.6% [23]. Recently Dermauw et  al. [24] reviewed the distribution of T. saginata taeniosis/cysticercosis in Eastern and Southern Africa with an inter-country con- text. Hiko and Seifu [25] also gave an overview of differ- ent reports in Ethiopia. © The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat​iveco​ mmons​.org/licen​ses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creat​iveco​mmons​.org/publi​cdoma​in/ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Jorga et al. Parasites Vectors (2020) 13:375 Page 2 of 17 Page 2 of 17 immunosorbent assays (ELISAs) detecting either specific antibodies or circulating antigens have been developed for BCC [10, 11]. While Ogunremi and Benjamin [11] estimated the sensitivity and specificity of their antibody detecting ELISA (based on excretory secretory antigen) at 92.9% and 90.6%, respectively, this could not be con- firmed in a later study, where a sensitivity of 13.8% and a specificity of 92.9% was estimated in animals with low levels of infection [8]. The genus-specific antigen detection ELISA has a high sensitivity (98.7%) for cattle infected with more than 50 viable cysticerci, but the sen- sitivity reduces significantly when less than 50 cysticerci are present (12.8%) [8, 9]. For the detection of human tae- niosis cases, the routinely used coprological techniques are also known to have low sensitivities [12] and lack species-specificity. Several copro-Ag ELISAs have been developed to detect Taenia spp. antigens in human stool samples [13–15]. Although these tests were originally developed for the detection of T. solium, most are genus- specific and can thus be used for T. saginata taeniosis as well, when further species identification by molecular tools is conducted. Copro PCR assays for direct detection in stool samples have been developed [16, 17], and dif- ferent PCR-based tests are available for the identification of proglottids or suspected cysticerci to Taenia species level, including PCR-restricted fragment length polymor- phism (RFLP) and multiplex PCR [16, 18, 19]. However, these ELISAs and PCR assays are not commercially avail- able and are not routinely used. Background Human taeniosis is associated with minor abdomi- nal discomfort, nausea, mild diarrhoea, weight loss, and anal pruritus, though serious digestive disorders such as intestinal blockage or perforation and peritonitis have been reported [6, 7]. The clinical effect of BCC is gener- ally insignificant in natural infections, but it accounts for considerable economic losses to the food industry due to condemnation, freezing and downgrading of infected carcasses [5]. In this systematic review, rather than a descriptive pres- entation of occurrence, a more detailed analysis of differ- ent factors influencing the varying prevalence estimates in Ethiopia was undertaken to gain more insight into the occurrence of T. saginata taeniosis and cysticercosis to date. With regard to the variation among the prevalence reports, different factors, such as the study set-up, study population, sample size, host and environmental factors, and the diagnostic strategy may all affect prevalence esti- mates. Therefore, the aim of this systematic review and Diagnosis and control of BCC is primarily based on meat inspection, which involves inspection for cystic lesions using palpation and incision of defined muscles, although the adopted inspection techniques and the final judgments vary greatly throughout the world [3]. Routine meat inspection generally has a low sensitivity (< 15%), especially so for low levels of infection as esti- mated recently in Belgium (0.76%) [8, 9]. As an alterna- tive, immuno-diagnostic tools such as enzyme-linked Jorga et al. Parasites Vectors (2020) 13:375 Jorga et al. Parasites Vectors (2020) 13:375 Page 3 of 17 Page 3 of 17 Inclusion and exclusion criteria meta-analysis was to present a qualitative and quantita- tive summary on the prevalence, distribution and risk factors of T. saginata taeniosis/cysticercosis in Ethio- pia based on the existing literature. Additionally, data on carcass condemnation, drug inventory records and taenicidal herbs were summarized from the retrieved manuscripts. Literature reporting the prevalence and/or risk fac- tors of BCC and/or human taeniosis, taenicidal reports including relevant information on economic loss esti- mation from Ethiopia were all included. The results of the search were not restricted by year of publication (until 27 March 2019), study design, journal, or status of publication. Review papers, book chapters, letters to the editor and editorials without original data and articles whose full text was not available or with insuf- ficient information in the abstract were excluded. Study selection Search results were combined to manage duplicates. Then a selection was made based on the title and abstract screening, followed by a full text reviewing. Articles were organized and grouped into five different topics as follows: (i) prevalence of BCC; (ii) prevalence of taeniosis, based on stool tests and questionnaires; (iii) organ and carcass condemnation reports; (iv) drug inventory reports; and (v) taenicidal and herbal research reports. Search strategiesh The search strategy includes the following databases: PubMed, ScienceDirect and Web of Science and a fur- ther search was carried out using Google Scholar and Google as well. An advanced search was followed and the search terms and Boolean operation combination used were as follows: For PubMed: (“Bovine cysticercosis” OR “Cysticercus bovis” OR Cysticercosis OR “C. bovis” OR “Metacestode” OR “Taenia saginata” OR “T. saginata” OR “Taenia spp.” OR “Taenia species” OR “Taeniasis” OR “Taeniosis”) AND “Ethiopia”. For Web of Science: (“Ethio- pia” AND (“Bovine cysticercosis” OR “Cysticercus bovis” OR “Cysticercosis” OR “C. bovis” OR “Metacestode” OR “Taenia saginata” OR “T. saginata” OR “Taenia spp.” OR “Taenia species” OR “Taeniasis” OR “Taeniosis) and for ScienceDirect “Bovine cysticercosis” OR “Cysticercus bovis” OR “C. bovis” OR “Taenia saginata” OR “T. sagi- nata” OR “Taenia species” OR “Taenia spp.” OR “Taenio- sis” OR “Taeniasis” AND “Ethiopia”. For ScienceDirect, the search was restricted to the title, abstract or author- specified keywords. In addition, the references cited in the published manuscripts were cross-checked to capture any relevant reports that might have been missed in the electronic search process. Similarly, Google and Google Scholar were used to search articles published in local journals such as the Ethiopian Veterinary Journal/Pro- ceeding and Bulletin of Animal Health and Production in Africa and grey literature. Additional searches for grey literature such as MSc thesis reports, were done at the repositories of universities and research centres of Ethio- pia and by contacting the original authors. Systematic review protocolh The review question was as follows: ‘What are the preva- lence, distribution, and risk factors for BCC and taeniosis in Ethiopia and which taenicidal herbs are used in Ethio- pia?’. This research question enabled defining the inclu- sion criteria, developing the search strategy and data collection. The approach for the review protocol followed the principles of the PRISMA guidelines for systematic reviews [26] (Additional file 1: Table S1). An overview of the literature searches and selection process is shown in Fig. 1. Data extraction Data from selected articles were recorded in Excel sheets for each of the topics. The following informa- tion was recorded for studies regarding the preva- lence of BCC: author names and year of publication; journal name and volume; data period; study region; study subjects; study objective; study setup; diagnostic method used; number of study sites/abattoirs; sample size; number of positive animals, muscles and organs where cysts were detected; total cysts collected; and proportion of viable cysts. Similarly, for data regard- ing questionnaire- and stool test-based human taenio- sis, author names and year of publication, journal name and volume, data period, study region, study objective, study set-up, diagnostic method used, sample size, and number of positives were recorded. For the evaluation of risk factors, the sample size, the number of positives and negatives in each category and the cut-off value points for variables such as age were recorded. Some variables were categorised in different levels by the different authors (such as occupation and educational status) and were re-grouped for the meta-analysis. The total number of carcasses and organs condemned and condemnation due to BCC with the estimated cost was recorded. The reported adult taenicidal dosages sold and the estimated cost were also recorded. Lastly, information such as study region, study setup, study period, taenicidal plant species and anthelmintic drug type used were described. Jorga et al. Parasites Vectors (2020) 13:375 Page 4 of 17 Fig. 1  Flow chart describing the output of the literature search and selection of articles g the output of the literature search and selection of articles Fig. 1  Flow chart describing the output of the literature search and selection of articles the sanitary status and possibility of open defecation in the country was obtained from World Bank report [28].h Results i The averaged zonal prevalence of BCC was maximum in Sidama zone, SNNP (24.6%) and the lowest in Buno Bedele zone, western Ethiopia (2.0%) (Fig. 3a). The over- all pooled prevalence estimate of BCC in Ethiopia was 7.8% (95% CI: 6.63–9.05%). The calculated Cochran Chi-square value (Q) of 3783 (df = 55, P < 0.001) and the inverse variance index value ­(I2) of 98.5% indicates a high degree of heterogeneity among the reports. Since the methodology of meat inspection varied among the reports, subgroup analysis was performed depending on the number of organs/tissues that were inspected. The pooled prevalence estimate was 19.4% (95% CI: 13.27–25.45%, df = 7) for studies inspecting eight or more Data analyses Due to this variation, studies were categorized in four different groups: (i) studies that included at least the following 8 organs/muscles: shoul- der, heart, masseter, tongue, diaphragm, thigh muscle, liver and intercostal muscle; (ii) studies that included at least the following 6 organs/muscles: shoulder, masse- ter, tongue, heart, liver and diaphragm/lung or studies that inspected at least seven organs/tissues in total; (iii) studies that included at least 5 of the following organs/ muscles: shoulder, masseter, tongue, heart and liver or diaphragm/thigh or at least six organs in total; and (iv) studies that include at most 4 of the following organs/ muscles including: shoulder, masseter, tongue, heart or other organs/muscles. For stool test-based human tae- niosis, subgroup analysis was done for the different study populations, so studies were categorized in the follow- ing groups: (i) general community; (ii) food handlers; (iii) patients; and (iv) school children. Risk factor analysis was performed for BCC and questionnaire-based taenio- sis using random effect models to pool the effect sizes of the individual studies. For each variable considered, the pooled odds ratio (OR) and its 95% confidence interval (CI) were calculated. The heterogeneity between studies was assessed by Cochran’s Q test and the percentage of the variation in the estimates attributable to heterogene- ity was quantified by the inverse variance index ­(I2) [29]. Higher values of ­I2 signify a greater degree of variation. Prevalence of BCC Out of the 62 articles, 61 provided information on meat inspection-based prevalence of BCC, and the remain- ing one was a molecular study. Ethiopia has 9 regions and two chartered cities (Addis Ababa and Dire Dawa) and BCC reports were identified from 6 regions and the two chartered cities i.e. all except Gambela, Benishah- gul Gumuz and Afar regions (Fig. 2a). The majority of the reports were from Oromia region (central Ethio- pia), Amhara region and parts of the Southern Nations and Nationalities People region (SNNP). Within these regions, BCC was reported in 27 different zones. Reports were obtained from 46 abattoirs; 42 were municipal abat- toirs slaughtering ruminants for local consumption and 4 were export abattoirs. Twelve reports were from East Shoa zone of Oromia region. The maximum number of reports per abattoir was six for Gondar (prevalence esti- mates range of 2.0–18.0%), five for Addis Ababa (1.9– 7.5%) and Jimma (2.9–5.1%). One seroprevalence report (25.6%) was obtained from Addis Ababa abattoir using an indirect hemagglutination test (IHAT) [30]. One molec- ular study from eastern and central Ethiopia identified that 92.7% (38/41) of the cysticerci from bovine carcasses were T. saginata, whereas 7.3% (3/41) of them were sug- gested to be Taenia spp. from wildlife (T. hyaena) [31] (Additional file 2: Table S2). Data analyses Prevalence reports for BCC from the same zonal area were converted to a zonal prevalence. An average zonal prevalence was preferred over a country-level estimate to account for potential regional differences in the preva- lence. The synthesized results were presented by maps using ArcGIS 10.4 (ArcGIS Inc., New York, USA), to visualize the zonal and regional distribution of taeniosis/ cysticercosis in Ethiopia. In addition, data on zonal cat- tle population of Ethiopia obtained from CSA 2014/2015 was converted to a map [27]. Similarly, a map showing The location of cysticerci in carcasses/predilection sites was recorded as the proportion of positive organs relative to the total number of positive cattle and the percent- age of viable cysts relative to the total number of cysts collected. Meta-analysis was performed using STATA ver- sion 14.0 (StataCorp 4905 Lakeway Drive College Sta- tion, Texas 77845, USA). The study level prevalence was Jorga et al. Parasites Vectors (2020) 13:375 Page 5 of 17 transformed to logit event estimate and the correspond- ing variances were calculated. The random effect model was used to pool the logit event estimates and later the pooled logit estimates were back-transformed to preva- lence estimates i.e. the pooled prevalence. To evaluate the effect of the number of organs/muscles that are inspected on BCC prevalence, a pooled prevalence was estimated for different subgroups. In most Ethiopian abattoirs the routine meat inspection regulation is not strictly fol- lowed. As a result, there is variation in the number of muscles/organs that are inspected. The most frequently inspected muscles/organs are the shoulder muscle, heart masseter and tongue. Due to this variation, studies were categorized in four different groups: (i) studies that included at least the following 8 organs/muscles: shoul- der, heart, masseter, tongue, diaphragm, thigh muscle, liver and intercostal muscle; (ii) studies that included at least the following 6 organs/muscles: shoulder, masse- ter, tongue, heart, liver and diaphragm/lung or studies that inspected at least seven organs/tissues in total; (iii) studies that included at least 5 of the following organs/ muscles: shoulder, masseter, tongue, heart and liver or diaphragm/thigh or at least six organs in total; and (iv) studies that include at most 4 of the following organs/ muscles including: shoulder, masseter, tongue, heart or other organs/muscles. Data analyses For stool test-based human tae- niosis, subgroup analysis was done for the different study populations, so studies were categorized in the follow- ing groups: (i) general community; (ii) food handlers; (iii) patients; and (iv) school children. Risk factor analysis was performed for BCC and questionnaire-based taenio- sis using random effect models to pool the effect sizes of the individual studies. For each variable considered, the pooled odds ratio (OR) and its 95% confidence interval (CI) were calculated. The heterogeneity between studies was assessed by Cochran’s Q test and the percentage of the variation in the estimates attributable to heterogene- ity was quantified by the inverse variance index ­(I2) [29]. Higher values of ­I2 signify a greater degree of variation. questionnaire-based taeniosis (n = 33), organ and carcass condemnation (n = 13), drug inventory records (n = 14) and taenicidal studies (n = 13). Sixty-one papers were used for zonal prevalence averaging of BCC, of which 54 (two articles reporting from two different regions were considered as each time two studies; n = 56) were selected for meat inspection-based meta-analysis of BCC and 41 of them were included for at least one BCC risk factor analysis. Of the BCC prevalence studies, 33 reported questionnaire-based prevalence of taeniosis and 14 included drug inventory records. An overview of the different regions and zones in Ethiopia where BCC and/ or taeniosis was reported is shown in Fig. 2a. transformed to logit event estimate and the correspond- ing variances were calculated. The random effect model was used to pool the logit event estimates and later the pooled logit estimates were back-transformed to preva- lence estimates i.e. the pooled prevalence. To evaluate the effect of the number of organs/muscles that are inspected on BCC prevalence, a pooled prevalence was estimated for different subgroups. In most Ethiopian abattoirs the routine meat inspection regulation is not strictly fol- lowed. As a result, there is variation in the number of muscles/organs that are inspected. The most frequently inspected muscles/organs are the shoulder muscle, heart masseter and tongue. Literature search result A total of 776 articles written in English were obtained from all data sources (including eight from the additional search), of which 39 were duplicates and 574 excluded as irrelevant based on the criteria following the screening of titles and abstracts (Fig. 1). One hundred sixty-three were passed for full article reading, out of which 31 were excluded, two of which were duplicate data. One hun- dred and thirty-two papers met the predefined criteria to be included. They were grouped as BCC prevalence (n = 62), stool test-based prevalence of taeniosis (n = 44), Jorga et al. Parasites Vectors (2020) 13:375 Page 6 of 17 muscles/organs, whereas it was the 3.4% when 4 or less muscles/organs were inspected (95% CI: 1.71–5.09%, df = 14) (Fig. 4; [32–81]). Nevertheless, the high ­I2 values (> 85%) for each of the subgroups indicate a high degree of heterogeneity between studies applying a similar methodology. and masseter muscle (14.5%). The detection rate was lower in the remaining predilection sites and the low- est was recorded in the intestinal mucosa and hump (0.02%). The proportion of viable cysticerci among the cysticerci collected was 55.8% (3523/6309) (Additional file 2: Table S4). The location of the cysticerci in predilection sites was reported in 54 articles (Additional file 2: Table S3). The heart and tongue were the most frequently inspected organs and were excluded from meat inspection in only one report each. The masseter muscle, shoulder mus- cle, and liver were not included by 3, 8, and 14 stud- ies, respectively. The neck muscle and longissimus dorsi muscle were inspected by only 3 and 2 reports, respectively, and the internal organs (kidney, spleen, intestinal mucosa) were inspected in less than 10 reports. In the 6481 infected cattle, 9935 organs were found infected and cysticerci were mostly found in the tongue (24.2%), heart (23.2%), shoulder muscle (18.8%) Meta‑analysis for risk factors of BCC Forty-one articles reporting risk factor for BCC were included for meta-analysis of at least one variable. Sex, age, breed and body condition score (poor/medium or good) of the slaughtered cattle and altitude (highland or lowland) from where the animals were brought were the variables analysed. The summary of the relation- ship between the studied variables and BCC is shown in Fig. 5. The reports were heterogenic for all the vari- ables that were tested ­(I2 > 45%). However, the calcu- lated odds ratios showing the risk of exposure to BCC did not differ significantly for any of the variables that Fig. 2  Map of Ethiopia showing regions and zones where a bovine cysticercosis (BCC), taeniosis (questionnaire-based (QB)), and taeniosis (stool test-based (STB)) is reported. b Cattle population density in Ethiopia (expressed as the number of cattle per ­km2). c The open defecation status in Ethiopia (expressed as the proportion of people without a latrine). Copyright: Creative Commons Attribution license (CC BY 3.0 IGO). Citation: World Bank. 2018. Maintaining the Momentum while Addressing Service Quality and Equity: A Diagnostic of Water Supply, Sanitation, Hygiene, and Poverty in Ethiopia. WASH Poverty Diagnostic. World Bank, Washington, DC [28] Fig. 2  Map of Ethiopia showing regions and zones where a bovine cysticercosis (BCC), taeniosis (questionnaire-based (QB)), and taeniosis (stool test-based (STB)) is reported. b Cattle population density in Ethiopia (expressed as the number of cattle per ­km2). c The open defecation status in Ethiopia (expressed as the proportion of people without a latrine). Copyright: Creative Commons Attribution license (CC BY 3.0 IGO). Citation: World Bank. 2018. Maintaining the Momentum while Addressing Service Quality and Equity: A Diagnostic of Water Supply, Sanitation, Hygiene, and Poverty in Ethiopia. WASH Poverty Diagnostic. World Bank, Washington, DC [28] Fig. 2  Map of Ethiopia showing regions and zones where a bovine cysticercosis (BCC), taeniosis (questionnaire-based (QB)), and taeniosis (stool test-based (STB)) is reported. b Cattle population density in Ethiopia (expressed as the number of cattle per ­km2). c The open defecation status in Ethiopia (expressed as the proportion of people without a latrine). Copyright: Creative Commons Attribution license (CC BY 3.0 IGO). Citation: World Bank. 2018. Maintaining the Momentum while Addressing Service Quality and Equity: A Diagnostic of Water Supply, Sanitation, Hygiene, and Poverty in Ethiopia. WASH Poverty Diagnostic. World Bank, Washington, DC [28] Jorga et al. Meta‑analysis for risk factors of BCC Parasites Vectors (2020) 13:375 Page 7 of 17 heterogeneity among the reports (Additional file  3: Figure S1). were tested. The highest OR was obtained for cattle of medium/poor condition having a 1.46 (pooled OR: 1.46, 95% CI: 0.88–2.45, P = 0.145) higher odds for BCC infection as compared to animals with a good body condition. Forty-four articles were obtained reporting human intestinal parasitic infections based on stool tests. Only one report was exclusively about taeniosis [71], whereas the remaining papers reported taeniosis together with other intestinal parasites. The overall stool test-based pooled prevalence of taeniosis in Ethiopia was 1.9% (95% CI: 1.6–2.2%, df = 43). Eight of the reports were based on a sampling of the general community, whereas the remaining papers targeted specific groups, such as hospital patients, food handlers and school children. The formol-ether concentration technique was most frequently used for detection of Taenia eggs, Kato- Katz and Modified Ziehl Neelsen staining techniques were also used. The average zonal prevalence shows that taeniosis is more common in central Ethiopia and parts of the Southern region (Fig. 3c, Additional file 2: Table S6). Region-based grouping showed 4.1% pooled prevalence at Addis Ababa (95% CI: 2.6–5.6%) and 3.2% Prevalence of taeniosis Thirty-three of the bovine cysticercosis studies reported also a questionnaire-based prevalence of human taeniosis. In all the reports, ‘having seen the proglottids in the stool and underwear’ was consid- ered as a positive finding. The highest average zonal prevalence was 70.0% obtained at Yirgalem, SNNP region of Ethiopia (Fig. 3b, Additional file 2: Table S5). Reinfection of up to two to six times per year was also reported [32, 41]. The country-wide pooled preva- lence for questionnaire-based taeniosis was 52.3% (95% CI: 46.4–58.2%, df = 32), and the Q and ­I2 values (Q = 719.7 and ­I2 = 95.6%, P < 0.001) are indicative of Fig. 3  Map of Ethiopia displaying the zonal prevalence of a bovine cysticercosis (BCC), b taeniosis (questionnaire-based) and c taeniosis (stool test-based) Fig. 3  Map of Ethiopia displaying the zonal prevalence of a bovine cysticercosis (BCC), b taeniosis (questionnaire-based) and c taeniosis (stool test-based) Jorga et al. Parasites Vectors (2020) 13:375 Page 8 of 17 Fig. 4  Forest plot showing an overview of studies reporting bovine cysticercosis (BCC) in Ethiopia, grouped by the number of organs/muscles that were inspected during post-mortem meat inspection. The box size shows the weight of the study and the middle of the box (dot) represents the point estimate of the study, the length of the horizontal lines indicates the 95% CI; the vertical broken line indicates the pooled estimate; the diamond-shaped box at the bottom represents the 95% CI; the solid line indicates the point of null assumption vine cysticercosis (BCC) in Ethiopia, grouped by the number of organs/muscles box size shows the weight of the study and the middle of the box (dot) represents s indicates the 95% CI; the vertical broken line indicates the pooled estimate; the solid line indicates the point of null assumption Fig. 4  Forest plot showing an overview of studies reporting bovine cysticercosis (BCC) in Ethiopia, grouped by the number of organs/muscles that were inspected during post-mortem meat inspection. Meta‑analysis of risk factors for questionnaire‑based taeniosis a common practice to purchase taenicides from a phar- macy following the finding of proglottids in the stool (self-diagnosis). Among the 16 studied variables, only 7 were included for risk factor analysis for the questionnaire-based human taeniosis. The remaining variables were reported by less than four researchers, such as knowledge about T. sagi- nata, use of latrines, residential area, use of spices in raw beef, raw beef preparation, community type, income, meat source and drug use; hence no clear trends were obtained for these variables (data not shown). For edu- cational status and occupational status, seven and four reports respectively, were omitted from the meta-analysis due to either unclear cut-off values or too diverse cat- egories. Because of the latter reason, educational status was thus limited to literacy (illiterate versus literate) only. Gender, religion, raw beef consumption habit, age, occu- pational group, literacy, and marital status were included in the final analyses (Fig. 7). The Cochran Q and ­I2 val- ues showed that studies for all variables except occupa- tion and literacy were heterogeneous. All the variables except marital status showed a significant association with taeniosis infection. Hence, males (pOR: 2.76, 95% CI: 2.13–3.59; P < 0.001), Christians (pOR: 2.00, 95% CI: 1.44–2.77), raw beef consumers (pOR: 10.35, 95% CI: 6.05–17.87), and older age groups (pOR: 2.50, 95% CI: 1.72–3.63) had a higher odd of self-reported taeniosis than females, Muslims, less frequent raw beef consumers and younger age groups, respectively. Fourteen reports were obtained regarding pharmacy inventory, originating from nine zones of Ethiopia. The number of pharmacies that were included was not speci- fied in five of the reports and in all the reports infor- mation was obtained from voluntary pharmacies only. Niclosamide and mebendazole were the most frequently sold drugs, followed by albendazole and praziquantel. The maximum number of adult taenicidal doses sold was 472,013 doses over a 5-year period at Hawasa, southern Ethiopia [42], the highest estimated taenicidal cost was US$ 93,310 during four years from seven pharmacy shops at Wolaita Sodo, southern Ethiopia [43] (Additional file 2: Table S7).h Thirteen meat inspection-based studies reported organ and carcass condemnation from different abattoirs of the country. According to these reports, the tongue, heart and carcass were most frequently condemned due to BCC. About 37.5% of the total condemned tongues were reported due to BCC, followed by the heart (15.1%) and carcass (5.3%). Prevalence of taeniosis The box size shows the weight of the study and the middle of the box (dot) represents the point estimate of the study, the length of the horizontal lines indicates the 95% CI; the vertical broken line indicates the pooled estimate; the diamond-shaped box at the bottom represents the 95% CI; the solid line indicates the point of null assumption 1.8% in hospital patients (95% CI: 1.4–2.1%) and it was 1.3% in school children (95% CI: 0.6–2.0%) (Fig. 6; [82–125]). The reports within each of the subgroups of patients were highly heterogeneous ­(I2 > 90%). at SNNP (95% CI: 2.2–4.2%). Study population-based subgroup analysis showed that the pooled prevalence was 3.0% in food handlers (95% CI: 1.7–4.4%), followed by 2.4% the general community (95% CI: 1.8–3.0%), Jorga et al. Parasites Vectors (2020) 13:375 Page 9 of 17 Fig. 5  Overview of the meta-analyses results for different risk factors for bovine cysticercosis (BCC) in Ethiopia. Abbreviations: OR, pooled odds ratio; n, number of studies that are included in the analysis; ­I2, inverse variance index representing the percentage of the variation in the estimates attributable to heterogeneity; Q, Cochrans Q test value representing heterogeneities of the study level estimates Fig. 5  Overview of the meta-analyses results for different risk factors for bovine cysticercosis (BCC) in Ethiopia. Abbreviations: OR, pooled odds ratio; n, number of studies that are included in the analysis; ­I2, inverse variance index representing the percentage of the variation in the estimates attributable to heterogeneity; Q, Cochrans Q test value representing heterogeneities of the study level estimates Meta‑analysis of risk factors for questionnaire‑based taeniosis One study from the Tigray region, north- ern Ethiopia estimated €2402.4 loss from carcasses and organ condemnation due to BCC within six-month time from three abattoirs, where the prevalence of BCC was 8.3% (308/3711) [54] (Additional file 2: Table S8). The economic losses of Taenia saginata infection Thirteen articles were obtained reporting either herbal taenicides or anthelmintic efficacy study. The Ethiopian indigenous tree Hagenica abyssinica, locally named kosso has been used traditionally as a remedy against T. sagi- nata infection before it was discovered by Europeans in the early seventeenth century [126]. Several plants were reported to be used by Amhara, Shinahsa and Agew-Awi Little information is available on the costs related to T. saginata in Ethiopia. Taeniosis patients are unlikely to seek health care unless the clinical signs and symptoms are very serious. Taenia saginata infection might be diag- nosed while patients are visiting health institutions for other intestinal illnesses in which case they are treated based on the physician’s prescription. In most cases, it is Jorga et al. Parasites Vectors (2020) 13:375 Page 10 of 17 Fig. 6  Forest plot showing an overview of studies reporting stool test-based human taeniosis prevalence in Ethiopia, grouped per study population. The box size shows the weight of the study and the middle of the box (dot) represents the point estimate of the study, the length of the horizontal lines indicates the 95% CI; the vertical broken line indicates the pooled estimate; the diamond-shaped box at the bottom represents the 95% CI; the solid line indicates the point of null assumption Fig. 6  Forest plot showing an overview of studies reporting stool test-based human taeniosis prevalence in Ethiopia, grouped per study population The box size shows the weight of the study and the middle of the box (dot) represents the point estimate of the study the length of the Fig. 6  Forest plot showing an overview of studies reporting stool test-based human taeniosis prevalence in Ethiopia, grouped per study population. The box size shows the weight of the study and the middle of the box (dot) represents the point estimate of the study, the length of the horizontal lines indicates the 95% CI; the vertical broken line indicates the pooled estimate; the diamond-shaped box at the bottom represents the 95% CI; the solid line indicates the point of null assumption Fig. 6  Forest plot showing an overview of studies reporting stool test-based human taeniosis prevalence in Ethiopia, grouped per study population. The economic losses of Taenia saginata infection The box size shows the weight of the study and the middle of the box (dot) represents the point estimate of the study, the length of the horizontal lines indicates the 95% CI; the vertical broken line indicates the pooled estimate; the diamond-shaped box at the bottom represents the 95% CI; the solid line indicates the point of null assumption 74, 76]. However, only a few in  vitro trials focusing on potency and the toxicity margin of some of these medic- inal plants were done. For instance, the in vivo trial on mice with crude hydroalcoholic extract of E. schimperi at 1000 mg/kg dose showed 100% clearance of the parasite Hymenolepis nana [131]. Desta [132] ranked 10 com- monly used taenicidal herbs on the basis of lower toxic- ity, higher potency and shorter worm expulsion time. The top three were Echinops giganteus, Embelia schimperi, ethnic groups in north-western Ethiopia [127]; Meinit people [128] and Sheko people [129] in south-western Ethiopia. Some authors reported frequently used herbs in central Ethiopia [66, 77, 130]. Generally, Hagenia abyssinica, Cucurbita pepo, Embelia schimperi, Glinus lotoides and Myrsine africana were the most frequently reported taenicidal plants in Ethiopia. Surveys showed that about 10–15% of people living in urban areas are still using traditional remedies for taeniosis [51, 66, Jorga et al. Parasites Vectors (2020) 13:375 Page 11 of 17 Fig. 7  Overview of the random effects meta-analyses results for different risk factors for questionnaire based taeniosis in Ethiopia. Abbreviations: OR, pooled odds ratio; n, number of studies that are included in the analysis; ­I2, inverse variance index representing the percentage of the variation in the estimates attributable to heterogeneity; Q, Cochrans Q test value representing heterogeneities of the study level estimates Fig. 7  Overview of the random effects meta-analyses results for different risk factors for questionnaire based taeniosis in Ethiopia. Abbreviations: OR, pooled odds ratio; n, number of studies that are included in the analysis; ­I2, inverse variance index representing the percentage of the variation in the estimates attributable to heterogeneity; Q, Cochrans Q test value representing heterogeneities of the study level estimates Hagenia abyssinica, with their medical effective dose in gram and worm expulsion time in hours of 7.84 ± 1.04 and 10.2 ± 2.0; 8.23 ± 1.50 and 10.8 ± 1.0; and 12.5 ± 2.2 and 11.3 ± 1.4, respectively. Demma et al. The economic losses of Taenia saginata infection [133] studied the toxicity margin of Glinus lotoides seeds and found it is safe at repeated doses. Past studies in Ethiopia showed that praziquantel was effective against T. saginata at a single oral dose (10 mg/kg body weight), whereas clinical and parasitological cures were obtained by a regimen of 2 g of niclosamide given on three consecutive days [134, 135]. inspection procedure may strongly affect the reported prevalence, thus hampering direct comparison of studies using different methodologies. A more harmonized meat inspection is consequently highly recommended to allow a more thorough assessment of zonal BCC prevalence. g p Furthermore, in this review most reports were based on routine meat inspection; as a result, the limitations of meat inspection, highlighted recently in a Belgian study determining a sensitivity of 0.76% [9], should also be con- sidered in these studies. This could be due to the fact that the cysticerci in light infections might not be evident on routine inspection of the predilection sites [3, 137]. Also, in early infections, viable cysts are inconspicuous in the red meat due to their translucent nature and pinkish-red colour [138]. It also depends on the technical ability and motivation of the meat inspector, for instance, the 4.8% retrospective prevalence report was much lower than the 19.5% reported from a prospective abattoir survey from the same abattoir [42]. Moreover, the speed of the slaughtering activity, the lighting system in the Ethiopian abattoirs and other factors might have contributed to the variation of prevalence reported in Ethiopia [139]. Thus, prevalence reported so far using routine meat inspection in Ethiopia is highly probable an underestimation of the true prevalence. Discussion According to this review, the environmental and human characteristics are more responsible for the prevalence variation of BCC in Ethiopia than the animal- related factors. Generally, the higher prevalence of BCC and taeniosis in Ethiopia as opposed to industrialised countries is due to a number of factors such as the deep-rooted raw beef consumption habit mentioned above, but also backyard slaughter lacking meat inspection, poor sanitary infrastruc- ture (Fig. 2c) and improper disposal of sewage. Eggs of T. saginata can remain infective for up to 9 months in the soil [150], pasture or water [151]. In Ethiopia, improper appli- cation of sewage effluent from sewage treatment plants, or even direct disposal of sewage sludge on the fields could be risky for cattle grazing on contaminated pasture/water. The animal husbandry practice in Ethiopia is mostly extensive. Taeniosis patients in Ethiopia are not regularly treated and the lack of feedback from the slaughterhouse to the farm after detection of a case so that the people on the farm can be tested and treated might also contribute. According to the joint monitoring programme report (UNICEF/WHO 2015) [152], Ethiopia has reduced the proportion of rural population practicing open defecation from 92% in 2000 to 39.1% by 2016. The differences and improvement of the sanitary situation in the rural communities in the recent years might have contributed to the variation in prevalence of changes over time, but none of the papers studied BCC/ taeniosis over a longer time period to assess this. Recent reports indicate that around 37% of the total population (over 35 million people) or 43% of the rural population still do not have access to any form of latrine and therefore def- ecate in the open [28], implying persistence of pasture con- tamination with taeniid eggs in places where infected people are living. According to the WHO [3, 144], human taeniosis can be detected by a well-structured questionnaire using the find- ing of proglottids in stool and underwear as a diagnostic sign. Moreover, as pork is rarely consumed in Ethiopia due to religious and cultural reasons, T. solium is less likely to occur, and the detected proglottids are assumed to be T. sag- inata. In this review, a higher prevalence was found using questionnaire-based taeniosis detection than via copro- logical techniques. Coprological techniques have fairly low sensitivities, related to the intermittent egg excretion and depending on the technique used [3, 15]. Discussion Taenia saginata taeniosis and cysticercosis are highly endemic in central and eastern African countries such as Ethiopia [3]. In this review, we observed a high preva- lence of BCC recorded in several studies, with substantial variation in the prevalence among the different zones of the country and within the same abattoir at different time points. The within-abattoir difference could be primarily attributed to variations in the application of the Ethiopian meat inspection regulation. The Ethiopian Meat Inspec- tion Regulation (1972) [136] recommends visual inspec- tion and palpation of all exposed surfaces, followed by incisions in the heart, triceps muscles, internal and exter- nal masseter muscle, tongue, the thigh muscles of both hind legs, the neck muscles, diaphragm, intercostal mus- cle, liver, lung, kidney and oesophagus. It also prescribes a thorough inspection of the whole carcass and offal if any cysticerci are found. However, the firmness by which this routine meat inspection procedure is implemented varies among meat inspectors and abattoirs in Ethiopia. This review identified that the prevalence differed among the reports with the number of organs and muscles inspected. Indeed, higher prevalences were reported by authors who inspected more muscles and organs [21, 32, 33] as compared to those authors who inspected fewer muscle and organs [20, 71, 81]. These variations in meat Despite the above-mentioned methodological prob- lems, there seems to be a variation in the prevalence of BCC and taeniosis in different regions of the country (Fig. 2a). Different factors could have affected these geo- graphical variations in the country; such as the cultural and religious differences in raw beef consumption, the agroecological condition, sanitation conditions (Fig. 2c) and human/cattle population density (Fig. 2b). For instance, the rural communities around Jimma, Borena, Arsi, Bale and south-eastern and north-eastern parts of the country rarely consume raw meat due to cultural and religious reasons [42, 52], which could lead to a lower number of tapeworm carriers potentially contaminating Page 12 of 17 Jorga et al. Parasites Vectors (2020) 13:375 the habit of raw or undercooked beef consumption. Discussion For instance, the following raw dishes are a potential source of taeniosis: ‘Kitfo’/ ‘Lebileb’ (finely minced beef) and ‘Kurt’ (cubes of beef) in Ethiopia [32], roasted beef over an open fire in Central and East Africa, semi-raw beef dish known as counters “basterma” in Egypt, Turkey and the Middle East [145], tartar shashlik in Russia [146], shish kebab in India [147], larb in Thailand [148] and raw meat in Cuba [149]. Moreover, the average zonal prevalence of BCC or taeniosis at Sidama, Gedeo, or Gurage zone and taenicidal doses/cost at Hawasa and Welayta zone of SNNP region was high, which might be related to the deep-rooted raw beef consumption habit in these communities. herds and grazing land with T. saginata eggs. The high prevalence reports seem to concur with the cattle den- sity (Fig. 2b) and highland altitude which is characterized by a more moderate, colder temperature, high moisture and vegetation coverage as compared to lowlands. Low temperatures are known to favour the survival of T. sagi- nata eggs in the environment [5]. However, there are still many data gaps with respect to the research coverage and agroecological factors contributing to parasite survival in the environment, thus urging for more research.hif herds and grazing land with T. saginata eggs. The high prevalence reports seem to concur with the cattle den- sity (Fig. 2b) and highland altitude which is characterized by a more moderate, colder temperature, high moisture and vegetation coverage as compared to lowlands. Low temperatures are known to favour the survival of T. sagi- nata eggs in the environment [5]. However, there are still many data gaps with respect to the research coverage and agroecological factors contributing to parasite survival in the environment, thus urging for more research.hif The risk factor analysis showed no significant differ- ence between the sex and age of slaughtered animals with the prevalence of BCC, which is also reported elsewhere [140, 141]. However, reports from some EU countries show significant associations of BCC with age [137], with higher odds in female, older cows [142] and a significant age-sex interaction [143]. Breed and body condition of slaughtered animals were not significantly associated with the odds of BCC, although here again, most of the animals slaughtered were local/zebu and of good body condition. Authors’ contributions EJ, BM and SG developed the selection criteria, data extraction criteria and search strategy. EJ, BM, IVD and SG drafted the manuscript. EJ, SG and IVD performed the analyses. All authors edited manuscript drafts and provided feedback. All authors read and approved the final manuscript. Conclusions Prevalence reports of BCC based on routine meat inspec- tion represent an underestimation of the actual preva- lence of the parasite in the country. Although the true prevalence of T. saginata infection cannot be confirmed based on the current literature, the existing reports are indicative of high prevalence and widespread occur- rence in the country. Taeniosis was strongly associated with raw or undercooked beef consumption, occupation, adults and males. An all-inclusive approach to break the life-cycle of the parasite through improving sanitary con- ditions and strengthening the meat inspection through a harmonized detection and reporting system among abattoirs is suggested. Besides, more research on the risk factors for such a high prevalence of the parasite and detailed investigation on the associated financial losses is needed. Discussion In developing countries like Ethiopia, farmers do not have access to an insurance for BCC, and as such do not have this cost. On the other hand, farmers/butchers do incur the value loss or total loss of the carcass in case of infection. Given the high prevalence of the parasite combined with a large human and cattle popula- tion, high economic losses in Ethiopia can be anticipated, though detailed up-to-date investigations on the economic impact of the parasite are currently lacking. Table S8. Condemnation of organs and carcasses due to BCC. Text S1. References. Table S8. Condemnation of organs and carcasses due to BCC. Text S1. References. Additional file 3: Figure S1. Overview of studies reporting human tae- niosis (questionnaire-based diagnosis) in Ethiopia. The forest plot contains a horizontal line representing the results of each study and the length of the straight line indicates the 95% CI, the box size the weight of the study and the middle of the box the point estimate of the study. A vertical bro- ken line is the pooled estimate and a diamond shaped box at the bottom is the CI, while the solid line shows the point of null assumption. Abbreviations Ag-ELISAs: Antigen-enzyme-linked immunosorbent assay; BCC: Bovine cysticercosis; CI: Confidence interval; I2: Inverse variance index; IHAT: Indirect hemagglutination test; pOR: Pooled odds ratio; PCR: Polymerase chain reaction; Q: Cochran’s Q test value; OIE: World Animal Health Organisation; RFLP: Restricted fragment length polymorphism; SNNP: Southern Nations and Nationalities and Peoples Region; US$: United States dollar; WHO: World Health Organisation. Funding The authors would like to acknowledge the financial support of the Flemish Inter-University Council-University Development Cooperation (VLIR-UOS). Acknowledgements The authors are thankful for those researchers who were devoted to produce data on bovine cysticercosis and taeniosis in Ethiopia. Emmanuel Abatih is acknowledged for the statistical advice and Moges Kidane and Wubneh Belete for the map advice. Discussion Also, the ques- tionnaire-based diagnosis is usually not restricted in time, while the stool examinations represent a one-time point. Moreover, in most of such reports, the study subjects were specific groups of the society such as patients visiting health institutions for other illnesses, who are not representative of the general community. Perhaps due to the reason that it is a self-diagnosed and treatable disease, taeniosis patients in Ethiopia visit the hospital less frequently. On the other hand, questionnaire-based studies could also give false positives due to the difficulty to differentiate between other gastroin- testinal worm infections and small T. saginata proglottids. In Ethiopia, a number of medicinal plants were docu- mented to have taenicidal effect and are still in use by some of the Ethiopian communities where modern health coverage is low or as an alternative to the mod- ern treatment in urban areas. However, the evaluation of the actual efficacy of these plants based on scientifically sound methodologies is not reported.h Human taeniosis was found strongly associated par- ticularly with raw beef consumption, adults, men and people working in the abattoir and butcher houses. In Ethiopia, mostly adults and particularly males visit restau- rants and butchers for beef consumption, often consumed raw. The high prevalence of taeniosis in African countries, some Asian countries, Thailand and Cuba is attributed to The economic impact of human taeniosis caused by T. saginata is due to treatment costs and/or the number of sick days, and long-term effects. Recently Jansen et al. [8] Jorga et al. Parasites Vectors (2020) 13:375 Page 13 of 17 reported a maximum estimated loss of €795,858 per year from 10,991 taeniosis patients in Belgium, which is related to medication cost, cost of diagnosis and consultation. Since self-medication for gastro-intestinal tract diseases is a com- mon practice in Ethiopia [153], these costs per person might be lower compared to the Belgian estimate. On the other hand, earlier reports from Africa showed a total loss from BCC in Botswana to be near £0.5 million per year while in Kenya it was £1 million [154]. In Iran the direct economic loss from BCC was estimated to be US$ 112,302 [155]. Jansen et al. [8] partitioned the loss incurred to BCC as due to value loss, cost of the inspection, cost of destruction and insurance cost on Belgian cattle. Supplementary information Supplementary information accompanies this paper at https​://doi. org/10.1186/s1307​1-020-04222​-y. Supplementary information accompanies this paper at https​://doi. org/10 1186/s13071-020-04222-y Supplementary information acc org/10.1186/s1307​1-020-04222​-y. Received: 11 March 2020 Accepted: 13 July 2020 Received: 11 March 2020 Accepted: 13 July 2020 Additional file 1: Table S1. PRISMA checklist. Additional file 2: Table S2. Prevalence data for bovine cysticercosis in Ethiopia extracted from included articles. Table S3. Extracted data for the distribution of cysticerci in organs and carcasses in infected cattle. Table S4. Extracted data for cyst viability and average viability propor- tion calculated. Table S5. Data extracted for the prevalence of human taeniosis from the questionnaire-based reports. Table S6. Average zonal prevalence of BCC, questionnaire-based and stool test-based taeniosis. Table S7. Taenicidal dose and cost based on pharmacy inventory records. Competing interests Competing interests The authors declare that they have no competing interests. Competing interests The authors declare that they have no competing interests. Competing interests The authors declare that they have no competing interests. Author details 1 Department of Veterinary Science, College of Agriculture and Veterinary Sciences, Ambo University, P.O. Box 19, Ambo, Ethiopia. 2 Department of Vet- erinary Public Health, Laboratory of Foodborne Parasitic Zoonoses, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium. Additional file 1: Table S1. PRISMA checklist. Additional file 2: Table S2. Prevalence data for bovine cysticercosis in Ethiopia extracted from included articles. Table S3. Extracted data for the distribution of cysticerci in organs and carcasses in infected cattle. Table S4. Extracted data for cyst viability and average viability propor- tion calculated. Table S5. Data extracted for the prevalence of human taeniosis from the questionnaire-based reports. Table S6. Average zonal prevalence of BCC, questionnaire-based and stool test-based taeniosis. Table S7. Taenicidal dose and cost based on pharmacy inventory records. Availability of data and materials y All datasets supporting the conclusions of this article are included within the article and its additional files. y All datasets supporting the conclusions of this article are included within the article and its additional files. Ethics approval and consent to participate Ethics approval and consent to participate Ethics approval and consent to participate Not applicable. 1. Ogunremi O, MacDonald G, Scandrett B, Geerts S, Brandt J. 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Survival of cysticercus in veal dishes prepared in the Buryat. ASSR. Med Parazitol. 1968;37:108–9. 147. Anataraman M. The prevalence and transmission of human taeniosis in India. Proceedings of the 3rd International Congress of Parasitology, Munich; 1974. p. 394–395. 147. Anataraman M. The prevalence and transmission of human taeniosis in India. Proceedings of the 3rd International Congress of Parasitology, Munich; 1974. p. 394–395. • fast, convenient online submission • thorough peer review by experienced researchers in your field • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research ? 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Quantifiable structure of professional language competence
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Abstract The competence-based approach in education has made it easier to use qualimetric procedures to assess learning outcomes. Given the fundamentally integrative nature of competencies, they tend to have more and more complex structures. This paper discusses the structure of one such competence, professional language competence, especially relevant to future technical university graduates who have to be able not only to solve professional problems but also use a foreign language to help them do this. The purpose of this study is to find out the component-wise structure of professional language competence of future technical university graduates and show experimentally that its development level can be assessed quantitatively, thus guaranteeing its development in students. The main research methods are pedagogical experiment, testing, method of expert group assessment. The pedagogical experiment involved 23 second-year students blogging in English on profession-related topics as part of their independent work organized with the author’s pedagogical technology. The results of the study show the effectiveness of this technology. Comparative assessments are provided of the level of different components of professional language competence as well as subcomponents of one such component. The monitoring of the changes in subcomponents and components of professional language competence allows a teacher to identify weaknesses in linguistic training and difficulties that students face in their independent work so that one address them in a timely and systematic manner. The results of the study show the effectiveness of this technology. Comparative assessments are provided of the level of different components of professional language competence as well as subcomponents of one such component. Th it i f th h i b t d t f f i l l t ll The results of the study show the effectiveness of this technology. Comparative assessments are provided of the level of different components of professional language competence as well as subcomponents of one such component. The monitoring of the changes in subcomponents and components of professional language competence allows a teacher to identify weaknesses in linguistic training and difficulties that students face in their independent work so that p p g g p p p The monitoring of the changes in subcomponents and components of professional language competence allows a teacher to identify weaknesses in linguistic training and difficulties that students face in their independent work so that one address them in a timely and systematic manner. Quantifiable structure of professional language competence Andrey A. Gareyev* (a) (a) M.T. Kalashnikov Izhevsk State Technical University, 426069, Izhevsk (Russia), 7 Studencheskaya street, andrei.gareeff@yandex.ru * Corresponding author. E-mail: andrei.gareeff@yandex.ru Proceedings IFTE-2021, 0491-0501 doi:10.3897/ap.5.e0491 Abstract Keywords: professional language competence, technical university, competence-based approach, qualimetric approach, students’ independent work. © 2021 Andrey A. Gareyev This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Published by Kazan federal university and peer-reviewed under responsibility of IFTE-2021 (VII International Forum on Teacher Education) © 2021 Andrey A. Gareyev This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Published by Kazan federal university and peer-reviewed under responsibility of IFTE-2021 (VII International Forum on Teacher Education) y y This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Published by Kazan federal university and peer-reviewed under responsibility of IFTE-2021 (VII International Forum on Teacher Education) Andrey A. Gareyev / Proceedings IFTE-2021 492 Introduction With the competence-based approach having been introduced in higher education, pedagogical science now considers students’ education as the development of their competencies. In Russia, the competence- based approach is typically considered an orientation towards the vector-goals of education: learning abilities, self-determination, self-actualization, socialization, and individuation (Zeer, 2005). The concept of competence has been introduced to empower the cooperation between higher education and labour market both of which have a number of discrepancies. To eliminate them, the idea of competence is used as a learning outcome that can be evaluated objectively. Also, the communication of educational institutions with their graduates’ employers is now conducted in the language of competencies both parties share and is aimed at building “an integrated educational, professional, and cultural environment which facilitates university graduates’ academic and professional mobility” (Baidenko, 2005). The new generation of federal state educational standards in the Russian Federation establishes as planned learning outcomes a set of universal, general-professional, and professional competencies, with more focus given to universal ones. However, due to the integral and multifaceted nature of competencies, it seems reasonable that the complex process of development of many different competencies simultaneously, in particular domain-specific ones, is more important (Gareyev, 2016). This paper analyzes the quantifiable structure of such competence especially relevant for the university training of engineers – professional language competence, which implies the ability of a future engineer to use a foreign language to solve professional problems. The main distinguishing feature of this research is that the level of a student’s professional language competence development can be assessed quantitatively. This is achieved by the qualimetric approach which, in this research, was used to evaluate the structure of the given competence and its development level in undergraduate students (Gareyev, Shikhova, & Shikhov, 2019). Literature review Russian researchers point out the integral nature of competence describing it as: “integration of knowledge, skills, and abilities that generate professional activity” (Zeer & Symanyuk, 2005), “ability and willingness to show flexibility under changing labour market conditions” (Galyamina, 2004), “generalized characteristic of personality” that expresses one’s knowledge, skills and experience “in a specific social or professional area” (Medvedev & Tatur, 2007), “a component of human quality, a group of one’s properties which determine one’s ability to perform a specific group of actions” (Subetto, 2009). The integral nature of competence is also evident due to various researchers including in its structure not only cognitive (knowledge) and operational (skills) components but also motivational, ethical, behavioural, and others. Moreover, historically the transition from knowledge-based to competence-based paradigm indicates a trend towards a gradual extension of the content of competence. For example, initially, the concept of competence was used only by linguists who mostly used the term “communication competence”. Later, in the 1970-90s, the ideas of professional and social competence became more common (Zimnyaya, 2004). Today it’s more common to encounter a more extensive competence like professional language competence used in this study. In the context of the given research, the definition by Subetto (2009) seems more adequate for a number of reasons. Not only does it represent the qualimetric aspect of competence in a detailed and precise manner but it also suits the technical character of engineering training. Another advantage of his definition lies in its connection of two distinct notions, of competence as “knowing the language” and performance as its actual use in different situations, originally introduced by Chomsky (1965), thereby adding strictness in how the concept can be used in real educational procedures and pedagogical studies. Thus, from this perspective and in the context of improving the quality of technical university students, their having a certain level of competence implies what professional activities they are able and willing to perform, as their activities to a greater extent are practical. Purpose and objectives of the study The purpose of the study is to find out the component-wise structure of professional language competence of future technical university graduates and show experimentally that its development level can be assessed quantitatively, thus guaranteeing its development in students. Andrey A. Gareyev / Proceedings IFTE-2021 493 Literature review Professional language competence, a term which was first introduced by Kruchinina & Mikhailova (2012), means “the ability and willingness of future specialists to solve communication problems in their professional area by communicating in a foreign language with native speakers, searching and analyzing the information needed to study a foreign experience, as well as dealing with scientific and technical literature and documentation in a foreign language in the area of the chosen specialization with the use of information and communication technologies”. Andrey A. Gareyev / Proceedings IFTE-2021 494 Let us focus on a few details present in this definition, as they help to refine the structure of professional language competence. Here, the competence: a) includes an often-used among language teachers “foreign language communication competence”; b) has an explicit practical orientation towards solving scientific or technical problems; c) implies developed analytical skills and critical thinking; d) requires basic skills of using information and communication technologies. Another aspect of this definition is concerned with the possibility of discerning invariant and variative parts for general and professional uses respectively which is suggested in educational standards of higher education. This aspect is of high importance to the given research, as it enables the use of individual educational trajectories for future graduates depending on their needs and goals, i.e. either going on to study for a Master’s degree or getting an engineering job. The disadvantage of structures of professional language competence in Kruchinina & Mikhailova’s (2012) and other scientists’ studies is the lack of objective, quantifiable evaluation of its components which is exactly what is suggested in this paper. Methodology The review of pedagogical literature and dissertation research has shown that the structure of professional language competence of technical university students must have three main components all of which meet the requirements of educational standards of higher education: - Communication component: characterizes the ability to comprehend texts in a foreign language and apply linguistic constructions with regards to a professional or learning situation; - Cognitive component: reflects the ability of self-education, self-organization, searching, analyzing, and synthesizing profession-oriented information, including one in a foreign language; - Domain-specific component: points to the ability to use one’s knowledge from a professional domain to perform communication tasks. This three-component structure does not mean that each of these components is developed separately: they have separate functions but by no means genetic roots. On the contrary, the processes of thinking (cognitive component), speaking (communication component), and professional activity (domain-specific component) are closely intertwined, interact with and influence each other (Vygotsky, 1997). Andrey A. Gareyev / Proceedings IFTE-2021 495 For example, when translating a technical text, if a student lacks the understanding of advanced grammar in a given sentence, his or her extensive professional knowledge can make up for it, so that this student becomes able to make sense of a grammatically unclear passage. For example, when translating a technical text, if a student lacks the understanding of advanced grammar in a given sentence, his or her extensive professional knowledge can make up for it, so that this student becomes able to make sense of a grammatically unclear passage. In addition, the domain-specific component comprises two individual educational trajectories which correspond to future engineers’ needs and goals: In addition, the domain-specific component comprises two individual educational trajectories which correspond to future engineers’ needs and goals: - Scientific trajectory: focuses on the development of scientific writing skills, abilities to analyze scientific texts and compose a scientific paper based on a task performed or research conducted; - Technical trajectory: implies the development of technical writing skills, abilities to translate and compose technical documentation. The student’s choice of one of these trajectories indicates a preference towards primarily scientific or technical professional activities each of which has its own features and, thus, requires the emphasis on different aspects of professional language training. It is worth noting that these two trajectories are in full accordance with the main professional tasks of engineers pointed out in educational standards of higher education. Methodology This three-component quantifiable structure of professional language competence with its subcomponents listed below has been approved using the method of expert group assessment. This three-component quantifiable structure of professional language competence with its subcomponents listed below has been approved using the method of expert group assessment. Table 1 below shows the subcomponents of each component in professional language competence. They help to identify how well each component of this competence is developed and are necessary to diagnose learning difficulties. Table 1 below shows the subcomponents of each component in professional language competence. They help to identify how well each component of this competence is developed and are necessary to diagnose learning difficulties. Table 1. Subcomponents of professional language competence Communication component (CM) Cognitive component (CG) Domain-specific component (DS) 1. Understanding of vocabulary and grammar (СМ-1). 2. Reading skills and analysis of profession- related written texts (СМ- 2). 3. Listening skills and analysis of profession- related audiovisual content 1. Ability to identify the essence of the text, including the main idea of profession-related information (СG-1). 2. Quality of searching profession-related content relevant to a given task (СG-2). 3. Ability to assess the importance of a piece of profession-related information 1. Understanding of processes and phenomena from a professional area (DS-1). 2. Experience in using professional terms in both oral and written communication (DS-2). 3. Willingness to use new professional terms (DS-3). 4. Experience in scientific or Table 1. Subcomponents of professional language competence Table 1. Subcomponents of professional language competence Andrey A. Gareyev / Proceedings IFTE-2021 496 (СМ-3). 4. Writing skills and clarity of written communication (СМ-4). 5. Speaking skills and clarity of oral communication (СМ-5). for a given task (СG-3). 4. Self-reflection, ability to assess the quality of one’s profession-related activity (СG- 4). 5. Ability to predict and produce hypotheses about a profession- related issue (СG-5) (СМ-3). 4. Writing skills and clarity of written communication (СМ-4). 5. Speaking skills and clarity of oral communication (СМ-5). technical writing (depending on the trajectory chosen) (DS-4). As a result, the developed pedagogical technology allows a researcher or teacher to assess the development level of both professional language competence as a whole and its components separately. In this paper, an example of one component’s assessment will be given. (СМ-3). 4. Writing skills and clarity of written communication (СМ-4). 5. Speaking skills and clarity of oral communication (СМ-5). for a given task (СG-3). 4. Self-reflection, ability to assess the quality of one’s profession-related activity (СG- 4). 5. Ability to predict and produce hypotheses about a profession- related issue (СG-5) Figure 1. Example of student blog post Methodology The levels of this competence development can be distinguished as follows: 1) threshold level: a student can convey a general idea of a text, comprehend basic professional vocabulary related to general-professional topics; 2) basic level: a student can use simple linguistic structures and professional vocabulary to communicate on few professional topics, correctly comprehend the essence of a professional text; 3) analytic-synthetic level: a student is able and willing to use complex grammar and extensive professional vocabulary to communicate on a few professional topics, willing to search and analyze profession-related information in a foreign language, synthesize logically coherent statements based on this analysis; 4) predictive level: a student is able and willing to critically assess statements based on one’s professional knowledge, rationalize and articulate one’s assessment to make fact-based predictions, ready for creative activity. A pedagogical experiment to see how students’ professional language competence, according to the aforementioned structure, might be developed was conducted in the 2018-2019 academic year. During the experiment, i.e. the four-month professional English course, a group of 23 second-year technical university students composed and published profession-related content for their individual blogs in English. Their participation was voluntary. Among the 23 student blog topics are mode structure control in laser cavities, effectiveness of new types of energy in Russia and the world (both – in scientific trajectory; for the example blog post on the latter, see Figure 1 below), development of simple systems on the Arduino platform, how to read electronic circuits and how electric vehicle hardware works (all three – in technical trajectory). Andrey A. Gareyev / Proceedings IFTE-2021 497 Results The results of the pedagogical experiment can be seen in Table 2. In this table, the assessments of all three components of professional language competence are given at an initial (pre-experiment, IS in Table 2) and final (post-experiment, FS in Table 2) testing stages. Table 2. Comparative assessments of the level of different components of professional language competence Professional language competence components Levels of competence development Threshold Basic Analytic- synthetic Predictive Number of students (%) IS FS IS FS IS FS IS FS nal language Levels of competence development Andrey A. Gareyev / Proceedings IFTE-2021 498 Communication 39 - 52 39 9 48 - 13 Cognitive 30 9 30 30 40 43 - 17 Domain-specific 48 - 48 48 4 30 - 22 As it can be seen from Table 2, the domain-specific component is the most challenging one to acquire: this is evident both at the initial testing stage (only 4% of the students are at analytic-synthetic level, 0% at predictive level) and at the final testing stage (52% of the students are at two higher levels with 60% and 61% of the students respectively for other components). This is likely to be caused by the lack of profession-related subjects and technical English language courses during the first two years of study at a technical university. Of all three components, the most developed one at an initial testing stage is the cognitive one (40% of the students are at the analytic-synthetic level while 4% and 9% of the students respectively are for other components). This might be explained by the analytical nature of most of the subjects studied in the first years of bachelor programs in a technical university. Communication and domain-specific components, being the least developed in students, thus show the highest growth rates (52% and 48% respectively at two higher levels compared with the 20% growth for the cognitive component). Also, differentiated assessment for subcomponents of each competence component has been made possible. As an example, Table 3 shows comparative test results at initial (IS), midpoint (MS), and final (FS) stages of different subcomponents for the domain-specific component. Table 3. Results Comparative assessments of the level of subcomponents for domain-specific component Domain-specific subcomponents Levels of competence development Threshold Basic Analytic- synthetic Predictive Number of students (%) IS MS FS IS MS FS IS MS FS IS MS FS DS-1 22 - - 48 27 22 13 43 43 17 30 35 DS-2 30 13 - 57 44 39 13 30 39 - 13 22 DS-3 35 13 - 61 48 48 4 22 22 - 17 30 DS-4 48 22 - 48 43 48 4 35 30 - - 22 3. Comparative assessments of the level of subcomponents for domain-specific component Table 3 demonstrates that DS-1 (see Table 1 for this and other subcomponents explanation) is the easiest subcomponent to acquire (only 22% of the students are at the initial stage, 0% at the final stage, growth from 30% to 78% at two higher levels). Andrey A. Gareyev / Proceedings IFTE-2021 499 This, i.e. understanding of the basics of a professional area, is expected of all students at technical training programs. DS-3 and DS-4 are more difficult to acquire. That is why both of them showed the highest growth rate (from 4% to 52% at two higher levels). The lack of willingness to use new concepts at the initial testing stage might be explained by a low number of profession-related subjects and, thus, students’ experience in their self-learning of new phenomena from their professional area and the terms which these phenomena are described with. Such labour-intensive students’ independent work with its further practical application (e.g., writing a course paper) begins, as a rule, at the third year of study. With that, at the midpoint testing stage about half of the students had no experience of scientific writing (48% with threshold level at the initial stage). This seems natural for second-year students, as usually very few of them have a chance to gain experience in writing scientific papers or technical documentation. These are the activities that are common only among either the most active students or those who already do profession-related work, the latter being extremely rare for second-year students. Positive dynamics are also seen in the DS-2 subcomponent (growth from 13% to 61% at two higher levels). The lack of experience in the use of professional terms can be explained by the lack of speaking practice on professional topics, even in Russian. Results This is possible since profession-oriented subjects in the first year of study are typically represented in the form of lectures where the main students’ activities are note-taking and listening but not the use of this material in new contexts. Discussion As it can be seen from the example above, the monitoring of the changes in subcomponents and components of professional language competence allows a teacher to identify weaknesses in linguistic training and difficulties that students face in their independent work. This, in turn, points out either the need for timely support from a teacher or possible corrections to be made in the way students’ independent work is organized. Thus, the results of students’ independent work, organized with the pedagogical technology developed in this study and based on student blogging in English, demonstrate high rates of development of their professional language competence which, in turn, implies the stimulation of their independent work and a higher quality of its organization. Andrey A. Gareyev / Proceedings IFTE-2021 500 Conclusion As we have seen, the use of qualimetric approach and quantitative analysis of changes in students’ competencies represents an active research area with a lot of potential for improvement of learning outcomes. This study, unfortunately, represents an outlier rather than a norm in how the results of teaching and learning are measured. It is partly due to the complexity of quantitative analysis, especially for ordinary non-technical university teachers, such as English teachers, partly because of the labour-intensive nature of qualimetric procedures that require the collaboration of experts from several university departments. Still, to support teachers and experts who participate in qualimetric procedures there is plenty of opportunities to automate a part of their job. Different software applications and the potential of artificial intelligence tools give hope for the elimination of routine tasks that people now have to perform. For example, Grammarly is an application that allows a teacher to automatically assess how accurate the grammar and vocabulary of a particular student’s text is without reading the whole piece. The results of this research are far from being conclusive. So far, it is just an example of how we can be more observant of educational processes and more specific in what outcome we want to achieve from students’ work. To refine and improve the technology and quantifiable competence structure suggested, more effort from other teachers and researchers is needed so that they could reproduce the results of this study and build their tools and models of how students’ work can be organized more efficiently. References Baidenko, V. I. (2005). Bologna process: the midway. Moscow: Research Center for Quality Problems in Specialist Training. Baidenko, V. I. (2005). Bologna process: the midway. Moscow: Research Center for Quality Problems in Specialist Training. Chomsky, N. (1965). Aspects of the Theory of Syntax. Cambridge, MA: MIT press. Galyamina, I. G. (2004). Design of educational standards of higher education of new generation with competency-based approach. Research Center for Quality Problems in Specialist Training, 1(49), 285-297. Gareyev, A. A. (2016). The necessity of professionally oriented foreign language training of vocational education teachers (the results of the survey). Bulletin of Kalashnikov ISTU, 2(19), 127-128. Gareyev, A. A. (2016). The necessity of professionally oriented foreign language training of vocational education teachers (the results of the survey). Bulletin of Kalashnikov ISTU, 2(19), 127-128. Andrey A. Gareyev / Proceedings IFTE-2021 501 Gareyev, A. A., Shikhova, O. F., & Shikhov, Yu. A. (2019). Profession-oriented blogging as the optimal teaching tool for the development of students’ professional language competence. ARPHA Proceedings, 1, 1213-1226. Kruchinina, G. А., & Mikhailova, Е. B. (2012). Forming professional foreign language competence in students of engineering specialities under the conditions of informatization of higher professional education. Bulletin of Lobachevsky Nizhny Novgorod University, 4(1), 26-33. Medvedev, V., & Tatur, Y. (2007). Training of university teachers: competence-based approach. Higher education in Russia, 11, 46-56. Subetto, A. I. (2009). The ontology of the competence-based approach in educational systemology. Siberian Pedagogical Journal, 1, 100-126. Vygotsky, L. S. (1997). The collected works: problems of the theory and history of psychology (Vol. 3). New York: Plenum Press. Zeer, E. F. (2005). Competency-based approach to education. The education and science journal, 3(33), 27-40. Zeer, E. F., & Symanyuk E. E. (2005). Competence-based approach to modernization of professional education. The higher education in Russia, 4, 23-30. Zimnyaya, I. A. (2004). Key competencies as a result-goal base for competency-based approach in education. Moscow: Research Center for Quality Problems in Specialist Training.
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https://jcannabisresearch.biomedcentral.com/track/pdf/10.1186/s42238-022-00123-2
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Massively parallel sequencing of Cannabis sativa chloroplast hotspots for forensic typing
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Abstract Background:  Marijuana (Cannabis sativa) is the most commonly used illicit drug in the USA, and the use of DNA bar- codes could assist drug trafficking investigations by indicating the biogeographical origin and crop type of a sample and providing a means for linking cases. Additionally, the legality of marijuana in the USA remains complicated with some states fully legalizing marijuana for recreational use while federally marijuana remains completely illegal. Mas- sively parallel sequencing (MPS) offers distinct advantages over capillary electrophoresis (CE), including more compre- hensive coverage of target loci, analysis of hundreds of markers simultaneously, and high throughput capabilities. Methods:  This study reports on the development of a MiSeq FGx® assay targeting seven “hotspot” regions in the Cannabis sativa chloroplast genome that are highly polymorphic and informative in attempts to determine biogeo- graphical origin and distinguishing between marijuana and hemp. Sequencing results were compared to previous studies that used CE-based genotyping methods. Results:  A total of 49 polymorphisms were observed, 16 of which have not been previously reported. Addition- ally, sequence data revealed isoalleles at one locus, which were able to differentiate two samples that had the same haplotype using CE-based methods. This study reports preliminary results from sequencing 14 hemp and marijuana samples from different countries using the developed MPS assay. Results:  A total of 49 polymorphisms were observed, 16 of which have not been previously reported. Addition- ally, sequence data revealed isoalleles at one locus, which were able to differentiate two samples that had the same haplotype using CE-based methods. This study reports preliminary results from sequencing 14 hemp and marijuana samples from different countries using the developed MPS assay. Conclusion:  Future studies should genotype a more comprehensive sample set from around the world to build a haplotype database, which could be used to provide investigative leads for law enforcement agencies investigating marijuana trafficking. Conclusion:  Future studies should genotype a more comprehensive sample set from around the world to build a haplotype database, which could be used to provide investigative leads for law enforcement agencies investigating marijuana trafficking. Keywords:  Cannabis sativa, Massively parallel sequencing, Chloroplast DNA, Genetic assay Keywords:  Cannabis sativa, Massively parallel sequencing, Chloroplast DNA, Genetic assay Keywords:  Cannabis sativa, Massively parallel sequencing, Chloroplast DNA, Genetic assay a time (Bruijns et al. 2018; Moorthie et al. 2011), com- pared to only about 25 loci by CE for a five-dye short tandem repeat (STR) kit (Lazaruk et al. © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. Roman et al. Journal of Cannabis Research (2022) 4:13 https://doi.org/10.1186/s42238-022-00123-2 Roman et al. Journal of Cannabis Research (2022) 4:13 https://doi.org/10.1186/s42238-022-00123-2 Journal of Cannabis Research Open Access Abstract 1998). Sequence data may elucidate isoalleles, alleles which have the same length and appear identical on CE but actually have dif- ferent sequences, leading to more discriminatory results. Isoalleles may differ in their repeat structure or contain variants in the flanking regions, and the International Society of Forensic Genetics (ISFG) has reported guide- lines to standardize the nomenclature for these sequence variable alleles (Parson et al. 2016). Costs and run times associated with MPS have dropped substantially, mak- ing targeted MPS assays a cost-effective approach for Madeline G. Roman, Ryan Gutierrez and Rachel Houston* Madeline G. Roman, Ryan Gutierrez and Rachel Houston* Background Massively parallel sequencing (MPS), also called next generation sequencing (NGS), is a high throughput technique capable of collecting DNA sequence data from multiple targets and multiple samples in paral- lel. It offers several distinct advantages over traditional DNA typing using capillary electrophoresis (CE), includ- ing providing more comprehensive coverage of target markers (sequence data in addition to length) and the ability to analyze hundreds or thousands of targets at *Correspondence: rmh034@shsu.edu Department of Forensic Science, Sam Houston State University, 1003 Bowers Blvd, Huntsville, TX 77340, USA Roman et al. Journal of Cannabis Research (2022) 4:13 Roman et al. Journal of Cannabis Research Page 2 of 10 characterizing samples for genetic individualization or identification (Moorthie et al. 2011). markers) gives better coverage and increased through- put capabilities compared to whole genome sequencing. The chloroplast genome is AT-rich (63%) and contains numerous homopolymeric stretches (Vergara et al. 2016). The MiSeq FGx® platform was chosen for sequencing in this study because has been shown to have a higher fidelity when sequencing homopolymeric stretches of DNA (Duke et al. 2015; Loman et al. 2012; McElhoe et al. 2014), and many of the polymorphisms identified in the previous studies (Roman et al. 2019; Roman and Houston 2020; Cheng and Houston, 2021) were homopolymeric STRs (hSTRs). i In forensics, MPS assays have been used for human identification purposes, including sequencing autoso- mal STRs and single nucleotide polymorphisms (SNPs) (Eduardoff et al. 2015; Guo et al. 2017; Kim et al. 2016; Seo et al. 2013; Wang et al. 2017), mitochondrial DNA analysis (Davis et al. 2015), phenotype prediction (Mehta et  al. 2016), and other purposes (Bruijns et  al. 2018; Budowle et al. 2017). While much of the forensic research on MPS has focused on human DNA, its use for forensic plant science has recently been investigated (Houston et al., 2018a, b). Houston et al. reported an MPS panel for the Ion S5™ consisting of twelve autosomal STRs in Cannabis sativa (marijuana). Results showed concord- ance with CE methods, and isoalleles were found at eight of the loci, providing a higher discriminatory power com- pared to CE (Houston et al., 2018a, b). MPS has been also been used for DNA barcoding studies in animals and has shown better recovery, reduced costs, and faster process- ing times compared to traditional Sanger sequencing (Shokralla et al. 2015). Studies involving the use of MPS for DNA barcoding in plants have been limited but do show the advantage of simultaneous analysis of multiple barcodes for enhanced phylogenetic resolution (Parks et al. 2009; Sucher et al. 2012). Recent studies reported chloroplast DNA barcoding markers in C. sativa that were informative for biogeographical origin and crop type prediction (Roman et  al. 2019; Roman and Hou- ston 2020). Since these regions represent the most highly polymorphic regions of the C. sativa chloroplast genome, they are referred to as “hotspots.” The polymorphisms were genotyped using CE-based methods, and Sanger sequencing revealed isoalleles at several loci with differ- ent repeat sequences or variations in the flanking regions that were not detected by CE (Roman et al. 2019; Roman and Houston 2020). This study seeks to expand upon pre- vious studies by incorporating the “hotspot” barcoding regions into an MPS assay to provide more discrimina- tory results and a high throughput method for building a database of C. sativa chloroplast haplotypes. This study seeks to design an MPS panel for the MiSeq FGx® consisting of seven highly polymorphic “hotspot” regions in the C. sativa chloroplast (trnK-matK-trnK, rps16, trnS-trnG, ycf3, accD-psaI, clpP, and rpl32-trnL) to provide additional sequence data, discover isoalleles, and provide a high throughput method for creating a hap- lotype DNA database for hemp and marijuana samples. This assay could provide important investigative leads for law enforcement agencies investigating marijuana traf- ficking into and within the USA. DNA samples l Hemp samples from Canada were purchased online from Badia Spices Inc. (Doral, FL, USA; N=1 (H2-4)) and Navitas Organics (Novato, CA, USA; N=1 (H3-3)). USA hemp samples were purchased from American Hemp Harvest (Boulder, CO, USA; N=4 (H5-4, NT H5-1, NT H5-2, NT H5-4)) and The Original Hemp Buds (OR or NY, USA; N=1; strain: Electra (H8-1)). THC-positive marijuana samples from the USA-Mexico border were obtained from U.S. Customs and Border Protection (N=4 from different seizures (10-A1, 12-A7, 16-B1, 21-A16)). Chile marijuana DNA extracts were received from the Policia de Investigaciones in southern Chile (N=2 from separate cases (35,41)). A Chilean medical marijuana DNA extract was provided by collaborators in Chile (N=1; strain: London Cheese (MedMJ10)). Data analysis Data analysis was performed using the Miseq Con- trol Software v1.4.0.0, MiSeq Reporter v2.5.1.3, and Real-Time Analysis v.1.18.54.0 software installed on the instrument. Variants were reported at a minimum cover- age of 10 reads, Q30 variant score, and a minimum vari- ant percentage of 20%. Sequences were compared to a C. sativa chloroplast reference genome (Yoruba Nigeria cul- tivar, GenBank accession NC_027223.1). Variants to the reference genome were reported in a variant call file, and .bam files were viewed in Integrative Genomics Viewer (IGV) 2.8.0 (Robinson et al. 2011). Target enrichment Following manual normalization, all 14 libraries and the NTC were pooled in equal amounts and denatured according to the manufacturer’s instructions, resulting in a 15 pM library (Illumina 2019). A denatured PhiX con- trol (20 pM) was spiked in at 5% volume. Sequencing on the MiSeq FGx® (Verogen, San Diego, CA) was performed using the MiSeq FGx® Reagent Micro Kit (Verogen). g Seven polymorphic “hotspot” regions in the chloroplast of C. sativa (trnK-matK-trnK, rps16, trnS-trnG, ycf3, accD- psaI, clpP, and rpl32-trnL) were amplified in single PCR (Roman et al. 2019). Primers were designed using Primer3 (Koressaar and Remm 2007) and checked for specific- ity using the Primer-BLAST tool (NCBI). The optimal annealing temperature for each primer set was deter- mined by gradient Polymerase Chain Reaction (PCR) as previously described (Roman et al. 2019). PCR was car- ried out on a Veriti™ 96-well Thermal Cycler (Thermo Fisher Scientific, Waltham, MA) using the TaKaRa LA PCR™ Kit Ver.2.1 (TaKaRa Bio Inc., Kusatsu, Shiga, Japan). Reactions consisted of 0.25 μL TaKaRa LA Taq polymerase, 2.5 μL 10X LA PCR Buffer II ­(Mg2+ free), 2.5 μL 25 μM ­MgCl2, 4 μL dNTP mix, 2.5 μL 2 μM primer mix (Table 1), 4 μL template DNA (20 pg/μL), and 9.25 μL water. Cycling conditions consisted of a 2 min initial denaturation at 94 °C; followed by 30 cycles of 98 °C for 10 s, the optimal annealing temperature (Table 1) for 1 min, and 68 °C for 2 min; and a 10 min final extension at 72 °C. A negative template control (NTC) was included. Results and discussion Sequencing metrics Following amplification, samples were quantified using the Qubit™ dsDNA HS Assay Kit™ (Invitrogen, Carlsbad, CA) on a Qubit™ 2.0 fluorometer (Invitrogen). The seven PCR targets were then diluted and pooled to a final con- centration of 1 ng/μL. A 25 μL aliquot of the mixed PCR products was moved to a new tube and incubated with 2 μL exonuclease I (10 U/μL, Invitrogen) at 35 °C for 72 min and 75 °C for 15 min to remove excess primers. All samples (n=14) were successfully sequenced. Sequencing quality was high, with 81.2% of base calls having a quality score of 30 or higher, which indicates at least 99.9% accuracy at each base. The error rate in the PhiX control was below 3%. The yield was 1.48 Mb, clus- ter density was 1045 ± 26 K/mm2, and 99.33% ± 0.26% of clusters passed the filter. Phasing/prephasing rate was 0.139/0.036. Coverage varied within each of the ampli- cons. The trnK-matK-trnK and rps16 amplicons consist- ently had the lowest coverage, and ycf3 consistently had the highest. The clusters passing filter (PF) and clusters aligned to the reference genome for each sample are shown in Supplemental Table 1. DNA extraction and quantification Full chloroplast genome sequences have been reported for several marijuana and hemp cultivars (Matielo et al. 2020; Oh et al. 2016; Vergara et al. 2016). The full genome is 153,871 bp (Carmagnola and Dagestani cultivars) and contains 83 genes (Vergara et al. 2016). In comparison, the human mitochondrial genome is 16,569 bp (Ander- son et al. 1981) (about a tenth of the size), and typically mitochondrial DNA analyses only involve sequencing a portion of the genome, usually the hypervariable regions (HV1 and HV2) (Ingman and Gyllensten 2006; Miller and Budowle 2001). Due to the large size of the C. sativa chlo- roplast genome, sequencing targeted regions (barcoding Total genomic and organelle DNA was extracted using the DNeasy® Plant Mini kit (QIAGEN, Hilden, Germany) (N=8 samples) or nexttec™ 1-Step DNA Isolation Kit for Plants (nexttec, Hilgertshausen, Germany) (N=3 sam- ples) according to the manufacturers’ protocols. Extrac- tion from USA-Mexico marijuana was performed on-site at CBP, and extraction from hemp was performed at Sam Houston State University. Chilean samples were provided by collaborators in the form of DNA extracts. Chloro- plast DNA was quantified using a real-time PCR method reported by Houston et al. (Houston et al., 2018a, b). Roman et al. Journal of Cannabis Research (2022) 4:13 Page 3 of 10 Roman et al. Journal of Cannabis Research Library preparation and sequencingh The Nextera XT DNA Library Preparation Kit (Illumina, San Diego, CA) was used for tagmentation and indexing of libraries. A 1 ng input of amplified, cleaned DNA was used (1 μL), and libraries were prepared according to the manu- facturer’s instructions with the exception that 15 cycles of PCR were used for indexing instead of 12 cycles to ensure adequate library quantity (Illumina 2018). Sample librar- ies were checked on the 2100 Bioanalyzer (Agilent, Santa Clara, CA) using the High Sensitivity DNA kit (Agilent). Due to the homopolymeric nature of many of the repeat units (hSTRs), forward and reverse stutter was observed. However, based on the sequence coverage, confident allele calls were made. Some reads aligned to Table 1  Primer sequences and optimal annealing temperatures for single PCR reactions Region Forward primer Reverse primer Product size (bp) Ta (°C) trnK-matK-trnK ACG​AGC​CAA​AGT​TTT​AAC​ACAGG​ TCG​GCT​TTT​AAG​TGC​GGC​TA 2111 69 rps16 AGA​AAA​GGG​TGT​AGA​CGA​ACG​ TCG​TTT​CTC​GGA​GGC​AAG​AAT​ 1398 66 trnS-trnG TCT​AAT​GAT​CCG​GGG​CGT​AA TGC​ATT​CAA​AAC​GAC​CTG​C 1668 66 ycf3 ACG​GCT​CAG​CAG​TCA​AGT​TC TTC​GAA​ATT​CAT​GAA​AGG​CCCC​ 2095 68 accD-psaI GGC​TGT​TCA​AAC​AGG​TAC​AGG​ TGC​CGG​AAA​TAC​TAA​GCC​CA 1424 68 clpP TAA​ATT​CCC​CTG​TCG​GTG​CC ATG​CCT​ATT​GGT​GTT​CCA​AAA​GTA​ 1984 66 rpl32-trnL GGA​AAA​ACC​CAC​ATA​CGG​CG TAA​CAC​TCG​GCG​CGG​TTA​TT 1964 69 Table 1  Primer sequences and optimal annealing temperatures for single PCR reactions Roman et al. Journal of Cannabis Research (2022) 4:13 Roman et al. Library preparation and sequencingh Journal of Cannabis Research Page 4 of 10 Table 2  Genotypes for polymorphisms in the trnK-matK-trnK region (2233–4337 bp) Italics indicate polymorphisms not analyzed previously by CE a Indicates new polymorphisms discovered in this study Sample name trnK-matK-trnK INDEL (GAA​TAC​) trnK-matK-trnK SNP (C/T) SNPa (T/C) SNPa (A/C) trnK-matK-trnK STR1 (A) trnK-matK- trnK STR2 (T) Start location (bp) 2984 3258 3561 3752 3809 4109 Yoruba Nigeria (NC_027223.1) Absent C T A 11 10 H2-4 Absent C T A 11 10 H3-3 Absent C T A 11 10 H5-4 Present C T A 11 10 NT H5-1 Absent C T A 11 10 NT H5-2 Present C T A 11 10 NT H5-4 Present C C C 15 9 H8-1 Present C T A 11 10 10-A1 Present C T A 11 10 12-A7 Present C T A 11 10 16-B1 Present C C C 13 9 21-A16 Present C T A 11 10 35 Present C C C 15 9 41 Present C C C 14 9 MedMJ10 Present C T A 11 10 Table 2  Genotypes for polymorphisms in the trnK-matK-trnK region (2233–4337 bp) portions of the genome outside of the hotspot regions and were not interpreted, and additionally, several reads misaligned within hotspot regions. These misalignments had low read depth and did not affect the interpretation of sequencing results. a Indicates new polymorphisms discovered in this study Italics indicate polymorphisms not analyzed previously by CE Sequence datah There were 33 known polymorphisms within the “hot- spot” amplicons (Roman et  al. 2019 and Cheng and Houston, 2021), and 16 more were discovered, bring- ing the total to 49 polymorphisms. Newly discovered Table 3  Genotypes for polymorphisms in the rps16 region (4803–6201 bp) Italics indicate polymorphisms not analyzed previously by CE Sample name INDELa (AAA​GTA​) trnK-rps16 hSTR (A) rps16 SNP1 (A/G) rps16 SNP2 (A/C) rps16 hSTR (C) rps16 SNP3 (G/A) Start location (bp) 4911 5197 5303 5517 5518 6103 Yoruba Nigeria (NC_027223.1) Absent 9 A A 12 G H2-4 Absent 9 A A 11 G H3-3 Absent 9 A A 11 G H5-4 Absent 10 A A 11 A NT H5-1 Absent 9 A A 11 G NT H5-2 Absent 10 A A 11 A NT H5-4 Present 11 A A 13 G H8-1 Absent 10 A A 11 A 10-A1 Absent 10 A A 11 A 12-A7 Absent 10 A A 11 A 16-B1 Present 11 A A 14 G 21-A16 Absent 10 A A 11 A 35 Present 11 A A 13 G 41 Present 11 A A 14 G MedMJ10 Absent 10 A A 11 A Table 3  Genotypes for polymorphisms in the rps16 region (4803–6201 bp) Table 3  Genotypes for polymorphisms in the rps16 region (4803–6201 bp) Page 5 of 10 Roman et al. Journal of Cannabis Research (2022) 4:13 Roman et al. Journal of Cannabis Research Table 4  Genotypes for polymorphisms in the trnS-trnG region (8300–9967 bp). Concordance Previously, 30 polymorphisms within the seven hotspot regions were analyzed in our laboratory using CE-based methods (Roman et al. 2019; Roman and Houston 2020; Cheng and Houston, 2021). The genotypes obtained by MPS are fully concordant with the CE-based genotypes with exceptions at the trnS-trnG hSTR2 and rpl32-trnL hSTR2 loci. In four samples (NT H5-4, 16-B1, 35, and 41), the sequence genotypes at both loci appeared to be off by 1 bp from the CE-based genotypes (indicated in Tables 4 and 8). The sequence data showed that the CE fragment assays for both of these loci amplified regions containing the locus of interest as well as an additional hSTR locus that was unknown at the time. Variation at this new hSTR locus explains the discrepancy between the CE and sequencing genotypes for all four samples at both loci. Sequence data for the two alleles observed at trnS-trnG hSTR2 were submitted to GenBank (accession numbers: MW010378–79), and these sequences include the newly observed hSTR. The genotypes at each locus for 14 samples are dis- played in Tables 2, 3, 4, 5, 6, 7, and 8. Samples H8-1 (USA hemp) and 12-A7 (USA-Mexico marijuana) were the only two that produced the same haplotype. These two samples were analyzed previously with CE-based meth- ods and also shown to have the same haplotype (Roman et  al. 2019; Roman and Houston 2020). Samples 10-A1 (USA-Mexico marijuana) and MedMJ 10 (Chile medi- cal marijuana) were also shown to have the same haplo- type in previous studies. However, using MPS, they were distinguished by their sequences at rpl32-trnL hSTR3; 10-A1 has a 6-bp allele with the sequence TAA​AAA​, and MedMJ10 has a 6 bp allele with the sequence AAA​ AAA​. Since they are the same size, these two isoalleles could not be distinguished in previous CE-based studies (Roman et al. 2019). Previously, rps16 hSTR and clpP hSTR3 alleles were reported by their bp size due to unclear results using Sanger sequencing (Roman and Houston 2020). However, as expected, the MPS method was able to elucidate the sequences of all alleles (GenBank accessions: rps16 hSTR (MW010380–83) and clpP hSTR3 (MW010383–86), and the fragment size and sequence data were determined to be concordant. Additionally, the 11 and 15 alleles at the rpl32-trnL hSTR3 locus were unable to be confirmed by Sanger sequencing in the previous study (Roman et  al. Sequence datah Discrepancies between the genotypes obtained by MPS and CE are indicated in parentheses Italics indicate polymorphisms not analyzed previously by CE a Indicates new polymorphisms discovered in this study Sample name psbI- trnS hSTR (T) hSTRa (A) trnS-trnG SNP1 (T/C) trnS-trnG SNP2 (A/T) hSTRa (T) INDELa (CAA​TAT​) SNPa (A/T) trnS-trnG hSTR1 (Variable) trnS-trnG hSTR2 (T) hSTRa (A) trnS-trnG SNP3 (T/A) SNPa (A/C) Start location (bp) 8359 8585 8595 8684 8685 8731 8877 9018 9104 9149 9360 9463 Yoruba Nigeria (NC_027223.1) 7 10 T A 9 Absent A 15 11 12 A A H2-4 7 10 T A 9 Absent A 15 11 12 A A H3-3 7 10 T A 9 Absent A 15 11 12 A A H5-4 8 10 C T 9 Absent A 15 11 12 T A NT H5-1 7 10 T A 9 Absent A 15 11 12 T A NT H5-2 8 10 C T 9 Absent A 15 11 12 T A NT H5-4 8 12 T A 8 Absent A 16 12 (CE: 11) 11 T C H8-1 8 10 C T 9 Absent A 15 11 12 T A 10-A1 8 10 C T 9 Absent A 15 11 12 T A 12-A7 8 10 C T 9 Absent A 15 11 12 T A 16-B1 8 10 T A 8 Absent T 16 12 (CE: 11) 11 T C 21-A16 8 10 C T 9 Absent A 15 11 12 T A 35 8 10 T A 8 Present A 16 12 (CE: 11) 11 T C 41 8 10 T A 8 Absent T 16 12 (CE: 11) 11 T C MedMJ10 8 10 C T 9 Absent A 15 11 12 T A Roman et al. Journal of Cannabis Research (2022) 4:13 Page 6 of 10 Page 6 of 10 Roman et al. Journal of Cannabis Research (2022) 4:13 Roman et al. Journal of Cannabis Research polymorphisms are indicated by asterisks in Tables  2, 3, 4, 5, 6, 7, and 8. Novel polymorphisms and new vari- ants were reported to GenBank (Accession numbers: MW010378–MW010419). The trnS-trnG and rpl32- trnL hotspots had the most new polymorphisms with 12 and 11, respectively, and ycf3 had the least with only three. Analysis of a higher number of polymorphic loci is expected to show increased differences between sam- ples from different populations. Sequence datah However, it is impor- tant to note that the genetic boundaries between hemp and drug-type marijuana have been extensively blurred due to the increased demand for high cannabidiol (CBD) strains, resulting in marijuana and hemp cross-breading. In addition, the relationship between cannabinoid syn- thase gene diversity and cannabinoid content is complex and not fully understood (Grassa et al. 2021). Concordance 2019), but MPS was able to provide sequence data for these alleles (GenBank accessions: MW010387–88). Table 5  Genotypes for polymorphisms in the ycf3 region (43,383–45,478 bp) Sample name ycf3 hSTR1 (T) ycf3 hSTR2 (T) ycf hSTR3 (A) Start location (bp) 43454 44007 45034 Yoruba Nigeria (NC_027223.1) 11 11 10 H2-4 11 11 10 H3-3 11 11 10 H5-4 10 12 10 NT H5-1 11 11 10 NT H5-2 9 12 10 NT H5-4 9 10 10 H8-1 10 12 10 10-A1 10 12 10 12-A7 10 12 10 16-B1 9 10 10 21-A16 10 12 10 35 9 10 10 41 9 10 10 MedMJ10 10 12 10 Table 5  Genotypes for polymorphisms in the ycf3 region (43,383–45,478 bp) Roman et al. Journal of Cannabis Research (2022) 4:13 Page 7 of 10 Roman et al. Journal of Cannabis Research Page 7 of 10 Table 6  Genotypes for polymorphisms in the accD-psaI region (58,173–59,596 bp) Italics indicate polymorphisms not analyzed previously by CE a Indicates new polymorphisms discovered in this study Sample name accD-psal SNP1 (A/G) accD-psal SNP3 (A/C) SNPa (G/A) SNPa (T/C) accD-psal SNP2 (T/G) accD-psal STR (A) Start location (bp) 58833 58851 58921 58924 58981 59141 Yoruba Nigeria (NC_027223.1) A A G T T 10 H2-4 A A G T T 10 H3-3 A A G T T 10 H5-4 G A G T G 11 NT H5-1 A A G T T 10 NT H5-2 G A G T G 11 NT H5-4 G C G T T 10 H8-1 G A G T G 11 10-A1 G A G T G 11 12-A7 G A G T G 11 16-B1 G C A C T 10 21-A16 G A G T G 11 35 G C A C T 10 41 G C A C T 10 MedMJ10 G A G T G 11 Table 6  Genotypes for polymorphisms in the accD-psaI region (58,173–59,596 bp) Italics indicate polymorphisms not analyzed previously by CE a Indicates new polymorphisms discovered in this study all seven regions in multiple samples, higher confidence for haplotype calls, and better discrimination through sequencing more polymorphisms and identifying isoal- leles. A preliminary set of 14 samples was sequenced, and a total of 49 polymorphisms were observed, 16 of which have not been previously published. The sequence data Conclusionsh The MPS assay developed in this study provided an effec- tive method for genotyping seven chloroplast regions previously shown to be informative for intra-species vari- ation of C. sativa. It provided multiple benefits over pre- vious CE-based assays, including simultaneous analysis of Table 7  Genotypes for polymorphisms in the clpP region (70,502–72,486 bp) Sample name clpP hSTR1 (A) clpP hSTR2 (Variable) clpP hSTR3 (T) clpP hSTR4 (T) clpP INDEL (TTC​AAT​ TTA) Start location (bp) 70,912 70,981 71,663 72,016 72,028 Yoruba Nigeria (NC_027223.1) 11 TATTT​ 14 13 Absent H2-4 11 TATTT​ 14 12 Absent H3-3 11 TATTT​ 14 12 Absent H5-4 10 TTTT​ 15 12 Present NT H5-1 11 TTTT​ 14 12 Absent NT H5-2 10 TTTT​ 15 11 Present NT H5-4 11 TTTT​ 12 11 Absent H8-1 10 TTTT​ 14 11 Present 10-A1 10 TTTT​ 15 11 Present 12-A7 10 TTTT​ 14 11 Present 16-B1 12 TTTT​ 11 10 Absent 21-A16 10 TTTT​ 15 11 Present 35 12 TTTT​ 11 10 Absent 41 12 TTTT​ 11 10 Absent MedMJ10 10 TTTT​ 15 11 Present Table 7  Genotypes for polymorphisms in the clpP region (70,502–72,486 bp) Page 8 of 10 Roman et al. Journal of Cannabis Research (2022) 4:13 Roman et al. Journal of Cannabis Research Table 8  Genotypes for polymorphisms in the rpl32-trnL region (112,153–114,100 bp). Conclusionsh Discrepancies between the genotypes obtained by MPS and CE are indicated in parentheses Italics indicate polymorphisms not analyzed previously by CE a Indicates new polymorphisms discovered in this study Sample name hSTRa (T) hSTRa (A) ndhF-rpl32 INDEL (Variable) rpl32-trnL hSTR1 (A) SNPa (A/T) rpl32-trnL hSTR2 (A) hSTRa (A) rpl32-trnL SNP (A/C) rpl32-trnL hSTR3 (Variable) rpl32-trnL INDEL (TAA​ AAA​) SNPa (A/G) Start location (bp) 112,294 112,551 112,562 112,830 112,900 112,961 113,017 113,044 113,149 113,246 113,459 Yoruba Nigeria (NC_027223.1) 8 12 Absent 12 A 11 10 A 8 (7T+A) Present A H2-4 8 12 Absent 12 A 11 10 A 9( 8T+A) Absent A H3-3 8 12 Absent 12 A 11 10 A 8 (7T+A) Absent A H5-4 8 13 GAA​TTG​+10A 11 A 11 10 C 6 (6A) Present A NT H5-1 8 11 Absent 12 A 12 10 A 6 (2T+4A) Present A NT H5-2 8 13 GAA​TTG​+11A 11 A 11 10 C 6 (6A) Present A NT H5-4 8 11 GAA​TTG​+10A 11 T 11 (CE:12) 11 A 11 (7T+4A) Present A H8-1 8 13 GAA​TTG​+12A 11 A 11 10 C 6 (6A) Present A 10-A1 8 13 GAA​TTG​+11A 11 A 11 10 C 6 (T+5A) Present A 12-A7 8 13 GAA​TTG​+12A 11 A 11 10 C 6 (6A) Present A 16-B1 7 11 GAA​TTG​+10A 11 T 11 (CE:12) 11 A 14 (6T+8A) Present G 21-A16 8 13 GAA​TTG​+11A 10 A 11 10 C 6 (6A) Present A 35 7 10 GAA​TTG​+10A 11 T 11 (CE:12) 11 A 15 (11T+4A) Present G 41 7 10 GAA​TTG​+10A 11 T 11 (CE:12) 11 A 14 (6T+8A) Present G MedMJ10 8 13 GAA​TTG​+11A 11 A 11 10 C 6 (6A) Present A Roman et al. Journal of Cannabis Research (2022) 4:13 Roman et al. Journal of Cannabis Research Page 9 of 10 were concordant with CE genotypes from previous stud- ies. The high throughput ability of MPS will allow for the creation of a worldwide haplotype database of C. sativa samples. Additionally, a comprehensive database is nec- essary to understand intra-species variation and could aid law enforcement trafficking cases. Cheng Y, Houston R. Evaluation of the trnK-matK-trnK, ycf3, and accD-psal chloroplast regions to differentiate crop type and biogeographical origin of Cannabis sativa. Int J Leg Med. 2021;135(4):1235–44. Davis C, Peters D, Warshauer D, King J, Budowle B. Acknowledgements Houston R, Mayes C, King JL, Hughes-Stamm S, Gangitano D. Massively parallel sequencing of 12 autosomal STRs in Cannabis sativa. Electrophoresis. 2018b;39:2906–11. The authors would like to acknowledge Jessica Cheng for her assistance in performing laboratory experiments. The authors would like to thank the US Customs and Border Protection LSSD Southwest Regional Science Center for providing marijuana specimens for this project. Additionally, the authors would like to thank the Policia de Investigaciones in Chile and Alejandra Figueroa for their kind donation of marijuana DNA extracts. Illumina. Nextera XT DNA Library Prep Kit Reference Guide: San Diego; 2018. Illumina. Nextera XT DNA Library Prep Kit Reference Guide: San Diego; 2018. Illumina. MiSeq System Denature and Dilute Libraries Guide: San Diego; 2019. Ingman M, Gyllensten U. mtDB: Human Mitochondrial Genome Database, a resource for population genetics and medical sciences. Nucleic Acids Res. 2006;34:D749–D51. resource for population genetics and medical sciences. Nucleic Acids Res. 2006;34:D749–D51. Availability of data and materials Matielo CBDO, Lemos RPM, Sarzi DS, et al. Whole Plastome Sequences of Two Drug-Type Cannabis: Insights Into the Use of Plastid in Forensic Analyses. J Forensic Sci. 2020;65:259–65. All data generated or analyzed during this study are included in this published article [and its supplementary information files]. McElhoe JA, Holland MM, Makova KD, et al. Development and assessment of an optimized next-generation DNA sequencing approach for the mtgenome using the Illumina MiSeq. Forensic Sci Int Genet. 2014;13:20–9. Additional file 1. Houston R, Birck M, LaRue B, Hughes-Stamm S, Gangitano D. Nuclear, chloro- plast, and mitochondrial data of a US cannabis DNA database. Int J Legal Med. 2018a;132(3):713-25. Abbreviations ll l bb e at o s CE: Capillary electrophoresis; ISFG: International Society of Forensic Genetics; MPS: Massively parallel sequencing; NGS: Next generation sequencing; NTC: Negative template control; PCR: Polymerase chain reaction; PF: Passing filter; STR: Short tandem repeat; hSTR: Homopolymeric short tandem repeat. Eduardoff M, Santos C, De La Puente M, et al. Inter-laboratory evaluation of SNP-based forensic identification by massively parallel sequencing using the Ion PGM™. Forensic Sci Int Genet. 2015;17:110–21. the Ion PGM™. Forensic Sci Int Genet. 2015;17:110–21. Grassa CJ, Weiblen GD, Wenger JP, et al. A new genome assembly associates elevated cannabidiol (CBD) with hemp introgressed into marijuana. New Phytol. 2021;230:1665–79. Conclusionsh Sequencing the hypervari- able regions of human mitochondrial DNA using massively parallel sequencing: enhanced data acquisition for DNA samples encountered in forensic testing. Leg Med. 2015;17:123–7. Duke J, Lind C, Mackiewicz K, et al. Towards allele-level human leucocyte antigens genotyping–assessing two next-generation sequencing plat- forms: Ion Torrent Personal Genome Machine and Illumina MiSeq. Int J Immunogenet. 2015;42:346–58. Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Loman NJ, Misra RV, Dallman TJ, et al. Performance comparison of benchtop high-throughput sequencing platforms. Nat Biotechnol. 2012;30:434. Declarations Ethics approval and consent to participate Not applicable Mehta B, Daniel R, Phillips C, Doyle S, Elvidge G, McNevin D. Massively parallel sequencing of customised forensically informative SNP panels on the MiSeq. Electrophoresis. 2016;37:2832–40. Authors’ contributions Kim EH, Lee HY, Yang IS, Jung S-E, Yang WI, Shin K-J. Massively parallel sequenc- ing of 17 commonly used forensic autosomal STRs and amelogenin with small amplicons. Forensic Sci Int Genet. 2016;22:1–7. Conceptualization: RH; Investigation: MRG, RG; Data Curation: MRG, RG; Writing – original draft preparation: MRG; Methodology: RH; Supervision: RH; Writing – reviewing and editing: RH, RG. The author(s) read and approved the final manuscript. Koressaar T, Remm M. Enhancements and modifications of primer design program Primer3. Bioinformatics. 2007;23:1289–91. Koressaar T, Remm M. Enhancements and modifications o program Primer3. Bioinformatics. 2007;23:1289–91. Lazaruk K, Walsh PS, Oaks F, et al. Genotyping of forensic short tandem repeat (STR) systems based on sizing precision in a capillary electrophoresis instrument. Electrophoresis. 1998;19:86–93. Competing interests Competing interests The authors declare that they have no competing interests. Moorthie S, Mattocks CJ, Wright CF. Review of massively parallel DNA sequenc- ing technologies. HUGO J. 2011;5:1–12. Oh H, Seo B, Lee S, et al. Two complete chloroplast genome sequences of Can- nabis sativa varieties. Mitochondrial DNA Part A. 2016;27:2835–7. Received: 17 September 2020 Accepted: 5 March 2022 Received: 17 September 2020 Accepted: 5 March 2022 Received: 17 September 2020 Accepted: 5 March 2022 Parks M, Cronn R, Liston A. Increasing phylogenetic resolution at low taxonomic levels using massively parallel sequencing of chloroplast genomes. BMC Biol. 2009;7:84. Parson W, Ballard D, Budowle B, et al. Massively parallel sequencing of forensic STRs: considerations of the DNA commission of the International Society for Forensic Genetics (ISFG) on minimal nomenclature requirements. Forensic Sci Int Genet. 2016;22:54–63. Ethics approval and consent to participate Not applicable Consent for publication All authors consent for manuscript publication. q p Miller KW, Budowle B. A compendium of human mitochondrial DNA control region: development of an international standard forensic database. Croat Med J. 2001;42:315–27. Moorthie S, Mattocks CJ, Wright CF. Review of massively parallel DNA sequenc- ing technologies. HUGO J. 2011;5:1–12. Miller KW, Budowle B. A compendium of human mitochondrial DNA control region: development of an international standard forensic database. Croat Med J. 2001;42:315–27. Miller KW, Budowle B. A compendium of human mitochondrial DNA control region: development of an international standard forensic database. Croat Med J. 2001;42:315–27. Moorthie S, Mattocks CJ, Wright CF. Review of massively parallel DNA sequenc- ing technologies. HUGO J. 2011;5:1–12. Oh H, Seo B, Lee S, et al. Two complete chloroplast genome sequences of Can- nabis sativa varieties. Mitochondrial DNA Part A. 2016;27:2835–7. Parks M, Cronn R, Liston A. Increasing phylogenetic resolution at low Supplementary Information The online version contains supplementary material available at https://​doi.​ org/​10.​1186/​s42238-​022-​00123-2. Guo F, Yu J, Zhang L, Li J. Massively parallel sequencing of forensic STRs and SNPs using the Illumina® ForenSeq™ DNA signature prep kit on the MiSeq FGx™ forensic genomics system. Forensic Sci Int Genet. 2017;31:135–48. References Anderson S, Bankier AT, Barrell BG, et al. Sequence and organization of the human mitochondrial genome. Nature. 1981;290:457–65. Bruijns B, Tiggelaar R, Gardeniers H. Massively parallel sequencing techniques for forensics: a review. Electrophoresis. 2018;39:2642–54. Budowle B, Schmedes SE, Wendt FR. Increasing the reach of forensic genetics with massively parallel sequencing. Forensic Sci Med Pathol. 2017;13:342–9. References References Anderson S, Bankier AT, Barrell BG, et al. Sequence and organization of the human mitochondrial genome. Nature. 1981;290:457–65. Bruijns B, Tiggelaar R, Gardeniers H. Massively parallel sequencing techniques for forensics: a review. Electrophoresis. 2018;39:2642–54. Budowle B, Schmedes SE, Wendt FR. Increasing the reach of forensic genetics with massively parallel sequencing. Forensic Sci Med Pathol. 2017;13:342–9. Robinson JT, Thorvaldsdóttir H, Winckler W, et al. Integrative genomics viewer. Nat Biotechnol. 2011;29:24–6. Roman MG, Gangitano D, Houston R. Characterization of new chloroplast markers to determine biogeographical origin and crop type of Cannabis sativa. Int J Leg Med. 2019;133:1721–32. Page 10 of 10 Roman et al. Journal of Cannabis Research (2022) 4:13 Roman et al. Journal of Cannabis Research (2022) 4:13 Roman MG, Houston R. Investigation of chloroplast regions rps16 and clpP for determination of Cannabis sativa crop type and biogeographical origin. Leg Med. 2020;47:101759. Roman MG, Houston R. Investigation of chloroplast regions rps16 and clpP for determination of Cannabis sativa crop type and biogeographical origin. Leg Med. 2020;47:101759. Seo SB, King JL, Warshauer DH, Davis CP, Ge J, Budowle B. Single nucleotide polymorphism typing with massively parallel sequencing for human identification. Int J Leg Med. 2013;127:1079–86. Shokralla S, Porter TM, Gibson JF, et al. Massively parallel multiplex DNA sequencing for specimen identification using an Illumina MiSeq platform. Sci Rep. 2015;5:9687. Sucher NJ, Hennell JR, Carles MC. DNA fingerprinting, DNA barcoding, and next generation sequencing technology in plants. Plant DNA Fingerprint- ing Barcoding. Methods Mol Biol. 2012;862:13–22. Vergara D, White KH, Keepers KG, Kane NC. The complete chloroplast genomes of Cannabis sativa and Humulus lupulus. Mitochondrial DNA Part A. 2016;27:3793–4. Wang Z, Zhou D, Wang H, et al. Massively parallel sequencing of 32 forensic markers using the Precision ID GlobalFiler™ NGS STR Panel and the Ion PGM™ System. Forensic Sci Int Genet. 2017;31:126–34. Publisher’s Note S N Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. • fast, convenient online submission • thorough peer review by experienced researchers in your field • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? Choose BMC and benefit from: ? 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Comparison of the efficacy and safety of 2% lidocaine HCl with different epinephrine concentration for local anesthesia in participants undergoing surgical extraction of impacted mandibular third molars
Medicine
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a Department of Dental Anesthesiology, b Department of Dental Anesthesiology and Dental Research Institute, Seoul National University School of Dentistry, c Department of Oral & Maxillofacial Surgery, Yonsei University College of Dentistry, Seoul, d Department of Dental Anesthesiology, Dankook University College of Dentistry, Dankook University, Cheonan-si, Chungnam, e Department of Oral and Maxillofacial Surgery, School of Dentistry, Pusan National University and Institute of Translational Dental Sciences, Pusan National University, f Department of Dental Anesthesia and Pain Medicine, School of Dentistry, Pusan National University and Dental Research Institute, Pusan National University Dental Hospital, Yangsan, Gyeongnam, g College of Dentistry, Wonkwang University, Iksan city, Jeonbuk, h Department of Oral and Maxillofacial Surgery, Dankook University College of Dentistry, Dankook University, Cheonan-si, Chungnam, i Department of Oral & Maxillofacial Surgery, Kyung Hee University Dental Hospital Kyung Hee University School of Dentistry, Seoul, j R&D Center, Huons Co. Ltd., College of Pharmacy, Hanyang University, Ansan-si, Kyeonggi-do, Republic of Korea. Received: 13 January 2017 / Received in final form: 30 March 2017 / Accepted: 3 April 2017 Abstract Abstract Background: The most commonly impacted tooth is the third molar. An impacted third molar can ultimately cause acute pain, infection, tumors, cysts, caries, periodontal disease, and loss of adjacent teeth. Local anesthesia is employed for removing the third molar. This study aimed to evaluate the efficacy and safety of 2% lidocaine with 1:80,000 or 1:200,000 epinephrine for surgical extraction of bilateral impacted mandibular third molars. Methods: Sixty-five healthy participants underwent surgical extraction of bilateral impacted mandibular third molars in 2 separate visits while under local anesthesia with 2% lidocaine with different epinephrine concentration (1:80,000 or 1:200,000) in a double- blind, randomized, crossover trial. Visual analog scale pain scores obtained immediately after surgical extraction were primarily evaluated for the 2 groups receiving different epinephrine concentrations. Visual analog scale pain scores were obtained 2, 4, and 6 hours after administering an anesthetic. Onset and duration of analgesia, onset of pain, intraoperative bleeding, operator’s and participant’s overall satisfaction, drug dosage, and hemodynamic parameters were evaluated for the 2 groups. Results:There were no statistically significant differences between the 2 groups in any measurements except hemodynamic factors (P>.05). Changes in systolic blood pressure and heart rate following anesthetic administration were significantly greater in the group receiving 1:80,000 epinephrine than in that receiving 1:200,000 epinephrine (P.01). Results:There were no statistically significant differences between the 2 groups in any measurements except hemodynamic factors (P>.05). Changes in systolic blood pressure and heart rate following anesthetic administration were significantly greater in the group receiving 1:80,000 epinephrine than in that receiving 1:200,000 epinephrine (P.01). Conclusion: The difference in epinephrine concentration between 1:80,000 and 1:200,000 in 2% lidocaine liquid does not affect the medical efficacy of the anesthetic. Furthermore, 2% lidocaine with 1:200,000 epinephrine has better safety with regard to hemodynamic parameters than 2% lidocaine with 1:80,000 epinephrine. Therefore, we suggest using 2% lidocaine with 1:200,000 epinephrine rather than 2% lidocaine with 1:80,000 epinephrine for surgical extraction of impacted mandibular third molars in hemodynamically unstable patients. Abbreviations: ASA = American Society of Anesthesiologist, BP = blood pressure, HR = heart rate, IP = investigational product, SD = standard deviation, TM = third molar, VAS = visual analog scale. Abbreviations: ASA = American Society of Anesthesiologist, BP = blood pressure, HR = heart rate, IP = investigational product, SD = standard deviation, TM = third molar, VAS = visual analog scale. Editor: Kazuo Hanaoka. Funding: This work was supported by Huons Co., Ltd. Pharmaceutical Company, Republic of Korea. The authors have no conflicts of interest to disclose. a Department of Dental Anesthesiology, b Department of Dental Anesthesiology and Dental Research Institute, Seoul National University School of Dentistry, c Department of Oral & Maxillofacial Surgery, Yonsei University College of Dentistry, Seoul, d Department of Dental Anesthesiology, Dankook University College of Dentistry, Dankook University, Cheonan-si, Chungnam, e Department of Oral and Maxillofacial Surgery, School of Dentistry, Pusan National University and Institute of Translational Dental Sciences, Pusan National University, f Department of Dental Anesthesia and Pain Medicine, School of Dentistry, Pusan National University and Dental Research Institute, Pusan National University Dental Hospital, Yangsan, Gyeongnam, g College of Dentistry, Wonkwang University, Iksan city, Jeonbuk, h Department of Oral and Maxillofacial Surgery, Dankook University College of Dentistry, Dankook University, Cheonan-si, Chungnam, i Department of Oral & Maxillofac Surgery, Kyung Hee University Dental Hospital Kyung Hee University School of Dentistry, Seoul, j R&D Center, Huons Co. Ltd., College of Pharmacy, Hanyang University, Ansan-si, Kyeonggi-do, Republic of Korea. ∗Correspondence: Hyun Jeong Kim, Department of Dental Anesthesiology and Dental Research Institute, Seoul National University School of Dentistry, Jongno-gu, Seoul, Republic of Korea (e-mail: dentane05@gmail.com). Copyright © 2017 the Author(s). Published by Wolters Kluwer Health, Inc. This is an open access article distributed under the Creative Commons Attribution-Share Alike License 4.0, which allows others to remix, tweak, and build upon the work, even for commercial purposes, as long as the author is credited and the new creations are licensed under the identical terms. Medicine (2017) 96:21(e6753) Received: 13 January 2017 / Received in final form: 30 March 2017 / Accepted: 3 April 2017 http://dx.doi.org/10.1097/MD.0000000000006753 Medicine ® Clinical Trial/Experimental Study OPEN Medicine ® Medicine ® py g ( ) y , This is an open access article distributed under the Creative Commons Attribution-Share Alike License 4.0, which allows others to remix, tweak, and build upon the work, even for commercial purposes, as long as the author is credited and the new creations are licensed under the identical terms. M di i ( ) ( ) Funding: This work was supported by Huons Co., Ltd. Pharmaceutical Company, Republic of Korea. Department of Oral & Maxillofacial Surgery, Yonsei University College of Dentistry, Seoul, Department of Dental Anesthesiology, Dankook University College of Dentistry, Dankook University, Cheonan-si, Chungnam, e Department of Oral and Maxillofacial Surgery, School of Dentistry, Pusan National University and Institute of Translational Dental Sciences, Pusan National University, f Department of Dental Anesthesia and Pain Medicine, School of Dentistry, Pusan National University and Dental Research Institute, Pusan National University Dental Hospital, Yangsan, Gyeongnam, g College of Dentistry, Wonkwang University, Iksan city, Jeonbuk, h Department of Oral and Maxillofacial Surgery, Dankook University College of Dentistry, Dankook University, Cheonan-si, Chungnam, i Department of Oral & Maxillofacia Surgery, Kyung Hee University Dental Hospital Kyung Hee University School of Dentistry, Seoul, j R&D Center, Huons Co. Ltd., College of Pharmacy, Hanyang University, Ansan-si, Kyeonggi-do, Republic of Korea. ∗Correspondence: Hyun Jeong Kim, Department of Dental Anesthesiology and Dental Research Institute, Seoul National University School of Dentistry, Jongno-gu, Seoul, Republic of Korea (e-mail: dentane05@gmail.com). Copyright © 2017 the Author(s). Published by Wolters Kluwer Health, Inc. This is an open access article distributed under the Creative Commons Attribution-Share Alike License 4.0, which allows others to remix, tweak, and build upon the work, even for commercial purposes, as long as the author is credited and the new creations are licensed under the identical terms. Medicine (2017) 96:21(e6753) Received: 13 January 2017 / Received in final form: 30 March 2017 / Accepted: 3 April 2017 ∗Correspondence: Hyun Jeong Kim, Department of Dental Anesthesiology and Dental Research Institute, Seoul National University Seoul, Republic of Korea (e-mail: dentane05@gmail.com). 2.3. Participants and study design Local anesthesia is employed for removing TMs. Local anesthesia is a reversible blockade of nerve conduction in a circumscribed area that produces loss of sensation.[4,5] Lidocaine is the most commonly used local anesthetic agent in dentistry and has excellent efficacy and safety.[6] The rare incidence of serious life-threatening hypersensitivity reactions associated with lido- caine is the biggest clinical advantage, due to the frequent use of local anesthesia in dentistry.[4] However, the possible neurotox- icity when used in high concentration is an important limitation of its use.[7,8] The study subjects included 65 participants with symmetrically located impacted mandibular TMs, as detected in panoramic radiographs. They were recruited by advertisements. The inclusion criteria were age over 19 years; physical grade 1 or 2 according to the American Society of Anesthesiologists (ASA);[21,22] requirement of bilateral surgical extraction of impacted mandibular TMs (either mesioangular or horizontal angulation of Winter’s classification) and similar degree of impaction in both sides; and subjects who agreed and signed written inform consent. The exclusion criteria were a history of hypersensitivity to lidocaine or this group of drugs; presence of active infection or abscess at the time of extraction; coagulation disorder, hyperthyroidism, atherosclerosis, heart failure, con- vulsions, uncontrolled hypertension, or diabetes mellitus; current use of vasoconstrictors, ergot alkaloids, phenothiazines, butyr- ophenones, tricyclic antidepressants, monoamine oxidase inhib- itors, sedatives, or anxiolytics; use of anticoagulants or antiplatelets, including aspirin, systemic corticosteroids, or nonsteroidal anti-inflammatory drugs, within 7 days before the extraction date; use of analgesics within 24hours before the extraction; requirement for sedatives or antianxiolytic drugs during the extraction; other operative plans requiring general or local anesthesia during the clinical trial period; other medical history that might affect the clinical trial (e.g., malignant tumor, immunodeficiency, kidney disease, liver disease, lung disease, or unstable psychiatric condition); pregnancy or breastfeeding; planned pregnancy or intention of using contraception during the clinical trial period; use of other investigated products or medical devices within 4 weeks before the extraction date; and a history of prior oral or maxillofacial surgery. There are several benefits of the addition of a vasoconstrictor to a local anesthetic: improvement in the duration and quality of anesthesia,[9] reduction of blood loss throughout the opera- tion,[10] reduction in the peak plasma concentration of the anesthetic agent,[11,12] and decrease of the minimum concentra- tion of anesthetic necessary for nerve block.[13,14] Epinephrine is the most studied and widely used vasoconstrictor. 1. Introduction An impacted tooth is one that either fails to develop in its natural location or is hindered from eruption by an overgrowth of soft tissue, compact bone, or neighboring teeth. The treatment for such an impacted tooth is extraction of either the obstructing interference or the tooth itself.[1] The most commonly impacted tooth is the third molar (TM). An impacted, partially erupted, or fully erupted TM can remain asymptomatic for many years but can ultimately cause acute pain, infection, tumors, cysts, caries, periodontal disease, and loss of adjacent teeth.[2] In addition, there are reports of relapse after orthodontic retention and orthodontic crowding caused by a retained or an impacted TM.[3] Local anesthesia is employed for removing TMs. Local anesthesia is a reversible blockade of nerve conduction in a circumscribed area that produces loss of sensation.[4,5] Lidocaine is the most commonly used local anesthetic agent in dentistry and has excellent efficacy and safety.[6] The rare incidence of serious life-threatening hypersensitivity reactions associated with lido- caine is the biggest clinical advantage, due to the frequent use of local anesthesia in dentistry.[4] However, the possible neurotox- icity when used in high concentration is an important limitation of its use.[7,8] An impacted tooth is one that either fails to develop in its natural location or is hindered from eruption by an overgrowth of soft tissue, compact bone, or neighboring teeth. The treatment for such an impacted tooth is extraction of either the obstructing interference or the tooth itself.[1] The most commonly impacted tooth is the third molar (TM). An impacted, partially erupted, or fully erupted TM can remain asymptomatic for many years but can ultimately cause acute pain, infection, tumors, cysts, caries, periodontal disease, and loss of adjacent teeth.[2] In addition, there are reports of relapse after orthodontic retention and orthodontic crowding caused by a retained or an impacted TM.[3] Medicine Medicine University Dental Hospital (2-2014-0017); and Wonkwang University Dental Hospital (WKDIRB 201410-02)]. Written informed consent was obtained from all participants. All aspects of participant privacy and confidentiality were preserved. The study was conducted in accordance to the Declaration of Helsinki.[20] 2.3. Participants and study design Epinephrine produces its vasoconstrictor effects by binding to and stimulating a1-adrenergic receptors in the walls of arterioles.[15] At low systemic concentrations usually employed in dental anesthesia, epinephrine can increase cardiac output, heart rate (HR), and peripheral vasodilation.[16] Lidocaine concentration affects the efficacy and safety of local anesthesia in patients undergoing surgical extraction of an impacted mandibular TM.[17,18] In addition, 2% lidocaine solution used for inferior alveolar nerve block in patients with symptomatic irreversible pulpitis had similar success rates when used with 1:80,000 or 1:200,000 epinephrine concentration.[19] This suggests that the efficacy of 2% lidocaine solution will not differ when it is used with 1:80,000 or 1:200,000 epinephrine concentration. However, the efficacy and safety of 2% lidocaine with 1:80,000 or 1:200,000 epinephrine in surgical extraction of bilateral impacted mandibular TMs has never been compared so far. Hence, we aimed in this study to evaluate the efficacy and safety of 2% lidocaine with 1:80,000 or 1:200,000 epinephrine in surgical extraction of bilateral impacted mandibular TMs. The statistician randomly assigned the participants using the block randomization method with SAS (SAS Institute, Cary, NC). The statistician delivered a list of random assignment codes to the pharmacy packager. 2% lidocaine with 1:200,000 epinephrine (2% Lidocaine HCL Injection, Huons Co., Ltd, Seongnam, Korea) and 2% lidocaine with 1:80,000 epinephrine (2% Lidocaine HCL:Epinephrin inj. , 3M, Seoul, Korea) were packaged so that they could not be recognized and were distributed to the trial institutes. The participants were randomly assigned to receive 2% lidocaine with epinephrine at a concentration of 1:80,000 (the L80 group) or 1:200,000 (the L200 group) at a 1:1 ratio. This study was double blinded; neither the operator nor the participant was aware of which anesthetic was administered. Each participant required equal surgical care on opposite sides of the mandible, which was conducted in 2 visits, 1 to 4 weeks apart. At the first visit, the participant was randomly assigned to receive 2% lidocaine with 1 of the 2 concentrations of epinephrine (L80 or L200). At the second visit, the participant received 2% lidocaine with epinephrine at the 2.2. Design The trial was a multicenter, randomized, double-blind, crossover, phase IV trial. Abstract Keywords: epinephrine, hemodynamics, lidocaine, local anesthesia, third molar Keywords: epinephrine, hemodynamics, lidocaine, local anesthesia, third molar ∗Correspondence: Hyun Jeong Kim, Department of Dental Anesthesiology and Dental Research Institute, Seoul National University School of Dentistry, Jongno-gu, Seoul, Republic of Korea (e-mail: dentane05@gmail.com). py g ( ) y , This is an open access article distributed under the Creative Commons Attribution-Share Alike License 4.0, which allows others to remix, tweak, and build upon the work, even for commercial purposes, as long as the author is credited and the new creations are licensed under the identical terms. 1 Karm et al. Medicine (2017) 96:21 2.4. Measurements The following parameters were assessed. Efficacy variables were divided into primary outcome and secondary outcomes. The primary outcome was “visual analog scale (VAS) pain score measured immediately after surgical extraction.” The secondary outcomes were “The onset time of local anesthesia,” “Duration of analgesia,” “VAS pain score recorded 2, 4, and 6hours after the administration of local anesthetics,” “The time interval between the administration of the local anesthetics and the first postoperative pain sensation,” “Perioperative bleeding assess- ment by the operator,” “Operator’s overall satisfaction,” “Participant’s overall satisfaction,” and “Drug dosage adminis- tered.” The safety variables were “Adverse event” and “Vital signs.” “VAS pain scores measured immediately after the surgical extraction” was measured by the VAS score. The VAS consisted of a horizontal row of light-emitting diodes of 100-mm length. The participants were told that the left end of the scale, labeled “minimum,” corresponded to “no pain at all,” and the right end, labeled “maximum,” corresponded to “maximum imaginable pain.”[24,25] The participants indicated their level of pain and the operator recorded their response. “The onset time of local anesthesia” was assessed by the loss of sensibility of the lower lip, the corresponding half of the tongue, and the mucosa. “Duration of analgesia” was measured by the lack of sensibility of the lower lip, tongue, and mucosa. The participants recorded the moment that the anesthesia had worn off. “Drug dosage administered” was measured by the total volume (mL) of anesthetic solution used during the operation. “Perioperative bleeding assessment by the operator,” “operator’s overall satisfaction,” and “partic- ipant’s overall satisfaction” were measured by the Likert scale.[26] The Likert scale scores 1–5 of “Perioperative bleeding assessment by the operator” were presented with 1 anchored by “a few bleeding” and 5 anchored by “very much bleeding.” The Likert scale 1–5 of “Operator’s overall satisfaction” and “participant’s overall satisfaction” were presented with 1 anchored by “very dissatisfied” and 5 anchored by “very satisfied.” “Adverse event” referred to an undesirable and unintended sign, symptom, or disease that occurred during the administration or use of a clinical trial drug. If an adverse event from the first visit continued to the second visit, it was not regarded as an adverse event resulting from the second visit. Any adverse event occurring before the extraction of the remaining mandibular TM was considered an adverse event resulting from the first visit. 2.5. Statistical analysis Categorical variables are presented as absolute numbers and percentages. Means with standard deviations (SD) were used when the continuous variables followed the normal distribution, and medians with interquartile range were used when the continuous variables did not follow the normal distribution. The statistical significance of the difference in parameters within each group was determined by the paired t-test or by the Wilcoxon signed-rank test when the distribution was not normal. Adverse events were assessed with Pearson’s x2 test or Fisher’s exact test to find the homogeneity between the 2 groups. Vital signs were assessed with the paired t-test or the Wilcoxon signed-rank test. A 2-tailed P-value of<.05 was considered to indicate a statistically significant difference. Analyses were performed using SPSS Statistics version 21 (SPSS, Chicago, IL). 2.6. Sample size calculation The sample size calculation was based on an a-level type I error of 0.025 for a 1-tailed test, a b-level type II error of 0.1, and a statistical power of 90%. Following research on the efficacy of 4% articaine with 1:100,000 epinephrine versus 1:200,000 epinephrine,[27–29] the VAS scores for postoperative pain, and considering a difference of 15mm as clinically significant (estimated mean standard deviation, 27mm), the sample size was calculated as 23 participants in each group. A dropout rate of 20% was assumed, and at least 29 participants were enrolled in each group. The upper limit of the VAS score was set at 15mm to determine the noninferiority tolerance limit with a confidence interval of 97.5%. If the measurement is below the upper limit, the result for the L200 group is noninferior to the result for the L80 group. 2.1. Study oversight The investigation was registered at http//www.clinicaltrials.gov (NCT 02696369) and performed according to the guidelines for the proper conduct of medical research on human participants. The study protocol was reviewed and approved by the research ethics boards of each participating hospital in Korea [Seoul National University Dental Hospital (CME 14002); Busan National University Dental Hospital (PNUDH-2014-001-MS); Kyung Hee University Dental Hospital (KHDIRB 1410-5); Dankook University Dental Hospital (H-1407/009/005); Yonsei 2 Karm et al. Medicine (2017) 96:21 www.md-journal.com adverse event was assessed by the following criteria. Mild: causes minimal inconvenience without interfering with the normal daily life (functioning) of the participant and is easily tolerated. Moderate: causes inconvenience that significantly interferes with the normal daily life (functioning) of the participant. Severe: makes daily life (functioning) of the participant impossible. “Vital signs” was the following: systolic, diastolic blood pressure (BP), and HR were measured by oscillometric BP measurement devices and pulse oximetry automatically and noninvasively. BP and HR were measured as follows: immediately before drug administration; up to 5 minutes after drug administration: 5 times every 1 minute; 5 minutes to 20 minutes after drug administra- tion: 5 times every 3 minutes; 20 minutes after drug administra- tion to 70 minutes: measured every 5 minutes. concentration that was not used in the first visit. To minimize factors affecting the efficacy and safety evaluation of the trial drugs, all operations, measurements, and postoperative controls were performed by a trained operator in each research institute. Initially, the participants were administered extraoral antisepsis with 0.2% chlorhexidine gluconate and intraoral antisepsis with 0.12% chlorhexidine gluconate. Afterward, the participants received a regional anesthetic blockade of the inferior alveolar nerves and long buccal nerve infiltration with each 1.8mL of the anesthetic solution. If the participant complained of pain during the extraction, an additional local anesthetic was administered, and the additional dosage was recorded in the case record. Extraction of the TM was performed by the buccal approach technique using rotation instruments.[23] At the end of the operation, the operating sites were thoroughly irrigated, suctioned, and sutured. 2.4. Measurements The frequencies and percentages of occurrence of adverse events and adverse drug reactions were calculated for each group. The severity of an 3.1. Participant flow Sixty-nine participants were recruited. Sixty-five were eligible and were randomly assigned to the L80 or the L200 group. Fifty-one (78.5%) of the 65 participants completed the study, including 24 of 31 participants in the initial L200 group (77.4%) and 27 of 34 participants in the initial L80 group (79.4%). The participants underwent their first operation with each concentration of local anesthesia and underwent operation with the other concen- trations of local anesthesia on the other side after 1 to 4 weeks. Ten participants dropped out because of randomization error. Four participants were omitted because the study drugs were not administered or the inclusion or exclusion criteria were violated. 3 Karm et al. Medicine (2017) 96:21 Medicine igure 1. Flow chart of clinical trial procedure and randomization of double-blind, crossover study with all participants receiving both treatments. IP=investig roduct, L80=2% lidocaine with 1:80,000 epinephrine, L200=2% lidocaine with 1:200,000 epinephrine, n=group size. Figure 1. Flow chart of clinical trial procedure and randomization of double-blind, crossover study with all participants receiving product, L80=2% lidocaine with 1:80,000 epinephrine, L200=2% lidocaine with 1:200,000 epinephrine, n=group size. Figure 1. Flow chart of clinical trial procedure and randomization of double-blind, crossover study with all participants receiving both treatments. IP=investigational product, L80=2% lidocaine with 1:80,000 epinephrine, L200=2% lidocaine with 1:200,000 epinephrine, n=group size. The total number of samples was 102, because 51 participants had anesthesia with 2 concentrations of epinephrine (Fig. 1). The baseline characteristics of the sample are depicted in Table 1. Table 1 Demographic characteristics of the participants (n=65). Parameters Total (n=65) Age, y 24.1±5.0 Gender Male 34 (52.31%) Female 31 (47.69%) Height, cm 167.6±8.0 Weight, kg 63.8±13.1 ASA class I 65 (100%) Data are expressed as means±standard deviation, numbers (%). ASA=American Society of Anesthesiologist. Table 1 Demographic characteristics of the participants (n=65). Parameters Total (n=65) Age, y 24.1±5.0 Gender Male 34 (52.31%) Female 31 (47.69%) Height, cm 167.6±8.0 Weight, kg 63.8±13.1 ASA class I 65 (100%) Data are expressed as means±standard deviation, numbers (%). ASA=American Society of Anesthesiologist. 3.2. Efficacy results Pain experienced during surgical extraction was measured immediately after the operation by VAS. The mean±SD VAS score was 13.7±1.9mm in the L80 group and 20.0±2.5mm in the L200 group (Table 2). Although the participants in the L200 group reported having experienced more intense pain than those in the L80 group (P=.055), the upper limit of the 1-sided confidence interval was 12.9mm, which is lower than the noninferiority tolerance of 15mm (Table 2). Therefore, the pain control of L200 was noninferior to that of L80. The secondary Data are expressed as means±standard deviation, numbers (%). ASA=American Society of Anesthesiologist. Data are expressed as means±standard deviation, numbers (%). ASA=American Society of Anesthesiologist. 4 Karm et al. Medicine (2017) 96:21 www.md-journal.com Table 3 Comparisons of secondary outcomes after the surgical extraction for L80 and L200 groups. Parameters L80 (n=51) L200 (n=51) P value Onset of anesthesia, min 4.9±4.1 5.2±4.1 .447 Duration of anesthesia, min 183.5±5.0 182.2±5.4 .758 VAS at 2 h after anesthetic injection, mm 17.2±2.3 21.04±2.2 .405 VAS at 4 h after anesthetic injection, mm 38.8±2.5 35.7±2.3 .433 VAS at 6 h after anesthetic injection, mm 34.8±2.6 38.0±2.7 .267 Onset of pain, min 255.5±11.0 237.5±14.9 .246 Bleeding assessment by operator (score 1–5) 2.0±0.1 2.2±0.1 .206 Operator’s overall satisfaction (score 1–5) 3.9±0.9 3.8±1.0 .548 Patient’s overall satisfaction (score 1–5) 3.6±0.1 3.7±0.1 .693 Drug dosage administered, mL 3.6±0.1 3.6±0.2 .163 Data are expressed means±standard deviation. L80=2% lidocaine with 1: 80,000 epinephrine, L200=2% lidocaine with 1: 200,000 epinephrine, VAS=visual analog scale. Table 2 Comparison of VAS after the surgical extraction for L80 and L200 groups. Parameters L80 (n=51) L200 (n=51) Mean (97.5% CI) P value VAS, mm 13.7±1.9 20.0±2.5 12.9 .055 Data are expressed means±standard deviation. CI=confidence interval, L80=2% lidocaine with 1: 80,000 epinephrine, L200=2% lidocaine with 1: 200,000 epinephrine, VAS=visual analog scale. Comparisons of secondary outcomes after the surgical extraction for L80 and L200 groups. efficacy analysis was carried out to compare the time of onset of anesthesia for the 2 concentrations of epinephrine. The mean± SD time of onset was 4.9±4.1minutes in the L80 group and 5.2 ±4.1minutes in the L200 group, with no significant difference between the groups (P=.447) (Table 3). The mean±SD duration of action of anesthesia was 183.5±5.0minutes in the L80 group and 182.2±5.4minutes in the L200 group, with no significant difference between the groups (P=.758) (Table 3). 3.2. Efficacy results There were no significant differences in the mean±SD VAS pain scores between the 2 groups at 2, 4, and 6hours after administration of the anesthetic (P>.05) (Table 3). The mean±SD time taken for the participant to report postoperative pain was 255.5±11.0 minutes in the L80 group and 237.5±14.9minutes in the L200 group, with no significant difference between the groups (P=.246) (Table 3). The mean±SD bleeding tendency was 2.0± 0.1 in the L80 group and 2.2±0.1 in the L200 group, with no significant difference between the groups (P=.206) (Table 3). The mean±SD overall satisfaction score of the operators was 3.9 ±0.9 in the L80 group and 3.8±1.0 in the L200 group, with no significant difference between the groups (P=.548) (Table 3). The mean±SD overall satisfaction score of the participants was 3.6±0.1 in the L80 group and 3.7±0.1 in the L200 group, with no significant difference between the groups (P=.693) (Table 3). The mean±SD drug dose administered was 3.6±0.1 in the L80 group and 3.6±0.2 in the L200 group, with no significant difference between the groups (P=.163) (Table 3). Data are expressed means±standard deviation. L80=2% lidocaine with 1: 80,000 epinephrine, L200=2% lidocaine with 1: 200,000 epinephrine, VAS=visual analog scale. and 3 participants (4.7%) in the L200 group. Alveolar osteitis occurred in 4 participants, 2 from each group, and inflammation of the administration site occurred in 1 participant in the L200 group. There was no statistically significant difference between the groups in the frequency of these adverse events (P=1.000). Adverse events away from the operating site occurred in 2 participants (3.1%) in the L80 group and 3 participants (4.7%) in the L200 group. These were diarrhea and headache in 1 participant each in the L80 group and lower abdominal pain, myalgia,and temporomandibular joint syndrome in 1 participant each in the L200 group. One mild and 1 moderate adverse event occurred in the L80 group. Of the 3 adverse events in the L200 group, 2 were considered mild and the other was considered moderate. There was no statistically significant difference between the groups in the frequency of these adverse events (P=1.000). Changes in the participants’ vital signs (systolic and diastolic BP and HR) from before and after administration of the anesthetics were analyzed. Vital signs measured after the administration of anesthetics were significantly increased compared with baseline measurements. 3.2. Efficacy results In the L80 group, the changes in vital signs were systolic BP 14.1±10.2mm Hg (P<.001), diastolic BP –10.8±12.9mm Hg (P<.001), and HR 14.8±11.1 (P<.001) (Table 4). In the L200 group, the changes in g p BP=blood pressure, L80=2% lidocaine with 1: 80,000 epinephrine, L200=2% lidocaine with 1: 200,000 epinephrine. References [1] Walters H. Lower third molar treatment. Br Dent J 1997;182:207. f l h f h d l h d l [2] Swift JQ, Nelson WJ. The nature of third molars: are third molars different than other teeth? Atlas Oral Maxillofac Surg Clin North Am 2012;20:159–62. [3] McCoy JM. Complications of retention: pathology associated with retained third molars. Atlas Oral Maxillofac Surg Clin North Am 2012;20:177–95. This study comparatively evaluated the efficacy and safety of 2% lidocaine with 1:80,000 or 1:200,000 epinephrine in surgical extraction of bilateral impacted mandibular TMs. Although there was a difference in VAS pain scores between the 2 groups after the operation, it was not statistically significant (P>.05) (Tables 2 and 3). There was also a difference between the groups in time of onset of pain after the operation, but it was also not statistically significant (P=.246) (Table 3). The changes in systolic BP and HR after the administration of the anesthetics were significantly greater in the L80 group than in the L200 group (P.01) (Table 4). There were no severe adverse events resulting from the anesthetics. [4] Becker DE, Reed KL. Local anesthetics: review of pharmacological considerations. Anesth Prog Summer 2012;59:90–101. quiz 102-103. [5] Ogle OE, Mahjoubi G. Local anesthesia: agents, techniques, and complications. Dent Clin North Am 2012;56:133–48. ix. [6] Haas DA, Lennon D. Local anesthetic use by dentists in Ontario. Can Dent Assoc 1995;61:297–304. [7] Moore PA, Hersh EV. Local anesthetics: pharmacology and toxicity. Dent Clin North Am 2010;54:587–99. [8] Kim DD, Asif A, Kataria S. Presentation of neurolytic effect of 10% lidocaine after perineural ultrasound guided injection of a canine sciatic nerve: a pilot study. Korean J Pain 2016;29:158–63. [9] Jastak JT, Yagiela JA. Vasoconstrictors and local anesthesia: a review and rationale for use. J Am Dent Assoc 1983;107:623–30. Our limit for noninferiority tolerance of 15mm is very strict, because 1 clinical study of articaine versus lidocaine for TM extraction reported that a difference of 20mm in the VAS pain score was clinically significant, with a sample size of 20 patients in each group, a type II error of 0.2, a type I error of 0.05, and a statistical power of 80%.[28] This is significant meaningful that there is no difference in VAS between the 2 groups in our study, despite the strict limit. [10] Sveen K. 3.3. Safety results After administration of the anesthetic, adverse events at the extraction site occurred in 2 participants (3.1%) in the L80 group Table 4 Comparisons of vital signs before and after the administration for L200 and L80 groups. Parameters L80 (n=51) P value within comparison L200 (n=51) P value within comparison P value between comparison Systolic BP, mm Hg <.001 ∗ P<.001 ∗ .002† Baseline 119.4±13.2 120.5±13.2 Maximum 133.5±14.6 129.8±12.5 Difference 14.1±10.2 9.3±7.3 Diastolic BP, mm Hg <.001‡ <.001 ∗ .205† Baseline 72.1±10.4 72.4±10.0 Maximum 61.3±10.9 64.0±8.6 Difference 10.8±12.9 -8.4±6.6 Heart rate, beats/min <.001 ∗ <.001 ∗ .010† Baseline 82.3±11.7 83.9±13.2 Maximum 97.1±15.6 94.4±13.0 Difference 14.8±11.1 10.5±12.5 Data are expressed as means±standard deviation. Comparisons of vital signs before and after the administration for L200 and L80 groups. 5 5 Karm et al. Medicine (2017) 96:21 Medicine vital signs were systolic BP 9.3±7 .3mm Hg (P<.001), diastolic BP –8.4±6.6mm Hg (P<.001), and HR 10.5±12.5 (P<.001) (Table 4). vital signs were systolic BP 9.3±7 .3mm Hg (P<.001), diastolic BP –8.4±6.6mm Hg (P<.001), and HR 10.5±12.5 (P<.001) (Table 4). studies should be conducted in patients with cardiovascular disease because of a lack of studies to date on the use of 2% lidocaine with 1:200,000 epinephrine in this patient population. 4. Discussion This work was supported by Huons Co., Ltd. Pharmaceutical Company, Republic of Korea. The TM is the most commonly impacted tooth, and local anesthesia is necessary for treatment. Lidocaine is the most commonly used local anesthetic, and epinephrine is an excellent vasoconstrictor that is added to lidocaine.[6,7] Efficacy and complications differ depending on the concentration of epineph- rine added to lidocaine.[9,15,16,30,31] Studies of the efficacy and safety of 2% lidocaine with 1:80,000 or 1:200,000 epinephrine for treatment of the impacted TM have not previously been conducted. This work was supported by Huons Co., Ltd. Pharmaceutical Company, Republic of Korea. References Effect of the addition of a vasoconstrictor to local anesthetic solution on operative and postoperative bleeding, analgesia and wound healing. Int J Oral Surg 1979;8:301–6. [11] Bromage PR, Robson JG. Concentrations of lignocaine in the blood after intravenous, intramuscular epidural and endotracheal administration. Anaesthesia 1961;16:461–78. [12] Wilkinson GR, Lund PC. Bupivacaine levels in plasma and cerebrospinal fluid following peridural administration. Anesthesiology 1970;33:482–6. [13] Luduena FP. Effect of some vasoconstrictors on intradermal anesthesia. J Dent Res 1960;39:947–54. In dentistry, epinephrine is the most commonly used vasocon- strictor for local anesthesia to provide excellent anesthesia and bleeding control.[9] Although epinephrine is an effective vasocon- strictor and is generally safe, it has many adverse effects depending on the dosage, such as hypertension, tachycardia, arrhythmia, and circulatory failure, especially in patients with cardiovascular diseases.[30,31] Focusing on this point, clinical trials using 6 to 8 cartridges of the anesthetic solution with 1:100,000 epinephrine concentration found increases in BP and HR.[32,33] In the present study, there were no differences between the 2 groups in adverse eventsafteradministrationoftheanesthetics.Therefore,epinephrine at 1:200,000 concentration is presumed to be equally effective as the 1:80,000 concentration and safer than the 1:80,000 concentration for patients with hemodynamically unstable patients. [14] Aberg G. Studies on the duration of local anesthesia: a possible mechanism for the prolonging effect of “vasoconstrictors” on the duration of infiltration anesthesia. Int J Oral Surg 1980;9:144–7. [15] Haas DA. An update on local anesthetics in dentistry. Can Dent Assoc 2002;68:546–51. [16] Sisk AL. Vasoconstrictors in local anesthesia for dentistry. Anesth Prog 1992;39:187–93. [17] Ping B, Kiattavorncharoen S, Durward C, et al. Hemodynamic changes associated with a novel concentration of lidocaine HCl for impacted lower third molar surgery. J Dent Anesth Pain Med 2015;15:121–8. [18] Ping B, Kiattavorncharoen S, Saengsirinavin C, et al. The efficacy of an elevated concentration of lidocaine HCl in impacted lower third molar surgery. J Dent Anesth Pain Med 2015;15:69–76. [19] Aggarwal V, Singla M, Miglani S, et al. Comparison of the anaesthetic efficacy of epinephrine concentrations (1: 80 000 and 1: 200 000) in 2% lidocaine for inferior alveolar nerve block in patients with symptomatic irreversible pulpitis: a randomized, double-blind clinical trial. Int Endod J 2014;47:373–9. p y y p A limitation of this study is that all participants were ASA class I. Since systolic BP and HR increased even in healthy participants, we can predict that vital signs would be unstable in unhealthy participants. References However, this study could not proceed to unhealthy participants in ASA class III or higher because of ethical issues. In conclusion, a difference in epinephrine concentration between 1:80,000 and 1:200,000 in 2% lidocaine liquid does not affect medical efficacy. Furthermore, 2% lidocaine with 1:200,000 epinephrine has better safety with regard to hemodynamic parameters than 2% lidocaine with 1:80,000 epinephrine. Therefore, we suggest using 2% lidocaine with 1:200,000 epinephrine in surgical extractions of impacted mandibular TMs rather than 2% lidocaine with 1:80,000 epinephrine in hemodynamically unstable patients. Further A limitation of this study is that all participants were ASA class I. Since systolic BP and HR increased even in healthy participants, we can predict that vital signs would be unstable in unhealthy participants. However, this study could not proceed to unhealthy participants in ASA class III or higher because of ethical issues. [20] WHOWorld Medical Association Declaration of Helsinki. Ethical principles for medical research involving human subjects. Bull World Health Organ 2001;79:373–4. [21] Dripps RD, Lamont A, Eckenhoff JE. The role of anesthesia in surgical mortality. JAMA 1961;178:261–6. In conclusion, a difference in epinephrine concentration between 1:80,000 and 1:200,000 in 2% lidocaine liquid does not affect medical efficacy. Furthermore, 2% lidocaine with 1:200,000 epinephrine has better safety with regard to hemodynamic parameters than 2% lidocaine with 1:80,000 epinephrine. Therefore, we suggest using 2% lidocaine with 1:200,000 epinephrine in surgical extractions of impacted mandibular TMs rather than 2% lidocaine with 1:80,000 epinephrine in hemodynamically unstable patients. Further [22] Bjorgul K, Novicoff WM, Saleh KJ. American Society of Anesthesiologist Physical Status score may be used as a comorbidity index in hip fracture surgery. J Arthroplasty 2010;25(6 suppl):134–7. [23] Vibha Singh KA, Pradhan R, Shadab Mohammad, et al. Techniques in the removal of impacted mandibular third molar: a comparative study. Eur J Gen Dent 2013;2:25–30. [24] Scott J, Huskisson EC. Graphic representation of pain. Pain 1976;2:175–84. [25] Wilson RC, Jones PW. A comparison of the visual analogue scale and modified Borg scale for the measurement of dyspnoea during exercise. Clin Sci 1989;76:277–82. 6 Karm et al. Medicine (2017) 96:21 www.md-journal.com [26] Jamieson S. Likert scales: how to (ab)use them. Med Educ 2004; 38:1217–8. [30] Murakawa T, Koh H, Tsubo T, et al. Two cases of circulatory failure after local infiltration of epinephrine during tonsillectomy. Masui 1998;47:955–62. [27] Lee W-Y, Park C-J, Seo K-S, et al. References Comparative study for the anesthetic efficacy between articaine HCl and lidocaine HCl during the surgical extraction of bilateral mandibular impacted third molars. J Korean Dent Soc Anesthesiol 2004;4:13–6. [31] Niwa H, Sugimura M, Satoh Y, et al. Cardiovascular response to epinephrine-containing local anesthesia in patients with cardiovascular disease. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2001;92:610–6. [28] Silva LC, Santos TD, Santos JA, et al. Articaine versus lidocaine for third molar surgery: a randomized clinical study. Med Oral Patol Oral Cir Bucal 2012;17:e140–5. [32] Hersh EV, Giannakopoulos H, Levin LM, et al. The pharmacokinetics and cardiovascular effects of high-dose articaine with 1:100,000 and 1:200,000 epinephrine. J Am Dent Assoc 2006;137:1562–71. [29] McEntire M, Nusstein J, Drum M, et al. Anesthetic efficacy of 4% Articaine with 1:100,000 epinephrine versus 4% articaine with 1:200,000 epinephrine as a primary buccal infiltration in the mandibular first molar. J Endod 2011;37:450–4. [33] Troullos ES, Goldstein DS, Hargreaves KM, et al. Plasma epinephrine levels and cardiovascular response to high administered doses of epinephrine contained in local anesthesia. Anesth Prog 1987;34:10–3. 7 7 7
https://openalex.org/W1560977520
http://eprints.lib.okayama-u.ac.jp/files/public/5/53771/20160528122807112460/K0005214_abstract_review.pdf
Japanese
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Intra-Arterial Transplantation of Allogeneic Mesenchymal Stem Cells Mounts Neuroprotective Effects in a Transient Ischemic Stroke Model in Rats: Analyses of Therapeutic Time Window and Its Mechanisms
PloS one
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氏 名 豊 嶋 敦 彦 授与した学位 博 士 専攻分野の名称 医 学 学位授与番号 博甲第 5214 号 学位授与の日付 平成27年 9月30日 学位授与の要件 医歯薬学総合研究科生体制御科学専攻 (学位規則第4条第1項該当) 学位論文題目 Intra-Arterial Transplantation of Allogeneic Mesenchymal Stem Cells Mounts Neuroprotective Effects in a Transient Ischemic Stroke Model in Rats: Analyses of Therapeutic Time Window and Its Mechanisms (一過性脳虚血モデルラットに対する経動脈的同種骨髄 幹細胞の神経保護効果: 最適な移植時期の検討) 論文審査委員 教授 阿部康二 教授 淺沼幹人 准教授 王 英正 氏 名 豊 嶋 敦 彦 授与した学位 博 士 専攻分野の名称 医 学 学位授与番号 博甲第 5214 号 学位授与の日付 平成27年 9月30日 学位授与の要件 医歯薬学総合研究科生体制御科学専攻 (学位規則第4条第1項該当) 学位論文題目 Intra-Arterial Transplantation of Allogeneic Mesenchymal Stem Cells Mounts Neuroprotective Effects in a Transient Ischemic Stroke Model in Rats: Analyses of Therapeutic Time Window and Its Mechanisms (一過性脳虚血モデルラットに対する経動脈的同種骨髄 幹細胞の神経保護効果: 最適な移植時期の検討) 論文審査委員 教授 阿部康二 教授 淺沼幹人 准教授 王 英正 氏 名 豊 嶋 敦 彦 授与した学位 博 士 専攻分野の名称 医 学 学位授与番号 博甲第 5214 号 学位授与の日付 平成27年 9月30日 学位授与の要件 医歯薬学総合研究科生体制御科学専攻 (学位規則第4条第1項該当) 学位論文題目 Intra-Arterial Transplantation of Allogeneic Mesenchymal Stem Cells Mounts Neuroprotective Effects in a Transient Ischemic Stroke Model in Rats: Analyses of Therapeutic Time Window and Its Mechanisms (一過性脳虚血モデルラットに対する経動脈的同種骨髄 幹細胞の神経保護効果: 最適な移植時期の検討) 論文審査委員 教授 阿部康二 教授 淺沼幹人 准教授 王 英正 氏 名 豊 嶋 敦 彦 授与した学位 博 士 専攻分野の名称 医 学 学位授与番号 博甲第 5214 号 学位授与の日付 平成27年 9月30日 学位授与の要件 医歯薬学総合研究科生体制御科学専攻 (学位規則第4条第1項該当) 学位論文内容の要旨 近年、脳虚血モデルラットに対する経動脈的骨髄幹細胞(MSC)移植の有効性が報 告されている。本研究では、一過性脳虚血モデルラットに対し、急性期の様々なタイミ ングで経動脈的同種MSC 移植を行い、神経保護効果と最適な移植時期について検討し た。Wistar 系雄性ラットに対し、90 分間右中大脳動脈を閉塞し、モデルを作製した。 移植MSC は1×106 個/ 1ml PBS とし右内頚動脈から投与した。移植群をモデル作製 1, 6, 24, 48 時間後移植群に分け、コントロール群はモデル作製1 時間後に1ml のPBS を投与した。行動学的評価をモデル作製0, 3, 7 日目に行い、7 日目に安楽死させ、脳梗 塞面積と生着細胞数を評価した。また、モデル作製7 日目に、コントロール群と24 時 間後移植群の両側皮質、線条体におけるbasic FGFとSDF-1αをELISA法で測定した。 結果として、行動学的評価で24 時間後移植群が他群に比べ有意な神経症状の改善を示 した。脳梗塞面積は24 時間後移植群で最も小さく、生着細胞数も24 時間後移植群で 有意に多く認められた。basic FGF とSDF-1α は24 時間後移植群の患側皮質において 有意に上昇した。これらの結果から、一過性脳虚血モデルラットに対する経動脈的同種 MSC 移植の神経保護効果が明らかとなった。治療効果の機序として、移植MSC から のbasicFGF およびSDF-1α 分泌の関与が示唆され、脳虚血モデル作製24 時間後の移 植が最適であった。 論文審査結果の要旨
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https://repository.library.noaa.gov/view/noaa/43196/noaa_43196_DS1.pdf
English
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Urban aerosol chemistry at a land-water transition site during summer – Part 1: Impact of agricultural and industrial ammonia emissions
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Correspondence: Christopher J. Hennigan (hennigan@umbc.edu) Correspondence: Christopher J. Hennigan (hennigan@umbc.edu) Received: 30 April 2021 – Discussion started: 18 May 2021 Revised: 29 July 2021 Accepted: 2 August 2021 Published: 2 September 2021 Received: 30 April 2021 – Discussion started: 18 May 2021 Revised: 29 July 2021 – Accepted: 2 August 2021 – Published: 2 September 2021 Abstract. This study characterizes the impact of the Chesa- peake Bay and associated meteorological phenomena on aerosol chemistry during the second Ozone Water-Land En- vironmental Transition Study (OWLETS-2) field campaign, which took place from 4 June to 5 July 2018. Measurements of inorganic PM2.5 composition, gas-phase ammonia (NH3), and an array of meteorological parameters were undertaken at Hart-Miller Island (HMI), a land–water transition site just east of downtown Baltimore on the Chesapeake Bay. The ob- servations at HMI were characterized by abnormally high NH3 concentrations (maximum of 19.3 µgm−3, average of 3.83 µgm−3), which were more than a factor of 3 higher than NH3 levels measured at the closest atmospheric Ammo- nia Monitoring Network (AMoN) site (approximately 45 km away). While sulfate concentrations at HMI agreed quite well with those measured at a regulatory monitoring station 45 km away, aerosol ammonium and nitrate concentrations were significantly higher, due to the ammonia-rich conditions that resulted from the elevated NH3. The high NH3 concentra- tions were largely due to regional agricultural emissions, in- cluding dairy farms in southeastern Pennsylvania and poultry operations in the Delmarva Peninsula (Delaware–Maryland– Virginia). Reduced NH3 deposition during transport over the Chesapeake Bay likely contributed to enhanced concentra- tions at HMI compared to the more inland AMoN site. Sev- eral peak NH3 events were recorded, including the maxi- mum NH3 observed during OWLETS-2, that appear to orig- inate from a cluster of industrial sources near downtown Baltimore. Such events were all associated with nighttime emissions and advection to HMI under low wind speeds (< 1 ms−1) and stable atmospheric conditions. Our results demonstrate the importance of industrial sources, including several that are not represented in the emissions inventory, on urban air quality. Together with our companion paper, which examines aerosol liquid water and pH during OWLETS-2, we highlight unique processes affecting urban air quality of coastal cities that are distinct from continental locations. Atmos. Chem. Phys., 21, 13051–13065, 2021 https://doi.org/10.5194/acp-21-13051-2021 © Author(s) 2021. This work is distributed under the Creative Commons Attribution 4.0 License. Nicholas Balasus1, Michael A. Battaglia Jr.1, Katherine Ball1, Vanessa Caicedo2, Ruben Delgado2, Annmarie G. Carlton3, and Christopher J. Hennigan1 icholas Balasus1, Michael A. Battaglia Jr.1, Katherine Ball1, Vanessa Caicedo2, Ruben Delgad nnmarie G. Carlton3, and Christopher J. Hennigan1 1Department of Chemical, Biochemical, and Environmental Engineering, University of Maryland Baltimore County, Baltimore, MD, 21250, USA 1Department of Chemical, Biochemical, and Environmental Engineering, University of Maryland, Baltimore County, Baltimore, MD, 21250, USA 2Joint Center for Earth Systems Technology, University of Maryland, Baltimore County, Baltimore, MD, 21250, USA 2Joint Center for Earth Systems Technology, University of Maryland, Baltimore County, Baltimore, MD, 21250, USA 3Department of Chemistry, University of California, Irvine, CA 92697, USA Urban aerosol chemistry at a land–water transition site during summer – Part 1: Impact of agricultural and industrial ammonia emissions Nicholas Balasus1, Michael A. Battaglia Jr.1, Katherine Ball1, Vanessa Caicedo2, Ruben Delgado2, Annmarie G. Carlton3, and Christopher J. Hennigan1 1 Introduction Inorganic salts, especially sulfate, nitrate, and ammonium, contribute a significant fraction of fine particulate matter (PM2.5) mass in the atmosphere. Ammonia (NH3) is an im- portant component in this system due to its reaction with acidic species, especially H2SO4 (or HSO− 4 ) and HNO3, to form secondary PM2.5. Volatility differences between H2SO4, HNO3, and NH3 result in a highly nonlinear sys- tem that is strongly influenced by the aerosol liquid water content and by small concentrations of nonvolatile aerosol species (Seinfeld and Pandis, 2016; Guo et al., 2018). The impact of NH3 on inorganic particulate matter formation has been extensively studied (e.g., Adams et al., 1999; Ansari and Pandis, 1998; Pinder et al., 2008; Bian et al., 2017). N. Balasus et al.: Urban aerosol chemistry at a land–water transition site during summer N. Balasus et al.: Urban aerosol chemistry at a land–water transition site during summer 13052 A common finding among these studies, and others, is that the NH3–H2SO4–HNO3–H2O system is characterized by “ammonia rich” and “ammonia poor” chemical regimes that produce substantial differences in aerosol composition and mass. These differences have direct implications for aerosol effects on public health (Pozzer et al., 2017; Paulot and Ja- cob, 2014), nutrient deposition and ecosystem health (Nenes et al., 2021; Bergström and Jansson, 2006), and global cli- mate change (Turnock et al., 2019). 2008). Further, meteorological processes associated with the land–water transition can exert a controlling effect on ur- ban air quality that are absent in continental cities. One such phenomenon is the land–sea breeze (or bay breeze), which can prevent or reduce dispersion and contribute to enhanced secondary pollutant formation by recirculating primary ur- ban emissions (Arya, 1999). This contributes to ozone ex- ceedances and increased PM concentrations in highly popu- lated coastal regions (Loughner et al., 2011; Caicedo et al., 2019). Understanding this process is critical since approxi- mately 40 % of the US population lives in “Coastal Shoreline Counties” (NOAA, 2013). The purpose of the National Aero- nautics and Space Administration (NASA) and Maryland Department of the Environment (MDE)-sponsored Ozone Water-Land Environmental Transitions Study (OWLETS-2) was to characterize the impacts of the Chesapeake Bay and its associated meteorological phenomena on air pollution for- mation and transport near Baltimore (Sullivan et al., 2019). In this study, we characterize secondary aerosol formation and sources of elevated NH3 measured at Hart-Miller Island, MD, a land–water transition site near Baltimore. In the com- panion paper, we analyze the effects of the Chesapeake Bay and elevated NH3 on aerosol liquid water content and aerosol pH during OWLETS-2 (Battaglia Jr. et al., 2021). Emissions of the secondary aerosol precursors SO2, NOx, and NH3 directly affect regional PM2.5 concentrations (Pin- der et al., 2007; Zhao et al., 2013). Air quality regulations in the US have substantially decreased SO2 and NOx emis- sions, leading to decreases in ambient PM2.5 (Hand et al., 2012a), while NH3 has remained largely unregulated. Re- ductions of NH3 emissions have been suggested as a cost- effective way to reduce PM2.5, as well (Pinder et al., 2007; Backes et al., 2016). Observations of wet deposition suggest that NH3 emissions have actually been rising over the past few decades (Keene et al., 2014). 2 Methods y pp ( ) While agriculture collectively emits a dominant fraction of NH3 globally, numerous other sources contribute to local or regional NH3 levels, especially in urban areas that are not in close proximity to major agricultural operations. Motor ve- hicles equipped with catalytic converters can reduce NO to NH3 in an undesired side reaction, resulting in significant NH3 emissions (Perrino et al., 2002; Sun et al., 2017; Kean et al., 2009). Anaerobic protein degradation produces NH3, as well, resulting in substantial atmospheric emissions from anthropogenic sources present in all urban areas, including landfills, composting, and wastewater treatment (Rittmann and McCarty, 2001; Artíñano et al., 2018). Numerous in- dustrial sources emit NH3 (Meng et al., 2017), while natu- ral sources (e.g., natural vegetation, wild animals, and large bodies of water) contribute to background NH3 levels (Paulot et al., 2015; Sutton et al., 2013). Biomass burning also emits NH3, though the effects are often seasonal and are most pro- nounced in regions with intense burning activities (Paulot et al., 2017). In the eastern US, biomass burning intensity is higher in spring and fall than in the summer (Washenfelder et al., 2015). N. Balasus et al.: Urban aerosol chemistry at a land–water transition site during summer Globally, agricultural activ- ity (from livestock and fertilizer application to crops) is the dominant source of NH3 emissions (Paulot et al., 2015; Pin- der et al., 2006; Sutton et al., 2013). Synthetic fertilizer pro- duction has recently been identified as an intense local NH3 source (Van Damme et al., 2018), though its contribution to global emissions is likely small compared to volatilization from synthetic fertilizer applications (Bouwman et al., 1997). Published by Copernicus Publications on behalf of the European Geosciences Union. ublished by Copernicus Publications on behalf of the European Geosciences Union. 2.2 NH3 measurements Speciated inorganic PM2.5 measurements were made with a particle-into-liquid sampler (PILS) coupled to dual anion- cation ion chromatographs (IC, model 850 Dual IC, Metrohm), collectively referred to as the PILS-IC (Orsini et al., 2003). Ambient air was sampled at 16.0 Lmin−1 through a PM2.5 cyclone (URG Corp.) and two annular de- nuders in series (URG Corp.) to remove acidic (e.g., HNO3, HCl) and basic gases (e.g., NH3). The PILS sample was split and simultaneously analyzed for concentrations of both cations (Na+, NH+ 4 , K+, Ca2+, and Mg2+) and anions (Cl−, NO− 3 , SO2− 4 , and C2O2− 4 ) according to Valerino et al. (2017). For anion analysis, the IC utilized a Metrosep A Supp 5 column (150 mm × 4.0 mm) with 1.0 mM NaHCO3 and 3.2 mM Na2CO3 eluent at a flow rate of 0.7 mLmin−1. For cation analysis, the IC utilized a Metrosep C 4 column (150 mm × 4.0 mm) that was run with 2.5 mM HNO3 and 0.5 mM C2H2O4 eluent at a flow rate of 0.9 mLmin−1. In the anion analysis, conductivity measurements were preceded by chemical suppression to reduce conductivity of the eluent. This configuration of the PILS-IC resulted in a limit of de- tection of 0.01 µgm−3 (air concentration) for each ion and a 5 min integrated sample every 20 min. An AiRRmonia analyzer (RR Mechatronics, the Nether- lands) was deployed for measurements of gas-phase am- monia (Norman et al., 2009). The analyzer was enclosed in a weatherproof aluminum box (non-temperature con- trolled) and placed on top of the UMBC trailer (∼4 m in- let height). The AiRRmonia uses a 5 cm stainless-steel in- let (i.d. = 0.2 cm) to minimize sampling artifacts associated with gas adsorption to inlet surfaces (Schmohl et al., 2001; Ellis et al., 2010; von Bobrutzki et al., 2010). The instru- ment samples air at a rate of 1 Lmin−1, giving a gas resi- dence time of ∼0.01 s in the inlet. Ammonia in the sampled air is absorbed through a gas-permeable polytetrafluoroethy- lene membrane into an acidic aqueous stripping solution with close to 100 % collection efficiency (Wyers et al., 1993). NaOH is then added to raise the pH so that the collected NH3 (present as NH+ 4 ) is shifted to the NH3 form, facilitating transfer of NH3 through another gas-permeable membrane into a separate flow of deionized water. 2.2 NH3 measurements Dual conductivity detectors measure the difference in conductivity between the deionized water before and after this transfer, enabling quan- tification of sampled NH3 (Norman et al., 2009; von Bo- brutzki et al., 2010). The temperature-dependent conductiv- ity was calibrated approximately daily during OWLETS-2 using aqueous NH+ 4 standards. The instrument’s operating principles, including gas collection efficiency and lack of re- sponse to aerosol NH+ 4 , have been characterized previously (Wyers et al., 1993; Erisman et al., 2001). The NH3 mea- surements during OWLETS-2 were conducted with 10 min resolution. Speciated inorganic PM2.5 measurements were made with a particle-into-liquid sampler (PILS) coupled to dual anion- cation ion chromatographs (IC, model 850 Dual IC, Metrohm), collectively referred to as the PILS-IC (Orsini et al., 2003). Ambient air was sampled at 16.0 Lmin−1 through a PM2.5 cyclone (URG Corp.) and two annular de- nuders in series (URG Corp.) to remove acidic (e.g., HNO3, HCl) and basic gases (e.g., NH3). The PILS sample was split and simultaneously analyzed for concentrations of both cations (Na+, NH+ 4 , K+, Ca2+, and Mg2+) and anions (Cl−, NO− 3 , SO2− 4 , and C2O2− 4 ) according to Valerino et al. (2017). Speciated inorganic PM2.5 measurements were made with a particle-into-liquid sampler (PILS) coupled to dual anion- cation ion chromatographs (IC, model 850 Dual IC, Metrohm), collectively referred to as the PILS-IC (Orsini et al., 2003). Ambient air was sampled at 16.0 Lmin−1 through a PM2.5 cyclone (URG Corp.) and two annular de- nuders in series (URG Corp.) to remove acidic (e.g., HNO3, HCl) and basic gases (e.g., NH3). The PILS sample was split and simultaneously analyzed for concentrations of both cations (Na+, NH+ 4 , K+, Ca2+, and Mg2+) and anions (Cl−, NO− 3 , SO2− 4 , and C2O2− 4 ) according to Valerino et al. (2017). 3 4 4 For anion analysis, the IC utilized a Metrosep A Supp 5 column (150 mm × 4.0 mm) with 1.0 mM NaHCO3 and 3.2 mM Na2CO3 eluent at a flow rate of 0.7 mLmin−1. For cation analysis, the IC utilized a Metrosep C 4 column (150 mm × 4.0 mm) that was run with 2.5 mM HNO3 and 0.5 mM C2H2O4 eluent at a flow rate of 0.9 mLmin−1. In the anion analysis, conductivity measurements were preceded by chemical suppression to reduce conductivity of the eluent. 2.2 NH3 measurements This configuration of the PILS-IC resulted in a limit of de- tection of 0.01 µgm−3 (air concentration) for each ion and a 5 min integrated sample every 20 min. N. Balasus et al.: Urban aerosol chemistry at a land–water transition site during summer Figure 1. Map of relevant locations for the field campaign. Yellow stars indicate sites used for primary comparison (HMI, Howard University- Beltsville, Beltsville), green stars are locations used for secondary comparisons (Arendtsville, Blackwater), and red stars are labeled for spatial reference (Baltimore, Washington D.C.). Figure 1. Map of relevant locations for the field campaign. Yellow stars indicate sites used for primary comparison (HMI, Howard University- Beltsville, Beltsville), green stars are locations used for secondary comparisons (Arendtsville, Blackwater), and red stars are labeled for spatial reference (Baltimore, Washington D.C.). 2.1 Site description The OWLETS-2 campaign was a field study with multiple measurement platforms coordinating simultaneous water and land measurements of trace gases, aerosols, and meteoro- logical parameters. This study presents analysis of inorganic aerosol composition and gas-phase ammonia measurements made at the Hart-Miller Island (HMI, Fig. 1) supersite. Hart- Miller Island is a state park located on the Chesapeake Bay, approximately 22 km from downtown Baltimore. All measurements were made in the University of Mary- land, Baltimore County (UMBC) trailer located on the south- eastern side of the island (39.2421◦, −76.3627◦). Continuous measurements were conducted from 4 June to 5 July 2018. Several gaps in data collection occurred due to periodic power outages on the island. Comparisons of the HMI measurements were made to two nearby inland sites: Beltsville and Howard Univer- sity Beltsville (HUB, denoted HU-Beltsville hereafter), ap- proximately 46 and 50 km southwest of HMI, respectively. Beltsville (39.0280◦, −76.8171◦) is a National Atmospheric Deposition Program Ammonia Monitoring Site (AMoN, http://nadp.slh.wisc.edu/amon/, last access: 28 July 2021) and was used for gas-phase NH3 comparisons. HUB (39.0553◦, −76.8783◦) is a regulatory monitoring site main- tained and operated by MDE and was used for comparisons of speciated PM2.5 concentrations. Urban air quality is controlled by emissions from multiple sectors, meteorology, and dynamic chemical processes that evolve throughout the day. Coastal cities often experience contributions from unique sources (e.g., ocean and shipping emissions), with chemical processes in the polluted marine boundary layer that are distinct from other regions in the at- mosphere (e.g., de Gouw et al., 2005; Vutukuru and Dabdub, Atmos. Chem. Phys., 21, 13051–13065, 2021 https://doi.org/10.5194/acp-21-13051-2021 13053 N. Balasus et al.: Urban aerosol chemistry at a land–water transition site during summer 3.1 Ammonia During the OWLETS-2 campaign, gas-phase ammonia was characterized by unusually high concentrations. The aver- age NH3 concentration (3.83 µgm−3) at HMI was more than 3 times greater than NH3 at the Beltsville AMoN site (1.20 µgm−3) during the summer of 2018 (Fig. 2a), a dif- ference that was statistically significant at the 99 % con- fidence level (p value < 0.01). While average summertime NH3 shows an increasing trend at Beltsville, the concentra- tions measured at HMI in 2018 far exceed recent summer- time levels at Beltsville. The HMI NH3 concentrations were also compared to two other AMoN sites (Fig. 1): Blackwater (∼90 km from HMI; 38.4449◦, −76.1112◦) and Arendtsville (∼110 km from HMI; 39.9231◦, −77.3078◦). Both of these sites are located in much closer proximity to strong agricul- tural NH3 emissions, as Blackwater is located on the East- ern Shore of Maryland and Arendtsville is located in south- eastern Pennsylvania. Their average summer NH3 concen- trations for 2018 were 57 % (Blackwater, 1.66 µgm−3) and 49 % (Arendtsville, 1.96 µgm−3) lower than those measured at HMI during OWLETS-2. In a previous deployment of the AiRRmonia analyzer at the Beltsville AMoN site (un- published work), the average concentrations measured by the AiRRmonia over a 2-week span (6–20 September 2016) were within ∼0.5 µgm−3 of the colocated AMoN NH3 mea- surements, suggesting that systematic measurement differ- ences are not responsible for the trends seen in Fig. 2a. While SO2− 4 concentrations were typical for the re- gion, the elevated NH3 did affect the particulate NH+ 4 and NO− 3 concentrations. An ammonia-rich environment, defined when [Total Ammonia] > 2× [Total Sulfate], can facilitate NH4NO3 aerosol formation (Seinfeld and Pandis, 2016). The system measured at HMI was ammonia rich for approxi- mately 25 % of the OWLETS-2 campaign. As shown in Fig. 2c, the average NH+ 4 concentration at HMI was more than 50 % higher than the summertime aver- age at HU-Beltsville (0.38 µgm−3 and 0.23 µgm−3, respec- tively), which is statistically significant at the 99 % confi- dence level (p value < 0.01). The NH+ 4 concentrations at HMI were higher than any of the past five summertime aver- ages measured at HU-Beltsville, demonstrating the influence of elevated NH3. Aerosol NO− 3 was also significantly ele- vated at HMI compared to historical and 2018 summertime levels at HU-Beltsville (Fig. 2d). 3.1 Ammonia The HMI average NO− 3 (0.36 µgm−3) was more than a factor of 2 higher than at HU- Beltsville (0.14 µgm−3), which is also significant at the 99 % confidence level (p value < 0.01). The NO− 3 concentrations at HMI were also higher than typical summertime levels in the eastern US (Sickles II and Shadwick, 2008). The high average NO− 3 concentration during OWLETS-2 was heavily influenced by several peak events where the NO− 3 concentra- tion exceeded 3 µgm−3 (Fig. S1). N. Balasus et al.: Urban aerosol chemistry at a land–water transition site during summer N. Balasus et al.: Urban aerosol chemistry at a land–water transition site during summer N. Balasus et al.: Urban aerosol chemistry at a land–water transition site during summer 13054 Rapid Refresh (HRRR) 3 km meteorological data were used in generating back-trajectories. The uncertainty and limita- tions of the HYSPLIT model are worth noting here, espe- cially in accounting for small-scale circulations. As a result, for all events discussed below, HYSPLIT trajectories were crosschecked with measured surface wind conditions and vertical wind fields measured from the meteorological sta- tion and a Doppler wind lidar (Leosphere Windcube 200S) deployed at HMI. Meteorological measurements of tempera- ture, relative humidity, and surface wind speed and direction were made with the Vaisala MAWS201 meteorological sta- tion at 1 min resolution throughout the study. sions (Hand et al., 2012a). This trend is evident at HU- Beltsville (Fig. 2b), where the average summertime SO2− 4 concentration has decreased by more than a factor of 8 since 2007 (8.23 µgm−3 versus 0.94 µgm−3). Note that the data shown in Fig. 2 only include the summer months (June, July, and August) to facilitate direct comparisons to the conditions during OWLETS-2. In contrast to the NH3 results, the av- erage SO2− 4 concentration at HMI (1.11 µgm−3) was quite close to the average concentration at HUB (0.94 µgm−3), a difference that was not significant at the 95 % confidence level (p value > 0.1). This suggests that the high NH3 con- centrations at HMI did not have a large effect on particulate sulfate mass concentrations, consistent with the understand- ing of SO2− 4 thermodynamics and results from a study in the southeast US (Weber et al., 2016). The agreement in SO2− 4 concentrations between HMI and HU-Beltsville is unsurpris- ing given the regional nature of SO2 emissions and the low spatial variability of SO2− 4 levels in the eastern US (Beyers- dorf et al., 2016). 2.4 HYSPLIT model The HYbrid Single-Particle Lagrangian Integrated Trajec- tory (HYSPLIT, https://www.ready.noaa.gov/HYSPLIT.php, last access: 28 July 2021) model was utilized to investigate source influences during OWLETS-2 (Stein et al., 2015). The model allows for air parcel trajectories, including the back- trajectory analyses used here, to infer regional source influ- ences for the air masses sampled at HMI. High-Resolution https://doi.org/10.5194/acp-21-13051-2021 Atmos. Chem. Phys., 21, 13051–13065, 2021 3.2 Impact on aerosol composition Another notable aspect of the NO− 3 observed during OWLETS-2 was the diurnal profile (Fig. S2a). Formation of NH4NO3 is favored by both lower temperature and higher relative humidity (Stelson and Seinfeld, 1982). Thus, the ob- served midday peak in NO− 3 coincided with the most un- favorable meteorological conditions (Fig. 1 in Battaglia Jr. et al., 2021). More typical summertime diurnal profiles of NO− 3 in the eastern US exhibit a maximum around 06:00– 07:00 LT and an afternoon minimum (Xu et al., 2015; Poulain et al., 2011; Weber, 2003; Wittig et al., 2004). The Before investigating the sources of such high NH3 concen- trations, we first characterize the effects on aerosol compo- sition, because NH3 contributes significantly to secondary aerosol formation (Seinfeld and Pandis, 2016). Note that our companion paper analyzes the effects of the high NH3 concentrations on aerosol liquid water and acidity (pH) (Battaglia Jr. et al., 2021). In the US, SO2− 4 concentrations have decreased due to reg- ulations and economic factors that have reduced SO2 emis- https://doi.org/10.5194/acp-21-13051-2021 Atmos. Chem. Phys., 21, 13051–13065, 2021 N. Balasus et al.: Urban aerosol chemistry at a land–water transition site during summer 13055 Figure 2. Summertime (June, July, and August) comparisons of (a) NH3 concentrations measured at HMI, Beltsville, Arendtsville, and Blackwater; (b) PM2.5 SO2− 4 measured at HMI and HUB; (c) PM2.5 NH+ 4 measured at HMI and HUB; and (d) PM2.5 NO− 3 measured at HMI and HUB. Error bars represent plus or minus the standard deviation. Figure 2. Summertime (June, July, and August) comparisons of (a) NH3 concentrations measured at HMI, Beltsville, Arendtsville, and Blackwater; (b) PM2.5 SO2− 4 measured at HMI and HUB; (c) PM2.5 NH+ 4 measured at HMI and HUB; and (d) PM2.5 NO− 3 measured at HMI and HUB. Error bars represent plus or minus the standard deviation. NO− 3 diurnal profile observed during OWLETS-2 indicates the important effect of the elevated NH3 during the campaign and will be discussed in more detail below. Figure 3. Scatter plot of 2× (SO2− 4 + NO− 3 ) versus NH+ 4 (all species in molar concentrations) for the OWLETS-2 campaign. NH+ 4 and NO− 3 concentrations were elevated at HMI com- pared to the historical data (Fig. 2). In the eastern US, the PM2.5 mass fraction of NH4NO3 peaks in the winter with a minimum in the summer (Wittig et al., 2004). 3.2 Impact on aerosol composition Ammo- nium sulfate, however, is consistently one of the largest frac- tions of PM2.5 mass in the eastern US (Hand et al., 2012b). For the OWLETS-2 study, the coefficient of determination (R2) for the linear correlation (least squares regression anal- ysis) between 2× (SO2− 4 + NO− 3 ) and NH+ 4 was 0.90 (Fig. 3), while the R2 value for the linear relationship between SO2− 4 and NH+ 4 was only 0.57 (not shown), indicating the impor- tance of NH4NO3 and the ammonia-rich conditions during OWLETS-2. Figure 3. Scatter plot of 2× (SO2− 4 + NO− 3 ) versus NH+ 4 (all species in molar concentrations) for the OWLETS-2 campaign. Finally, it should be noted that NO− 3 concentrations can be affected by interactions between nitric acid and sodium chlo- ride (Seinfeld and Pandis, 2016). This can have a large im- pact on aerosol composition in coastal urban areas (Athana- sopoulou et al., 2008). However, sodium chloride aerosols, while observed periodically in the fine mode during the cam- paign, were not frequent enough or high enough in magni- tude (max Na + Cl = 0.91 µgm−3) to suggest a strong influ- ence of primary aerosol emissions from the Chesapeake Bay. N. Balasus et al.: Urban aerosol chemistry at a land–water transition site during summer Eleven distinct events had peak NH3 concentrations above the 95th percentile (> 7.96 µgm−3) for the OWLETS- 2 study (Fig. 5). Back-trajectories for each event are shown in Fig. 6. Altitudes of all back-trajectories were below 1 km over the postulated source region. The potential NH3 sources for event nos. 8–10 include the Back River Wastewater Treatment Plant (A in Fig. 8), the Patapsco Wastewater Treatment Plant (B), a W.R. Grace Chemical Production Facility (C), the Quarantine Road Landfill (D), a large-scale composting facility (E), and Yara Fertilizer Distributor (F). Of these facilities, only W.R. Grace (224.1 tyr−1) and the composting facility (1.03 tyr−1) are permitted to release NH3 by the state of Maryland (MDE, 2019a, b). However, the non-permitted sites are likely ma- jor NH3 emitters, based upon urban NH3 sources previously identified in the literature. It is likely that emissions from multiple sources collectively contributed to NH3 levels dur- ing these events; however, the potential sources are too close together to distinguish or estimate individual contributions. Agriculture represents the largest source of atmospheric NH3 emissions globally (Sutton et al., 2013). Agricultural NH3 emissions stem from fertilizer and livestock manage- ment (Pinder et al., 2006). Previous studies have linked high livestock NH3 emissions with particulate nitrate formation (Xu et al., 2019; Paulot et al., 2016; Sorooshian et al., 2008). HMI is located in relatively close proximity to several ar- eas of concentrated livestock production (Fig. 7). To the east and southeast of HMI, the Delmarva Peninsula (Delaware– Maryland–Virginia) is a large poultry producing region. The emissions from these chicken houses have been characterized by ground measurements (Siefert et al., 2004) and captured by satellite measurements (Warner et al., 2017). Additionally, there are large concentrations of dairy cows in southeastern Pennsylvania, also shown in Fig. 7. The NH3 emissions in this area are some of the highest in the country for dairy cows (Pinder et al., 2004b). While dairy cow populations are relatively minor in Maryland, poultry emissions could be a significant emitter in the same region of Pennsylvania where dairy cows are concentrated (Fig. S3). The back-trajectories identify events 1, 2, 4, 5, 6, 7, and 11 as having likely agri- cultural influence (Table 1), consistent with the agricultural hotspots identified here (Figs. 7, S3). Additionally, the source for event no. 3 is ambiguous, but has the potential to be the poultry sources discussed above. N. Balasus et al.: Urban aerosol chemistry at a land–water transition site during summer N. Balasus et al.: Urban aerosol chemistry at a land–water transition site during summer 13056 trajectories were initialized from HMI (50 m altitude) at the time corresponding to each NH3 measurement. Air mass back-trajectories were run based upon a recent study’s es- timate of 15 h for the average NH3 lifetime (Hauglustaine et al., 2014), though other studies have estimated much lower NH3 lifetimes for point sources (Dammers et al., 2019). Fig- ure 4 shows the ensemble of all back-trajectories for the cam- paign colored by the measured NH3 concentration. Overall, the back-trajectory origins were consistent with predominant surface winds during the campaign. Figure 4 also shows that elevated NH3 concentrations were not limited to trajectories originating from a single direction or region. ban and industrial NH3 sources are known, including mo- tor vehicles (Bishop and Stedman, 2015; Sun et al., 2017) and fertilizer production plants (Van Damme et al., 2018). Ammonia is produced when proteins are broken down under anaerobic conditions (Rittmann and McCarty, 2001), causing landfills (Sutton et al., 2000), waste composting and process- ing (Sutton et al., 2013; Pagans et al., 2006), and wastewater treatment plants (Reche et al., 2015; Artíñano et al., 2018) to emit significant quantities of NH3, as well. The contribution of traffic to event nos. 8–10 was likely quite small due to a combined R2 value of 0.201 between NH3 and CO for the duration of the three events (based on CO measurements colocated at HMI). NH3 and CO are correlated in vehicle emissions (Kean et al., 2009; Perrino et al., 2002); however, for these events, high NH3 was ob- served without a spike in CO. We acknowledge that order- of-magnitude differences in the atmospheric lifetimes of CO and NH3 would alter correlations downwind from sources co-emitting both pollutants, but in this case, the species with the longer lifetime (CO) did not show an enhancement dur- ing the NH3 events, suggesting mobile sources were not a significant factor here. The trajectories for event nos. 8–10 identify a cluster of potential industrial NH3 sources (Fig. 8). A subset of the trajectories in Fig. 4 was further in- vestigated to identify the potential sources of peak NH3 events. When both particle-phase and gas-phase ammonia were measured, total ammonia and gas-phase ammonia cor- related strongly (R = 0.9927), showing that gas-phase am- monia is an effective tracer for total ammonia for this cam- paign. 3.3 Characterization of ammonia sources Source locations of the high NH3 events were investigated using the NOAA HYSPLIT model. Trajectories produced by the model were checked for consistency with vertical wind fields measured by Doppler wind lidar (when avail- able, with the one discrepancy noted in Table 1). Back- https://doi.org/10.5194/acp-21-13051-2021 Atmos. Chem. Phys., 21, 13051–13065, 2021 N. Balasus et al.: Urban aerosol chemistry at a land–water transition site during summer N. Balasus et al.: Urban aerosol chemistry at a land–water transition site during summer It is important to note that numerous back-trajectories also passed over this cluster of industrial sources during times when NH3 was not elevated. However, event nos. 8–10 share characteristics that explain the high NH3 levels at HMI. The peak concentration of each event occurred between 07:00– 08:00 LT (Table 1). Additionally, the events had extremely low wind speeds of 0.8, 0.7, and 0.6 ms−1, respectively, all well below the campaign average of 2.9 ms−1 (Table 1). These values were below the 10th percentile of all wind speeds measured at HMI during OWLETS-2. With such low wind speeds, transport time for the ∼ 18 km distance between the cluster of NH3 sources near downtown Baltimore and HMI would be ∼7–8 h for event nos. 8–10, suggesting that the original emissions occurred near midnight. Low wind speeds occurring at night corre- spond to the most stable atmospheric conditions and to a low boundary layer, which together enable elevated pollu- tant concentrations downwind of point sources (Arya, 1999). While agricultural emissions were the likely source of many peak events, several events – including the highest NH3 concentration measured during the campaign – appear to have a nonagricultural origin. Specifically, the trajectories for event nos. 8–10 pass directly over the city of Baltimore and do not appear to intersect with any major agricultural sources in the 15 h prior to arrival at HMI. A number of ur- https://doi.org/10.5194/acp-21-13051-2021 Atmos. Chem. Phys., 21, 13051–13065, 2021 N. Balasus et al.: Urban aerosol chemistry at a land–water transition site during summer N. Balasus et al.: Urban aerosol chemistry at a land–water transition site during summer 13057 Figure 4. Aggregate 15 h HYSPLIT back-trajectories initialized at HMI (50 m altitude) and colored by NH3 concentrations measured at HMI utilizing 3 km HRRR meteorology. Every other back-trajectory is displayed for clarity. Figure 4. Aggregate 15 h HYSPLIT back-trajectories initialized at HMI (50 m altitude) and colored by NH3 concentrations measured at HMI utilizing 3 km HRRR meteorology. Every other back-trajectory is displayed for clarity. Figure 5. Time series of NH3 measurements at HMI during OWLETS-2. The 11 NH3 peak events are labeled and represent the concentrations above the 95th percentile for the entire data set (7.96 µgm−3). Gaps in data are due to power outages that occurred on the island. Events 2 and 3 were cut off by power outages. N. Balasus et al.: Urban aerosol chemistry at a land–water transition site during summer The blue dashed line represents the campaign-average NH3 concentration (3.83 µgm−3). Figure 5. Time series of NH3 measurements at HMI during OWLETS-2. The 11 NH3 peak events are labeled and represent the concentrations above the 95th percentile for the entire data set (7.96 µgm−3). Gaps in data are due to power outages that occurred on the island. Events 2 and 3 were cut off by power outages. The blue dashed line represents the campaign-average NH3 concentration (3.83 µgm−3). For example, event no. 10 had a peak NH3 concentration ∼ 15.5 µgm−3 higher than the campaign average (3.8 µgm−3). Under Pasquill–Gifford stability class F (nighttime, low winds), the required point source emission rate is 2 orders of magnitude lower (1 gs−1 vs. 172 gs−1) than it is under stabil- ity class B (slight daytime solar radiation, low winds) to give the same 15.5 µgm−3 NH3 enhancement at HMI. This illus- trative example shows the potential for atmospheric stabil- For example, event no. 10 had a peak NH3 concentration ∼ 15.5 µgm−3 higher than the campaign average (3.8 µgm−3). Under Pasquill–Gifford stability class F (nighttime, low winds), the required point source emission rate is 2 orders of magnitude lower (1 gs−1 vs. 172 gs−1) than it is under stabil- ity class B (slight daytime solar radiation, low winds) to give the same 15.5 µgm−3 NH3 enhancement at HMI. This illus- trative example shows the potential for atmospheric stabil- ity to impact downwind surface concentration enhancement. Specifically, for this study, urban sources with moderate NH3 emissions can have a profound effect on downwind NH3 lev- els under the most stable atmospheric conditions. Further, event nos. 8–10 also occurred shortly before on- sets of the Chesapeake Bay breeze. The convergence between an offshore synoptic flow and the breeze creates a period of stagnation or calm winds that favor the accumulation of pol- https://doi.org/10.5194/acp-21-13051-2021 Atmos. Chem. Phys., 21, 13051–13065, 2021 13058 N. Balasus et al.: Urban aerosol chemistry at a land–water transition site during summer N. Balasus et al.: Urban aerosol chemistry at a land–water transition site during summer Figure 6. HYSPLIT back-trajectories initialized at HMI using 3 km HRRR meteorology and an altitude of 50 m for the peak NH3 events identified in Fig. 5. There are three trajectories for each event: one for the highest concentration of the event, one 10 min earlier, and one 10 min later. Figure 6. N. Balasus et al.: Urban aerosol chemistry at a land–water transition site during summer HYSPLIT back-trajectories initialized at HMI using 3 km HRRR meteorology and an altitude of 50 m for the peak NH3 events identified in Fig. 5. There are three trajectories for each event: one for the highest concentration of the event, one 10 min earlier, and one 10 min later. Figure 7. Inventories of dairy cows in Pennsylvania and broiler and other meat-type chickens in Maryland and Delaware. Data are from the 2017 Census of Agriculture (USDA, 2017). Figure 7. Inventories of dairy cows in Pennsylvania and broiler and other meat-type chickens in Maryland and Delaware. Data are from the 2017 Census of Agriculture (USDA, 2017). Natural sources of NH3 can represent the dominant source of emissions in some regions (Paulot et al., 2015; Sutton et al., 2013). The Chesapeake Bay can be a source or sink for NH3 in the summertime (Larsen et al., 2001); however, the emission factors reported by Larsen et al. (2001) suggest a relatively minor influence of Chesapeake Bay emissions compared to the agricultural and industrial sources discussed above. lutants (Caicedo et al., 2019; Loughner et al., 2014). Addi- tionally, the early morning occurrence of event nos. 8–10 also suggests that a low dilution volume as the marine boundary layer is only beginning its convective regime (Stull, 1988). Combined, calm winds and relatively shallow boundary lay- ers are important factors contributing to the high NH3 con- centrations. lutants (Caicedo et al., 2019; Loughner et al., 2014). Addi- tionally, the early morning occurrence of event nos. 8–10 also suggests that a low dilution volume as the marine boundary layer is only beginning its convective regime (Stull, 1988). Combined, calm winds and relatively shallow boundary lay- ers are important factors contributing to the high NH3 con- centrations. Atmos. Chem. Phys., 21, 13051–13065, 2021 https://doi.org/10.5194/acp-21-13051-2021 13059 N. Balasus et al.: Urban aerosol chemistry at a land–water transition site during summer N. Balasus et al.: Urban aerosol chemistry at a land–water transition site during summer Figure 8. Back-trajectories initialized at HMI (star) using 3 km HRRR meteorology for event nos. 8 (yellow), 9 (dark blue), and 10 (teal). Potential industrial sources of NH3 are (A) Back River Wastewater Treatment Plant, (B) Patapsco Wastewater Treatment Plant, (C) W.R. Grace Facility, (D) Quarantine Road Landfill, (E) a composting facility, and (F) Yara Fertilizer Distributor. Figure 8. Back-trajectories initialized at HMI (star) using 3 km HRRR meteorology for event nos. 3.4 Temperature effects NH3–temperature over the entire campaign). In the absence of strong local sources of NH3 emissions, ambient observa- tions are most impacted by transport. At HMI, this includes diverse source sectors from different areas that correspond to various time lags in emission to observation. This confounds strict interpretation of temperature-dependent emission rela- tionships. Temperature plays an important role in NH3 emissions from fertilizer and animal waste (Robarge et al., 2002; Pleim et al., 2013). This contributes to strong seasonal and diurnal pro- files of agricultural NH3 emissions (Warner et al., 2017; Zhu et al., 2015; Pinder et al., 2004a). The average diurnal profile of NH3 (Fig. 9a) shows enhanced concentrations between the hours of 07:00–15:00 LT, consistent with temperature-driven emissions. Overall, NH3 concentrations rose with increasing temperature (Fig. 9b); however, there was a lot of scatter in the data (R2 = 0.05 for individual (10 min) measurements of Table 1. Description of peak NH3 events. Event no. Peak NH3 Time (local time) Temperature Wind speed previous 8 h average Source (µgm−3) (◦C) (ms−1) 1 11.0 6 June 2018 23:40 18.9 2.68 Poultry 2 8.89 9 June 2018 14:45 24.6 2.09 Dairy 3 10.3 20 June 2018 13:44 27.7 2.33 Ambiguous 4 16.2 21 June 2018 19:29 25.3 2.16 Dairy 5 8.16 22 June 2018 08:19 22.5 4.50 Poultry 6 9.93 23 June 2018 14:49 22.7 3.18 Poultry 7 8.42 23 June 2018 23:39 23.9 2.08 Poultry 8 8.02 24 June 2018 07:19 24.5 0.79 Industrial 9 10.4 30 June 2018 07:15 26.3 0.67 Industrial 10 19.3 1 July 2018 07:35 27.8 0.61 Industrial 11 8.77 3 July 2018 13:42 31.7 1.77 Dairy Events 2 and 3 were cut short by power outages. Event 2 (one of the two events where wind lidar data were not available for crosschecking the back-trajectory) is the only event with a discrepancy between HYSPLIT (predicts wind direction coming from NNE) and observations (measured surface wind direction coming from E). Events 2 and 3 were cut short by power outages. Event 2 (one of the two events where wind lidar data were not available for crosschecking the back-trajectory) is the only event with a discrepancy between HYSPLIT (predicts wind direction coming from NNE) and observations (measured surface wind direction coming from E). N. Balasus et al.: Urban aerosol chemistry at a land–water transition site during summer 8 (yellow), 9 (dark blue), and 10 (teal). Potential industrial sources of NH3 are (A) Back River Wastewater Treatment Plant, (B) Patapsco Wastewater Treatment Plant, (C) W.R. Grace Facility, (D) Quarantine Road Landfill, (E) a composting facility, and (F) Yara Fertilizer Distributor. Table 1. Description of peak NH3 events. 4 Conclusions Semicontinuous measurements of aerosol chemical compo- sition and gas-phase NH3 were carried out during summer 2018 as part of the OWLETS-2 field campaign. The mea- surement site was located in close proximity to the land– water transition between the Chesapeake Bay and downtown Baltimore, MD. Sulfate levels were in line with historical trends and with concentrations measured at the same time in other locations near Baltimore. However, average aerosol NO− 3 and NH+ 4 concentrations were significantly elevated compared to the average concentrations at a regulatory mon- itoring site ∼45 km away. The elevated aerosol concentra- tions were likely driven by gas-phase NH3, as higher back- ground levels and episodic spikes contributed to average NH3 concentrations that were more than 3 times higher than the average levels measured at a nearby (∼45 km) AMoN site. 3 ( ) The most likely explanation for the higher concentrations at HMI is a dramatic difference in the bidirectional flux of NH3 during transport to each site. The path from the Delmarva Peninsula to Hart-Miller Island consists primar- ily of transport over water (the Chesapeake Bay), while the path for transport to Beltsville includes significantly more time and distance over land (∼40 km). The dry deposition velocity of gases is typically lower over water than over land, especially forested regions like the land surrounding the Beltsville AMoN site (Li et al., 2019). Additionally, the Chesapeake Bay can be a net source of NH3 during the sum- mer (Larsen et al., 2001), suggesting deposition can be, on a net basis, quite small over the body of water. A recent model- ing study found that reactive nitrogen undergoes much higher rates of dry deposition to the coastline bordering the Chesa- peake Bay compared to rates over water (Loughner et al., 2016). The NH3 measurements at Beltsville are 2-week inte- grated samples, precluding a direct comparison to the HMI measurements and quantitative estimates of NH3 lost to dry deposition during transport. However, dry deposition of NH3 is a key uncertainty in accurate NH3 predictions (Pleim et al., 2013), so future studies should examine this question di- rectly. The dry deposition of NH3 and HNO3 are affected by An array of sources likely contributed to the elevated NH3 concentrations. Agricultural emissions from poultry opera- tions in the Delmarva Peninsula and dairy cattle in south- eastern Pennsylvania were the most frequent sources of high NH3 at HMI. N. Balasus et al.: Urban aerosol chemistry at a land–water transition site during summer 13060 13060 N. Balasus et al.: Urban aerosol chemistry at a land–water transition site during summer Figure 9. (a) The diurnal profile of NH3 (error bars represent plus or minus the standard deviation) and (b) box plot showing variations in the NH3 concentration with temperature. Lines in (b) represent the 5th, 25th, 50th, 75th, and 95th percentiles; diamonds represent the mean, while numbers beneath each box represent the number of observations in each temperature interval. Figure 9. (a) The diurnal profile of NH3 (error bars represent plus or minus the standard deviation) and (b) box plot showing variations in the NH3 concentration with temperature. Lines in (b) represent the 5th, 25th, 50th, 75th, and 95th percentiles; diamonds represent the mean, while numbers beneath each box represent the number of observations in each temperature interval. the aerosol liquid water content and particle pH (Nenes et al., 2021), and this will be analyzed in our companion paper. observed at Beltsville (Fig. 2a). Due to differences in proxim- ity to the city of Baltimore (Beltsville is ∼45 km from down- town Baltimore) and to the Chesapeake Bay (e.g., bay breeze circulation), Beltsville was not affected by the identified industrial areas as severely as Hart-Miller Island. Further, when the wind direction may have facilitated transport from downtown Baltimore to Beltsville, the atmospheric condi- tions were not typically stable, giving rise to much lower downwind concentrations. However, the trajectory analysis (not shown) indicates that Beltsville frequently received air masses that had recently passed over the agricultural areas of the Delmarva Peninsula, the most common source of extreme NH3 concentrations at HMI (Table 1). 3.5 Deposition effects Several factors likely explain why the NH3 concentrations observed at HMI were more than 3 times higher than those https://doi.org/10.5194/acp-21-13051-2021 Atmos. Chem. Phys., 21, 13051–13065, 2021 N. Balasus et al.: Urban aerosol chemistry at a land–water transition site during summer References Adams, P. J., Seinfeld, J. H., and Koch, D. M.: Global concentra- tions of tropospheric sulfate, nitrate, and ammonium aerosol sim- ulated in a general circulation model, J. Geophys. Res.-Atmos., 104, 13791–13823, https://doi.org/10.1029/1999JD900083, 1999. Code availability. Publicly available code (https://github.com/ AirChem/HYSPLITcontrol, Wolfe, 2021) was used to run back- trajectories in bulk and scale their color by user-input data (code courtesy of Glenn Wolfe, NASA/GSFC and UMBC/JCET). Ansari, A. S. and Pandis, S. N.: Response of inorganic PM to pre- cursor concentrations, Environ. Sci. Technol., 32, 2706–2714, https://doi.org/10.1021/es971130j, 1998. Artíñano, B., Pujadas, M., Alonso-Blanco, E., Becerril-Valle, M., Coz, E., Gómez-Moreno, F. J., Salvador, P., Nuñez, L., Palacios, M., and Diaz, E.: Real-time monitoring of atmospheric ammonia during a pollution episode in Madrid (Spain), Atmos. Environ., 189, 80–88, https://doi.org/10.1016/j.atmosenv.2018.06.037, 2018. Data availability. Data are available at https://www-air.larc.nasa. gov/cgi-bin/ArcView/owlets.2018 (NASA, 2021). Data availability. Data are available at https://www-air.larc.nasa. gov/cgi-bin/ArcView/owlets.2018 (NASA, 2021). Supplement. The supplement related to this article is available on- line at: https://doi.org/10.5194/acp-21-13051-2021-supplement. Arya, S. P.: Air pollution meteorology and dispersion, vol. 6, Ox- ford University Press, New York, 1999. Athanasopoulou, E., Tombrou, M., Pandis, S. N., and Russell, A. G.: The role of sea-salt emissions and heterogeneous chemistry in the air quality of polluted coastal areas, Atmos. Chem. Phys., 8, 5755–5769, https://doi.org/10.5194/acp-8-5755-2008, 2008. Author contributions. CJH, AGC, and RD conceived the analysis and study participation. MAB Jr., NB, and KB collected and ana- lyzed the PILS-IC and NH3 data. NB and CJH conducted the tra- jectory and source identification analyses. VC and AGC provided analytical input and interpretation. NB and CJH wrote the article. All authors provided feedback and revisions to the article. Backes, A. M., Aulinger, A., Bieser, J., Matthias, V., and Quante, M.: Ammonia emissions in Europe, part II: How ammonia emission abatement strategies af- fect secondary aerosols, Atmos. Environ., 126, 153–161, https://doi.org/10.1016/j.atmosenv.2015.11.039, 2016. Battaglia Jr., M. A., Douglas, S., and Hennigan, C. J.: Effect of the urban heat island on aerosol pH, Environ. Sci. Technol., 51, 13095–13103, https://doi.org/10.1021/acs.est.7b02786, 2017. Competing interests. The contact author has declared that neither they nor their co-authors have any competing interests. Battaglia, M. A., Balasus, N., Ball, K., Caicedo, V., Delgado, R., Carlton, A. G., and Hennigan, C. J.: Urban aerosol chem- istry at a land-water transition site during summer – Part 2: Aerosol pH and liquid water content, Atmos. Chem. Phys. Dis- cuss. [preprint], https://doi.org/10.5194/acp-2021-368, in review, 2021. Disclaimer. N. Balasus et al.: Urban aerosol chemistry at a land–water transition site during summer Prior studies have shown that sulfate is regional in nature in the eastern US, exhibiting low spatial variability over tens of kilometers (Beyersdorf et al., 2016). Our results are con- sistent with these findings, suggesting that meteorological phenomena such as the bay breeze circulation or urban heat islands (Battaglia et al., 2017) do not substantially alter sul- fate levels in this region. On the contrary, our results suggest strong gradients of reactive nitrogen species in the aerosol and gas phases, which will also produce periodic spatial gra- dients (including episodic spikes) in PM2.5 mass. The rela- tively short duration of these events and their dependence on proximity to water suggests they may not be well captured by regulatory monitoring (or longer-term monitoring for de- position networks). These results likely have implications for atmospheric chemistry and air quality in other coastal cities, including those outside of the eastern US. Our results suggest that analyses of urban air quality may be more illuminating if the urban areas are, themselves, segregated into coastal and inland locations. For example, the AeroCom study evaluated a number of global aerosol models using predictions in ur- ban and remote locations as key indicators of model predic- tive skill (Tsigaridis et al., 2014). Our results highlight im- portant differences in sources and processes affecting coastal versus inland urban areas that may have a determining effect on aerosol composition and concentrations. Acknowledgements. Annmarie G. Carlton and Christopher J. Hen- nigan acknowledge funding from the National Science Foun- dation, AGS-1719252 and AGS-1719245. Ruben Delgado and Vanessa Caicedo acknowledge support by the Maryland De- partment of the Environment (contract no. U00P8400651). Nicholas Balasus and Katherine Ball received support through the NOAA Office of Education, Educational Partnership Program with Minority Serving Institutions (EPP/MSI) and the Cooperative Sci- ence Center for Earth System Sciences and Remote Sensing Tech- nologies (grant no. NA16SEC4810008). Acknowledgements. Annmarie G. Carlton and Christopher J. Hen- nigan acknowledge funding from the National Science Foun- dation, AGS-1719252 and AGS-1719245. Ruben Delgado and Vanessa Caicedo acknowledge support by the Maryland De- partment of the Environment (contract no. U00P8400651). Nicholas Balasus and Katherine Ball received support through the NOAA Office of Education, Educational Partnership Program with Minority Serving Institutions (EPP/MSI) and the Cooperative Sci- ence Center for Earth System Sciences and Remote Sensing Tech- nologies (grant no. NA16SEC4810008). Financial support. N. Balasus et al.: Urban aerosol chemistry at a land–water transition site during summer This research has been supported by the Na- tional Science Foundation (grant nos. AGS-1719252 and AGS- 1719245), Maryland Department of the Environment (contract no. U00P8400651), and the National Oceanic and Atmospheric Ad- ministration (grant no. NA16SEC4810008). Review statement. This paper was edited by James Allan and re- viewed by two anonymous referees. Review statement. This paper was edited by James Allan and re- viewed by two anonymous referees. References Publisher’s note: Copernicus Publications remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Atmos. Chem. Phys., 21, 13051–13065, 2021 4 Conclusions Low dry deposition velocities over the Chesa- peake Bay enabled efficient transport of NH3 (and likely HNO3, as well) to the site. 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https://openalex.org/W2883066305
https://europepmc.org/articles/pmc6180092?pdf=render
English
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The AHR represses nucleotide excision repair and apoptosis and contributes to UV-induced skin carcinogenesis
Cell death and differentiation
2,018
cc-by
10,705
Abstract Ultraviolet B (UVB) radiation induces mutagenic DNA photoproducts, in particular cyclobutane pyrimidine dimers (CPDs), in epidermal keratinocytes (KC). To prevent skin carcinogenesis, these DNA photoproducts must be removed by nucleotide excision repair (NER) or apoptosis. Here we report that the UVB-sensitive transcription factor aryl hydrocarbon receptor (AHR) attenuates the clearance of UVB-induced CPDs in human HaCaT KC and skin from SKH-1 hairless mice. Subsequent RNA interference and inhibitor studies in KC revealed that AHR specifically suppresses global genome but not transcription-coupled NER. In further experiments, we found that the accelerated repair of CPDs in AHR-compromised KC depended on a modulation of the p27 tumor suppressor protein. Accordingly, p27 protein levels were increased in AHR- silenced KC and skin biopsies from AHR−/−mice, and critical for the improvement of NER. Besides increasing NER activity, AHR inhibition was accompanied by an enhanced occurrence of DNA double-strand breaks triggering KC apoptosis at later time points after irradiation. The UVB-activated AHR thus acts as a negative regulator of both early defense systems against carcinogenesis, NER and apoptosis, implying that it exhibits tumorigenic functions in UVB-exposed skin. In fact, AHR−/−mice developed 50% less UVB-induced cutaneous squamous cell carcinomas in a chronic photocarcinogenesis study than their AHR+/+ littermates. Taken together, our data reveal that AHR influences DNA damage-dependent responses in UVB-irradiated KC and critically contributes to skin photocarcinogenesis in mice. 3 Department of Dermatology and Allergology, Kiel University, 24105 Kiel, Germany 4 University Grenoble Alpes, INAC, CNRS, SyMMES UMR 3819, F-38000 Grenoble, France 5 Department of Environmental Toxicology and Center for Health and the Environment, University of California, Davis, CA 95616, USA Cell Death & Differentiation https://doi.org/10.1038/s41418-018-0160-1 ARTICLE ARTICLE ARTICLE The AHR represses nucleotide excision repair and apoptosis and contributes to UV-induced skin carcinogenesis Marius Pollet1 ●Siraz Shaik1 ●Melina Mescher1 ●Katrin Frauenstein1 ●Julia Tigges1 ●Stephan A. Braun2 ● Kevin Sondenheimer1 ●Mana Kaveh1 ●Anika Bruhs3 ●Stephan Meller2 ●Bernhard Homey2 ●Agatha Schwarz3 ● Charlotte Esser1 ●Thierry Douki4 ●Christoph F. A. Vogel5 ●Jean Krutmann1 ●Thomas Haarmann-Stemmann1 Received: 22 December 2017 / Revised: 18 June 2018 / Accepted: 25 June 2018 © ADMC Associazione Differenziamento e Morte Cellulare 2018. This article is publi Received: 22 December 2017 / Revised: 18 June 2018 / Accepted: 25 June 2018 © ADMC Associazione Differenziamento e Morte Cellulare 2018. This article is published with open access / / p © ADMC Associazione Differenziamento e Morte Cellulare 2018. This article is published with open access Introduction p NER is divided into two distinct sub-pathways: transcription-coupled repair (TCR), which quickly removes DNA adducts in actively transcribed genes, and global genome repair (GGR), which removes DNA lesions throughout the entire genome [3]. Both subpathways only differ in their way of DNA damage recognition. In case of TCR, Cockayne Syndrome (CS)-A and CSB proteins recognize the stalled RNA polymerase II and serve as damage sensors. In GGR, a complex consisting of XPC, RAD23B, and centrin-2 is responsible for DNA damage recognition. Upon damage verification by XPA, DNA is unwinded by the helicases XPB and XPD, two components of the general transcription factor TFIIH. Subsequently, the damage-containing DNA fragment is excised by the endo- nucleases XPF-ERCC1 and XPG and the remaining gap is filled by DNA polymerases. The pivotal role of NER in skin photocarcinogenesis is illustrated by the autosomal reces- sive NER disorder Xeroderma Pigmentosum (XP). XP patients with a deficiency in GGR have a dramatically increased risk of developing SCC and other skin cancers [5]. In contrast to the repair process, the precise regulation of NER in response to UVB radiation is still not completely understood. In the present study, we directly tested this hypothesis by assessing the impact of AHR on the removal of UVB- induced CPDs in human HaCaT KC and SKH-1 hairless mice. We found that AHR inhibition enhances the removal of CPDs in vitro and in vivo. Mechanistic studies revealed that AHR specifically dampens GGR activity by decreasing the protein level of the tumor suppressor p27KIP1 (p27), which probably affects DNA repair independently from its main function, i.e., induction of cell-cycle arrest. In addition to its positive effect on NER, AHR inhibition enhances the apoptotic clearance of remaining CPD-positive KC at later time points and thus enforces both early defense mechan- isms against photocarcinogenesis. Accordingly, chronic UVB irradiation experiments conducted in AHR+/+ and AHR−/−SKH-1 mice revealed a dramatically reduced SCC formation in the AHR−/−animals. The aryl hydrocarbon receptor (AHR) is a ligand- activated transcription factor that mediates the toxic effects of dioxins, polycyclic aromatic hydrocarbons and related environmental chemicals [6, 7]. In its inactive state, AHR rests in a cytosolic multiprotein complex. Upon ligand-binding, AHR shuttles in the nucleus, dimerizes with AHR nuclear translocator and binds to xenobiotic- responsive elements in the enhancer of target genes to initiate their transcription. Introduction the most frequent malignancies in humans [1, 2]. When skin is exposed to solar radiation, high-energy UVB rays pene- trate into the epidermis where they are mainly absorbed by the DNA of keratinocytes (KC). The resulting DNA pho- toproducts, in particular cyclobutane pyrimidine dimers (CPDs), are highly mutagenic and critically involved in the development of SCC [1, 2]. Depending on the extent of DNA damage, KC either initiate nucleotide excision repair (NER) or apoptosis to preserve genomic integrity [3, 4]. Failure of these early defense mechanisms may give rise to initiated cells, whose spread is further promoted by UVB- activated signaling pathways facilitating proliferation, Exposure to ultraviolet B (UVB) radiation is the major risk factor for cutaneous squamous cell carcinoma (SCC), one of Edited by S. Kaufmann. These authors contributed equally: Marius Pollet, Siraz Shaik, Melina Mescher. * Thomas Haarmann-Stemmann thomas.haarmann-stemmann@iuf-duesseldorf.de 1 IUF—Leibniz-Research Institute for Environmental Medicine, 40225 Düsseldorf, Germany 2 Department of Dermatology, Medical Faculty, Heinrich-Heine University, 40225 Düsseldorf, Germany Electronic supplementary material The online version of this article (https://doi.org/10.1038/s41418-018-0160-1) contains supplementary material, which is available to authorized users. Edited by S. Kaufmann. These authors contributed equally: Marius Pollet, Siraz Shaik, Melina Mescher. * Thomas Haarmann-Stemmann thomas.haarmann-stemmann@iuf-duesseldorf.de 1 IUF—Leibniz-Research Institute for Environmental Medicine, 40225 Düsseldorf, Germany 2 Department of Dermatology, Medical Faculty, Heinrich-Heine University, 40225 Düsseldorf, Germany Electronic supplementary material The online version of this article (https://doi.org/10.1038/s41418-018-0160-1) contains supplementary material, which is available to authorized users. These authors contributed equally: Marius Pollet, Siraz Shaik, Melina Mescher. Electronic supplementary material The online version of this article (https://doi.org/10.1038/s41418-018-0160-1) contains supplementary material, which is available to authorized users. * Thomas Haarmann-Stemmann thomas.haarmann-stemmann@iuf-duesseldorf.de * Thomas Haarmann-Stemmann thomas.haarmann-stemmann@iuf-duesseldorf.de 1 IUF—Leibniz-Research Institute for Environmental Medicine, 40225 Düsseldorf, Germany 2 Department of Dermatology, Medical Faculty, Heinrich-Heine University, 40225 Düsseldorf, Germany 2 Department of Dermatology, Medical Faculty, Heinrich-Heine University, 40225 Düsseldorf, Germany M. Pollet et al. contributes to photocarcinogenesis [12]. This idea is sup- ported by our recent observations that AHR triggers immunosuppression [13] and anti-apoptosis in response to UVB exposure [14]. Interestingly, independent reports revealed that AHR-antagonizing polyphenols, such as epigallocatechin-3-gallate [15], stimulate the repair of UVB-induced CPDs [16], suggesting that AHR signaling may influence NER. apoptosis resistance and impairment of host immune responses [1, 2]. Introduction AHR target genes encode drug- metabolizing enzymes, such as cytochrome P450 1A1, as well as proteins controlling cell division, differentiation and apoptosis [6, 7]. Another target is the AHR repressor (AHRR), a negative feedback regulator, which, depending on cell-type and tissue, may compete with AHR for both AHR nuclear translocator- and xenobiotic-responsive ele- ment-binding [8]. Beside the canonical AHR pathway, AHR activation often affects other signal transduction pathways, including NF-κB and EGFR signaling [6, 7]. AHR inhibition accelerates the removal of UVB- induced CPDs in human KC and murine skin c HaCaT-EV and HaCaT-shAHR KC were 2 based analysis of CPDs in the DNA of skin samples from AHR+/+ and AHR-/- SKH-1 mice 0.5 h and 48 h after exposure to a single dose o 185 mJ/cm2 UVB. f HaCaT KC were exposed to 200 J/m2 and treated with MNF (20 µM) and caspase inhibitor Ac-DEVD-CHO (20 µM alone or in combination. After 4 h, CPD content was measured. To assess if AHR affects TCR and/or GGR, we transiently silenced the expression of XPA (g), CSB (h) and XPC (i) and 24 h later irradiated the cells with 200 J/m2 UVB. Subsequently, the KC were treated fo AHR represses nucleotide excision repair and apoptosis and contributes to UV-induced skin. . . The AHR represses nucleotide excision repair and apoptosis and contributes to UV-induced skin. . . Fig. 1 Chemical and genetic inhibition of AHR enhances the removal of UVB-induced CPDs by modulating GGR. a Time-dependent clearance of CPDs in HaCaT KC irradiated with 200 J/m2 UVB. Cells were harvested at 0 (directly), 2, 4, 8, and 24 h after irradiation. Below the diagram, a representative SWB result is shown. b HaCaT KC were irradiated with 50 J/m2 (left panel) and 200 J/m2 (right panel) UVB and immediately treated with 0.1% DMSO or 20 µM MNF. After 4 h, CPD content was determined. c HaCaT-EV and HaCaT-shAHR KC were exposed to 200 J/m2 UVB and 4 h later the CPD content was mea- sured. d HaCaT KC were transfected with Ctrl. vector or an AHRR overexpression vector and 24 h later irradiated with 200 J/m2 UVB. After 4 h, the amount of CPDs was determined. e HPLC-MS/MS- based analysis of CPDs in the DNA of skin samples from AHR+/+ and AHR-/- SKH-1 mice 0.5 h and 48 h after exposure to a single dose of 185 mJ/cm2 UVB. f HaCaT KC were exposed to 200 J/m2 and treated with MNF (20 µM) and caspase inhibitor Ac-DEVD-CHO (20 µM) alone or in combination. After 4 h, CPD content was measured. To assess if AHR affects TCR and/or GGR, we transiently silenced the expression of XPA (g), CSB (h) and XPC (i) and 24 h later irradiated the cells with 200 J/m2 UVB. Subsequently, the KC were treated for 4 h with MNF (20 µM) or 0.1% DMSO and CPD content was ana- lyzed. Measurement of CPD content was carried out by SWB. AHR inhibition accelerates the removal of UVB- induced CPDs in human KC and murine skin To assess if AHR affects the removal of CPDs, we irra- diated human HaCaT KC with 200 J/m2 UVB and mon- itored CPD clearance by southwestern slot blot (SWB) analysis over time. CPD content stayed elevated in irra- diated cells for the first 4 h after irradiation and then gra- dually declined to only 10% after 24 h (Fig. 1a). As expected (and true for all of the following CPD SWB analyses), we did not detect a CPD signal in sham-exposed KC. Next, we exposed HaCaT KC to 50 J/m2 and 200 J/m2 UVB (approximately equivalent to 0.25 and 1 minimal erythema dose for a fair-skinned individual [17]) and sub- sequently treated the cells with the AHR antagonist 3′-methoxy-4′-nitroflavone (MNF, 20 µM) to exclude putative UV-filtering effects. 4 h after irradiation, the CPD content in MNF-treated cells was 37 and 33% lower than in solvent controls (Fig. 1b). Similar results were obtained when primary human epidermal KC were treated with MNF (data not shown). In stable AHR-knockdown cells (HaCaT- shAHR), the amount of UVB-induced CPDs was reduced by 37% 4 h after irradiation, as compared to irradiated empty vector control cells (HaCaT-EV) (Fig. 1c), thus excluding putative off-target effects of MNF. In addition, AHR is expressed in all cutaneous cell-types and con- tributes to physiological as well as pathophysiological processes [9]. In epidermal KC, AHR activation results from the absorbance of UVB rays by tryptophan and the subsequent generation of 6-formylindolo [3,2-b] carbazole [10]. This tryptophan photoproduct is a high-affinity ligand for AHR [11] and, among others, induces the expression of cytochrome P450 1A1 and cyclooxygenase-2 [9, 10]. As these enzymes play critical roles in tumor initiation and promotion, it was proposed that the UVB-activated AHR 1 Chemical and genetic inhibition of AHR enhances the removal UVB-induced CPDs by modulating GGR. a Time-dependent rance of CPDs in HaCaT KC irradiated with 200 J/m2 UVB. Cells e harvested at 0 (directly), 2, 4, 8, and 24 h after irradiation. Below diagram, a representative SWB result is shown. b HaCaT KC were diated with 50 J/m2 (left panel) and 200 J/m2 (right panel) UVB and mediately treated with 0.1% DMSO or 20 µM MNF. After 4 h, CPD tent was determined. AHR inhibition accelerates the removal of UVB- induced CPDs in human KC and murine skin For each experiment, the CPD content of the respective UVB-exposed controls was set to 100%. ns not significant. *p ≤0.05 AHR+/+ and AHR−/−SKH-1 hairless mice were irradiated once with 185 mJ/cm2 UVB and 0.5 h and 48 h thereafter skin biopsies were taken. CPD content was quantified in ectopic overexpression of AHR’s feedback inhibitor AHRR accelerated the removal of UVB-induced CPDs (Fig. 1d). To confirm an influence of AHR on CPD removal in vivo, M. Pollet et al. Fig. 2 AHR inhibits GGR by modulating the protein level of the tumor suppressor p27. a Western blot analysis of p27, pCDK2, and CDK2 in untreated HaCaT-EV and HaCaT-shAHR KC (representative blots). b HaCaT KC were transiently transfected with p27-targeted siRNA and Ctrl. siRNA. After 24 h, the cells were irradiated with 200 J/m2 UVB and treated with 20 µM MNF or 0.1% DMSO. After 4 h, the CPD content was compared by SWB. c HaCaT KC were transiently transfected with Ctrl. vector or a p27 expression plasmid. After 24 h, the cells were exposed to 200 J/m2 UVB and 4 h later the CPD content was determined. d HaCaT KC were irradiated with 200 J/m2 UVB and subsequently treated with 1 µM roscovitine, 500 nM SU9516, 125 nM BS-181 or 0.1% DMSO. After 4 h, the CPD content was analyzed by SWB. e Protein lysates from skin samples of AHR+/+ and AHR−/−SKH-1 mice were analyzed for p27 protein content by SDS-PAGE/western blotting. f Protein lysates from skin samples of AHR+/+, AHRR Tg and AHR−/−B6 mice were analyzed for p27 and AHRR protein content by SDS-PAGE/western blotting. g HaCaT KC were transiently transfected with an overexpression plasmid for rat AHRR or empty vector. After 24 h, the p27 protein level was compared by SDS-PAGE/western blot analysis (representative blot). ns not significant. *p ≤0.05 Fig. 2 AHR inhibits GGR by modulating the protein level of the tumor suppressor p27. a Western blot analysis of p27, pCDK2, and CDK2 in untreated HaCaT-EV and HaCaT-shAHR KC (representative blots). b HaCaT KC were transiently transfected with p27-targeted siRNA and Ctrl. siRNA. After 24 h, the cells were irradiated with 200 J/m2 UVB and treated with 20 µM MNF or 0.1% DMSO. After 4 h, the CPD content was compared by SWB. c HaCaT KC were transiently transfected with Ctrl. vector or a p27 expression plasmid. AHR regulates NER In all of the in vitro experiments, CPD content was deter- mined in adherent KC. Also, co-exposure of MNF-treated HaCaT KC to Ac-DEVD-CHO (20 µM), an inhibitor of effector caspases, did not affect the MNF-mediated accel- eration of CPD removal (Fig. 1f). These results indicate that the reduced CPD levels observed in AHR-compromised UVB-irradiated human KC were not due to an early apop- totic demise of damaged cells, but likely resulted from AHR inhibition accelerates the removal of UVB- induced CPDs in human KC and murine skin After 24 h, the cells were exposed to 200 J/m2 UVB and 4 h later the CPD content was determined. d HaCaT KC were irradiated with 200 J/m2 UVB and subsequently treated with 1 µM roscovitine, 500 nM SU9516, 125 nM BS-181 or 0.1% DMSO. After 4 h, the CPD content was analyzed by SWB. e Protein lysates from skin samples of AHR+/+ and AHR−/−SKH-1 mice were analyzed for p27 protein content by SDS-PAGE/western blotting. f Protein lysates from skin samples of AHR+/+, AHRR Tg and AHR−/−B6 mice were analyzed for p27 and AHRR protein content by SDS-PAGE/western blotting. g HaCaT KC were transiently transfected with an overexpression plasmid for rat AHRR or empty vector. After 24 h, the p27 protein level was compared by SDS-PAGE/western blot analysis (representative blot). ns not significant. *p ≤0.05 altered NER. To assess whether acceleration of CPD removal induced by AHR inhibition was NER-dependent, we silenced the expression of XPA. In contrast to control cells, MNF exposure of UVB-exposed XPA-silenced KC failed to affect CPD removal (Fig. 1g), strongly indicating that AHR modulates NER activity. We next investigated whether AHR antagonism affects GGR and/or TCR. Therefore, we transiently transfected HaCaT KC with siR- NAs targeted against XPC and CSB. After 24 h, transfected cells were irradiated with 200 J/m2 UVB and treated with 20 µM MNF or solvent. We found that MNF significantly increased CPD clearance in CSB-knockdown KC (Fig. 1h), but not in XPC-silenced KC (Fig. 1i). These results provide evidence that AHR inhibition accelerates CPD removal by specifically modulating GGR. complete biopsies by HPLC-MS/MS. In the skin of both genotypes, the distribution between TT, TC and CT CPDs was approximately 55:35:10 (Supplementary Figure S1), which is in accordance with previously published results [18]. Immediately after UVB exposure, CPD content did not significantly differ between AHR genotypes. However, after 48 h approximately 50% less CPDs were present in the skin of AHR−/−mice as compared to AHR+/+ mice (Fig. 1e), thus demonstrating that the negative effect of AHR on CPD clearance is also present in vivo. p27 is causally involved in the AHR-dependent inhibition of NER We have previously reported that AHR-compromised KC contain an increased level of the CDK inhibitor p27 [14]. he AHR represses nucleotide excision repair and apoptosis and contributes to UV-induced skin. . . Accordingly, the p27 protein level was strongly elevated in HaCaT-shAHR KC as compared to HaCaT-EV KC, and associated with a reduced phosphorylation of CDK2 T-160) (Fig. 2a). As enhanced p27 levels were reported to correlate with DNA repair capacity in human peripher blood lymphocytes [19], we assessed whether elevated p2 levels may be causative for the acceleration of NER. W silenced p27 expression using transient RNAi an ngly, the p27 protein level was strongly elevated in shAHR KC as compared to HaCaT-EV KC, and ed with a reduced phosphorylation of CDK2 correlate with DNA repair capacity in human peripheral blood lymphocytes [19], we assessed whether elevated p27 levels may be causative for the acceleration of NER. We Accordingly, the p27 protein level was strongly elevated in HaCaT-shAHR KC as compared to HaCaT-EV KC, and associated with a reduced phosphorylation of CDK2 (T-160) (Fig. 2a). As enhanced p27 levels were reported to correlate with DNA repair capacity in human peripheral blood lymphocytes [19], we assessed whether elevated p27 levels may be causative for the acceleration of NER. We silenced p27 expression using transient RNAi and M. Pollet et al. sham- and UVB-exposed HaCaT KC did not reveal a direct protein–protein interaction of p27 and CDK7 (data not shown). Thus, p27 may either affect GGR independently from CDK7 or through an indirect modulation of CDK7 function. Fig. 3 AHR-dependent modulation of the p27 protein level. HaCaT KC were irradiated with 0 and 200 J/m2 UVB and treated with 20 µM MNF or 0.1% DMSO. After 2 h and 3 h, cells were fixed and antibody stained for a p27 and b pp27 T-187 (scale: 10 µm). c HaCaT KC were treated with 2.5 µM BaP and 20 µM MNF alone or in combination. Control cells were treated with 0.2% DMSO. Protein content of p27 was assessed by SDS-PAGE/western blot analysis (right). d HaCaT- cells were exposed to 2.5 µM BaP, 20 µM MNF or both. After 24 h, copy numbers of p27 were determined by qPCR. Data are shown as fold of DMSO ctrl. e HaCaT KC were treated with 2.5 µM BaP, 10 µM MG132 and 0.2% DMSO alone or in combination. AHR regulates the proteasomal degradation of p27 We found that in contrast to respective control cells, knockdown of p27 abolished the MNF-mediated increase in CPD removal (Fig. 2b). In addition, transient overexpression of p27 in HaCaT KC resulted in an enhanced clearance of UVB-induced CPDs (Fig. 2c), demonstrating that p27 can per se stimulate NER activity. investigated if MNF exposure still accelerates NER. We found that in contrast to respective control cells, knockdown of p27 abolished the MNF-mediated increase in CPD removal (Fig. 2b). In addition, transient overexpression of p27 in HaCaT KC resulted in an enhanced clearance of UVB-induced CPDs (Fig. 2c), demonstrating that p27 can per se stimulate NER activity. AHR regulates the proteasomal degradation of p27 To assess the in vivo relevance of the observed AHR- dependent effects on p27 level, we determined the p27 protein content in the skin of two strains of AHR+/+ and AHR−/−mice. As expected, protein levels of p27 were higher in the skin of AHR−/−SKH-1 mice (Fig. 2e) and AHR−/−B6 mice (Fig. 2f) as compared to littermate con- trols. Also, overexpression of AHRR increased p27 protein content in the skin of transgenic B6 (AHRR Tg) mice [21] (Fig. 2f) as well as in transiently transfected HaCaT KC (Fig. 2g). We next tested whether transcriptional or post- transcriptional events were responsible for these effects. As expected, immunofluorescence (IF) staining of HaCaT KC revealed a nuclear accumulation of AHR in response to UVB irradiation, which was attenuated by MNF treatment (Supplementary Figure S3A). Exposure of HaCaT KC to MNF alone resulted in an increase of nuclear p27 (Fig. 3a). In comparison to irradiated HaCaT KC, an increased amount of nuclear p27 was also present in MNF-treated HaCaT KC 2 h and 3 h after exposure to 200 J/m2 UVB (Fig. 3a). The increased levels of p27 were accompanied by a reduced phosphorylation of CDK2 (Supplementary Fig- ure S3B). The differences in nuclear p27 were reflected by IF stainings using a phospho-p27 (T-187) antibody (Fig. 3b). The amino acid T-187 serves as substrate for CDK2 and targets p27 to proteolysis [22]. Further mechanistic studies in HaCaT cells using chemical AHR ligands confirmed that AHR activity affected p27 on the protein level. Treatment of HaCaT KC with the AHR agonist benzo[a]pyrene (BaP) reduced the p27 protein level, whereas exposure to MNF increased it (Fig. 3c). These changes were not retrievable on the mRNA level (Fig. 3d). Accordingly, the BaP-induced reduction of p27 protein was abrogated by co-treatment with the proteasome inhibitor MG-132 (Fig. 3e). Interestingly, exposure of HaCaT- shAHR KC to EGF attenuated the elevated p27 protein level (Fig. 3f), whereas treatment of HaCaT-EV KC with the EGFR inhibitor PD153035 was sufficient to increase it (Fig. 3g). Importantly, treatment of HaCaT KC with inhi- bitors for EGFR (PD153035) and its effector pathways PI3K/AKT (Wortmannin) and MEK/ERK (PD98059) accelerated the CPD removal 4 h after UVB irradiation (Fig. 3h). These results are consistent with the assumption that AHR attenuates GGR in UVB-irradiated KC by pro- moting proteolysis of p27, most likely involving EGFR and downstream signal transduction. investigated if MNF exposure still accelerates NER. p27 is causally involved in the AHR-dependent inhibition of NER Protein content of p27 was assessed by SDS-PAGE/western blotting (top: quantification, bottom: representative WB). f HaCaT-shAHR KC were treated for 2 h with 50 ng/ml EGF or solvent. Subsequently, p27 protein level was determined by SDS-PAGE/western blot analysis (top: quantification, bottom: representative WB). g HaCaT-EV KC were treated for 2 h with 10 µM of the EGFR inhibitor PD153035 or 0.1% DMSO. Sub- sequently, p27 protein level was determined by SDS-PAGE/western blotting (top: quantification, bottom: representative WB). h HaCaT KC were irradiated with 200 J/m2 UVB and immediately treated with 0.1% DMSO, 10 µM PD153035 (EGFR inhibitor), 1 µM Wortmannin (PI3K inhibitor) or 10 µM PD98059 (MEK inhibitor). After 4 h, CPD content was determined by SWB. *p ≤0.05 p27 enhances GGR activity independently from cell- cycle arrest After 24 h, the KC were exposed to 200 J/m2 UVB and another 24 h later, the amount of dead cells was determined by Annexin V/PI staining. c Western blot analysis of CHK1 in HaCaT KC 24 h after irradiation with 0 and 200 J/m2 UVB (representative blot). d HaCaT KC were irradiated with 0 and 200 J/m2 UVB. After 18 h, γH2AX levels were assessed by SDS-PAGE/western blotting. e HaCaT KC were irradiated with 0 and 200 J/m2 UVB and immedi- ately treated with MNF (20 µM) and DMSO alone or in combination with the caspase inhibitor Ac-DEVD-CHO (20 µM). After 18 h, DSBs were detected by neutral comet assay analyses. *p ≤0.05 p27 enhances GGR activity independently from cell- cycle arrest Interestingly, FACS analyses of UVB-exposed HaCaT KC treated with DMSO or MNF did not show any significant differences in the cell-cycle profiles, at least within the first 4 h after irradiation (supplementary figure S2), suggesting that p27 affects GGR independently from CDK2/CDK4 inhibition. To elucidate if p27 alters GGR activity in a CDK-dependent manner, we tested the potential of various chemical CDK inhibitors on their ability to alter CPD removal in HaCaT KC. In fact, treatment of UVB-exposed KC with roscovitine, an inhibitor of CDK1, CDK2, CDK5, and CDK7, resulted in an enhanced removal of CPDs 4 h after UVB irradiation (Fig. 2d). In contrast, exposure to SU9516, an inhibitor of CDK1, CDK2, and CDK4, had no effect on the cellular amount of UVB-induced CPDs (Fig. 2d), again supporting our notion that p27 alters GGR activity independently from CDK2 and CDK4. Interest- ingly, treatment of UVB-irradiated cells with BS-181, a specific inhibitor of CDK7, mimicked the positive effect of p27 overexpression on CPD repair (Fig. 2d). Although CDK7 is known to be involved in the regulation of NER [20], subsequent co-immunoprecipitation analyses in Fig. 4 AHR inhibition increases UVB-induced apoptosis and is associated with DSB formation. a HaCaT-EV and HaCaT-shAHR KC were irradiated with 0 and 200 J/m2 UVB. After 24 h, the amount of dead cells was analyzed by Annexin V/PI staining. b HaCaT KC were transiently transfected with an expression vector for rat AHRR or empty vector. After 24 h, the KC were exposed to 200 J/m2 UVB and another 24 h later, the amount of dead cells was determined by Annexin V/PI staining. c Western blot analysis of CHK1 in HaCaT KC 24 h after irradiation with 0 and 200 J/m2 UVB (representat blot). d HaCaT KC were irradiated with 0 and 200 J/m2 UVB. A 18 h, γH2AX levels were assessed by SDS-PAGE/western blotti e HaCaT KC were irradiated with 0 and 200 J/m2 UVB and imme ately treated with MNF (20 µM) and DMSO alone or in combinat with the caspase inhibitor Ac-DEVD-CHO (20 µM). After 18 h, DS were detected by neutral comet assay analyses. *p ≤0.05 The AHR represses nucleotide excision repair and apoptosis and contributes to UV-induced skin. . . Fig. 4 AHR inhibition increases UVB-induced apoptosis and is associated with DSB formation. a HaCaT-EV and HaCaT-shAHR KC were irradiated with 0 and 200 J/m2 UVB. p27 enhances GGR activity independently from cell- cycle arrest After 24 h, the amount of dead cells was analyzed by Annexin V/PI staining. b HaCaT KC were transiently transfected with an expression vector for rat AHRR or empty vector. After 24 h, the KC were exposed to 200 J/m2 UVB and another 24 h later, the amount of dead cells was determined by Annexin V/PI staining. c Western blot analysis of CHK1 in HaCaT KC 24 h after irradiation with 0 and 200 J/m2 UVB (represent blot). d HaCaT KC were irradiated with 0 and 200 J/m2 UVB. A 18 h, γH2AX levels were assessed by SDS-PAGE/western blot e HaCaT KC were irradiated with 0 and 200 J/m2 UVB and imm ately treated with MNF (20 µM) and DMSO alone or in combina with the caspase inhibitor Ac-DEVD-CHO (20 µM). After 18 h, D were detected by neutral comet assay analyses. *p ≤0.05 The AHR represses nucleotide excision repair and apoptosis and contributes to UV-induced skin. . . The AHR represses nucleotide excision repair and apoptosis and contributes to UV-induced skin. . . Fig. 4 AHR inhibition increases UVB-induced apoptosis and is associated with DSB formation. a HaCaT-EV and HaCaT-shAHR KC were irradiated with 0 and 200 J/m2 UVB. After 24 h, the amount of dead cells was analyzed by Annexin V/PI staining. b HaCaT KC were transiently transfected with an expression vector for rat AHRR or empty vector. After 24 h, the KC were exposed to 200 J/m2 UVB and another 24 h later, the amount of dead cells was determined by Annexin V/PI staining. c Western blot analysis of CHK1 in HaCaT KC 24 h after irradiation with 0 and 200 J/m2 UVB (representative blot). d HaCaT KC were irradiated with 0 and 200 J/m2 UVB. After 18 h, γH2AX levels were assessed by SDS-PAGE/western blotting. e HaCaT KC were irradiated with 0 and 200 J/m2 UVB and immedi- ately treated with MNF (20 µM) and DMSO alone or in combination with the caspase inhibitor Ac-DEVD-CHO (20 µM). After 18 h, DSBs were detected by neutral comet assay analyses. *p ≤0.05 Fig. 4 AHR inhibition increases UVB-induced apoptosis and is associated with DSB formation. a HaCaT-EV and HaCaT-shAHR KC were irradiated with 0 and 200 J/m2 UVB. After 24 h, the amount of dead cells was analyzed by Annexin V/PI staining. b HaCaT KC were transiently transfected with an expression vector for rat AHRR or empty vector. AHR inhibition increases UVB-induced apoptosis in KC by enhancing DNA double strand break formation The DSBs still occurred when the cells were co-exposed to a caspase inhibitor, indicating that they were not produced by apoptosis-related DNases (Fig. 4e). These results indicate that the enhanced UVB-induced apoptosis in AHR- compromised cells is driven by an increased formation of DSBs and that it occurs independently of NER, thus reconciling our seemingly contradictory observations on NER and apoptosis in AHR-deficient KC. Fig. 5 AHR contributes to UVB-induced skin carcinogenesis in SKH- 1 hairless mice. a Ten AHR+/+ and eight AHR−/−SKH-1 mice were chronically exposed to UVB radiation as described in Materials and Methods section. 2 weeks after the last irradiation, total numbers of SCCs were compared. b Time-course of UVB-induced SCC devel- opment in the skin of AHR+/+ and AHR−/−SKH-1 mice. c Repre- sentative histological pictures from primary SCCs from AHR+/+ and AHR−/−mice (scale: 200 µm). *p ≤0.05. d Immunohistochemical staining of lesional and adjacent non-lesional skin of AHR+/+ and AHR−/−SKH-1 mice with an antibody detecting mutant p53 protein (scale: 300 µm). e Protein lysates from lesional (T) and irradiated non- lesional (NS) skin of AHR+/+ and AHR−/−SKH-1 mice were ana- lyzed for phosphorylation (at Y-705) and expression of STAT3 by SDS-PAGE/western blotting. *p ≤0.05 AHR inhibition increases UVB-induced apoptosis in KC by enhancing DNA double strand break formation AHRR-overexpressing HaCaT KC resulted in a more pro- nounced apoptotic response, as compared to control cells (Fig. 4b, Supplementary Figure S4C). In our previous study we showed that the anti-apoptotic action of AHR is due to the loss of checkpoint kinase-1 (CHK1) expression [14]. CHK1 is a stress kinase that is directly activated in response to DNA damage to prevent cell-cycle progression [24]. In fact, exposure of HaCaT KC to 20 µM MNF resulted in a reduced expression of CHK1 protein, both in sham and UVB-irradiated cells (Fig. 4c). As CHK1 is also required for homologous recombination repair (HRR) [25], we next asked if DNA double-strand breaks (DSBs) may be responsible for the enhancement of apoptosis observed in AHR-compromised KC. Accordingly, phosphorylation of An increase in NER should partially reduce UVB-induced KC apoptosis [23]. This is in contrast to our previous report that AHR inhibition enhances UVB-induced KC apoptosis [14]. Accordingly, FACS-based analyses of Annexin V/PI- stained cells revealed a stronger apoptotic response in HaCaT-shAHR KC exposed to 200 J/m2 UVB as compared to HaCaT-EV cells (Fig. 4a, Supplementary Figure S4A). The AHR-dependent difference in UVB-induced apoptosis was confirmed on the level of caspase-3 activity (Supplementary Figure S4B). Also, UVB irradiation of M. Pollet et al. Fig. 5 AHR contributes to UVB-induced skin carcinogenesis in SKH- 1 hairless mice. a Ten AHR+/+ and eight AHR−/−SKH-1 mice were chronically exposed to UVB radiation as described in Materials and Methods section. 2 weeks after the last irradiation, total numbers of SCCs were compared. b Time-course of UVB-induced SCC devel- opment in the skin of AHR+/+ and AHR−/−SKH-1 mice. c Repre- sentative histological pictures from primary SCCs from AHR+/+ and AHR−/−mice (scale: 200 µm). *p ≤0.05. d Immunohistochemical staining of lesional and adjacent non-lesional skin of AHR+/+ and AHR−/−SKH-1 mice with an antibody detecting mutant p53 protein (scale: 300 µm). e Protein lysates from lesional (T) and irradiated non- lesional (NS) skin of AHR+/+ and AHR−/−SKH-1 mice were ana- lyzed for phosphorylation (at Y-705) and expression of STAT3 by SDS-PAGE/western blotting. *p ≤0.05 histone H2AX (γH2AX), an established marker for DSBs, was significantly stronger in UVB-exposed MNF-treated HaCaT as compared to irradiated control KC (Fig. 4d). Moreover, neutral comet assay analyses demonstrated an enhanced occurrence of DSBs in MNF-treated HaCaT KC 18 h after UVB irradiation (Fig. 4e). Reduced photocarcinogenesis in AHR−/−mice Fig. 5 AHR contributes to UVB-induced skin carcinogenesis in SKH- 1 hairless mice. a Ten AHR+/+ and eight AHR−/−SKH-1 mice were chronically exposed to UVB radiation as described in Materials and Methods section. 2 weeks after the last irradiation, total numbers of SCCs were compared. b Time-course of UVB-induced SCC devel- opment in the skin of AHR+/+ and AHR−/−SKH-1 mice. c Repre- sentative histological pictures from primary SCCs from AHR+/+ and AHR−/−mice (scale: 200 µm). *p ≤0.05. d Immunohistochemical staining of lesional and adjacent non-lesional skin of AHR+/+ and AHR−/−SKH-1 mice with an antibody detecting mutant p53 protein (scale: 300 µm). e Protein lysates from lesional (T) and irradiated non- lesional (NS) skin of AHR+/+ and AHR−/−SKH-1 mice were ana- lyzed for phosphorylation (at Y-705) and expression of STAT3 by SDS-PAGE/western blotting. *p ≤0.05 Our results demonstrate that the UVB-activated AHR is a negative regulator of GGR and apoptosis. We therefore speculated that AHR-deficiency would protect mice against the UVB-induced development of SCC. To test this hypothesis, we conducted a chronic photocarcinogenesis study in AHR+/+ and AHR−/−SKH-1 mice. As shown in Fig. 5a, AHR−/−mice developed approximately 50% less skin tumors than their AHR+/+ littermates. The animals started to develop skin tumors after 15–17 weeks of UVB exposure (Fig. 5b). There were no genotype-dependent dif- ferences in tumor histology (Fig. 5c). As previously reported for hairless mice [26] all UVB-induced skin tumors were SCC ranging from well to poorly differentiated in situ car- cinomas to deeply infiltrating tumors. This was confirmed by immunohistochemical analyses revealing an accumulation of mutant p53 protein, a hallmark of UVB-induced SCCs [27, 28], in lesional but not adjacent non-lesional skin from mice of both AHR genotypes (Fig. 5d). In addition, immu- noblot analyses exhibited an elevated activation of STAT3 (phosphorylation at Y-705), an established key driver of UVB-induced SCC development [29], in tumor samples from both, AHR+/+ and AHR−/−mice, as compared to irradiated non-lesional skin (Fig. 5e). Notably, sham- exposed control animals of both genotypes did not develop any skin tumors (data not shown). Taken together, these data reveal a crucial role of AHR in UVB-induced skin carcinogenesis. Discussion The major findings of the present study are that AHR attenuates the clearance of UVB-induced CPDs by specifi- cally repressing GGR in a p27-dependent manner, and that AHR-deficiency largely protects mice against UVB-induced skin carcinogenesis. Although an effect on p27 transcription was observed in extracutaneous tissues and cells [43], we propose that AHR affects p27 post-translationally, i.e., by activating signal transduction pathways that phosphorylate p27 and target it to the pro- teasome. The precise underlying mechanism has not yet been identified, however our data clearly point to an involvement of EGFR and downstream PI3K/AKT and MEK/ERK signal transduction. Importantly, AKT [44, 45] and ERK [46, 47] are known to phosphorylate p27 and thereby affect its sub-cellular localization and associated proteasomal degradation. As indicated by our IF stainings, the latter process may involve a CDK2-mediated phos- phorylation of p27 at T-187 [22]. Our hypothesis is further supported by previous reports from others and us, showing that the UVB-activated AHR stimulates EGFR and down- stream MEK/ERK and PI3K/AKT signaling [10, 48]. A role of EGFR in p27 regulation is also underscored by clinical studies reporting elevated cutaneous p27 protein levels in cancer patients under systemic EGFR inhibitor therapy [49]. As reported earlier [14], AHR inhibition did not reduce but rather enhance UVB-induced apoptosis in HaCaT KC (Fig. 6). The increase of p27 protein upon AHR antagonism resulted in a decreased expression of CHK1, which is required for the initiation of DNA damage responses, i.e., Our results are consistent with the view that an upregu- lation of the cutaneous p27 protein is causative for the increased GGR in AHR-compromised KC (Fig. 6). The underlying molecular mechanism is quite enigmatic and seems to be independent from p27′s capability to inhibit CDK2/CDK4 and induce cell-cycle arrest. Interestingly, an inhibition of CDK7 seemed to mimic the positive effect of p27 overexpression on NER. CDK7 is the active subunit of the CDK-activating complex, which is part of the multi- faceted transcription factor TFIIH. It has been shown that upon UV exposure the CDK-activating complex dissociates from the TFIIH core complex [36], which then switches its function from transcription factor to NER factor [20, 37]. In fact, chemical inhibition of CDK7 has been shown to spe- cifically increase GGR activity [20], thus making it a likely candidate being involved in the stimulation of this repair pathway in AHR-compromised KC. The AHR represses nucleotide excision repair and apoptosis and contributes to UV-induced skin. . . CPDs are primarily responsible for the onset of UVB- induced skin carcinogenesis [28] and their forced repair has been demonstrated to efficiently reduce the incidence of skin cancer in mice [30] and humans [31]. We therefore believe that the reduced SCC development in AHR−/−mice is, at least to a major extent, the consequence of elevated NER activity. This assumption is further supported by the fact that AHR inhibition specifically increased GGR, which is the pivotal DNA repair system restraining photo- carcinogenesis. In fact, XP patients suffering from GGR- deficiency have a greatly increased risk of developing skin cancer [5]. In contrast, TCR-deficiency (CS) is not asso- ciated with an increased incidence of skin cancer, which is probably due to an enhanced cytotoxicity (but not muta- genicity) in response to UV exposure [32]. Since CPDs are the major trigger for UVB-induced immunosuppression [33], their accelerated repair in AHR−/−mice may have also amplified antitumor immune responses. In addition to the beneficial effect on GGR, the observed increase in UVB- induced apoptosis and the associated clearance of damaged cells may have also contributed to the reduced SCC development in AHR−/−mice. Indeed, an enhancement of epidermal apoptosis, for instance by topical application of caffeine or resveratrol, has been shown to reduce UVB- induced skin carcinogenesis in mice [34, 35]. Thus, an enhanced stimulation of both defense mechanisms, GGR and apoptosis, is most probably responsible for the reduced SCC occurrence in AHR−/−mice. Fig. 6 Hypothetical model: AHR inhibition increases p27 protein content resulting in an acceleration of GGR and reduction of muta- genic CPDs and associated SCC development. The increased p27 level results in a reduced CHK1 expression and an attenuation of HRR. Accordingly, CPD-positive AHR-compromised KC are sensitive towards mitosis-related DSBs and subsequent apoptotic cell death Fig. 6 Hypothetical model: AHR inhibition increases p27 protein content resulting in an acceleration of GGR and reduction of muta- genic CPDs and associated SCC development. The increased p27 level results in a reduced CHK1 expression and an attenuation of HRR. Accordingly, CPD-positive AHR-compromised KC are sensitive towards mitosis-related DSBs and subsequent apoptotic cell death correlate with cancer proneness. Hence, downregulation of p27 by oncogenic signal transduction occurs frequently in various solid cancers [41], including SCC [42]. However, in accor- dance with a previous report [38], we were not able to show a direct protein–protein interaction between CDK7 and p27 in our cell system, indicating that both proteins either interact indirectly via an additional yet to be identified factor or affect GGR through independent mechanisms. As reported earlier [14], AHR inhibition did not reduce but rather enhance UVB-induced apoptosis in HaCaT KC (Fig. 6). The increase of p27 protein upon AHR antagonism resulted in a decreased expression of CHK1, which is required for the initiation of DNA damage responses, i.e., The p27 gene (CDKN1B) is haplo-insufficient for tumor suppression [39, 40], indicating that low p27 levels M. Pollet et al. cell-cycle arrest [24] and HRR [25]. Inhibition of CHK1 was shown to enhance UVB-induced KC apoptosis and to prevent photocarcinogenesis [34]. In fact, we have pre- viously observed reduced CHK1 protein levels not only in AHR-compromised human KC, but also in the skin of AHR −/−mice [14]. When UVB-irradiated KC enter mitosis, remaining CPDs may cause a collapse of the replication fork leading to the formation of DSBs [50, 51]. It is therefore highly likely that AHR-compromised KC, due to reduced CHK1 levels, do not properly repair these potent apoptosis-inducing DNA lesions [4], resulting in elevated cell death at later time points (Fig. 6). Accordingly, expo- sure of CHO cells to dioxin has been shown to accelerate HRR in an AHR-dependent manner [52]. In addition, we have previously reported that AHR is required for proper repair of DSBs induced by ionizing radiation [53] and that reconstitution of CHK1 expression neutralizes the enhanced susceptibility of AHR-silenced KC towards UVB-induced apoptosis [14]. Accordingly, the increased DSB formation observed in AHR-deficient KC is most probably due to an attenuated HRR. Thus, AHR inhibition prevents photo- carcinogenesis by accelerating GGR as well as by pro- moting lethal DSBs in remaining CPD-positive KC (Fig. 6). and Cell Cultures (Braunschweig, Germany). The cultiva- tion of HaCaT KC and the generation and cultivation of HaCaT-EV and HaCaT-shAHR KC has been previously described [10]. The source for UVB irradiation was a TL20W/ 12RS lamp (Philips, Eindhoven, The Netherlands), which emits most of its energy in the UVB range (290–320 nm) with an emission peak at 310 nm. For both UVB and sham exposure, culture medium was replaced by PBS. Southwestern slot–blot analyses Equal amounts of isolated DNA were diluted in TE10/1- buffer (pH 8), incubated for 5 min in boiling water and cooled down for 2 min on ice. Samples were spotted on positively charged nitrocellulose membrane (GE Healthcare, Little Chalfont, UK) using a slot–lot chamber coupled to a vacuum manifold. The membrane-bound DNA was dena- tured for 45 min on Whatmann paper soaked with 0.4 N NaOH. Membranes were blocked overnight in 5% skim milk in TBS-Tween-20 (0.5%; TBS-T) at 4 °C. Membranes were incubated for 2 h at 4 °C with a HRP-conjugated thy- mine dimer antibody (Kamiya Biomedical Company, Tuk- wila, WA, USA) in 5% skim milk/TBS-T. Membranes were washed and signals were detected using the WesternBright ECL substrate (Advansta, Menlo Park, CA, USA). The signal intensity of the irradiated control sample was defined as 100%. A serial dilution of this sample was spotted and stained for CPDs to ensure linearity of the signal intensity (example shown in Supplementary Figure S5). The SWB-based CPD detection in sham-exposed KC did not produce any detectable signals and therefore is not shown. Overexpression of AHRR increased p27 protein level, CPD removal and apoptosis, implying that AHRR not only attenuates AHR/xenobiotic-responsive element-dependent responses but also AHR-triggered protein kinase activities. In fact, we have recently reported that the dioxin-induced nuclear accumulation and DNA-binding of C/EBPβ and NF-κB, which is mediated via non-canonical AHR signal- ing, is reduced in tissues of AHRR Tg mice [21]. However, as AHRR abrogates the growth and malignancy of various human cancers [8], a further elucidation of the link between AHRR and p27 may help to better understand AHRR’s tumor suppressive properties. In summary, we provide evidence that AHR represses GGR and apoptosis in UVB-exposed KC and critically contributes to skin photocarcinogenesis. The translational relevance of these findings is highlighted by a recent two- stage genome-wide association study identifying AHR as a novel susceptibility locus for SCC in humans [54]. As we have previously shown that AHR antagonism in human skin in vivo is feasible [55], AHR may be a suitable target for topical chemoprevention of UVB-induced skin malignancies. SDS-PAGE and western blot analyses Protein isolation, SDS-PAGE and western blot analyses were carried out as described previously [14]. Primary antibodies used in this study were: p27, CDK2, γH2AX, β- actin, GAPDH, STAT3, pSTAT3 Y-705 (all from Cell Signaling Technology, Dancers, MA, USA), XPA (Sigma- Aldrich), α-tubulin (ExBio, Vestec, Czech Republic), AHRR (Novoprotein Scientific, Summit, NJ, USA), AHR, pp27 T-187, pCDK2 T-160, XPC and CSB (all from Santa Cruz Biotechnology). For cell treatment, Wortmannin, PD153035, PD98059, MG-132, BaP (all from Sigma-Aldrich, Munich, Germany), roscovitine (Enzo Life Sciences, Loerrach, Germany), BS-181 (Selleckchem, Houston, TX, USA), SU9516 (Tocris Bioscience, Bristol, UK) and MNF (pro- vided by I. Meyer, Symrise AG, Holzminden, Germany) were dissolved in DMSO. EGF (Sigma-Aldrich) and Ac-DEVD-CHO (Enzo Life Sciences) were dissolved in water. cell-cycle arrest [24] and HRR [25]. Inhibition of CHK1 was shown to enhance UVB-induced KC apoptosis and to prevent photocarcinogenesis [34]. In fact, we have pre- viously observed reduced CHK1 protein levels not only in AHR-compromised human KC, but also in the skin of AHR −/−mice [14]. When UVB-irradiated KC enter mitosis, remaining CPDs may cause a collapse of the replication fork leading to the formation of DSBs [50, 51]. It is therefore highly likely that AHR-compromised KC, due to reduced CHK1 levels, do not properly repair these potent apoptosis-inducing DNA lesions [4], resulting in elevated cell death at later time points (Fig. 6). Accordingly, expo- sure of CHO cells to dioxin has been shown to accelerate HRR in an AHR-dependent manner [52]. In addition, we have previously reported that AHR is required for proper repair of DSBs induced by ionizing radiation [53] and that reconstitution of CHK1 expression neutralizes the enhanced susceptibility of AHR-silenced KC towards UVB-induced apoptosis [14]. Accordingly, the increased DSB formation observed in AHR-deficient KC is most probably due to an attenuated HRR. Thus, AHR inhibition prevents photo- carcinogenesis by accelerating GGR as well as by pro- moting lethal DSBs in remaining CPD-positive KC (Fig. 6). Overexpression of AHRR increased p27 protein level, CPD removal and apoptosis, implying that AHRR not only attenuates AHR/xenobiotic-responsive element-dependent responses but also AHR-triggered protein kinase activities. In fact, we have recently reported that the dioxin-induced nuclear accumulation and DNA-binding of C/EBPβ and NF-κB, which is mediated via non-canonical AHR signal- ing, is reduced in tissues of AHRR Tg mice [21]. However, as AHRR abrogates the growth and malignancy of various human cancers [8], a further elucidation of the link between AHRR and p27 may help to better understand AHRR’s tumor suppressive properties. Cell culture, UVB irradiation and treatment HaCaT KC were provided by P. Boukamp (DKFZ/IUF) and authenticated by the German Collection of Microorganisms Quantiative real-time PCR RNA isolation, cDNA synthesis, quantitative real-time PCR and primer sequences were described previously [14]. Immunofluorescence staining of cells KC were grown on collagen I-coated coverslips until subconfluency and fixed with either ice-cold MeOH or 70% EtOH for 10 min at −20 °C. EtOH-fixed cells were permeabilized with 0.1% Triton/PBS for 10 min at RT. Cells were blocked with 5% BSA for 1 h at RT and subsequently incubated with primary antibodies (AHR, p27, pp27 T-187, pCDK2 T-160) diluted in blocking solution. Next day, cells were incubated with AlexaFluor 488- and 568-conjugated secondary antibodies and DAPI for 1 h at RT. Antibodies were diluted in blocking solu- tion and immunostained cells were mounted in Mowiol (Sigma-Aldrich). Animals and chronic UVB irradiation Generation, breeding and genotyping of AHR−/−and AHR+/+ SKH-1 hairless mice was described previously [14]. The generation, breeding and characterization of transgenic AHRR B6 (AHRR Tg) mice is described in Ref. [21]. All animals were housed in our specific pathogen-free animal facility. For the photocarcinogenesis study, 8 AHR−/−SKH-1 mice and 10 AHR+/+ littermates were irradiated over a period of 24 weeks. Sham-exposed mice of each genotype were used as control groups: In week 1 the animals were irradiated 3-times with 90 mJ/cm2 UVB, followed by one irradiation-free week. From week 3 on, the mice were irradiated 3-times/week with an initial dose of 60 mJ/cm2 UVB, followed by a weekly increase of 10 mJ/cm2 until a maximum dose of 150 mJ/cm2 UVB was reached (week 12). Mice were further irradiated with this dose until the end of week 24.2 weeks later, animals were sacrificed and tumor numbers were assessed. Tumor samples were embedded in paraffin and the resulting slices were H&E stained and number-coded for blinded histopathological evaluation. Animal experiments were performed according to the national animal care guidelines. The AHR represses nucleotide excision repair and apoptosis and contributes to UV-induced skin. . . Neutral comet assay HaCaT KC were detached with trypsin/EDTA and 20 µl of the cell suspension (approx. 1000 cells) plus 120 µl of 0.5% low melting agarose solution were pipetted on pre-coated superfrost slides, sealed with a cover slip and incubated for 5 min on ice. Slides were transferred to vertical staining jars containing lysis buffer (pH 9.5; 2.5 M NaCl, 100 mM EDTA, 10 mM Tris, 10% DMSO, 1% Triton X-100). After 1 h, slides were washed 3-times with dH2O. Next, slides were placed in electrophoresis buffer (pH 8.5; 300 mM NaOH, 1 mM, EDTA) for 20 min followed by 10 min electrophoresis. Slides were washed 3-times with ice-cold neutralization buffer (pH 7.5; 0.4 M Tris), kept in absolute ethanol for 5 min and then air-dried. Slides were stained with ethidium bromide and sealed with cover slips. Fifty cells per treatment were analyzed using a camera-connected microscope (Olympus BX60). Tail moment was used as parameter to characterize the extent of DNA damage. Transient RNA interference Transient transfection of HaCaT KC with XPA, XPC, CSB, p27 and non-silencing siRNA (all from Santa Cruz Bio- technology) was done using INTERFERin reagent (Poly- plus Transfection, Illkirch, France). Apoptosis assay Hematoxylin and eosin (H&E) staining and immunohis- tochemistry of skin tumors were performed on paraformaldehyde-fixed 7-µm paraffin sections. H&E stain- ings were mounted in Vectashield medium (Biozol, Eching, Germany). For immunohistochemical staining of tissues, paraffin sections were deparaffinized. Antigens were retrieved by boiling in pH 6 citrate buffer for 12 min. After washing, endogenous mouse Ig's were blocked using M.O.M Blocking Reagent (Biozol) according to the manufacturer´s protocol. Primary antibody (mutant p53, clone PAb 240, Thermo Sci- entific, Dreieich, Germany) was applied overnight at 4°C in a humidified chamber. Next day, staining was visualized using a peroxidase-based detection kit (Vector AEC Substrate Kit, Biozol) before mounting in gelatine. Apoptosis was determined by using the Annexin V-FITC Apoptosis kit (BioVision, Mountain View, CA, USA) and a FACSCalibur II device (BD Biosciences, San Jose, CA, USA). In addition, caspase-3 activity was measured by using the Caspase-3 Fluorometric Assay Kit (PromoCell, Heidel- berg, Germany) according to the manufacturer’s instructions. Overexpression experiments Transient transfection of HaCaT KC with pCMV5p27 (provided by J. Massagué, Addgene plasmid #14049 [56]), pcDNA5-rAHRR (provided by Y. Inouye [57]), and respective empty vectors was done using JetPEI reagent (Polyplus Transfection). Efficiency of rat AHRR overexpression was assessed by semi-quantitative PCR. M. Pollet et al. Quantification of CPD by HPLC/MS-MS 6. Denison MS, Soshilov AA, He G, DeGroot DE, Zhao B. Exactly the same but different: promiscuity and diversity in the molecular mechanisms of action of the aryl hydrocarbon (dioxin) receptor. Toxicol Sci. 2011;124:1–22. The HPLC-MS/MS-based detection of CPD in DNA iso- lated from UVB-irradiated murine skin was performed as described previously [18]. 7. Nebert DW. 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The cell-cycle checkpoint kinase Chk1 is required for mammalian homologous recombination repair. Nat Cell Biol. 2005;7:195–201. p 43. Kolluri SK, Weiss C, Koff A, Gottlicher M. p27(Kip1) induction and inhibition of proliferation by the intracellular Ah receptor in developing thymus and hepatoma cells. Genes Dev. 1999;13:1742–53. 26. Winkelmann R, Zollman P, Baldes E. Squamous cell carcinoma produced by ultraviolet light in hairless mice. J Invest Dermatol. 1963;40:217–24. 44. Fujita N, Sato S, Katayama K, Tsuruo T. Akt-dependent phos- phorylation of p27Kip1 promotes binding to 14-3-3 and cyto- plasmic localization. J Biol Chem. 2002;277:28706–13. 27. Berg RJ, van Kranen HJ, Rebel HG, de Vries A, van Vloten WA, van Kreijl CF, et al. Early p53 alterations in mouse skin carci- nogenesis by UVB radiation: immunohistochemical detection of mutant p53 protein in clusters of preneoplastic epidermal cells. Proc Natl Acad Sci USA. 1996;93:274–8. 45. 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Lenferink AE, Simpson JF, Shawver LK, Coffey RJ, Forbes JT, Arteaga CL. Blockade of the epidermal growth factor receptor tyrosine kinase suppresses tumorigenesis in MMTV/Neu + MMTV/TGF-alpha bigenic mice. Proc Natl Acad Sci USA. 2000;97:9609–14. 30. Yarosh D, Alas LG, Yee V, Oberyszyn A, Kibitel JT, Mitchell D, et al. Pyrimidine dimer removal enhanced by DNA repair liposomes reduces the incidence of UV Cancer Res. 1992;52:4227–31. 48. Wu R, Zhang L, Hoagland MS, Swanson HI. 54. Chahal HS, Lin Y, Ransohoff KJ, Hinds DA, Wu W, Dai HJ, et al. Genome-wide association study identifies novel susceptibility loci for cutaneous squamous cell carcinoma. Nat Commun. 2016;7:12048. q 55. Tigges J, Haarmann-Stemmann T, Vogel CF, Grindel A, Hubenthal U, Brenden H, et al. The new aryl hydrocarbon receptor antagonist E/Z-2-benzylindene-5,6-dimethoxy-3,3-dime- thylindan-1-one protects against UVB-induced signal transduc- tion. J Invest Dermatol. 2014;134:556–9. 56. Luo Y, Hurwitz J, Massague J. 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Pri- mary structure and organ-specific expression of the rat aryl hydrocarbon receptor repressor gene. Biol Pharm Bull. 2006; 29:640–7.
https://openalex.org/W3012377039
https://www.degruyter.com/document/doi/10.1515/zrp-2020-0001/pdf
German
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Zur Motivation des französischen Wortakzents
Zeitschrift für romanische Philologie
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ZrP 2020; 136(1): 2–24 Korrespondenzadresse: Prof. Dr. Ulrich Wandruszka, Alpen-Adria-Universität Klagenfurt, Universitätsstraße 65–67, A-9020 Klagenfurt, E-Mail: Ulrich.Wandruszka@aau.at Open Access. © 2020 Ulrich Wandruszka, publiziert von De Gruyter. Dieses Werk ist lizensiert unter der Creative Commons Attribution 4.0 Lizenz. Open Access. © 2020 Ulrich Wandruszka, publiziert von De Gruyter. Dieses Werk ist Korrespondenzadresse: Prof. Dr. Ulrich Wandruszka, Alpen-Adria-Universität Klagenfurt, Universitätsstraße 65–67, A-9020 Klagenfurt, E-Mail: Ulrich.Wandruszka@aau.at Ulrich Wandruszka Zur Motivation des französischen Wortakzents https://doi.org/10.1515/zrp-2020-0001 https://doi.org/10.1515/zrp-2020-0001 Open Access. © 2020 Ulrich Wandruszka, publiziert von De Gruyter. Dieses Werk ist lizensiert unter der Creative Commons Attribution 4.0 Lizenz. Korrespondenzadresse: Prof. Dr. Ulrich Wandruszka, Alpen-Adria-Universität Klagenfurt, Universitätsstraße 65–67, A-9020 Klagenfurt, E-Mail: Ulrich.Wandruszka@aau.at https://doi.org/10.1515/zrp-2020-0001 https://doi.org/10.1515/zrp-2020-0001 Abstract: Studies of individual languages rarely discuss in any depth the question of how, or to what extent, the positioning of word stress in polysyllabic words is motivated functionally although addressing this issue would mean relating the structural levels of phonetics/phonology, morphology, syntax, semantics and pragmatics systematically to each other. Given that in French practically all (Lat- in) syllables following the syllable which carries the main stress have been mut- ed, words and word forms are oxytones, generally carrying stress on the last syl- lable. As a result, endings and suffixes which constitute the morphological head of a word and are, as such, normally unstressed, now also carry stress. This can be seen in faisait or nageur, which in English would correspond to was *doíng or *swimmér or in German to *machté or *Schwimmér respectively. Since stress is normally positioned on the lexical stem, which carries more information, a mor- phoprosodic mismatch leading to a countericonic stress pattern obtains in French. Interestingly, this phenomenon has hardly attracted serious attention in linguis- tics so far, and speakers of French have apparently remained undisturbed by the mismatch. The same is true of the counter-intuitive stress placed on enclitic pro- nouns in prends-lé! – táke it – nímm(e)s! or vient-íl? – is he cóming? – kómmt er? Apparently, language as an instrument also works when the encoding is subopti- mal or dysfunctional. The reason for this must be that language is also supported by the (lexical) content of the signs, which nevertheless makes an appropriate interpretation possible. Keywords: morphological head, word (group) stress, secondary stress, fixed/free stress, falling/rising accent, logical word structure, informational importance, contra-iconic, posttonic syllable Dieses Werk ist Zur Motivation des französischen Wortakzents 3 Schlagwörter: morphologischer Kopf, Wort(gruppen)Akzent, Nebenakzent, fes- ter/freier Akzent, fallender/steigender Akzent, logische Wortstruktur, informatio- nelle Wichtigkeit, kontraikonisch, nachtonige Silbe 1 Voraussetzungen Mehrsilbige Endungen wie -ávit sind daher phonologisch regelkonform akzentuiert, einmal abgesehen davon, dass der Themavokal ā noch Teil des Stammes ist. Durch die Reduktion der Auslautsilbe im Altspanischen wird die Verbform dann endbetont, in prinzipiellem Unterschied zum Lateinischen, wo die Ultima von der Betonung allgemein ausgeschlossen und immer als unbetonte Nachtonsilbe erscheint.1 Pho- nologisch regulär hingegen ist im Spanischen die Akzentuierung der Form (yo) termíno, da auf einen Vokal endende Wörter dort tendenziell auf der Pänultima betont sind (im Unterschied zu lat. término und ital. (io) término, daneben freilich auch span. (el) término, cf. supra). Entsprechendes gilt nun für ein französisches passé simple (il) terminá gegenüber dem Präsens (il) termíne mit dem Unter- schied, dass hier das Endresultat der Lautentwicklung in beiden Fällen ein Oxy- tonon ist, da im Französischen mit gewissen Ausnahmen alle Nachtonsilben ver- stummen. dem Prinzip der informationellen Wichtigkeit konkurrierenden, phonologischen Regularitäten des Spanischen motiviert ist. In der lateinischen Entsprechung ter- minávit liegt der Akzent zwar ebenfalls auf der dritten Silbe, was aber durch die allgemeine Silbenstrukturregel des Lateinischen begründet ist, nach der grund- sätzlich die schwere Pänultima, hier das lange ā, den Akzent trägt. Mehrsilbige Endungen wie -ávit sind daher phonologisch regelkonform akzentuiert, einmal abgesehen davon, dass der Themavokal ā noch Teil des Stammes ist. Durch die Reduktion der Auslautsilbe im Altspanischen wird die Verbform dann endbetont, in prinzipiellem Unterschied zum Lateinischen, wo die Ultima von der Betonung allgemein ausgeschlossen und immer als unbetonte Nachtonsilbe erscheint.1 Pho- nologisch regulär hingegen ist im Spanischen die Akzentuierung der Form (yo) termíno, da auf einen Vokal endende Wörter dort tendenziell auf der Pänultima betont sind (im Unterschied zu lat. término und ital. (io) término, daneben freilich auch span. (el) término, cf. supra). Entsprechendes gilt nun für ein französisches passé simple (il) terminá gegenüber dem Präsens (il) termíne mit dem Unter- schied, dass hier das Endresultat der Lautentwicklung in beiden Fällen ein Oxy- tonon ist, da im Französischen mit gewissen Ausnahmen alle Nachtonsilben ver- stummen. Auch im Französischen kann folglich der Wortakzent nicht durchgängig die Grundfunktion der informationellen Gewichtung erfüllen, wobei jedoch eine neue relativ einheitliche und einfache Regelung Platz greift: abgesehen von der be- dingten Realisierung eines finalen Schwa-Lautes des Typs chambre sind mehrsil- bige französische Wörter prinzipiell auf der Ultima betont. 1 Lateinschüler fallen hingegen gern in die Endbetonung, wenn sie ein Paradigma memorieren, also «cantó, cantás, cantát» etc., wobei aus mnemotechnischen Gründen die Silbe akzentuiert wird, die sich verändert und auf die deshalb das Augenmerk gerichtet werden muss – sie ist in diesem pragmatischen Kontext die wichtigere. 1 Voraussetzungen Zumal in einzelsprachlich ausgerichteten Studien zum Wortakzent wird die Fra- ge, wie bzw. inwieweit die Akzentverteilung innerhalb mehrsilbiger Wörter funk- tionell motiviert ist, meist nicht grundsätzlich erörtert, obgleich mit dieser Frage die Strukturebenen der Phonetik/Phonologie, der Morphologie, der Syntax, der Semantik und Pragmatik systematisch miteinander in Beziehung zu setzen sind. Geht man einmal davon aus, dass die Funktion des (Haupt-)Akzents innerhalb der Domäne des Wortes – wie auch in der des syntaktischen Konstrukts – darin besteht, das informationell jeweils wichtigste Segment als solches zu markieren und hervorzuheben, muss gefragt werden, inwieweit dies für die einzelnen Wort- formen mit ihrem aktuellen historisch entwickelten und verfestigten Akzentprofil tatsächlich (noch) zutrifft. Negativenfalls ist zu untersuchen, wie die Dysfunktio- nalität entstanden ist und welche Kräfte, d. h. welche Entwicklungstendenzen im- stande waren, diese natürliche Grundfunktion des Akzents in bestimmten Fällen außer Kraft zu setzen. Bekannte Beispiele (cf. z. B. Meisenburg/Selig 2008, 152) aus Sprachen mit sog. freiem Wortakzent wie span. término ‘Termin’, termíno ‘ich beende’ und terminó ‘er/sie beendete’ zeigen schnell, dass diese Grundfunktion nicht mehr durchgehend erfüllt sein kann; denn warum sollte die akzentuierte Perfektendung in termin-ó informationell wichtiger sein als die unbetonte Prä- sensendung in termín-o. Nimmt man an, dass der Verbstamm termin- aufgrund seines spezifischen lexikalischen Inhalts ein größeres informationelles Gewicht aufweist als die abstraktere Personalendung -o (cf. dazu Wandruszka 2011), dann entspricht die stammbetonte Präsensform der angenommenen Funktion des Ak- zents genau, während das endungsbetonte termin‑ó dem klar zuwiderläuft. Diese Akzentuierung ist zwar historisch mit der Silbenstruktur der zugrundeliegenden lateinischen Form terminávit zu erklären, synchronisch betrachtet jedoch ist sie willkürlich und kontraikonisch, gewissermaßen ein morphoprosodischer mis- match. In der Linguistik kann man unter mismatches Phänomene verstehen, in denen sprachliche Merkmale auf verschiedenen Strukturierungsebenen nicht zu- einander zu passen scheinen (cf. Pomino/Stark 2011) – man kann in diesem Sinne auch einfach von Diskrepanzen oder Inkongruenzen zwischen Inhalt und Form sprechen. Die Akzentuierung der Flexionsendung in span. terminó erscheint vor allem auch deswegen als mismatch, weil sie nicht durch irgendwelche anderen, mit Ulrich Wandruszka 4 dem Prinzip der informationellen Wichtigkeit konkurrierenden, phonologischen Regularitäten des Spanischen motiviert ist. In der lateinischen Entsprechung ter- minávit liegt der Akzent zwar ebenfalls auf der dritten Silbe, was aber durch die allgemeine Silbenstrukturregel des Lateinischen begründet ist, nach der grund- sätzlich die schwere Pänultima, hier das lange ā, den Akzent trägt. 5 bestehendes einsilbiges Wort, wie das volkstümlich entwickelte neufranzösische frêle über afz. fraile(s). Wenn aber lat. frágile im 14. Jahrhundert als gelehrtes Wort in das Französische integriert wird, muss der Akzent der zweisilbigen Basis nach rechts rücken, um schließlich die neufranzösische jambische End- und da- mit Suffixbetonung fragíle zu erreichen. In span./port. frágil hingegen bleibt der Initialakzent bewahrt und das ital. frágile entspricht sogar noch voll dem lateini- schen Proparoxytonon. Die deutsche Entlehnung fragíl übernimmt das französi- sche Muster, wie etwa auch in diffizíl (←difficíle) vs. ital. diffícile, span./port. difícil u. a. Daktylische Folgen im Lateinischen werden im Französischen also ent- weder einsilbig wie frêle oder cálidu →chaud, dann auch hómine →homme und fráxinu →frêne, oder der Akzent wird versetzt wie etwa auch in lat. grácile →frz. grêle und gracíle oder cólubra →couléuvre gegenüber ital. cólubro (daneben auch colúbro), oder auch fúnebris →funèbre u. a. Dieser Zwang zur Akzentverschie- bung gilt auch für diverse Entlehnungen aus anderen Sprachen mit fallendem Akzent, wie etwa für zweisilbige englische Nomina agentis des Typs speaker, das zu suffixbetontem speakér mit gerundetem [-œ:R] wird, so wie auch leader → leadér. Von einer semantisch-informationell unangemessenen oder wenn man so will «sinnwidrigen» Betonung der letzten Silbe des französischen Wortes kann man also nicht nur im Fall der Flexionsendungen sprechen, sondern generell auch bezüglich der Wortbildungssuffixe, die ja ebenfalls normalerweise unbetont sind und nicht den Hauptakzent tragen, wie dies z. B. in deverbalem nag-éur oder cour-éur der Fall ist. Dieses Akzentmuster, dem im Deutschen ein abwegiges kon- traikonisches *Schwimm-ér/*Läuf-ér entspräche, wurde im Laufe der diachronen Entwicklung von den Sprachbenutzern offenbar nicht als störender mismatch empfunden, und auch in der romanistischen Forschung ist es meines Wissens kaum explizit thematisiert worden (cf. infra). Suffixbetonte Ableitungen kommen freilich etwa auch im Spanischen vor, wie gerade in nadadór (aus lat. natatorem) oder corredór. Die Akzentuierung dieser Silbe ist dort allerdings eher systeminte- griert als im Französischen, da sie silbenstrukturbedingt regulär im Plural nada- dóres und in der femininen Variante nadadóra mit Pänultima-Betonung erscheint. Im Spanischen sind die Nachtonsilben ja keineswegs immer, sondern nur in spe- ziellen lautlichen Umgebungen verstummt, so dass dort im Unterschied zum Französischen das trochäische fallende Akzentmuster grundsätzlich erhalten bleibt. Endbetont sind im Spanischen auch Ableitungen wie natación/nataciónes oder ingratitúd, sinceridád und in der Flexion sämtliche Infinitivendungen wie in nad-ár, com-ér etc., sowie die Futurformen mandaré/mandarás, entsprechend ital. manderò/manderai. 1 Voraussetzungen Diese Regelung ist nun grundsätzlich nicht mehr intern morphologisch motiviert, weder durch die kom- munikative Wichtigkeit noch durch die Silbenstruktur, sondern allein durch die Einheit des Wortes/der Wortform als solcher. Diese bedingungslose Endbeto- nung, die historisch nicht aus einem Positionswechsel des Akzents resultiert, son- dern indirekt aus einer Veränderung der lautlichen Umgebung, eben aus der ge- nerellen Reduktion der Nachtonsilben, ist als Tendenz im Französischen so domi- nant, dass sie gegebenenfalls die Akzentstelle tatsächlich auch versetzt. Dieses lautgeschichtlich eher seltene Phänomen – der Wortakzent verharrt in der Regel konstant auf einer Silbe – lässt sich vor allem bei Proparoxytona beobachten. Da diese jeweils zwei Nachtonsilben haben, wie z. B. lat. frágilis, müssten im Franzö- sischen im Fall der Beibehaltung der Akzentstelle beide Silben verstummen. Da- durch entstünde bzw. entsteht ein praktisch allein aus der akzenttragenden Silbe Zur Motivation des französischen Wortakzents Es gibt im Spanischen also differenzierte Wortbeto- nungsregeln, die auf die jeweilige lautliche Umgebung Bezug nehmen, wie z. B. die, dass auf einen Vokal oder die Konsonanten n/s auslautende Wörter im un- Ulrich Wandruszka 6 markierten Fall Paroxytona sind und dass in Wörtern, die auf einen Konsonanten außer n/s enden, die Ultima akzentuiert ist. Im Italienischen hingegen finden wir in diesen Fällen ein auslautendes ‑e wie in: nuotatore/-ori, corridore und natazio- ne/-ioni, ingratitudine oder dann nuotare, vedere, dormire etc., die weitgehend dem lateinischen bzw. dem vulgärlateinischen Silbenstand entsprechen. Endbe- tonung findet man in Ableitungen wie sincer-ità, qual-ità und dergleichen mit ver- stummtem intervokalischen -t- im Lateinischen. Auf weitere syn- oder diachrone romanische Details und Differenzen soll hier jedoch nicht eingegangen werden. Die unmotivierte sinnwidrige Betonung der französischen Endungen und Suffixe wird deswegen nicht als störend empfunden, weil mit der Akzentuierung nicht das Endungsmorphem als solches gemeint ist, sondern einfach nur die letz- te Silbe des Wortes. Da diese prinzipiell akzentuiert ist, liegt der Akzentuierung keine Intention auf der Basis einer freien Wahlmöglichkeit zugrunde und hat da- mit auch keine semiotische Relevanz. In diese Richtung zielt schon Pierre Guiraud (1980, 118–119) mit seinem Hinweis auf die «situation illogique qui fait porter l’accent sur une détermination secondaire et qui trouble la structuration du système. Car l’accentuation oxytonique du suffixe est une des causes qui contribue au caractère arbitraire du mot français en affaiblissant le sentiment de la dériva- tion; dans des mots comme vérité, blancheur etc. le suffixe accentué n’est pas senti comme un morphème, ni la forme comme un dérivé». Auch wenn das Prädikat «arbitraire» für Suffixableitungen wie blancheur oder nageur etc., einfach aufgrund ihrer Bedeutung und morphologischen Durchsich- tigkeit, überzogen ist, kann man annehmen, dass ein betontes Suffix im Franzö- sischen deswegen nicht «auffällt» und stört, weil es der normalen Wortbetonung entspricht. Wie bereits angemerkt ist der französische Wortakzent inhaltlich grundsätzlich nicht motiviert, einmal abgesehen von seiner delimitativen Funk- tion, durch die das Ende des Wortes bzw. des mot phonique (cf. infra) markiert wird. Aus diesem Grund stören auch die anderen im Französischen allgegenwär- tigen «falschen» bzw. nicht intendierten Betonungen, auf die wir noch zurück- kommen werden, nicht wirklich. 2 Daher kann etwa im Kreolischen einfach ein endungsloser Stamm in Verbindung mit dem Sub- jektpronomen als Verbform verwendet werden: mwe sav «je sais» (Haiti). 2 Akzentmuster und logische Struktur des Wortes Geht man also davon aus, dass der Hauptakzent polymorphemischer bzw. mehr- silbiger Wörter natürlicherweise auf die Silbe mit der höchsten informationellen Wichtigkeit zu liegen kommt, sind unterschiedliche Arten von Wichtigkeit an- zunehmen; denn keines der interdependenten Bestandteile eines Wortes kann in jeder Hinsicht unwichtig sein. Wir unterscheiden die informationelle Wichtigkeit, Zur Motivation des französischen Wortakzents 7 die Flexionsendungen, praktisch keine neue Information mehr für den Emp- fänger enthalten.2 Eben deswegen können die Endungen im Französischen, so- Ulrich Wandruszka 8 8 weit sie nicht den Akzent tragen, vollständig verstummen, so dass die Verbform, morphologisch «kopflos», (lautlich) allein durch den Stamm repräsentiert wird: je/tu/il(s)/on/les enfants mange(nt) gegenüber nous mangeons/vous mangez. weit sie nicht den Akzent tragen, vollständig verstummen, so dass die Verbform, morphologisch «kopflos», (lautlich) allein durch den Stamm repräsentiert wird: je/tu/il(s)/on/les enfants mange(nt) gegenüber nous mangeons/vous mangez. Normalerweise trägt also der als Komplement/Determinans des flexivischen oder affixalen Kopffunktors fungierende Stamm den Hauptakzent, während der Funktor als die weniger betonte Vor- oder Nachtonsilbe erscheint – als Vortonsil- be bei Postdetermination, wie etwa in frz. dedáns oder dt. hin-únter und als Nach- tonsilbe bei Prädetermination mit fallendem Akzentmuster, wie in dt. Schwímm- er, síngen und ital./span./port. cánta, lat. cántor oder proparoxyton ital. cántano. Die Tatsache, dass die Endungen und Affixe als kategoriebestimmende Köpfe na- türlicherweise den weniger betonten Bestandteil bilden, kann andersherum auch als prima-facie-Kriterium für die theoretisch nicht immer ganz einfache Bestim- mung des Kopfes eines mehrgliedrigen Ausdrucks verwendet werden. Diese Kor- relation besteht aber, wie gesagt, in den romanischen Sprachen nicht mehr durchgehend und im Französischen nach dem Verstummen der lateinischen Nachtonsilben überhaupt nicht mehr, weswegen wir diesbezüglich von mismatch sprechen. Phonetisch erklärt sich dieses Verstummen damit, dass Nachtonsilben gene- rell besonders schwach ausgeprägt sind und daher auch Funktoren in dieser Po- sition, nach einer klitischen Phase, in hohem Maße reduktionsanfällig sind (cf. Wandruszka 1992). Durch ein trochäisch fallendes Akzentmuster charakterisierte prädeterminierende (OV-)Sprachen mit ausgeprägter Endungs- und Suffixmor- phologie tragen somit den Keim ihrer Auflösung, d. h. ihres typologischen Wan- dels bereits in sich. Generell ist ein lautlicher Schrumpfungsprozess zu beobach- ten, der in bestimmten Konstellationen zu einer Verkehrung des typologisch be- dingten Wortakzent-Grundmusters einer Sprache führen kann, in unserem Fall von einem fallenden zu einem steigenden jambischen Betonungsmuster. Wäh- rend zweisilbige Paroxytona mit einsilbiger Endung schlicht einsilbig werden, wie múru →mur, vénit →vient, entstehen aus drei- und mehrsilbigen Paroxytona Oxytona, die mit der akzentuierten Silbe enden, wie in murále →murál oder mo- numéntum →monumént, cantávit →chantá, womit man auch innerhalb des Wor- tes zu einem für postdeterminierende Sprachen charakteristischen jambischen Akzentmuster gelangt. Aus den zweisilbigen lateinischen Suffixen/Endungen mit fallendem Akzent entstehen dabei die kontraikonisch akzentuierten einsilbigen Suffixe und Endungen des Französischen. 7 womit die relative Menge an neuer nicht zu erwartender Information gemeint ist, von der strukturellen Wichtigkeit, die für die grammatische Struktur und syntakti- sche Kategorie eines Ausdrucks von Bedeutung ist (cf. Wandruszka 2011 und 2015). Der die Kategorie eines Ausdrucks bestimmende Bestandteil ist dessen Kopf-(Funktor), während der jeweils andere als Komplement bzw. Attribut fun- giert, das den Kopf determiniert und inhaltlich spezifiziert. Ersterer ist in der Re- gel (cf. unten) ein Funktionselement, wie eben eine Flexionsendung oder ein Wortbildungssuffix, und Letzterer normalerweise der lexikalische Kern eines komplexen Wortes oder auch ein Präfix. Der kategoriebestimmende Kopf steht sowohl für die Wortart als auch für die syntaktische Funktion eines Ausdrucks. In einer Form wie chant-ons identifiziert die Endung den Ausdruck als Verbum und dessen funktionale Kategorie als Prä- dikat oder Satz, im Unterschied etwa zur Verbform chant-ant mit der Funktion des Attributes eines Nomens wie in accent chantant, im Unterschied auch zu einer Ableitung wie chant-eur, wo die Endung den Ausdruck als Nomen kennzeichnet, das nun nicht mehr der Wortart Verbum angehört und somit kein direktes Objekt zu sich nehmen aber von einem Adjektiv determiniert und mit einem Artikel ver- bunden werden kann, wie in un chanteur allemand. Insofern sind die Endungen und das Suffix von entscheidender struktureller Wichtigkeit, während der Stamm chant- nicht notwendigerweise ein Indiz für die Wortart oder die syntaktische Funktion des Gesamtausdrucks ist. Dies illustriert einerseits eben das deverbale Nomen chanteur und andererseits etwa ein deadjektivisches Nomen wie lent-eur bzw. ein denominales Verbum wie huil-er ‘ölen’. Der Stamm bezeichnet indessen den Vorgang, die Eigenschaft oder das Objekt, d. h. die Entität, mit der das Deno- tat des Gesamtausdrucks etwas zu tun hat. Der Stamm repräsentiert jeweils das spezielle lexikalische Individuum als Element einer theoretisch unendlichen Menge, während die Endung die abstrak- tere allgemeine Kategorie und Funktion des Ausdrucks markiert und damit einen deutlich geringeren Mitteilungswert besitzt. Ein Wert, der sich zudem dadurch reduziert, dass Kategorie und Funktion eines Ausdrucks normalerweise in den jeweils vorausgehenden sprachlichen Einheiten antizipiert sind. So ist in einem Satz wie Demain il partira die Kategorie Verbum, dessen Finitheit und die Futuri- tät, also Person und Tempus, im Unterschied zur lexikalischen Bedeutung des Stammes, eindeutig vorhersehbar, womit die entsprechenden Trägermorpheme, d. h. 9 zents mit seiner markanten Differenz zwischen betonter und unbetonter Silbe (cf. Geisler, 1982) – gibt es praktisch keinen fallenden Akzent mehr, denn auf eine akzentuierte Silbe kann nun im Prinzip nichts mehr folgen: ... s-ś-s →... s-ś. Die konsequente Endbetonung der französischen Wörter ist historisch vor allem da- durch bedingt, dass das Lateinische zu den sog. gewichtssensitiven Sprachen ge- hört, bei denen zur Bestimmung der Akzentsilbe auch das Silbengewicht eine Rolle spielt (Meisenburg/Selig 2008, 151–152). Im Unterschied zum Deutschen fällt der Akzent im Lateinischen bei entsprechendem Silbengewicht immer auf die Pänultima und rückt daher bei Suffixableitung jeweils um eine oder mehrere Sil- ben nach rechts: páter →patérnus →paternális →paternalitátem, was schließ- lich zur französischen Oxytonie führt: père →patérne →paternél →paternalité. Im Deutschen hingegen ist der wortinitiale Akzent hier unverrückbar: Váter → väterlich →Väterlichkeit →väterlicherseits. Da das Ende bzw. die letzte Silbe eines Wortes nun aber per se keine akzen- togene Größe darstellt, gibt es für die Position des neufranzösischen Wortakzents keine direkte (mor)phonologische Motivation. Eine indirekte Motivation oder Re- gel, auch für den Sprecher, kann aber einfach in dem Umstand gesehen werden, dass mit dem Ausschluss fallender Akzentmuster jedes Wort auf eine betonte Sil- be enden muss bzw. aus einer betonten Silbe besteht. Dies gilt tendenziell auch für Wörter mit auslautendem Schwa bzw. e-muet wie faible, concombre, quatre oder signe. Da die neufranzösischen Verhältnisse auf historische Silbenstruktur- bedingungen zurückgehen, wie eben die Akzentuierung der schweren Pänultima im Lateinischen und das spätere zur Oxytonie führende Verstummen der Nach- tonsilben, sind darüber hinaus für den neufranzösischen Wortakzent (wie für den des sog. mot phonique, s. u.) keine weiteren synchronen phonologischen Regeln mehr zu formulieren. Das steigende jambische Akzentmuster ist auch in der wortübergreifenden Syntax charakteristisch für postdeterminierende Sprachen mit dem Akzent auf dem nachgestellten Determinans, also dem Adjektiv in voiture rouge, dem Adverb in parler lentement, der NP in derrière la maison oder dem Nomen in la maison. In dem diesbezüglich strikten Französisch gibt es daher keine betonten Determinan- tien in Voranstellung mehr und keine unbetonten Determinantien in Postpositi- on, etwa nach Art der enklitischen Objektpronomina im Italienischen oder Spa- nischen wie in farlo, dartelo, vedendoti und dergleichen. Infolge der Generalisie- rung des jambischen Akzentmusters im Französischen müssen derartige Klitika dem verbalen Kopf vorangehen: pour le faire, pour te le donner, en te voyant. Hier ergibt sich folglich ein «klassischer» Konflikt zwischen der Tendenz zur Post- determination, d. h. Dadurch erhält das Französische eine Vielzahl von Wörtern/Wortformen mit betontem vokalischem Auslaut, während unbetonte Vokale am Wortende, außer Schwa, im Prinzip nicht mehr vorkom- men. Ab dem Zeitpunkt des völligen Verlustes der Nachtonsilben – im Französi- schen möglicherweise durch den Einfluss des dynamischen germanischen Ak- Zur Motivation des französischen Wortakzents 3 Die besonders enge Bindung enklitischer Morpheme an ihre Bezugseinheit zeigt sich auch in der Zusammenschreibung und Univerbierung italienischer Kombinationen wie farlo/fallo/dimmi und entsprechend imSpanischenoderPortugiesischen.SoistauchderBindestrichinfrz.prends-le!etc. motiviert. zur VO-Folge einerseits, und dem Verlust des fallenden tro- chäischen Akzents andererseits – ein Konflikt, der im Französischen im Unter- schied zu anderen romanischen Sprachen ausnahmslos zugunsten des Akzent- Ulrich Wandruszka 10 musters und zu Lasten der typkonformen Determinationsrichtung gelöst wird. Wenn das Französische hier OV-Strukturen aufweist, dann eben nicht, wie mit- unter behauptet wurde (cf. z. B. Sasse 1977), weil es in Resten noch, wie das Latei- nische, eine OV-Sprache wäre, so wie das Deutsche in Strukturen der Art ... dass Maria ihn besucht/um es zu tun (oder auch nominal in ... dass Maria einen Fréund besucht), sondern weil es als besonders ausgeprägte VO-Sprache keinen fallen- den Akzent mehr duldet. Aus diesem Grund ist andererseits die Akzentuierung des Objektpronomens in frz. prends-lé nicht darauf zurückzuführen, dass es ein (betontes) Determinans ist, sondern allein darauf, dass es die letzte Silbe bildet. Prends-le nimmt hier also die Gestalt eines oxytonen Wortes an, dessen Endsilbe freilich sinnwidrig akzen- tuiert ist, da sie pragmatisch nicht etwa kontrastiv betont werden soll. Prends-lé entspricht nicht einem akzentuell analogen dt. nimm íhn, sondern einem norma- len unmarkierten nímm ihn/es mit enklitischem Pronomen. Le [ˈlœ] ist folglich ein akzentuiertes Enklitikon und als solches ein in sich widersprüchliches Gebilde, ein mismatch im eigentlichen Sinn des Wortes, prinzipiell vergleichbar dem ak- zentuierten Suffix des Nomens nagéur. Die Betonung des Enklitikons ist eben nur deshalb möglich, weil es als letzte Silbe eines Wortes prends-le, das als solches im Französischen regulär oxyton ist, interpretiert wird.3 Außerhalb eines solchen morphonologischen Kontextes, also etwa in proklitischer Position, ist ein Klitikon nicht akzentuierbar, da der ihm inhärente satzphonetische Status dort nicht auf- gehoben werden kann. Wenn also der Imperativ unmittelbar mit dem Verbum einsetzen soll, muss entweder gegen die Unzulässigkeit fallender Akzentmuster im Französischen verstoßen werden (*prénds-le) oder gegen die Unbetonbarkeit von Klitika bzw. gegen eine inhaltlich nicht-gegebene und nicht-gemeinte (kon- trastive) Betonung des Pronomens. Im zweiten Fall verliert das Pronomen nun zwar phonetisch seinen Status als Klitikon, wird aber dennoch als solches ver- standen, wobei die Betonung interessanterweise einfach überhört wird. Man sieht unbewusst davon ab und perzipiert das Pronomen in seiner eigentlichen pragma- tischen Funktion als vorerwähntes thematisches Element, das aber als letzte Silbe eines Wortes den Akzent übernehmen muss. 11 nomen als komplexes Wort wird dadurch befördert, dass das Pronomen als «ur- sprüngliches» Klitikon sehr eng mit seiner lexikalischen Bezugseinheit verbun- den ist, was in besonderem Maß – wie bereits erwähnt – für nachtonige enkliti- sche Elemente gilt (cf. Wandruszka 1992). Die Entwicklung zur Entklitisierung und Akzentuierung datiert übrigens erst seit dem Mittelfranzösischen, denn das Objektpronomen konnte bis zur Renaissance auch in einer derartigen Position seinen klitischen Status bei fallendem Akzent beibehalten, wie Maurice Grevisse (1975, 461) anmerkt: «le pronom le, après un impératif sans négation, pouvait être inaccentué: perd-LE, par exemple, pouvait rimer avec perle». Wo es eine betonte Variante des Objektpronomens gibt, wird diese in unserer Position verwendet, wie etwa in crois-moi! oder approche-toi!. In solchen Fällen besteht der mismatch nur zwischen der formal prinzipiell korrekten (und obliga- torischen) Akzentuierung des Pronomens und der nicht-intendierten Betonung, im Unterschied etwa zu einer Präpositionalphrase pour toi. Dasselbe gilt für ein approchez-vous!, dessen Pronomen formal jedenfalls korrekt akzentuierbar ist, hier aber akzentuiert werden muss, auch wenn es nicht betont werden soll. Im Italienischen wird in diesem Kontext gerade die klitische Variante ti und nicht die betonte te verwendet, also z. B. avvicinati! und credimi (a me)!, entsprechend auch im Spanischen: créeme (a mí)! und acércate!. Eine inhaltlich widersinnige bzw. nicht-intendierte Versprachlichung, näm- lich die Akzentuierung von nicht zu Betonendem und eigentlich nicht Beton- barem liegt auch in der Inversionsfrage mit pronominalem Subjekt wie in vient-íl? vor, womit ebenfalls kein kontrastives kommt ér gemeint ist, sondern unmarkier- tes kómmt er? mit enklitischem Subjekt, so wie etwa auch in einem qu’elle heure est-íl? mit seinem nicht-referentiellen und daher im Prinzip absolut unbetonbaren Pronomen. Auch diese Unstimmigkeiten sind überhörbar, weil die Akzentuierung des dem Verb folgenden Pronomens gemäß den neufranzösischen Intonations- regeln obligatorisch ist und daher nicht als intendierter, einer freien Wahl ent- sprungener Zeichenwert interpretiert wird. Ebenfalls nur in enklitischer Position und nicht etwa in Isolation kann das Subjektpronomen der 2. Pers. Sing. den Akzent des Wortes bzw. des mot phonique tragen, wie in einem où vas-tú?, statt der betonten Variante wie z. B. in c’est toi. Die Frequenz des konkurrierenden où tu vas? mit proklitischem Pronomen ist frei- lich heute schon bedeutend höher, was entsprechend auch für où va-t-on? gegen- über où on va? gilt oder für que fera-t-on? vs. qu’est-ce qu’on fera. Auch die Pro- nominaladverbien en und y sind in Postposition akzentuiert: prenez-en! oder pen- ses-y! Die Klitika werden also gemäß der dominanten Akzentstruktur französischer Wörter in der Enklise akzentuiert, wobei der Konflikt zwischen einer inhaltlich- pragmatischen Motivation und einer solchen des (Wort-)Akzentmusters zuguns- ten der Letzteren entschieden wird. Die Interpretation der Folge Verbum-Pro- Zur Motivation des französischen Wortakzents 12 angelehnt werden kann. Klitisch bleibt in der Position nach dem Verbum auch das neutrale Demonstrativpronomen ce, wie z. B. in qui ést-ce? – wer íst das?, im Unterschied zu qui est-íl?. Wie je in puis-je kann ce in est-ce als nicht-silbischer frikativer Auslautkonsonant artikuliert werden [ki ˈæs] und insoweit ebenfalls als Teil des Bezugswortes erscheinen. Die beiden Pronomina sind hier also keine nachtonigen Enklitika mehr und somit auch keine Ausnahme von der allgemei- nen Regel, die derartige fallende Akzentmuster im Neufranzösischen ausschließt. Auf der Basis dieser Zusammenhänge lässt sich nun auch das Faktum erklä- ren, warum die Inversionsfrage im Neufranzösischen nur noch mit pronominalem Subjekt akzeptabel ist, wie in vient-íl? oder pourquoi pars-tú?, und seit dem Mittel- französischen nicht mehr mit nominalem Subjekt, wie in part Michel? oder part votre père?. Da das Subjekt in Sätzen wie vient-íl?/pars-tú? pragmatisch gerade nicht betont ist bzw. nicht betont sein kann und daher einem dt. kómmt er/géhst du? und nicht etwa einem kontrastiven kommt ér/gehst dú? entspricht, müsste die nominale Ausführung – dt. kómmt/géht Michael? – eine im Französischen inakzep- table fallende Intonation aufweisen, nämlich eben párt Michel? oder párt votre père?, statt votre père, part-il?. Diese kommunikative Struktur mit nachgestelltem Subjekt kann aus den genannten Gründen nur durch ein pronominales part-íl? wie- dergegeben werden, während einnominales Subjekt in dieserPosition auch inhalt- lich-pragmatisch betont wäre: vient/part Michél, was heute aber durch ein est-ce/ c’est Michel qui part? bzw. pronominal est-ce/c’est lui qui part? dargestellt wird. (mit eingefügtem [z] wie in vas-y!). Nur das Pronomen der 1. Pers. Sing. bleibt in Postposition klitisch tonlos: puís-je?, ai-je, suis-je und pourrais-je vs. pourrais-tú. Auffällig sind auch literarische Formen wie dussé-je, wo das e-muet der Endung von dusse silbisch und akzentuiert wird, damit das klitische je daran Ulrich Wandruszka Zur Motivation des französischen Wortakzents 13 konstruktion, wie in la vache qui rit statt la vache riante. Ersetzung der Inversions- frage durch die Fragekonstruktion mit est-ce que. Als nicht ganz erklärliche Aus- nahme erscheint ihm hingegen die zweigliedrige Negation mit nachgestelltem pas in elle ne mange pas, auch ohne vorangestelltes ne in je (ne) sais pas (cf. infra). konstruktion, wie in la vache qui rit statt la vache riante. Ersetzung der Inversions- frage durch die Fragekonstruktion mit est-ce que. Als nicht ganz erklärliche Aus- nahme erscheint ihm hingegen die zweigliedrige Negation mit nachgestelltem pas in elle ne mange pas, auch ohne vorangestelltes ne in je (ne) sais pas (cf. infra). Hier ist Determination offensichtlich nicht in einem logisch-syntaktischen Sinn gemeint, d. h. als Relation zwischen Determinatum (Operand) und Determinans (Operator). Gemeint ist vielmehr Determination in einem morphologisch-paradig- matischen Sinn: «Was determiniert, muss Morphem und bound form sein» (Wan- druszka 1980, 91). Diese charakteristische Struktur des Neufranzösischen hat Ha- rald Weinrich (1966) mit folgender deskriptiven Formulierung zusammengefasst (sinngemäß): Für das Nfz. gilt tendenziell: Morphem vor Lexem. So stehen im Neu- französischen tendenziell gerade die Elemente voran, die ein finites grammati- sches Paradigma bilden und den Einheiten, auf die sie sich beziehen, eine be- stimmte Position innerhalb dieses Paradigmas und damit bestimmte Funktionen und Merkmale zuweisen (cf. Wandruszka 1980, 58).4 Dies sind nun aber eben die Elemente, die als klassen- oder kategoriebestimmende Konstituenten in unserem Sinn den determinierten Funktorkopf eines komplexen Ausdrucks darstellen und daher gerade für die postdeterminierende Struktur des Neufranzösischen stehen, wie z. B. die Präposition oder das finite Auxiliar in periphrastischen Verbformen. Betrachtet man das jeweils akzentuierte Glied eines komplexen Ausdrucks als dessen Determinans, wie etwa in maison blanche oder maison mère, müsste man sowohl im Fall der Endungen (außer im Französischen) als auch in dem der Prä- position oder des Auxiliars – wenn man diese mit Baldinger oder Geisler (1982) als Determinantien definiert – eine Ausnahme von dieser Konstante ansetzen, da die- se Elemente ja gerade unbetont sind. 3 Das Begriffspaar Post-/Prädetermination in der deutschen Romanistik Theoriegeschichtlich bemerkenswert ist die inkonsistente Verwendung des zen- tralen Begriffs der ‘Determination’ in der (Morpho-)Syntax bei deutschen Roma- nisten, die eine gewisse Verwirrung gestiftet hat und dies offensichtlich bis heute tut (cf. Wandruszka 1980). So spricht etwa Kurt Baldinger (1968) von einem struk- turellen Wandel von der lateinischen Postdetermination zur neufranzösischen Prädetermination. Als Zeugen für diesen Wandel führt Baldinger u. a. folgende Phänomene an: Die syntaktische Funktion, Numerus etc. des Nomens werden durch Artikel in Verbindung mit einer Präposition markiert statt durch eine En- dung. Ersetzung des suffixalen Komparativs durch Gradadverbien und des Dimi- nutivsuffixes ‑ette durch vorangestelltes Adjektiv petit. Ersetzung der Personalen- dungen beim Verbum durch ein obligatorisches Subjektpronomen und die Erset- zung des passé simple und des Futurs durch periphrastische Formen, wie il a mangé und il va manger. Ersetzung des attributiven Partizips durch eine Relativ- Geisler (ib., 97) sieht sich daher veranlasst, eine Unterscheidung zwischen lexikalischer und morphologischer Determination zu treffen: «Im Gegensatz zur lexikalischen Determination [Nomen-Adjektiv und dergleichen] trägt bei morphologischer Determination nicht das Determinans den Hauptakzent, sondern genau umgekehrt das Determinatum, während die Determinantien unbetont sind (was gleichzeitig Voraussetzung für die Klitisierung bei Ausbildung der Morpheme ist)».5 4 So etwa noch bei Stefan Barme (2012, 18), der davon spricht, dass bei den Verben «die Markie- rung der grammatischen Kategorie Person in der geschriebenen Sprache überwiegend prä- und postdeterminierend erfolgt» – d. h. durch Subjektpronomen und Verbalendung. Hinzukommt, dass zwischen Subjekt und Verbum im logischen Sinn gar keine Determinations- sondern eine Prä- dikationsrelation besteht. 5 In diesem Sinn auch Weinrich (1976, 227), der von der Grundstruktur der französischen Sprache spricht, «nach der in der Regel die determinierenden Morpheme vor und die determinierenden Lexeme hinter dem Substantiv stehen». 14 Ulrich Wandruszka Als Beispiele hierfür werden lat. pátrem und dt. Kíndheit, spársam oder spáren genannt, deren Endungen und Suffixe als Determinantien interpretiert werden. Die Annahme einer solchen Umkehrung erscheint freilich nicht plausibel, da das die Determinationsverhältnisse widerspiegelnde Akzentmuster nicht von der morphologischen Gestalt der Konstituenten abhängig ist und sich prinzipiell ein- heitlich darstellen sollte. Gerade Suffixe sind in der Regel eindeutig als determi- nierte Köpfe zu identifizieren (cf. supra), da sie die Träger der Kategorie des Ge- samtausdrucks sind, also in Kindheit der Merkmale [Nomen, Femininum, Singu- lar]. Darüber hinaus auch semantisch, da das Wort nicht ein Kind bezeichnet, sondern eine Lebens- und Entwicklungsphase, wofür das Suffix ‑heit steht. In sparsam steht das Suffix für die Wortart, denn es macht als Kopffunktor mit einem Verbstamm spar- ein Adjektiv. Im Fall von Flexionsendungen wie bei patr-em ist die Situation vielleicht weniger eindeutig aber auch hier ist die Endung klassen- bestimmender Kopf, der das Wort als singularisches Nomen im Akkusativ katego- risiert. Offenbar ist der Begriff Kopf bei Baldinger und Geisler aber auch bei Wein- rich und verschiedenen anderen Romanisten einfach mit der Vorstellung eines selbständigen Vollwortes bzw. des lexikalischen Kerns eines mehrgliedrigen Wor- tes verbunden, so dass eine Präposition oder gar ein Suffix bzw. eine Endung nicht als Kopf in Erwägung gezogen werden. Es gibt dort also keine Umkehrung der Akzentmusters und damit keinen mis- match in Gestalt eines betonten Determinatums, da eben auch in diesen Fällen weiterhin das Determinans, hier in Gestalt des lexikalischen Kerns, den Haupt- akzent trägt. 15 was eben mit ihrer Unbetontheit übereinstimmt. Als prä- und postdeterminierend wird die periphrastische Verbform j’ai chanté gewertet, wobei sich ersteres auf das Verhältnis von Auxiliar und Partizip bezieht, das aber zweifellos postdetermi- nierend ist; denn das kategoriebestimmende Auxiliar wird von dem Partizip, das von diesem strukturell abhängt, näher spezifiziert und nicht umgekehrt. Als post- determinierend werden sowohl Suffixableitungen wie die Nomina fad-eur und popre-té, als auch die Verbformen chant-ais und chant-ons!, sowie die Folge sais pas betrachtet. Letzteres ist nun insofern interessant, als die nachgestellte Nega- tionspartikel pas – was meist übersehen wird – wie z. B. ein Adverb tatsächlich ein Determinans (des Prädikats) ist und als solches funktionsgerecht den Akzent trägt, im Unterschied zu den Funktorköpfen ‑ais und ‑ons der beiden Verbformen, die den angesprochenen prosodischen mismatch zwischen Betonung und Funk- tion aufweisen. Im Französischen ist es ja gerade nicht so, dass das nachgestellte morphologische «Determinans» im Sinne der Autoren unbetont wäre und der Hauptakzent auf dem Stamm läge. Wie wir gesehen haben, ist der Grund dafür in externen Faktoren wie eben dem Verstummen der nachtonigen Auslautsilben im Fall der französischen Suffixe und Endungen zu finden. Aufgrund der Linearität und Eindimensionalität des sprachlichen Mediums ist der Konflikt zwischen Determinationsrichtung und Akzentuierung system- immanent und nicht auflösbar, weil gewisse Determinantien/Komplemente, zu- mal pronominale und klitische, grundsätzlich unbetont sind und daher nur «ver- kehrt» stehen können, jedenfalls wenn ein Akzentmuster so dominant ist wie das jambische im Französischen. Die Voranstellung in einem le faire verstößt gegen die Determinationsrichtung und die Nachstellung in *faire le gegen den verbind- lichen Finalakzent. Entsprechendes gilt natürlich für prädeterminierende Spra- chen mit trochäischem Akzentmuster, in denen die Voranstellung, wie etwa in dt. dass Maria ihn besúcht, gegen die Initialbetonung eines fallenden Akzents, wie in ... einen Fréund besucht, verstößt, und die Nachstellung in einem ... *dass Maria besucht ihn gegen die Determinationsrichtung. Es ist freilich zu vermerken, dass der deutsche Hauptsatz hinsichtlich der Position (nicht der Reihenfolge) der Ob- jekte zum Verbum postdeterminierend ist, so dass in diesem Fall mit einem Maria besucht ihn gegen das jambische Akzentmuster verstoßen wird, das bei nomina- len Objekten gegeben ist: Maria besucht einen Fréund. Zusammenfassend lässt sich also festhalten, dass verbabhängige Pronominalobjekte weder in prä- noch in postdeterminierenden Sprachen systemkonform «korrekt» serialisiert werden können. Da das determinierende Glied im unmarkierten Fall den Akzent trägt, sind unbetonte bzw. Es ist auch nicht so, wie Geisler schreibt, dass die lateinische Syntax prädeterminierend wäre, also O-V, und die Morphologie postdeterminierend und sich daher «lexikalische und morphologische Determination in der Linearisie- rungsrichtung unterscheiden» (Geisler 1982, 53). Die reiche Endungs- und Suffix- morphologie des Lateinischen ist vielmehr gerade ein Indiz für seine prädetermi- nierende Grundstruktur mit trochäischem Akzentmuster. Hier offenbart sich ein tatsächlicher Nachteil eines solchen Determinationsbegriffs, mit dem die struktu- relle Isomorphie zwischen «lexikalischer» und «morphologischer» Determinati- on, d. h. die Parallelität von freier Syntax und Morphosyntax nicht nur nicht zu erfassen ist, sondern direkt verschleiert wird. Das eigentliche Problem dieser Ter- minologie ist aber darin zu sehen, dass bei der Unterscheidung von «lexika- lischer» und «morphologischer» Determination der Begriff Determination nicht einheitlich verwendet wird: im ersten Fall nämlich im logischen Sinn, z. B. in mai- son blanche, und im zweiten Fall, z. B. in Kindheit, im paradigmatischen Sinn, da das Suffix -heit den Stamm Kind- ja nicht determiniert. Als weitere Kronzeugen für französische Prädetermination werden Präposi- tionalphrasen wie par le livre (Geisler 1982, 214, und Baldinger 1968, 92ss.) auf- geführt, wiewohl die Präposition hier eindeutig als Kopf der Struktur fungiert, Zur Motivation des französischen Wortakzents klitische Determinantien diesbezüglich in sich widersprüch- liche Entitäten – eben Stellvertreter, die keine neue Information vermitteln. Oder unter anderem Aspekt betrachtet: unbetonte Determinantien bewirken auto- matisch eine Akzentverlagerung auf das jeweilige Determinatum, den jeweiligen Ulrich Wandruszka 16 Kopf, also auf die Konstituente, die normalerweise gerade unbetont ist: dt. síeht ihn/ihn síeht und frz. le vóit. Die meisten der hier betrachteten Sprachen sind freilich nicht so konsequent einem Akzentmuster verpflichtet wie das Französische. Im Deutschen können un- betonte Objektpronomina enklitisch und proklitisch zum Verbum auftreten, wie in den genannten deutschen Beispielen (besúcht ihn/ihn besúcht), wobei freilich postdeterminierender Hauptsatz und prädeterminierender Nebensatz, wie gesagt, unterschiedlichen Serialisierungstypen angehören. Im Gegensatz zu den Pro- nomina stehen betonte nominale Objekte im Deutschen immer «richtig», d. h. sys- temkonform: steigend postdeterminierend im VO-Hauptsatz, Maria besucht einen Fréund und prädeterminierend fallend im OV-Nebensatz, dass Maria einen Fréund besucht. Was die Pronominalobjekte angeht, kann man auch einfach sa- gen, dass sie im Deutschen, im Unterschied zum Französischen, immer dort ste- hen, wo auch die Nominalobjekte auftreten, wobei das Akzentmuster nicht von Relevanz ist. Wie wir gesehen haben, verhält es sich in den romanischen Sprachen und zumal im Französischen gerade nicht so. Gemäß dem zum Determinationstyp passenden Akzentmuster, in unserem Fall dem jambischen, geht das unbetonte klitische Objektpronomen im Unterschied zum nominalen dem Verbum voran. Entsprechendes gilt für die Pronominaladverbien wie z. B. ital. ci und ne in einem non ci vado mai. Die Folge Pronomen-Verbum, wie in Marie le voit, verbindet mit dem steigenden Akzentmuster auch die natürliche Reihung Thematisches vor Rhematischem. Das betonte Pronomen steht entsprechend nach dem Verbum wie typisch in span. te quiere a ti bzw. ital. pensa a te, ama te und frz. il pense à toi oder das bereits angesprochene imperativische assieds-toi! Romanische Objekt- pronomina wie lo oder le(s)/la, frz. te sind als echte Klitika nicht betonbar im Gegensatz zu den deutschen ihn, sie, dich etc. in Maria liebt íhn/mích/dích. Aus- nahmen bilden lediglich die französischen Objektpronomina le(s)/la in imperati- vischem prends-le/la! Im Unterschied zum Deutschen also ist der Akzent in den romanischen Sprachen für die Position von Pronomina relevant, womit Enklitika und damit ein fallender Akzent verhindert werden. Variation findet sich aller- dings in den südromanischen Sprachen mit der Möglichkeit einer enklitischen Position nach infiniten Formen des Verbums, wie in ital. non posso farlo gegen- über non lo posso fare. Wie erwähnt gibt es in diesen Sprachen auch innerhalb des Wortes sowohl steigende als auch vor allem fallende Akzentmuster, mehrheitlich Paroxytona (cf. u. a. Christine Kaschny 2011), also cantó vs. canto/cantano etc. Wir werden darauf zurückkommen. 17 Zur Motivation des französischen Wortakzents 18 Subjekt-Prädikat-Abfolge – unterstützt, so dass es auch innerhalb einer mehr oder weniger vorherrschenden Prädetermination immer seinen Platz hat.6 Subjekt-Prädikat-Abfolge – unterstützt, so dass es auch innerhalb einer mehr oder weniger vorherrschenden Prädetermination immer seinen Platz hat.6 Fallende Akzentmuster innerhalb des Wortes und solche innerhalb syntakti- scher Konstruktionen sind differenziert zu betrachten. Im Wortinneren findet sich dieses Betonungsmuster sowohl in eher prädeterminierenden Sprachen wie dem Deutschen oder dem Englischen, wie in Sänger, Kinder, lachend bzw. singer, chil- dren, laughing etc., als auch – außer im Französischen – in eher postdeterminie- renden Sprachen wie dem Italienischen oder Spanischen: canta, in zweisilbigen Endungen wie in cantate oder in parole sdrucciole wie in cantano oder mangiabile etc., jedenfalls in einer Unmenge von Wörtern mit unbetonter Endsilbe wie cane, porta, orto etc. Solche Endungen und Suffixe fungieren, wie wir gesehen haben, in der Regel als Kopf des Wortes bzw. der Wortform und werden von dem lexika- lischen Nukleus determiniert; so auch in Wortbildungen wie pescatore, bufalaio, parlamento und dergleichen, in denen zwar das Suffix aus silbenstrukturellen Gründen den Akzent trägt, aber in sich wieder ein fallendes Muster aufweist. In- nerhalb syntaktischer Konstruktionen hingegen haben unbetonte bzw. nicht-be- tonbare Elemente in enklitischer Postposition meist eine determinierende Funk- tion als Objekt oder Adverb. Dort ist jedenfalls die Wortfolge postdeterminierend, wie z. B. in ital. farlo, facendolo, fattolo, in imperativischem fammelo! oder auch in andarci und dergleichen. Als unmittelbare Konstituente eines Satzes allerdings werden enklitische Pronomina kaum mehr verwendet, sondern nur noch als Teil eines Satzgliedes, wie eben in farlo, facendolo oder in vistolo als adverbialer Aus- druck, wie in ....vistolo, fu mosso a pietà, keinesfalls jedoch *ho vistolo. Dies gilt auch für andere romanische Sprachen mit Ausnahme u. a. des Portugiesischen in Sätzen wie Chamo-me Enrique neben Não me chamo Enrique (gemäß Tobler-Mus- safia). Man muss sich nun die Frage stellen, warum Enklitika fast nur noch als Teil eines Satzglieds und speziell als Komplement einer nicht-finiten Verbform vor- kommen. Betrachtet man Wortformen wie parlo/parla/parlano etc. und infinite Verbformen mit enklitischem Objektpronomen wie farlo/facendolo/fattolo gegen- über heute inkorrektem (Maria) *facevalo/*fecelo oder auch *ha fattolo mit fini- tem Auxiliar, lässt sich festhalten, dass fallender Akzent nur im Wortinneren und in der Syntax eben nur noch in Kombination mit subordinierten nicht-finiten Verbformen auftritt. 6 Hubert Haider (2018) meint dazu generell: «Die SVO-Strukturierung ist daher ein Attraktor in der kognitiven Evolution menschlicher Grammatiksysteme». 4 Fallender Akzent im Wortinneren und in der Satzsyntax Der Konflikt zwischen Akzentmuster und Determinationstyp ist im Französi- schen allgegenwärtig, tritt aber, wie gesagt, auch im Deutschen auf, sowohl in Konstruktionen der freien Syntax als auch im Wortinneren. Morphosemantische mismatches, wie wir sie genannt haben, finden sich in deutschen Wortbil- dungsprodukten mit prädeterminierenden nicht-betonten Elementen, wie z. B. in bestimmten Präfixableitungen. Wenn Präfixe als determinierende Attribute fungieren, sollten sie entsprechend auch den Hauptakzent tragen, wie etwa in úmfahren, aúsgehen, aúfwischen, eínheizen etc. In Fällen wie umfáhren, über- sétzen, unterláufen hingegen trägt, wie auch sonst tendenziell im Deutschen, der lexikalische Kern des Wortes den Akzent. Die Betonung des Präfixes im Typ úmfahren mit dem für Prädetermination typischen fallenden Muster passt im Übrigen zum Faktum der Trennbarkeit dieser Präfixe, die auch vom lexika- lischen Kopf isoliert den Hauptakzent tragen: Der Bagger fuhr den Baum úm. In dieser Konstruktion lässt sich erkennen, dass das Präfix in einer zugrunde liegenden syntaktischen Struktur als eine Art Adverb fungiert. In Bildungen des Typs úmfahren/éinfahren und dergleichen hat man es mit Quasikomposita zu tun, innerhalb deren die determinierende erste Konstituente regulär den Ton trägt, so wie in einem herum- oder hereinfahren. In den stammbetonten Ablei- tungen liegt dem Präfix eher eine präpositionale Funktion zugrunde: Er um- fáehrt den Baum ←Er fährt um den Baum. Der Konflikt ergibt sich im Wort- inneren also aus der Tendenz des Deutschen, den jeweiligen lexikalischen Kern zu akzentuieren und andererseits aus dem Prinzip des akzenttragenden Deter- minans. Unbetonte prädeterminierende Elemente erzeugen ein jambisches und da- mit ein typisch postdeterminierendes Akzentmuster, wie in (er) übersétzt/um- fáehrt. Bei derartigen Präfixen handelt es sich zunächst (cf. Wells 1990, 254) um regulär akzentuierte Determinantien, wie noch in den erwähnten úebersetzen, úmfahren etc., deren Akzent dann durch den lexikalischen Kopf attrahiert wur- de. Eine derartige Entwicklung lässt sich im romanischen Bereich bei der Ak- zentverschiebung von lat. récipit zu vlat. recípit zu frz. reçoit oder von lat. réti- net zu vlat. reténet und frz. retient oder von lat. dísplicet zu vlat. displácet und frz. déplaît (sog. Rekomposition, cf. Rheinfelder 1987 [1953], 11, und Geisler 1982, 57) beobachten. Die Abschwächung determinierender Präfixe könnte als ein Anstoß für den Typwandel von trochäischer OV- zu jambischer VO-Akzentu- ierung gewertet werden. Das jambische Muster wird freilich darüber hinaus durch verschiedene Faktoren – wie etwa die Thema-Rhema-Progression und die Ulrich Wandruszka tolo sowie auch pensandoci oder ricordarsene. Hier sind Verbum und Pronomen besonders eng miteinander verbunden, so dass Letzteres nach Art einer Endung zu einem Teil des Wortes werden kann (auch durch die Graphie widergespiegelt). In einem *facciolo wäre eine so enge Bindung nicht möglich, da hier das Objekt- pronomen vom Verbstamm durch eine Subjektendung getrennt ist. Darüber hi- naus könnte diese Endung vielfach variieren, also z. B. *fannolo, *fareilo, *faceva- lo etc. Das Pronomen wäre hier auch nicht Teil eines Satzglieds, sondern selbst unmittelbares Satzglied, wie übrigens auch in einem heute nicht mehr akzepta- blen *ho fattolo, in dem das Pronomen nicht nur die Partizipform determinierte, sondern das ganze Prädikat ho fatto, wie in korrektem l’ho fatto. tolo sowie auch pensandoci oder ricordarsene. Hier sind Verbum und Pronomen besonders eng miteinander verbunden, so dass Letzteres nach Art einer Endung zu einem Teil des Wortes werden kann (auch durch die Graphie widergespiegelt). In einem *facciolo wäre eine so enge Bindung nicht möglich, da hier das Objekt- pronomen vom Verbstamm durch eine Subjektendung getrennt ist. Darüber hi- naus könnte diese Endung vielfach variieren, also z. B. *fannolo, *fareilo, *faceva- lo etc. Das Pronomen wäre hier auch nicht Teil eines Satzglieds, sondern selbst unmittelbares Satzglied, wie übrigens auch in einem heute nicht mehr akzepta- blen *ho fattolo, in dem das Pronomen nicht nur die Partizipform determinierte, sondern das ganze Prädikat ho fatto, wie in korrektem l’ho fatto. *Facciolo wäre also nicht so etwas wie ein komplexes Wort sondern eine syn- taktische Konstruktion, ein zweigliedriger vollständiger Satz mit zwei Vorgangs- beteiligten, wie lo faccio, während die Enklitika, wie gesagt, heute nur noch als Teile von untergeordneten Satzgliedern auftreten. Vorangestellte Elemente wie das Objektpronomen im letzten Beispiel sind grundsätzlich weniger eng mit ihrer Bezugseinheit verbunden als nachgestellte (cf. Wandruszka 1992 zur sog. «Suffix- präferenz»), zumal sie erst sekundär in Anteposition gelangen, während das den infiniten Verbformen folgende Pronomen, historisch betrachtet, einfach dort ste- hen bleibt und auch phonetisch eng mit der Verbform verschmelzen kann, wie speziell in farlo, das im Altitalienischen sogar assimilierte Varianten wie fallo kennt. Hinzukommt, dass das finite jambische lo faccio einer Thema-Rhema-Pro- gression folgt, die in nicht-satzhaltigen infiniten Formen wie farlo oder facendolo keine wesentliche Rolle spielt. Demnach sollte ein vollständiger Satz im Italie- nischen mit einem akzenttragenden Wort im definierten Sinn enden und nicht mit einem unbetonbaren unmittelbaren Satzglied wie in *facciolo. Zur Motivation des französischen Wortakzents 19 Man kann nun folgern, dass ein farlo quasi als komplexes Wort behandelt wird und somit einem im Wortinneren möglichen und geläufigen Akzentmuster entspricht, ebenso wie gerundiales facendolo und partizipiales fat- Zur Motivation des französischen Wortakzents Die italienische Syntax zeigt sich auch in diesem Fall jambisch strukturiert, im Unterschied zu einem altital. fecelo ‘er machte es’. Wie im Französischen erfährt im Italienischen auch der Imperativ eine geson- derte Behandlung, wenn das Verbum in Spitzenposition gebracht werden soll und das Pronomen daher enklitisch wird bzw. bleibt. Dies wird gleichfalls durch die Annahme erklärlich, dass Konstruktionen wie prendilo! und scrivetelo! oder vacci! ‘geh hin!’ als komplexe Wörter betrachtet werden, worauf im Übrigen auch die Zusammenschreibung hindeutet (cf. auch das infinitivische non prenderlo!). Eine Konstruktion wie prendilo! präsentiert sich nach Form und Akzentmuster wie etwa das proparoxytone Nomen cóndilo ‘Gelenkkopf’ bzw. wie die dreisilbige Verbform prendono. Wenn im Französischen und im Italienischen bei solchen Ausdrücken die Postposition des Pronomens bewahrt werden kann, weil die Kon- struktion als ein Wort gesehen wird, muss im Französischen das Enklitikon ak- zentuiert werden, prends-lé, während im Italienischen das Pronomen enklitisch unbetont bleiben kann. Das heißt, frz. prends-lé! ist endbetont wie ein zweisilbi- Ulrich Wandruszka 20 ges französisches Wort und ital. prendilo ist initialbetont wie ein einfacher italie- nischer Dreisilber. ges französisches Wort und ital. prendilo ist initialbetont wie ein einfacher italie- nischer Dreisilber. 21 nique ist darin zu sehen, dass durch die akzentuierte Endsilbe das Ende der Wort- gruppe eindeutig markiert wird, während mit einer unbetonten Endsilbe der In- tonationsbogen nicht mit dieser Deutlichkeit geschlossen werden könnte. Das Ende ist deutlich wahrnehmbar, weil sich die akzentuierte letzte Silbe von allen vorangehenden nicht-betonten Silben abhebt, während sich die letzte Silbe bei fallendem Akzent nicht von allen ihr vorausgehenden, sondern im Prinzip nur von der allerersten betonten Silbe unterscheiden würde. Um die oft als «schwebend» bezeichnete Intonation innerhalb des französi- schen mot phonique herzustellen, müssen die Wortakzente, außer dem letzten, nivelliert, d. h. abgesenkt werden. Die betonte Silbe eines Wortes wird an die je- weils vorangehende nicht (mehr) betonte Silbe angeglichen oder jedenfalls ange- nähert. Diese Silben sind dann von etwa gleicher Länge, Höhe und Intensität (cf. Meunier 2017, 34), im Unterschied zur jeweils letzten Silbe am Ende des mot pho- nique, wie z. B. in dem Satz: Maman a perdu une dent, wobei so etwas wie ein überlanger Anapäst mit betonter und gelängter Schlusssilbe entsteht (È È È ´—). Bei trochäisch fallender Akzentuierung hingegen können akzentuierte Silben nicht ohne weiteres an vorangehende nachtonige Silben angeglichen werden, wie etwa in dem Satz Peter wollte Schuhe kaufen. Hier müsste die erste Silbe von wollte so schwach betont sein wie die zweite Silbe von Peter (mit ihrem Schwa) und dann auch wie die zweite Silbe von wollte selbst. Entsprechend müssten die bei- den Silben von Schuhe gleich betont bzw. unbetont und jedenfalls gleichgewich- tig sein, wie auch die von kaufen. Dies stellt vor allem deshalb ein Problem dar, weil die Differenz zwischen akzentuierter und nachtoniger Silbe im Deutschen größer ist als die zwischen vortoniger und akzentuierter Silbe. Wenn nun diese Differenz zwischen haupttoniger Silbe, wie eben in wóllte oder Schúhe, und be- sonders schwacher nachfolgender Silbe nivelliert wird und letztere so ihre cha- rakteristische Nachtonigkeit verliert, entsteht durch einen akustischen Kippeffekt ein tendenziell jambisches Akzentmuster, also in etwa wólltè oder Schúhè. Durch die Deakzentuierung der Haupttonsilbe nähert sich diese der unbetonten Silbe an (und umgekehrt), wobei im Deutschen auch ein schwacher Nebenakzent, etwa in einem Schwímmèr als unangemessene Akzentuierung des Suffixes empfunden wird. 5 Das mot phonique Das Phänomen des mot phonique hat wesentlich mit der Generalisierung des fran- zösischen Wortakzents auf der letzten Silbe zu tun. Das einzelne Wort kann hier seinen Akzent weitgehend aufgeben, weil es keinen individuell unterschiedlichen silbenstrukturell oder morphosemantisch motivierten Eigenakzent besitzt. Das heißt, jedes Wort hat einen vorhersehbaren Akzent, der in einem vorhersehbaren gleichgerichteten Wortgruppenakzent aufgehen kann, was eben genau dann ge- schieht, wenn alle Wörter im Prinzip auf der gleichen Silbe betont werden und zwar auf der letzten. Die Satzglied- oder Wortgruppenbetonung hat mit der letzten Silbe einen übergreifenden Flucht- oder Zielpunkt, auf den die gespannte Intona- tion zuläuft und der einen markanten Abschluss bildet. Es ist also nicht nur das letzte Wort, wie im Italienischen und anderen postdeterminierenden Sprachen, sondern gerade die letzte Silbe, die den Satz(glied)-Akzent trägt, was perfekt der natürlichen Tendenz zur Betonung des Endes einer Äußerung gemäß einer un- markierten Thema-Rhema-Progression entspricht – wenngleich im Französischen die natürliche Übereinstimmung zwischen Rhematizität und internem Wort- akzent, wie gezeigt, nachhaltig gestört ist: Der Wortakzent verläuft parallel zum Satzgruppenakzent jambisch steigend, während die innere Struktur polymorphe- matischer Wörter eben nicht postdeterminierend ist. In einer Sprache hingegen, in der die Wörter nicht nur auf der letzten sondern auch auf der vorletzten oder vorvorletzten Silbe betont sein können, kann eine Wortgruppe nicht prinzipiell das gleiche Akzentmuster aufweisen wie die darin enthaltenen einzelnen Wörter. Die letzte Silbe eines Wortes ist immer die letzte, während die erste Silbe eines Wortes die letzte (a), die vorletzte (b) oder die vor- vorletzte (c) sein kann: (a) arbeitet mit Fléiß, (b) ... mit Fréude, (c) ... mit Áusdauer Das mot phonique könnte also selbst in einer Sprache mit durchgehendem Initi- alakzent nicht immer mit dem gleichen Betonungsmuster enden wie die einzel- nen vorausgehenden Wörter mit ihrer je unterschiedlichen Akzentposition. Wenn die Initialbetonung nicht durchgängig ist – man denke etwa an ein deutsches ... arbeitet mit Verstánd – kann es ohnehin zu keiner völligen Übereinstimmung zwischen Wort und Wortgruppe kommen. Bei durchgehender Oxytonie hingegen endet im Prinzip jedes mot phonique so wie die einzelnen darin enthaltenen Wör- ter auf eine akzentuierte Silbe. Ein wesentliches Charakteristikum des mot pho- Zur Motivation des französischen Wortakzents Solange die letzte Silbe schwach betont oder jedenfalls nicht wirklich unbe- tont ist und allgemein, wenn zwei aufeinanderfolgende Silben akzentuell annä- hernd gleichwertig sind, entsteht tendenziell ein Jambus, weil eine nicht deutlich abgesenkte Nachtonsilbe zu viel Gewicht relativ zur Tonsilbe erhält und so den genannten Kippeffekt auslöst. Auf vortonigen Silben hingegen, die normalerweise ebenfalls nicht oder nur sehr schwach betont sind, ist ein Nebenakzent nicht nur eher möglich, sondern gerade im Französischen durchaus geläufig, wie z. B. in den von Meisenburg/Se- lig (2008, 159) erwähnten bàteau-citérne, la màjeure partíe, àcculturatión u. a. Die- 22 Ulrich Wandruszka ser auch als contre-accent bezeichnete Initialakzent tritt insbesondere in silben- zählenden Sprachen wie dem Französischen auf, in denen der Unterschied zwi- schen akzentuierter und nicht akzentuierter Silbe nicht so ausgeprägt ist wie im akzentzählenden Deutschen. Dies führt zu der angesprochenen «schwebenden» Betonung/Intonation des Französischen, die sich aus der Gleichgewichtigkeit der Silben ergibt und die Bildung von mots phoniques befördert. Deutsche Sprecher neigen interessanterweise dazu, die letzte Silbe französischer Wörter zuunguns- ten der jeweils vorangehenden besonders nachdrücklich zu betonen, wodurch wie im Deutschen wieder ein starker Wortakzent produziert wird. 6 Schlussbemerkung Formale signifikantenseitige Entwicklungen, hervorgerufen durch äußere Ein- flüsse oder einfach bedingt durch die Natur des sprachlichen Mediums, können dysfunktionale Modifikationen der inneren Struktur der Sprache zur Folge haben. Es kann sich eine funktionale Entkoppelung von Form und Inhalt vollziehen, oh- ne dass ein solcher mismatch vom Sprachbenutzer wahrgenommen und als be- sonders störend empfunden werden muss. Autokorrektur und «Selbstheilungs- kräfte» der Sprache nach der Vorgabe form follows function sind nicht immer stark genug, um derartige Störungen des Zusammenspiels der verschiedenen Ebenen der Sprache zu verhindern oder zu beheben. Das Instrument Sprache funktioniert offenbar auch bei suboptimaler oder dysfunktionaler Ausdrucksgestaltung, da es prinzipiell auch durch den (lexikalischen) Inhalt der Zeichen gestützt wird, der die passende Interpretation eines komplexen Ausdrucks gleichwohl ermöglicht. Schlagende Beispiele hierfür sind die nachgestellten betonten Pronomina, in frz. vient-il, donne-le-lui, vas-y und dergleichen, oder die oxytonen Wortformen wie chanter, chanta, chanteur etc. als Ergebnis des Verstummens der lateinischen Nachtonsilben. Dies ist umso bemerkenswerter, als das Phänomen im Französi- schen keineswegs nur vereinzelt sondern als Grundprinzip quasi ausnahmslos auftritt, was dazu führt, dass das System an dieser Stelle regelrecht kippt und einen massiven Typwandel erfährt, hier in Form des Verlustes fallender trochäi- scher Akzentmuster und ihrer Ersetzung durch jambische. Diese nicht funktional-inhaltlich motivierte Veränderung auf der Signifikan- tenseite zieht im Wortinneren keine dem jambischen Akzentmuster entsprechen- de postdeterminierende Organisation nach sich, in der das jeweilige Determinans betont wäre; denn der Akzent kommt in der Regel auf die betonte Silbe eines ur- sprünglich mehrsilbigen Funktorkopfs zu stehen und damit prinzipiell auf das dann einsilbige Flexiv oder Suffix wie in cantavit →chanta oder ligamentum → ligament usw. Die Endbetonung der Wörter und Wortformen passt formal zur Zur Motivation des französischen Wortakzents 23 postdeterminierenden Struktur der französischen Syntax, nicht jedoch die Kopf- funktion der betonten Silbe. Solche systematischen Verwerfungen ergeben sich in unserem Fall daraus, dass durch die lineare Bewegung der Sprachproduktion po- sitionsbedingte Asymmetrien in der Akzentstärke entstehen. So ist die Nach- tonsilbe in der Regel noch schwächer als die Vortonsilbe, weshalb sie zu Reduk- tion oder Tilgung tendiert, gewissermaßen im Windschatten des Hauptakzents und zur Erhaltung der Gesamtenergie der beiden unmittelbar aufeinanderfolgen- den Silben. Da die Nachtonsilben am Ende des Wortes normalerweise die Flexive und Suffixe enthalten, die als Köpfe des Ausdrucks fungieren, bedeutet ihr Ver- stummen den Verlust des morphologischen Kopfes, jedenfalls des unbetonten Teils eines mehrsilbigen Kopfes. 6 Schlussbemerkung Identifiziert man den Kopf lateinischer Wörter mit der am Wortende befindlichen Konstituente, die unbetont ist oder jedenfalls diese unbetonte Konstituente enthält, dann besitzen die französischen Entspre- chungen keinen derartigen Kopf (mehr). Was bleibt, ist der von uns als mismatch bezeichnete betonte Kopf oxytoner Wortformen, wie in chantons, nageur oder bar- rage, fragile und generell im Französischen. Wenn der Kopf im Lateinischen ein- silbig war, wird die französische Nachfolgeform morphologisch vollständig «kopflos» wie in vient ←venit bzw. in Nomina wie chant ←cantus, fils ←filius oder einem Adjektiv wie las ←lassus, die nur aus dem betonten ursprünglichen Komplement bestehen. Immerhin trägt in diesem Fall nicht der lateinische Kopf, sondern das einstige Komplement den Akzent. 7 Bibliographie Baldinger, Kurt, Post- und Prädeterminierung im Französischen, in: id. (ed.), Festschrift Walther von Wartburg, vol. 1, Tübingen, Niemeyer, 1968, 86–106. Barme, Stefan, Gesprochenes Französisch, Berlin/Boston, De Gruyter, 2012. Eckert, Gabriele, Sprachtypus und Geschichte. Untersuchungen zum typologischen Wandel des Französischen, Tübingen, Narr, 1986. Eisenberg, Peter, Grundriss der deutschen Grammatik, Stuttgart, Metzler, 42013. Geisler, Hans, Studien zur typologischen Entwicklung. Lateinisch – Altfranzösisch · Neufranzö- sisch, München, Fink, 1982. Grevisse, Maurice, Le bon usage. Grammaire française, Gembloux, Duculot, 101975. Guiraud, Pierre, La syntaxe du français, Paris, Presses Universitaires de France, 61980. Haider, Hubert, Endstation SVO? Warum viele Sprachen zu SVO-Sprachen wurden und viele an- gebliche «SVO»-Sprachen keine sind, Berlin, Humboldt-Universität, 2018, <https://www. uni-salzburg.at/fileadmin/multimedia/Linguistik/2018-Feb-HUB-Berlin-HA.pdf> [letzter Zu- griff 30.07.2019]. Kaschny, Christine, Primärer Wortakzent und morphologische Struktur im Italienischen. Eine theoriebezogene, quantitativ und empirisch angelegte Untersuchung, Konstanz, Bibliothek der Universität Konstanz, 2011. 24 Ulrich Wandruszka 24 Meisenburg, Trudel/Maria Selig, Phonetik und Phonologie des Französischen, Stuttgart, Klett, 2008. Meunier, Christian, Grammaire participative: enseignée avec la participation active des appre- nants, Marseille, Les Editions du FLE, 2017. Pomino, Natascha/Stark, Elisabeth (edd.), Proceedings of the V Nereus International Workshop on «Mismatches in Romance», Universität Konstanz, Arbeitspapier Nr. 125, Fachbereich Sprachwissenschaft, 2011. Rheinfelder, Hans, Altfranzösische Grammatik, vol. 1: Lautlehre, München, Hueber, 51987 [1953]. Rheinfelder, Hans, Altfranzösische Grammatik, vol. 1: Lautlehre, München, Hueber, 51987 [1953]. Sasse, Hans-Jürgen, Gedanken über Wortstellungsveränderung, Papiere zur Linguistik 13:14 (1977), 82–142. Sasse, Hans-Jürgen, Gedanken über Wortstellungsveränderung, Papiere zur Linguistik 13:14 (1977), 82–142. Wandruszka, Ulrich, Post- oder Prädetermination in den romanischen Sprachen, Romanistisches Jahrbuch 31 (1980), 56–72. Wandruszka, Ulrich, Klassemisch versus lexemisch. Zwei Grundtypen sprachlicher Strukturbil- dung, Papiere zur Linguistik 41:2 (1989), 77–100. Wandruszka, Ulrich, Zur Suffixpräferenz. Prolegomena zu einer Theorie der morphologischen Abgeschlossenheit, Papiere zur Linguistik 46:1 (1992), 3–27. Wandruszka, Ulrich, Figure et fond en morphosyntaxe et la notion d’importance, in: Dufter, An- dreas/Jacob, Daniel (edd.), Syntaxe, structure informationnelle et organisation du discours dans les langues romanes, Bern/Berlin, Lang, 2011, 173–188. Wandruszka, Ulrich, Sprache – linearisierte Struktur in Bewegung. Eine Einführung in die Me- chanik der Sprache auf der Basis der Kategorialgrammatik, Tübingen, Stauffenburg, 2015. chanik der Sprache auf der Basis der Kategorialgrammatik, Tübingen, Stauffenburg, 2015. Weinrich, Harald, La place de l’adjectif en français, Vox Romanica 25 (1966), 82–89. Weinrich, Harald, La place de l’adjectif en français, Vox Romanica 25 (1966), 82–89. Weinrich, Harald, Sprache in Texten, Stuttgart, Klett, 1976. Wells, Christopher, J., Deutsch. Eine Sprachgeschichte bis 1945, Tübingen, Niemeyer, 1991. Wells, Christopher, J., Deutsch. 7 Bibliographie Eine Sprachgeschichte bis 1945, Tübingen, Niemeyer, 1991. Wunderli, Peter/Benthin, Karola/Karasch, Angela, Französische Intonationsforschung. Kritische Bilanz und Versuch einer Synthese, Tübingen, Narr, 1978. Wunderli, Peter/Benthin, Karola/Karasch, Angela, Französische Intonationsforschung. Kritis Bilanz und Versuch einer Synthese, Tübingen, Narr, 1978. Wunderli, Peter, Intonationsforschung und Prosodie, in: Holtus, Günter/Metzeltin, Michael/ Schmitt, Christian (edd.), Lexikon der Romanistischen Linguistik (LRL), vol. 5/1: Französisch, Tübingen, Niemeyer, 1990, 34–46.
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Photoresponse of the Al/n-Si Schottky diode with nanorod ZnO interface layer prepared by hydrothermal method
Gazi Üniversitesi Fen Bilimleri dergisi
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Photoresponse of the Al/n-Si Schottky Diode with Nanorod ZnO Interface Layer Prepared Using Hydrothermal Method Gazi University, Faculty of Science, Department of Physics, 06500, Yenimahalle/Ankara/TÜRKİYE Gazi University, Faculty of Science, Department of Physics, 06500, Yenimahalle/Ankara/TÜRKİYE GU J Sci, Part C, 10(4): 1059-1069 (2022) GU J Sci, Part C, 10(4): 1059-1069 (2022) Gazi University e-mail: neslihanturan@gazi.edu.tr Abstract Article Info Research article Received: 7.10.2022 Revision: 1.11.2022 Accepted: 9.11.2022 Keywords Hydrothermal Zinc oxide Photovoltaic nanorod Article Info Research article Received: 7.10.2022 Revision: 1.11.2022 Accepted: 9.11.2022 Keywords Hydrothermal Zinc oxide Photovoltaic nanorod In this study, ZnO nanorods (ZnO-NR) were prepared on n-Si wafer by hydrothermal method. Structural and morphologic properties of ZnO nanostructures were investigated through XRD and SEM method. The illumination impacts on the current-voltage (I-V) measurements of the prepared Al/ZnO-NR/n-Si diode were explored in the dark and different illumination intensities (20–100 mW/cm2) between ± 1.5 V bias voltage range. The Schottky diode barrier height value had an increasing trend with increasing illumination intensity from 20 to 100 mW/cm2 while the ideality factor had a decreasing trend with the increase of photocurrent. The temporary photocurrent increases as illumination intensity increases. The slope (α) of the logIph-logP curve was obtained as 0.618 and this slope confirmed that this ZnO nanorod shows photoconducting behavior. The short-circuit current (Isc) and open-circuit voltage (Voc) values were obtained to be 774.08 μA and 0.24 V under 100 mW/cm2 illumination intensity, respectively. The prepared Al/ZnO-NR/n-Si diode showed both photodiode and photovoltaic properties under different light intensities. It was concluded that the prepared Al/ZnO-NR/n-Si diode can be used in the optoelectronic applications, especially for the photodiode industry. 1. INTRODUCTION In metal-semiconductor (MS) structures, the interfacial layer between the MS structure will affect many parameters of the diode, and changes in the thickness and concentration of the interface material affect the electrical behavior of the structure. The study of changes in the properties of MS structures using many materials as interfaces still attracts great interest. By using different production techniques and various materials, doped or layered structures are obtained and the changes in the electrical properties of these structures are examined. ZnO is a well-known semiconductor and functional material used in many places such as chemical sensors, conductivity electrodes, photoelectric devices, and solar cells, etc. The development of photovoltaic technology is to increase the efficiency of cells and reduce the cost of production, making them more suitable for various applications. It can be produced by many methods such as physical [1–4] (ball milling, chemical and physical vapor deposition, lithographic, laser ablation, etc.), chemical (solution-oriented synthesis, gas phase reaction) and biological methods, and the properties of nanostructures change according to the production methods. Compared to other methods, solution-oriented methods show advantages such as low production cost, scalability, ease of application, and relatively low temperatures (<200 °C) are required for production. Due to these advantages, solution-oriented methods attract attention and ZnO can be produced by hydrothermal/solvothermal [5–7], precipitation, sol-gel [8], chemical bath deposition [9, 10], and electrochemical deposition [11]. Because there are too many effects in solution-oriented methods, the nanoscale effects are different, and ZnO can be produced in different structures such as nanorods [12], nanoflowers [13, 14], nanoarrays [15, 16], and nanospheres [17, 18]. The electrical conductivity and optical properties of ZnO change with the change of their morphological structure. ZnO nanorod structures attract research attention due to their large surface area, low cost and ease of use in potential applications in optoelectronic fields. ZnO nanorods can be produced effectively using the hydrothermal method [19]. ZnO DOI: 10.29109/gujsc.1185766 1060 Neslihan TURAN / GU J Sci, Part C, 10(4):1059-1069(2022) nanowires/nanorods are used in various nanodevice applications such as solar cells [20], nanogenerator [21], field emitters [22], gas sensors [23], and UV detectors [24]. nanowires/nanorods are used in various nanodevice applications such as solar cells [20], nanogenerator [21], field emitters [22], gas sensors [23], and UV detectors [24]. 1. INTRODUCTION In this article, the Al/ZnO-NR/n-Si/Al device was prepared using the hydrothermal method, and it was investigated whether it is practical for photovoltaic devices with I-V measurements in the range of ± 1.5 V in the dark and various illumination intensities. 2. MATERIALS AND METHODS In this study, ZnO-NR were synthesized on n-Si wafer by hydrothermal method. Before the growth of ZnO- NR, a thin ZnO seed layer was coated on the surface of n-Si wafer. First n˗type Si wafer (orientation 100, thickness 525 μm, and resisivity 1-20 Ω∙cm) was cleaned with an ultrasonic cleaner using cleaning steps with chloroform, acetone, and methanol. The oxide structure was removed using a mixture of HF-deionized water (1:10), rinsed with deionized water, and dried. Aluminum (99.99% purity) was evaporated as a ohmic contact on the back of the wafer under 10˗6 Torr vacuum using the thermal evaporation system. Secondly, the seed layer was prepared by spin coating technique using zinc acetate dihydrate (Zn(CH3COO)2•2H2O), methanol, and NaOH precursors. Zinc acetate dihydrate was dissolved in 100 ml methanol on the magnetic stirrer. Then, NaOH (33%) was dropped slowly to the 0.02 M zinc acetate dihydrate solution. The solution was stirred at 60°C for 2 h on magnetic stirrer. The resultant transparent solution was cooled room temperature and was dropped on n-Si wafer. 500 rpm for 5 s and 3000 rpm for 30 s were chosen as the spin coating parameters. After the coating, the substrate was annealed at 130°C for 5 min in oven and this coating procedure was repeated four times. For the hydrothermal process, ZnO was prepared using Zinc acetate dihydrate, deionized water, and ammonia (25%). Zinc acetate dissolved in deionized water (0.05 M), then 1.5 ml ammonia dropped slowly in the solution on magnetic stirrer. Wafer inserted into teflon container with the seed layer side down, and the solution was added to hydrothermal system which was set to 90 °C for 12 h. After the system cooled down to room temperature naturally, wafer was taken out of the hydrothermal system, rinsed with deionized water, and dried. Finally, For the Al/ZnO-NR/n-Si/Al structure, the top contact was obtained by deposition of Al with a thickness of 150 nm and a diameter of 2.75 mm, under 10-6 torr pressure, with a thermal evaporation system. Schmetic representation of experimental procedure is shown in Fig. 1. Fig. 1. Schematic representation of experimental procedure Fig. 1. Schematic representation of experimental procedure 1061 Neslihan TURAN / GU J Sci, Part C, 10(4):1059-1069(2022) The crystallographic and morphologic properties of ZnO-NR were characterized by Bruker D8 Advance diffractometer and FE-SEM (Hitachi SU5000) respectively. 2. MATERIALS AND METHODS The (I-V) measurements of the Al/ZnO-NR/n- Si/Al in ± 1.5 V bias voltage range was performed under the dark and various illumination intensities using the FYTRONIX solar simulator and Keithley 2400 at room temperature. 3. RESULTS The surface morphology of the sample identified by FE-SEM is shown in Fig 2. a and b are the surface views above, and c is the cross-section view. It was observed that nanorods were formed on the surface and their orientation was random in the c direction. When the c-oriented nanorods in Figure 2b were examined, it was seen that the ZnO nanorods had a hexagonal structure. The thickness of the ZnO nanorod layer on the surface is approximately 350–400 nm, as can be seen in the cross-sectional view in Figure 2c. Fig. 2. SEM images of the Al/ZnO-NR/n-Si structure a) X50k b) X100k c) cross-section view Fig. 3 shows the XRD pattern of ZnO-NR grown on n-Si recorded in the range of 2θ=10–80°. The diffraction peak of the (002) of ZnO-NR was marked in the diffraction pattern. The peak is compatible with the JCPDS 36–1451 pdf card belonging to ZnO hexagonal wurtzite (space group P63mc). The peak belonging to the n-Si in the XRD diffraction pattern were also observed. a b c b a Fig. 2. SEM images of the Al/ZnO-NR/n-Si structure a) X50k b) X100k c) cross-section view c Fig. 2. SEM images of the Al/ZnO-NR/n-Si structure a) X50k b) X100k c) cross-section view Fig. 3 shows the XRD pattern of ZnO-NR grown on n-Si recorded in the range of 2θ=10–80°. The diffraction peak of the (002) of ZnO-NR was marked in the diffraction pattern. The peak is compatible with the JCPDS 36–1451 pdf card belonging to ZnO hexagonal wurtzite (space group P63mc). The peak belonging to the n-Si in the XRD diffraction pattern were also observed. Fig. 3 shows the XRD pattern of ZnO-NR grown on n-Si recorded in the range of 2θ=10–80°. The diffraction peak of the (002) of ZnO-NR was marked in the diffraction pattern. The peak is compatible with the JCPDS 36–1451 pdf card belonging to ZnO hexagonal wurtzite (space group P63mc). The peak belonging to the n-Si in the XRD diffraction pattern were also observed. 1062 Neslihan TURAN / GU J Sci, Part C, 10(4):1059-1069(2022) Fig. 3. XRD pattern of the Al/ ZnO-NR/n-Si structure Fig. 3. XRD pattern of the Al/ ZnO-NR/n-Si structure Fig. 3. XRD pattern of the Al/ ZnO-NR/n-Si structure The current-voltage (I–V) measurements of the fabricated Al/ZnO-NR/n-Si Schottky diode according to thermionic emission (TE) theory were investigated from in dark and various illumination intensities. 3. RESULTS The current-voltage equation in reverse and forward bias can be defined by the following relation [25–29] 𝐼= 𝐼0𝑒𝑥𝑝( 𝑞𝑉 𝑛𝑘𝑇)[1 −𝑒𝑥𝑝(−𝑞𝑉 𝑘𝑇)] (1) 𝐼0 = 𝐴𝐴∗𝑇2𝑒𝑥𝑝[−𝑞𝛷𝐵 𝑘𝑇] (2) 𝐼= 𝐼0𝑒𝑥𝑝( 𝑞𝑉 𝑛𝑘𝑇)[1 −𝑒𝑥𝑝(−𝑞𝑉 𝑘𝑇)] (1) Where Where 𝐴𝐴∗𝑇2𝑒𝑥𝑝[−𝑞𝛷𝐵 𝑘𝑇] (2) 𝐼0 = 𝐴𝐴∗𝑇2𝑒𝑥𝑝[−𝑞𝛷𝐵 𝑘𝑇] (2) where 𝛷𝐵 is the Schottky barrier height, k is the Boltzmann constant, q is the charge of electron, T is the absolute temperature, 𝑛 is the ideality factor, 𝐴∗ is the effective Richardson constant of 112 Acm-2K-2 for n-Si, A is the area of effective diode and 0I is the saturation current of reverse. From the forward bias 𝑙𝑛𝐼– 𝑉 measurement by using Eq. 1 and Eq. 2, the barrier height and ideality factor of the fabricated sample can be estimated using the Eq. 3 and Eq. 4 [30–32] 𝑛= 𝑞 𝑘𝑇 𝑑𝑉 𝑑(𝑙𝑛𝐼) (3) 𝛷𝐵= 𝑘𝑇 𝑞𝑙𝑛(𝐴𝐴∗𝑇2 𝐼0 ) (4) 𝑛= 𝑞 𝑘𝑇 𝑑𝑉 𝑑(𝑙𝑛𝐼) 𝛷𝐵= 𝑘𝑇 𝑞𝑙𝑛(𝐴𝐴∗𝑇2 𝐼0 ) (3) 𝛷𝐵= 𝑘𝑇 𝑞𝑙𝑛(𝐴𝐴∗𝑇2 𝐼0 ) (4) 𝛷𝐵= 𝑘𝑇 𝑞𝑙𝑛(𝐴𝐴∗𝑇2 𝐼0 ) (4) The logarithmic current-voltage (I-V) measurements of the ZnO-NR film deposited in Al/n-Si interface, including the illustration of the between ±1.5 V and in the illumination intensity range of 20–100 mW/cm2 and in dark are indicated in Fig 4. Neslihan TURAN / GU J Sci, Part C, 10(4):1059-1069(2022) 1063 Fig. 4. Current-voltage characteristics of Al/ZnO-NR/n-Si dark and under various illumination intensities Fig. 4. Current-voltage characteristics of Al/ZnO-NR/n-Si dark and under various illumination intensities As can be seen from the forward bias part of Fig. 4, the I-V curve shifts toward the positive voltage as the illumination intensity increases. This situation corresponds to photovoltaic behavior. The dark characteristics of the produced Al/ZnO-NR/n-Si diode showed good rectification behavior. While the ratios of the forward current and reverse current in the dark and 100mW/cm2 intensity are 50.9 and 8.9 at 1.5 V, respectively. As can be seen from the different illumination characteristics in Fig. 4, the produced diode showed both photodiode properties in reverse bias and photovoltaic properties in forward bias. Using Eq. 3 and Eq. 4, 𝑛, 𝛷𝐵 , and 𝐼0 values were calculated in the dark and at various illumination intensities and are given in Table 1. Table. 1. 3. RESULTS Variation of electrical parameters with various illumination intensity of Al/ZnO-NR/n-Si Schottky diode Illumunation intensity (mW/cm2) 𝑛 𝛷𝐵 (eV) 𝐼0 (𝜇A) 𝐼𝑝ℎ (𝜇A) 𝑅𝑠 (Ω) 𝑅𝑠ℎ (Ω) 𝑉𝑜𝑐 (V) 𝐼𝑠𝑐 (𝜇𝐴) 0 2.92 0.631 15.40 ------ 84.4 5955 ------ ------ 20 2.49 0.672 3.13 613 89.5 5462 0.16 244 40 2.28 0.693 1.38 887 91.2 5206 0.20 417 60 2.05 0.713 0.64 1161 92.8 5003 0.22 566 80 1.97 0.728 0.37 1423 93.5 4844 0.22 685 100 1.66 0.758 0.11 1646 94.4 4628 0.24 774 tion of electrical parameters with various illumination intensity of Al/ZnO-NR/n-Si Schottky de Table. 1. Variation of electrical parameters with various illumination intensity of Al/ZnO-NR/n diode The fourth region data of the I–V measurements are shown in Fig. 5. The open circuit voltage (𝑉𝑜𝑐) and short circuit current (𝐼𝑠𝑐) values are determined from the points where the curves of I–V intersect the voltage and current axis, respectively. As observed in Fig. 5 and Table 1, the 𝐼𝑠𝑐 and 𝑉𝑜𝑐 values increase as illumination intensity increases. For example, the values of 𝐼𝑠𝑐 and 𝑉𝑜𝑐 are found 244 𝜇A and 0.16 V for 20 mW/cm2 and 774 𝜇A and 0.24 V for 100 mW/cm2, respectively. This depicts that the Au/ZnO-NR/n-Si Schottky diode is completely sensitive to illumination intensity and shows photovoltaic behavior. The fourth region data of the I–V measurements are shown in Fig. 5. The open circuit voltage (𝑉𝑜𝑐) and short circuit current (𝐼𝑠𝑐) values are determined from the points where the curves of I–V intersect the voltage and current axis, respectively. As observed in Fig. 5 and Table 1, the 𝐼𝑠𝑐 and 𝑉𝑜𝑐 values increase as illumination intensity increases. For example, the values of 𝐼𝑠𝑐 and 𝑉𝑜𝑐 are found 244 𝜇A and 0.16 V for 20 mW/cm2 and 774 𝜇A and 0.24 V for 100 mW/cm2, respectively. This depicts that the Au/ZnO-NR/n-Si Schottky diode is completely sensitive to illumination intensity and shows photovoltaic behavior. Neslihan TURAN / GU J Sci, Part C, 10(4):1059-1069(2022) 1064 Fig. 5. I–V plots for the Al/ZnO-NR/n-Si Schottky diode Fig. 5. I–V plots for the Al/ZnO-NR/n-Si Schottky diode As the illumination intensity increases, the 𝑛 and 𝐼0 values decrease, while the 𝛷𝐵 value increases. For example, The 𝑛 values decreased from 2.92 in the dark to 1.66 at 100 mW/cm2 and the 𝛷𝐵 values increased from 0.631 eV in the dark to 0.758 eV at 100 mW/cm2. It was determined from Fig. 3. RESULTS 4 that the diode produced due to the photocurrents (𝐼𝑝ℎ) increasing in reverse bias as illumination intensity increases was a photodiode. 𝐼𝑝ℎ values for 20 mW/cm2 and 100 mW/cm2 were found as 613 𝜇A and 1646 𝜇A, respectively. Both the series resistance (𝑅𝑠) in the forward bias and the shunt resistance (𝑅𝑠ℎ) in the reverse bias affect the electrical properties of the produced diode. 𝑅𝑠 and 𝑅𝑠ℎ values were found by Ohm's law (𝑅𝑖= 𝑑𝑉𝑑𝐼 ⁄ ) and are illustrated in Table 1 and Fig. 6. Fig. 6. The Ri-V plots at dark and under various illumination intensities Fig. 6. The Ri-V plots at dark and under various illumination intensities Neslihan TURAN / GU J Sci, Part C, 10(4):1059-1069(2022) 1065 While 𝑅𝑠 values almost do not change under illumination, 𝑅𝑠ℎ values decrease with increasing illumination intensity. Hence, 𝑅𝑠 values in dark and 100 mW/cm2 were found as 84 Ω and 94 Ω at 1.5V for the produced diode, respectively. 𝑅𝑠ℎ values in dark and 100 mW/cm2 were found as 5748 Ω and 2578 Ω at -0.4V for the produced diode, respectively. In addition to the I-V measurements, current-time (I-t) measurements were performed under different illumination intensities to investigate the photosensitivity of the produced diode at -1V and are illustrated in Fig 7. From Fig. 7, it has been observed that the current increases when the light is on and decreases when the light is off. That is, the photocurrent for Al/ ZnO-NR /n-Si diode was observed when the light was on. This photocurrent increased as the light intensity increased from 20 to 100 mW/cm2. This behavior is due to charge trapping at deep levels [33]. Fig. 7. Transient photocurrent characteristics of Al/ZnO-NR/n-Si at various illumination intensity. Fig. 7. Transient photocurrent characteristics of Al/ZnO-NR/n-Si at various illumination intensity. Fig. 7. Transient photocurrent characteristics of Al/ZnO-NR/n-Si at various illumination intensity To determine the variation of the photoconduction via the various illumination intensity, the double- logarithmic photocurrent (𝐼𝑝ℎ) versus illumination intensity (𝑃) at -1.5V was plotted and shown in Fig. 8. The mechanism of photoconduction can be found by the Eq. 5 [34]. To determine the variation of the photoconduction via the various illumination intensity, the double- logarithmic photocurrent (𝐼𝑝ℎ) versus illumination intensity (𝑃) at -1.5V was plotted and shown in Fig. 8. The mechanism of photoconduction can be found by the Eq. 5 [34]. Where, 𝛼, 𝐼𝑝ℎ, and 𝐴 are a power exponent, the photocurrent, and a constant, respectively. 𝐼𝑝ℎ= 𝑃𝛼 (5) 3. RESULTS 𝐼𝑝ℎ= 𝑃𝛼 (5) Where, 𝛼, 𝐼𝑝ℎ, and 𝐴 are a power exponent, the photocurrent, and a constant, respectively. Where, 𝛼, 𝐼𝑝ℎ, and 𝐴 are a power exponent, the photocurrent, and a constant, respectively. 1066 Neslihan TURAN / GU J Sci, Part C, 10(4):1059-1069(2022) Fig. 8. log (Iph) vs log (P) plot for the Al/ZnO-NR/n-Si The 𝛼 value was found from the slope of the log𝐼𝑝ℎ-log𝑃 curve and obtained as 0.618 The linear variation of the photocurrent from Fig. 8 is due to trapping of charge carriers at deep levels [35] Fig. 8. log (Iph) vs log (P) plot for the Al/ZnO-NR/n-Si The 𝛼 value was found from the slope of the log𝐼𝑝ℎ-log𝑃 curve and obtained as 0.618 The linear variation of the photocurrent from Fig. 8 is due to trapping of charge carriers at deep levels [35] The 𝛼 value was found from the slope of the log𝐼𝑝ℎ-log𝑃 curve and obtained as 0.618 The linear variation of the photocurrent from Fig. 8 is due to trapping of charge carriers at deep levels [35] 4.CONCLUSIONS ZnO nanorods were successfully produced on n-Si by hydrothermal method. It has been observed by SEM images that the diameter of the hexagonal ZnO nanorods is around 60 nm and the thickness on the surface is approximately 350–400 nm. In XRD analysis, the characteristic 002 peak of the hexagonal wurtzite structure of ZnO nanorods was observed. The reverse and forward bias current-voltage (I-V) characteristics of the prepared Al/ZnO-NR/n-Si photodiode have been analyzed under dark, 20, 40, 60, 80, and 100 mW/cm2 illumination intensity at room temperature. The main electrical parameters such as,𝑛 𝛷𝐵,𝐼0,𝐼𝑝ℎ, 𝑅𝑠, 𝑅𝑠ℎ,𝑉𝑜𝑐, and 𝐼𝑠𝑐 of the fabricated Schottky barrier diode were investigated. It was observed that as the illumination intensity increased, the n values decreased and the 𝛷𝐵 values increased. The results showed that the electrical parameters are sensitive to illumination intensity and the Al/ZnO-NR/n-Si Schottky barrier diode has photovoltaic and photodiode properties. 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https://periodicos.ufsb.edu.br/index.php/paubrasilia/article/download/49/32
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família Solanaceae Juss. no município de Vitória da Conquista, Bahia, Brasil
Paubrasilia
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16,752
Paubrasilia Paubrasilia Artigo Original doi: 10.33447/paubrasilia.2021.e0049 2021;4:e0049 Abstract Solanaceae is one of the largest families of vascular plants, with 100 genera and ca. 2,500 species, with subcosmopolitan distribution and greater diversity in the Neotropical region. This work carried out a floristic survey of Solanaceae species in the municipality of Vitória da Conquista, Bahia, in an ecotonal area between Caatinga and Atlantic Forest. Weekly collections were carried out from August/2019 to March/2020, totaling 30 specimens, deposited in the HUESBVC and HVC herbaria. Nineteen species were registered, distributed in nine genera: Brunfelsia (2 spp.), Capsicum (1 sp.), Cestrum (1 sp.), Datura (1 sp.), Iochroma (1 sp.), Nicandra (1 sp.), Nicotiana (1 sp.), Physalis (1 sp.) and Solanum (10 spp.). Among the species collected, five are endemic to Brazil and 11 were new records for the municipality. Our results demonstrate that Solanaceae is a family of high species richness in the municipality, contributing to the knowledge of the local flora. Recebido em: 08/07/2020 Aceito em: 15/12/2020 Editor responsável: Gleidson V. Marques (UFSB) eISSN: 2595-6752 Recebido em: 08/07/2020 Aceito em: 15/12/2020 A família Solanaceae Juss. no município de Vitória da Conquista, Bahia, Brasil The family Solanaceae Juss. in the municipality of Vitória da Conquista, Bahia, Brazil Jerlane Nascimento Moura1 & Claudenir Simões Caires 1 Jerlane Nascimento Moura1 & Claudenir Simões Caires 1 Resumo 1. Universidade Estadual do Sudoeste da Bahia, Departamento de Ciências Naturais, Vitória da Conquista, Bahia, Brasil Solanaceae é uma das maiores famílias de plantas vasculares, com 100 gêneros e ca. de 2.500 espécies, com distribuição subcosmopolita e maior diversidade na região Neotropical. Este trabalho realizou um levantamento florístico das espécies de Solanaceae no município de Vitória da Conquista, Bahia, em área ecotonal entre Caatinga e Mata Atlântica. Foram realizadas coletas semanais de agosto/2019 a março/2020, totalizando 30 espécimes, depositados nos herbários HUESBVC e HVC. Foram registradas 19 espécies, distribuídas em nove gêneros: Brunfelsia (2 spp.), Capsicum (1 sp.), Cestrum (1 sp.), Datura (1 sp.), Iochroma (1 sp.) Nicandra (1 sp.), Nicotiana (1 sp.), Physalis (1 sp.) e Solanum (10 spp.). Dentre as espécies coletadas, cinco são endêmicas para o Brasil e 11 foram novos registros para o município. Nossos resultados demonstram que Solanaceae é uma família de elevada riqueza de espécies no município, contribuindo para o conhecimento da flora local. Palavras-chave Solanales. Taxonomia. Florística. Nordeste. Keywords Solanales. Taxonomy. Floristics. Northeast. Palavras-chave Solanales. Taxonomia. Florística. Nordeste. Keywords Solanales. Taxonomy. Floristics. Northeast. Introdução Solanaceae Juss. é reconhecida dentro da ordem Solanales (APG, 2016) constituída com cerca de 2.500 espécies distribuídas em 100 gêneros, que apresentam grande diversidade morfológica e química (Olmstead et al., 2008; Olmstead, 2013). As principais características que definem a família são a forma de vida herbácea a lenhosa, inerme ou aculeada; folhas alternas, simples, inteiras a pinatissectas, sem estípulas; inflorescências cimosas, em fascículos ou condensadas, raro reduzidas a uma flor, comumente hermafroditas, actinomorfas ou zigomorfas, gamossépalas, Paubrasilia 2021;4:e0049 Moura & Caires 2 de 33 (2009), Giacomin (2010), Knapp et al. (2015a; 2015b) e Costa-Silva e Agra (2018) são relevantes para a compreensão da família e de representantes da mesma. Mundialmente falando, a obra mais robusta sobre Solanaceae diz respeito aos estudos de Särkinen et al. (2013). gamopétalas, estreladas, rotáceas, rotáceo-estreladas, tubulares, infundibuliformes ou hipocrateriformes, pequenas a grandes; anteras com deiscência longitudinal ou poricida, ovário súpero e frutos do tipo baga, drupa ou cápsula (Barboza et al., 2016; Giacomin; Gomes, 2018). Para o Nordeste do Brasil, poucos trabalhos envolvem a taxonomia da família Solanaceae, visto que se trata de uma das famílias mais representativas, com elevado número de espécies, tornando o trabalho florístico muito mais complexo. Dentre os estudos taxonômicos já realizados, a maioria refere-se a representantes e não à família em sua totalidade. Alguns dos trabalhos que fazem menção à região Nordeste referem-se aos estudos de Agra (1992, 1999a,b, 2006, 2009), Silva et al. (2002, 2003), Zappi et al. (2003), Agra et al. (2009), Moraes et al. (2009), Barbosa et al. (2011), Giacomin e Stehmann (2011) e Sampaio et al. (2019), em síntese, apresentando a taxonomia e a diversidade da família e de espécies do gênero Solanum. Além destas, outras obras, como estudos etnobotânicos (Moreira et al., 2002; Silva et al., 2012; Pereira et al., 2016; Silva et al., 2017), farmacológicos (Lopes et al., 2006; Santos et al., 2012; Santos et al., 2016) e de composição florística (Rodal; Sales, 2007; Amorim et al., 2009; Santos; Melo, 2010), evidenciam a importância econômica, comercial e a riqueza da família. Dentre estes, os trabalhos de Agra (1992, 1999a), Moreira et al. (2002), Silva et al. (2002, 2003), Zappi et al. (2003), Amorim et al. (2009), Moraes et al. (2009), Giacomin e Stehmann (2011) e Silva et al. (2012) englobam o estado da Bahia. É uma das famílias mais importantes economicamente, em virtude do potencial alimentício, medicinal e ornamental. Introdução Culturas como tomate (Solanum lycopersicum L.), batata (Solanum tuberosum L.), pimenta- malagueta (Capsicum frutescens L.) e berinjela (Solanum melongena L.), exibem uma extensa importância na economia mundial. Grande parte das espécies alimentares da família estão distribuídas entre os gêneros Solanum L., Capsicum L. e Physalis L. (Samuels, 2015). Muitas espécies apresentam potencial farmacológico de grande valia para a medicina – como Capsicum annum L., Capsicum frutescens L. (Al-Snafi, 2015); Nicandra physalodes (L.) Gaertn., Physalis angulata L. (Silva; Agra, 2005); Solanum nigrum L. (Giorgetti; Negri, 2011) e Solanum sisymbriifolium Lam. (More, 2019). Algumas espécies, como Datura stramonium L., são tóxicas, possuem princípios ativos que causam alterações fisiológicas, intoxicações em animais e humanos que podem levar à morte (Singh; Singh, 2013); outras, são utilizadas como plantas ornamentais – como Brunfelsia uniflora (Pohl.) D.Don. (Althaus-Ottmann et al., 2006); Capsicum annum L. (Stommel; Bosland, 2007); Nicotiana bonariensis Lehm. e Nicotiana forgetiana Hemsl. (Vignoli-Silva; Mentz, 2005). A distribuição da família Solanaceae é subcosmopolita, ocorrendo principalmente na região Neotropical, e está presente em todos os domínios fitogeográficos do Brasil, sendo mais expressiva na Mata Atlântica, na Amazônia e no Cerrado (Agra et al., 2009; Flora do Brasil, 2020), colonizando geralmente áreas abertas, como clareiras, bordas de estradas ou florestas. Muitas espécies são consideradas invasoras ou daninhas, como Solanum paniculatum L. e Nicandra physalodes, as quais apresentam comportamento ruderal e ocupam ambientes desde pastagens a terrenos baldios (Guaraná et al., 2011). Nesse contexto, o presente estudo vem apresentar um levantamento de espécies da família Solanaceae para o município de Vitória da Conquista, contribuindo para um melhor entendimento da família, gêneros e espécies que compõem a flora local. Inclui descrições, chave de identificação e pranchas fotográficas, tendo em vista que as informações nos bancos de dados mostram-se ainda bastante escassas para algumas regiões do Estado, incluindo sua porção sudoeste. Estudos apontam a América do Sul como centro de origem, endemismo e maior diversidade de Solanaceae (Hunziker, 2001; Agra et al., 2009). Estima-se que, para o continente, ocorram aproximadamente 50 gêneros endêmicos (Carvalho; Bovini, 2006). No Brasil, a família está representada por 36 gêneros e 504 espécies, das quais 237 são endêmicas. Na região Nordeste, ocorrem cerca de 21 gêneros e 189 espécies (Flora do Brasil, 2020). Dentre os gêneros que constituem a família, Solanum destaca-se por ser o mais expressivo, morfologicamente mais diversificado e economicamente importante (Sampaio et al., 2019). Área de estudo O município de Vitória da Conquista (Figura 1a–b) encontra-se na mesorregião Centro Sul Baiana e microrregião de Vitória da Conquista, tendo grande parte do município situada na região do Planalto da Conquista. Vitória da Conquista está a 509 km da capital baiana, Salvador, e ocupa a terceira posição em densidade populacional da Bahia. Encontra-se entre as coordenadas geográficas –40,83981ºS, – 14,84800ºW, ocupando 3.705,838 km² de extensão territorial e 923 metros de altitude média (IBGE, 2015). Segundo a tipologia climática definida por Köppen, Vitória da Conquista está sujeita a um clima subúmido a seco ou semiárido, sendo que, para o sul do município, predomina o clima úmido a subúmido (tipo Aw e o BSwh) (SEI, 1998), com temperaturas médias mensais ou anuais entre 20 ºC e 24 ºC, em média, e os índices pluviométricos são relativamente baixos, com média anual de precipitação equivalente a 733,9 mm (PMVC, 2019). Os principais tratamentos taxonômicos sobre as espécies brasileiras de Solanaceae referem-se aos trabalhos de Vellozo (1831), na Flora Fluminensis, Sendtner (1846), na Flora Brasiliensis, e Dunal (1852), em Prodromus Systematis Naturalis Regni Vegetabilis, de Alphonso de Candolle. Outras contribuições importantes foram dadas à família, como os estudos de Hunziker (2001), com uso de caracteres morfológicos, anatômicos, químicos e citológicos para a classificação das espécies, e Olmstead et al. (2008), o qual apresentou a revisão de 94% dos gêneros, com enfoque na filogenia molecular da família. No Brasil, trabalhos como Stehmann (1999), Agra (2007), Vignoli-Silva 3 de 33 Paubrasilia 2021;4:e0049 3 de 33 Moura & Caires Figura 1. Mapa de distribuição da família Solanaceae para o estado da Bahia (a) e para o município de Vitória da Conquista (b). Os dados de distribuição são oriundos dos herbários presentes no banco de dados do SpeciesLink no CRIA (2020). Figura 1. Mapa de distribuição da família Solanaceae para o estado da Bahia (a) e para o município de Vitória da Conquista (b oriundos dos herbários presentes no banco de dados do SpeciesLink no CRIA (2020). Todo material coletado foi fotografado em campo, analisado em laboratório com auxílio de microscópio estereoscópio e óptico, em seguida, foram elaboradas pranchas fotográficas das espécies. O relevo mostra-se como pouco acidentado e os biomas ocorrentes no município são representados pela Caatinga e Mata Atlântica (PMVC, 2019). A cobertura vegetal do município encontra-se bastante modificada, restando apenas fragmentos florestais (Maia, 2005). Área de estudo Destes, destacamos a Reserva Florestal do Poço Escuro, último remanescente de Mata Atlântica presente na cidade e que integra a Unidade de Conservação do Parque Municipal da Serra do Periperi. Outras duas áreas de proteção ambiental estão presentes no município: Parque Urbano Lagoa das Bateias e Parque Urbano da Lagoa do Jurema (PMVC, 2007). A identificação taxonômica foi baseada na literatura especializada, como Sendtner (1846), Knapp (2002), Silva et al. (2003), Agra et al. (2009), Moraes et al. (2009), Barboza et al. (2016), Giacomin e Gomes (2018) e Sampaio et al. (2019), e através de consultas aos acervos digitais dos herbários presentes no banco de dados do CRIA 2020 (Centro de referência e Informação Ambiental – https://splink.cria.org.br/) e na Flora do Brasil 2020 (http://floradobrasil.jbrj.gov.br/). Estes bancos de dados também foram consultados para informações sobre a distribuição das espécies, endemismo, hábito e novos registros. Coleta de dados e tratamento taxonômico Expedições e observações de campo foram realizadas semanalmente no município de Vitória da Conquista, entre os meses de agosto de 2019 e março de 2020, seguindo a metodologia de Filgueiras et al. (1994), em áreas de bordas, clareiras e interior de fragmentos florestais, bem como em locais antropizados. As amostras obtidas foram coletadas em estágio reprodutivo (flor e/ou fruto), herborizadas seguindo técnicas usuais (Mori et al., 1989), georreferenciadas e incorporadas aos acervos dos herbários HUESBVC e HVC. Dentre as áreas de estudo, as unidades de conservação também foram visitadas. A terminologia morfológica está de acordo com Jaeger (1985), Knapp (2002), Simpson (2010), Barboza et al. (2016) e Sampaio et al. (2019). As classificações simpodiais (unidades di, tri ou plurifoliadas), utilizadas para Solanum, seguiram o trabalho de Knapp (2002). Em Solanaceae, após o início da floração, o crescimento passa a ser simpodial, logo, cada gema apical é substituída por uma gema lateral de onde surgirá uma nova inflorescência e assim sucessivamente, sempre de cada axila da última folha. Dessa forma, todas as unidades diferenciadas em uma inflorescência fazem referência a uma unidade simpodial (Jaeger, 1985; Knapp, 2002). 4 de 33 4 de 33 Paubrasilia 2021;4:e0049 Moura & Caires Chave de identificação para as Solanaceae do município de Vitória da Conquista 1. Ervas, subarbustos, arbustos ou arvoretas armados ............................................................................................................................................................. 2 Ervas, subarbustos, arbustos ou arvoretas inermes ........................................................................................................................................................... 14 2. Unidade simpodial plurifoliada ................................................................................................................................................................................................ 3 Unidade simpodial difoliada ..................................................................................................................................................................................................... 7 3. Cálice fortemente armado e acrescente no fruto; epicarpo glabro, vermelho na maturidade ..................................................... Solanum sisymbriifolium Cálice inerme não acrescente; epicarpo pubescente a tomentoso, marrom escuro ou verde na maturidade ............................................................. 4 4. Face adaxial da folha glabra ou tomentosa, verde escura; face abaxial densamente tomentosa; tricomas estrelados, sésseis ou pediculados, indumento alvo; corola lilás ................................................................................................................................................................................................... 5 Face adaxial da folha velutina a tomentosa escabra, verde ferrugínea; face abaxial velutina a tomentosa; tricomas porrecto-estrelados; corola alva a creme .............................................................................................................................................................................................................................. 6 5. Face adaxial da folha glabra; face abaxial densamente tomentosa; tricomas estrelados, sésseis ou pediculados ......................... Solanum paniculatum Face adaxial da folha tomentosa, hirsuta; face abaxial tomentosa; tricomas glandulares e porrecto-estrelados ............................ Solanum megalonyx 6. Ramos não alados; acúleos retos; epicarpo piloso a pubescente ..................................................................................................... Solanum rhytidoandrum Ramos alados; acúleos recurvos; epicarpo tomentoso .............................................................................................................................. Solanum robustum 7. Corola estrelada, branca, esverdeada ou lilás ......................................................................................................................................................................... 8 Corola rotáceo-estrelada, esverdeada, branca, lilás ou púrpura ........................................................................................................................................ 11 8. Corola e anteras esverdeadas ......................................................................................................................................................................... Coleta de dados e tratamento taxonômico Solanum agrarium Corola branca ou lilás; anteras amarelas ou brancas ............................................................................................................................................................. 9 9. Caule e ramos tomentosos a esparso-pubescentes; tricomas glandulares e estrelados; corola lilás ........................................... Solanum palinacanthum Caule e ramos pilosos a pubescentes; tricomas simples, glandulares; corola branca a esbranquiçada ....................................................................... 10 10. Folhas pilosas; corola revoluta; epicarpo amarelo na maturidade ............................................................................................................. Solanum viarum Folhas pubescentes a esparso-pubescentes; corola não revoluta; epicarpo vermelho na maturidade .......................................... Solanum stenandrum 11. Corola 4,5–5,0 cm diâm., lilás ou púrpura; baga 5,0 × 4,7 cm, epicarpo não glanduloso ................................................................... Solanum crinitum Corola 1,7–2,0 cm diâm., esverdeadas a brancas; baga 0,6‒1,2 × 0,4‒1,1 cm, epicarpo glabro a glabrescente ....................................................... 12 12. Folhas geminadas; cálice armado .......................................................................................................................................................... Solanum asterophorum Folhas não geminadas; cálice inerme ................................................................................................................................................................................. 13 13. Cálice não ampliado no fruto; corola sem lacíneas reflexas; baga 1,2 × 1,1 cm ......................................................................... Solanum thomasiifolium Cálice ampliado no fruto; corola com lacíneas reflexas; baga 0,6 × 0,4 cm .......................................................................................... Solanum gardneri 14. Anteras com deiscência poricida ......................................................................................................................................................................................... 15 Anteras com deiscência longitudinal .................................................................................................................................................................................... 20 15. Folhas geminadas, dando aspecto de opostas; cálice acrescente no fruto ............................................................................................ Solanum didymum Folhas não geminadas, claramente alternas; cálice não acrescente .................................................................................................................................. 16 16. Baga pubescente, epicarpo verde-cinéreo quando jovem, marrom na maturidade; anteras poricidas sem fendas; com pseudoestípulas ......... 17 Baga glabra, epicarpo verde quando jovem, negro na maturidade; anteras com deiscência poricida ou terminando em fendas tardias; sem pseudoestípulas ..................................................................................................................................................................................................................... 18 17. Folhas grandes, 12‒30 × 5‒10 cm; ovário tomentoso .................................................................................................................................................... 19 Folhas médias, 4‒10 × 2‒3 cm; ovário glabro ...................................................................................................................................... Solanum stipulaceum 18. Inflorescências laterais às folhas; corola alva ........................................................................................................................................ Solanum americanum Inflorescência terminal; corola lilás a púrpura .......................................................................................................................................... Solanum flaccidum 19. Indumento congesto; venação conspícua através dos indumentos; lobos do cálice sem nervuras aparentes; corola não exerta .................................................................................................................................................................................................................... Solanum mauritianum Indumento flocoso; venação inconspícua através dos indumentos; lobos do cálice com nervuras aparentes; corola exerta ............................................................................................................................................................................................................. Solanum granulosoleprosum 20. Ervas ou arbustos .................................................................................................................................................................................................................. 21 Arvoretas ................................................................................................................................................................................................................................ 32 21. Corola com lóbulos intermediários; 2‒4 estames férteis ................................................................................................................................................. 22 Corola sem lóbulos intermediários; 5 estames férteis ....................................................................................................................................................... 23 22. Folha ovado-lanceolada a cordada; corola amarela ............................................................................................................................. Schwenckia molissima Folha linear-lanceolada a oblonga; corola esverdeada a violácea ..................................................................................................... Schwenckia americana 23. Cálice acrescente e persistente no fruto ............................................................................................................................................................................. 24 Cálice não acrescente no fruto, persistente ou decíduo .................................................................................................................................................. 26 24. Coleta de dados e tratamento taxonômico Caules e ramos cilíndricos; cálice livre na maior parte do comprimento; corola campanulada, lilases a alva; ovário tetralocular ........................................................................................................................................................................................................................Nicandra physalodes Caules e ramos angulosos; cálice fusionado na maior parte do comprimento; corola rotada-estrelada, amarela com mácula marrom; ovário bilocular .................................................................................................................................................................................................................................. 25 25. Caules em geral glabros; lâminas ovado-lanceoladas a oblongas; corola com máculas marrons ........................................................ Physalis angulata Caules sempre com tricomas; lâminas deltoides; corola com máculas vináceas ................................................................................. Physalis pubescens 26. Cálice circunciso; corola tubular, ovário espinescente .......................................................................................................................... Datura stramonium Cálice não circunciso; corola rotada ou hipocrateriforme; ovário não espinescente .................................................................................................. 27 27. Cálice truncado com cinco lacínias filiformes ......................................................................................................................................... Capsicum baccatum Cálice não truncado com lacínias triangulares (regulares ou irregulares) ..................................................................................................................... 28 Chave de identificação para as Solanaceae do município de Vitória da Conquista 1. Ervas, subarbustos, arbustos ou arvoretas armados ............................................................................................................................................................. 2 Ervas, subarbustos, arbustos ou arvoretas inermes ........................................................................................................................................................... 14 2. Unidade simpodial plurifoliada ................................................................................................................................................................................................ 3 Unidade simpodial difoliada ..................................................................................................................................................................................................... 7 3. Cálice fortemente armado e acrescente no fruto; epicarpo glabro, vermelho na maturidade ..................................................... Solanum sisymbriifolium Cálice inerme não acrescente; epicarpo pubescente a tomentoso, marrom escuro ou verde na maturidade ............................................................. 4 4. Face adaxial da folha glabra ou tomentosa, verde escura; face abaxial densamente tomentosa; tricomas estrelados, sésseis ou pediculados, indumento alvo; corola lilás ................................................................................................................................................................................................... 5 Face adaxial da folha velutina a tomentosa escabra, verde ferrugínea; face abaxial velutina a tomentosa; tricomas porrecto-estrelados; corola alva a creme .............................................................................................................................................................................................................................. 6 5. Face adaxial da folha glabra; face abaxial densamente tomentosa; tricomas estrelados, sésseis ou pediculados ......................... Solanum paniculatum Face adaxial da folha tomentosa, hirsuta; face abaxial tomentosa; tricomas glandulares e porrecto-estrelados ............................ Solanum megalonyx 6. Ramos não alados; acúleos retos; epicarpo piloso a pubescente ..................................................................................................... Solanum rhytidoandrum Ramos alados; acúleos recurvos; epicarpo tomentoso .............................................................................................................................. Solanum robustum 7. Corola estrelada, branca, esverdeada ou lilás ......................................................................................................................................................................... 8 Corola rotáceo-estrelada, esverdeada, branca, lilás ou púrpura ........................................................................................................................................ 11 8. Corola e anteras esverdeadas ......................................................................................................................................................................... Solanum agrarium Corola branca ou lilás; anteras amarelas ou brancas ............................................................................................................................................................. 9 9. Caule e ramos tomentosos a esparso-pubescentes; tricomas glandulares e estrelados; corola lilás ........................................... Solanum palinacanthum Caule e ramos pilosos a pubescentes; tricomas simples, glandulares; corola branca a esbranquiçada ....................................................................... 10 10. Folhas pilosas; corola revoluta; epicarpo amarelo na maturidade ............................................................................................................. Solanum viarum Folhas pubescentes a esparso-pubescentes; corola não revoluta; epicarpo vermelho na maturidade .......................................... Solanum stenandrum 11. Corola 4,5–5,0 cm diâm., lilás ou púrpura; baga 5,0 × 4,7 cm, epicarpo não glanduloso ................................................................... Coleta de dados e tratamento taxonômico Solanum crinitum Corola 1,7–2,0 cm diâm., esverdeadas a brancas; baga 0,6‒1,2 × 0,4‒1,1 cm, epicarpo glabro a glabrescente ....................................................... 12 12. Folhas geminadas; cálice armado .......................................................................................................................................................... Solanum asterophorum Folhas não geminadas; cálice inerme ................................................................................................................................................................................. 13 13. Cálice não ampliado no fruto; corola sem lacíneas reflexas; baga 1,2 × 1,1 cm ......................................................................... Solanum thomasiifolium Cálice ampliado no fruto; corola com lacíneas reflexas; baga 0,6 × 0,4 cm .......................................................................................... Solanum gardneri 14. Anteras com deiscência poricida ......................................................................................................................................................................................... 15 Anteras com deiscência longitudinal .................................................................................................................................................................................... 20 15. Folhas geminadas, dando aspecto de opostas; cálice acrescente no fruto ............................................................................................ Solanum didymum Folhas não geminadas, claramente alternas; cálice não acrescente .................................................................................................................................. 16 16. Baga pubescente, epicarpo verde-cinéreo quando jovem, marrom na maturidade; anteras poricidas sem fendas; com pseudoestípulas ......... 17 Baga glabra, epicarpo verde quando jovem, negro na maturidade; anteras com deiscência poricida ou terminando em fendas tardias; sem pseudoestípulas ..................................................................................................................................................................................................................... 18 17. Folhas grandes, 12‒30 × 5‒10 cm; ovário tomentoso .................................................................................................................................................... 19 Folhas médias, 4‒10 × 2‒3 cm; ovário glabro ...................................................................................................................................... Solanum stipulaceum 18. Inflorescências laterais às folhas; corola alva ........................................................................................................................................ Solanum americanum Inflorescência terminal; corola lilás a púrpura .......................................................................................................................................... Solanum flaccidum 19. Indumento congesto; venação conspícua através dos indumentos; lobos do cálice sem nervuras aparentes; corola não exerta .................................................................................................................................................................................................................... Solanum mauritianum Indumento flocoso; venação inconspícua através dos indumentos; lobos do cálice com nervuras aparentes; corola exerta ............................................................................................................................................................................................................. Solanum granulosoleprosum 20. Ervas ou arbustos .................................................................................................................................................................................................................. 21 Arvoretas ................................................................................................................................................................................................................................ 32 21. Corola com lóbulos intermediários; 2‒4 estames férteis ................................................................................................................................................. 22 Corola sem lóbulos intermediários; 5 estames férteis ....................................................................................................................................................... 23 22. Folha ovado-lanceolada a cordada; corola amarela ............................................................................................................................. Schwenckia molissima Folha linear-lanceolada a oblonga; corola esverdeada a violácea ..................................................................................................... Schwenckia americana 23. Cálice acrescente e persistente no fruto ............................................................................................................................................................................. 24 Cálice não acrescente no fruto, persistente ou decíduo .................................................................................................................................................. 26 24. Caules e ramos cilíndricos; cálice livre na maior parte do comprimento; corola campanulada, lilases a alva; ovário tetralocular ........................................................................................................................................................................................................................Nicandra physalodes Caules e ramos angulosos; cálice fusionado na maior parte do comprimento; corola rotada-estrelada, amarela com mácula marrom; ovário bilocular .................................................................................................................................................................................................................................. 25 25. Caules em geral glabros; lâminas ovado-lanceoladas a oblongas; corola com máculas marrons ........................................................ Physalis angulata Caules sempre com tricomas; lâminas deltoides; corola com máculas vináceas ................................................................................. Physalis pubescens 26. Cálice circunciso; corola tubular, ovário espinescente .......................................................................................................................... Datura stramonium Cálice não circunciso; corola rotada ou hipocrateriforme; ovário não espinescente .................................................................................................. 27 27. Cálice truncado com cinco lacínias filiformes ......................................................................................................................................... 1. Brunfelsia brasiliensis (Spreng.) L.B.Sm. & Downs, Fl. Ilustr. Catarin. [R. Reitz] 1966;1:303. Arbusto 15 cm alt., ereto, inerme. Caules e ramos cilíndricos, glabrescentes, pubescência amarelada, tricomas simples. Folhas simples, discolores, sub-coriáceas, lâmina 2,5–6,4 × 1,2–4,0 cm, oblonga- lanceolada, ápice agudo a acuminado, base atenuada, margem inteira, face adaxial pubescente ao longo da nervura, face abaxial glabrescente, ambas as faces com tricomas simples ou glandulares esparsos, diminutos, distribuídos na lâmina foliar; pecíolo 0,3–0,6 mm de compr., cilíndrico, puberulento. Inflorescência em cimeira, terminal, pluriflora. Botões florais não observados. Flores hermafroditas, pediceladas. Cálice campanulado, não anguloso, tubo 0,7–1,0 cm de compr., pubescente, lacínias triangulares. Corola alva a violeta, hipocrateriforme, tubo da corola 1,2–1,5 cm de compr., glabrescente, lobos arredondados. Anteras oblongas, deiscência longitudinal. Estigma capitado, ovário cônico. Frutos e sementes não observados. O gênero de maior riqueza foi Solanum, com 10 espécies. Os demais gêneros foram representados por uma única espécie, exceto Brunfelsia, que se apresentou com duas espécies. A maior representatividade de Solanum é similar aos resultados observados por Moraes et al. (2009), onde o gênero representou 58% do total das espécies de Solanaceae catalogadas. Em Alves et al. (2015), Solanum obteve maior representatividade, quando comparado aos 139 gêneros listados; nos estudos de Feliciano e Salimena (2011), das 26 espécies de solanáceas registradas, 19 eram de Solanum. Fenologia: coletada com flores em novembro. De acordo com os dados do CRIA (2020), existem 17 registros de Solanaceae para Vitória da Conquista. Contudo, um estudo sobre plantas ruderais no Planalto Conquistense (Soares-Filho et al., 2016) identificou a presença de mais sete espécies de Solanaceae (Tabela 1). Dessa forma, 11 das 19 espécies registradas no presente estudo foram novas ocorrências para o município, sendo elas: Brunfelsia obovata, Cestrum axillare Vell., Iochroma arborescens (L.) J.M.H.Shaw, Nicandra physalodes (L.) Gaertn., Nicotiana glauca Graham, Physalis angulata L., Solanum agrarium, S. crinitum Lam., S. rhytidoandrum Sendtn., S. sisymbriifolium Lam. e S. viarum Dunal. Material examinado – BRASIL, Bahia: Vitória da Conquista, estrada da Barra após a entrada do Alphaville, 19/XI/2019, Moura & Caires 15 (HUESBVC, HVC). Material examinado – BRASIL, Bahia: Vitória da Conquista, estrada da Barra após a entrada do Alphaville, 19/XI/2019, Moura & Caires 15 (HUESBVC, HVC). Brunfelsia brasiliensis é uma espécie endêmica do Brasil, distribuída pelas regiões Nordeste (BA, PI), Centro-Oeste (DF, GO) e Sudeste (ES), em domínios fitogeográficos do Cerrado e da Mata Atlântica (Flora do Brasil, 2020). Estudos evidenciam também a capacidade de adaptação da espécie na Caatinga (Carvalho et al., 2001). Coleta de dados e tratamento taxonômico Capsicum baccatum Cálice não truncado com lacínias triangulares (regulares ou irregulares) ..................................................................................................................... 28 Moura & Caires Paubrasilia 2021;4:e0049 5 de 33 28. Flores em fascículos axilares; lobos do cálice irregulares; corola rotado-cupular, alva com manchas esverdeadas internamente ........................................................................................................................................................................................................................... Athenaea velutina Flores em cimeiras não fasciculadas, axilares e/ou terminais; lobos do cálice regulares; corola hipocrateriforme, alternando de lilás a alva entre a antese e a senescência ....................................................................................................................................................................................................... 29 29. Cálice anguloso, glabro, inflado; lâminas obovadas ou oblongo-obovadas ................... ..................................................................... Brunfelsia obovata Cálice não anguloso com tricomas, não inflado; lâminas oblongas a lanceoladas ....................................................................................................... 30 30. Folhas lanceoladas; tricomas amarelos; 5 a muitas flores ................................................................................................................... Brunfelsia brasiliensis Folhas oblongas a oblongo-elípticas; tricomas alvos; 1‒11 flores ................................................................................................................................. 31 31. Pedúnculo 1‒6 mm de compr.; flores solitárias com tubo da corola entre 15‒25 mm de compr. .................................................. Brunfelsia uniflora Pedúnculo 11‒25 mm de compr.; várias flores com tubo da corola > 25 mm de compr. ............................................................. Brunfelsia pauciflora 32. Corola amarela; frutos secos ........................................................................................................................................................................... Nicotiana glauca Corola branca ou creme esverdeada; frutos carnosos ..................................................................................................................................................... 33 33. Inflorescência fasciculada a glomeruliforme; corola branca com manchas esverdeadas; frutos globosos, alaranjados ............. Iochroma arborescens Inflorescência em cimeiras, plurifloras, não glomeruliformes; corola branco-arroxeada a creme-esverdeada; frutos ovoides, alongados, roxo- nigrescente ......................................................................................................................................................................................................... Cestrum axilare 28. Flores em fascículos axilares; lobos do cálice irregulares; corola rotado-cupular, alva com manchas esverdeadas internamente ........................................................................................................................................................................................................................... Athenaea velutina Flores em cimeiras não fasciculadas, axilares e/ou terminais; lobos do cálice regulares; corola hipocrateriforme, alternando de lilás a alva entre a antese e a senescência ....................................................................................................................................................................................................... 29 29. Cálice anguloso, glabro, inflado; lâminas obovadas ou oblongo-obovadas ................... ..................................................................... Brunfelsia obovata Cálice não anguloso com tricomas, não inflado; lâminas oblongas a lanceoladas ....................................................................................................... 30 30. Folhas lanceoladas; tricomas amarelos; 5 a muitas flores ................................................................................................................... Brunfelsia brasiliensis Folhas oblongas a oblongo-elípticas; tricomas alvos; 1‒11 flores ................................................................................................................................. 31 31. Pedúnculo 1‒6 mm de compr.; flores solitárias com tubo da corola entre 15‒25 mm de compr. .................................................. Brunfelsia uniflora Pedúnculo 11‒25 mm de compr.; várias flores com tubo da corola > 25 mm de compr. ............................................................. Brunfelsia pauciflora 32. Corola amarela; frutos secos ........................................................................................................................................................................... Nicotiana glauca Corola branca ou creme esverdeada; frutos carnosos ..................................................................................................................................................... 33 33. Inflorescência fasciculada a glomeruliforme; corola branca com manchas esverdeadas; frutos globosos, alaranjados ............. Iochroma arborescens Inflorescência em cimeiras, plurifloras, não glomeruliformes; corola branco-arroxeada a creme-esverdeada; frutos ovoides, alongados, roxo- nigrescente ......................................................................................................................................................................................................... Cestrum axilare o CRIA (2020), S. Coleta de dados e tratamento taxonômico stipulaceum é a espécie com maiores amostragens, com sete registros, ao passo que Brunfelsia brasiliensis, Datura stramonium L. e Solanum thomasiifolium apresentam os menores valores – três, duas e duas coletas, respectivamente. Resultados e Discussão Foram registrados no presente estudo 19 espécies distribuídas em nove gêneros (Brunfelsia L., Capsicum L., Cestrum L., Datura L., Iochroma Benth., Nicandra Adans., Nicotiana L., Physalis L. e Solanum L.), pertencentes aos estratos herbáceo-subarbustivo e arbustivo-arbóreo em ambientes de mata ciliar, mata-de-cipó ou antropizados (por exemplo, terrenos baldios e beira de estradas), com maior predominância do hábito arbustivo. Dentre os 19 táxons, Brunfelsia brasiliensis (Spreng.) L.B.Sm. & Downs, B. obovata Benth., Solanum agrarium Sendtn., S. stipulaceum Willd. ex Roem. & Schult. e S. thomasiifolium Sendtn. são considerados endêmicos do Brasil (Flora do Brasil, 2020). 1. Brunfelsia brasiliensis (Spreng.) L.B.Sm. & Downs, Fl. Ilustr. Catarin. [R. Reitz] 1966;1:303. 1. Brunfelsia brasiliensis (Spreng.) L.B.Sm. & Downs, Fl. Ilustr. Catarin. [R. Reitz] 1966;1:303. Os acrônimos dos herbários seguem Thièrs (2020): ALCB = Herbário da Universidade Federal da Bahia, Campus de Ondina; CEPEC = Herbário do Centro de Pesquisas do Cacau; HUESBVC = Herbário da Universidade Estadual do Sudoeste da Bahia, Campus de Vitória da Conquista; HURB = Herbário da Universidade Federal do Recôncavo da Bahia; HVC = Herbário da Universidade Federal da Bahia, Campus Anísio Teixeira; MBM = Herbário do Museu Botânico Municipal de Curitiba; NY = Herbário de New York (USA); UB = Herbário da Universidade de Brasília; US = Herbário do Instituto Smithsonian (USA). Espécies (* novas ocorrências) Voucher (herbário) Endemismo Hábito Domínios Fitogeográficos Athenaea velutina (Sendtn.) D'Arcy1 Santos 2504 (CEPEC) EN Arbusto CAA, CER, MAT Brunfelsia brasiliensis (Spreng.) L.B.Sm. & Downs1 Moura & Caires 15 (HVC) EN Arbusto CER, MAT Brunfelsia obovata Benth.* Moura & Caires 21 (HVC) EN Arbusto CAA, CER, MAT Brunfelsia pauciflora (Cham. & Schltdl.) Benth.1 Azevedo 963 (HVC) EN Arbusto MAT Brunfelsia uniflora (Pohl) D.Don1 Santos 2511 (CEPEC) NEN Arbusto AMA, CAA, CER, MAT Capsicum baccatum L.2 Moura & Caires 25 (HVC) NEN Arbusto Todos os domínios Cestrum axillare Vell.* Moura & Caires 23 (HVC) NEN Árvore Exceto Pampa Datura stramonium L.1 Moura & Caires 24 (HVC) NEN Arbusto CAA, CER, MAT Iochroma arborescens (L.) J.M.H.Shaw* Moura & Caires 29 (HVC) NEN Árvore MAT Nicandra physalodes (L.) Gaertn.* Moura & Caires 10 (HVC) NEN Arbusto CAA, CER, MAT Nicotiana glauca Graham* Moura & Caires 16 (HVC) NEN Arbusto CAA, CER, MAT, PAM Physalis angulata L.* Moura & Caires 06 (HVC) NEN Erva Todos os domínios Physalis pubescens L.2 HUESBVC7610 NEN Erva Todos os domínios Schwenckia americana L.1,2 Santos 2484 (CEPEC) NEN Erva AMA, CAA, CER, MAT Schwenckia molissima Nees & Mart.1 Torrend s.n. (ALCB03728) NEN Subarbusto AMA, CAA, MAT Solanum agrarium Sendtn.* Moura & Caires 20 (HVC) EN Erva CAA, CER, MAT Solanum americanum Mill.2 Moura & Caires 07 (HVC) NEN Erva Todos os domínios Solanum asterophorum Mart.2 HUESBVC7692 EN Arbusto MAT Solanum crinitum Lam.* Moura & Caires 26 (HVC) NEN Arbusto AMA, CAA, CER, MAT Solanum didymum Dunal1 Mori 9476 (CEPEC) NEN Arbusto AMA, CER, MAT Solanum flaccidum Vell.1 Mori 9429 (NY) NEN Liana MAT, PAM Solanum gardneri Sendth.1 Barbosa et al. 251 (HURB) EN Arbusto CAA, MAT Solanum granulosoleprosum Dunal1 Carvalho et al. 1. Brunfelsia brasiliensis (Spreng.) L.B.Sm. & Downs, Fl. Ilustr. Catarin. [R. Reitz] 1966;1:303. Na Bahia, a espécie é pouco amostrada, com registros apenas para porções da região sudoeste e sul do Estado (CRIA, 2020). A espécie coloniza frequentemente clareiras e margens de estradas e possui potencial ecológico para sobreviver mesmo com o desmatamento de florestas onde estão inseridas (Plowman, 1998; Carvalho et al., 2001). Na área em estudo, a espécie foi encontrada em um terreno aberto. As espécies com melhores amostragens no estado da Bahia são Solanum stipulaceum, S. paniculatum L. e S. thomasiifolium (CRIA, 2020). Esses dados corroboram os resultados encontrados em campo, visto que essas espécies sempre se apresentavam em grandes concentrações e em diversos ambientes, com exceção de S. thomasiifolium, encontrado apenas em mata-de-cipó. Para Vitória da Conquista, ainda conforme 6 de 33 Paubrasilia 2021;4:e0049 Moura & Caires Tabela 1. Listagem das espécies de Solanaceae ocorrentes no município de Vitória da Conquista, Bahia, Brasil, complementadas com os dados do Species-Link do CRIA (2020)1 e do trabalho de Soares-Filho et al. (2016)2. Os dados de Endemismo (EN = Endêmica do Brasil; NEN =Não endêmica), Hábito e Domínios Fitogeográficos (AMA = Amazônia; CAA = Caatinga; CER = Cerrado; MAT = Mata Atlântica; PAM = Pampa) estão seguindo a Flora do Brasil 2020 em construção. Os acrônimos dos herbários seguem Thièrs (2020): ALCB = Herbário da Universidade Federal da Bahia, Campus de Ondina; CEPEC = Herbário do Centro de Pesquisas do Cacau; HUESBVC = Herbário da Universidade Estadual do Sudoeste da Bahia, Campus de Vitória da Conquista; HURB = Herbário da Universidade Federal do Recôncavo da Bahia; HVC = Herbário da Universidade Federal da Bahia, Campus Anísio Teixeira; MBM = Herbário do Museu Botânico Municipal de Curitiba; NY = Herbário de New York (USA); UB = Herbário da Universidade de Brasília; US = Herbário do Instituto Smithsonian (USA). Tabela 1. Listagem das espécies de Solanaceae ocorrentes no município de Vitória da Conquista, Bahia, Brasil, complementadas com os dados do Species-Link do CRIA (2020)1 e do trabalho de Soares-Filho et al. (2016)2. Os dados de Endemismo (EN = Endêmica do Brasil; NEN =Não endêmica), Hábito e Domínios Fitogeográficos (AMA = Amazônia; CAA = Caatinga; CER = Cerrado; MAT = Mata Atlântica; PAM = Pampa) estão seguindo a Flora do Brasil 2020 em construção. 1. Brunfelsia brasiliensis (Spreng.) L.B.Sm. & Downs, Fl. Ilustr. Catarin. [R. Reitz] 1966;1:303. 2598 (UB) NEN Árvore CER, MAT, PAM Solanum mauritianum Scop.1 Duarte 9299 (MBM) NEN Arbusto MAT, PAM Solanum megalonyx Sendtn.1 Schury 615 (US) EN Arbusto AMA, CAA, CER Solanum palinacanthum Dunal2 Moura & Caires 22 (HVC) NEN Arbusto Todos os domínios Solanum paniculatum L.2 Moura & Caires 28 (HVC) NEN Arbusto Exceto Pantanal Solanum rhytidoandrum Sendtn.* Moura & Caires 30 (HVC) NEN Arbusto AMA, CAA, CER Solanum robustum H.L.Wendl.1 Schury 618 (US) NEN Arbusto MAT Solanum sisymbriifolium Lam.* Moura & Caires 19 (HVC) NEN Subarbusto Todos os domínios Solanum stenandrum Sendtn.1 Mori 15045 (CEPEC) EN Subarbusto CAA, CER Solanum stipulaceum Willd. ex Roem. & Schult.1,2 Moura & Caires 01 (HVC) EN Arbusto CAA, CER, MAT Solanum thomasiifolium Sendtn.1,2 Moura & Caires 04 (HVC) EN Arbusto CAA, CER, MAT Solanum viarum Dunal* Moura & Caires 09 (HVC) NEN Arbusto Exceto Caatinga 2. Brunfelsia obovata Benth., Prodr. [A.P. de Candolle] 1846;10:199. Figura 2a–j. Anteras pardas, deiscência longitudinal. Estigma bífido, ovário cônico a ovoide. Frutos e sementes não observados. Anteras pardas, deiscência longitudinal. Estigma bífido, ovário cônico a ovoide. Frutos e sementes não observados. Fenologia: coletada com flores em fevereiro. Arbusto 30 cm alt., ereto, inerme. Caules e ramos cilíndricos, glabros. Folhas simples, discolores, subcoriáceas, lâmina 2,7–4,0 × 1,9–2,5 cm, obovada a elíptica, ápice agudo, base aguda, margem inteira, ciliada a puberulenta, face adaxial verde escuro, lisa, com nervura principal pubescente, com tricomas simples, face abaxial glabra, verde claro amarelado; pecíolo 0,2–0,5 mm de compr., cilíndrico, pubescente, com tricomas simples. Inflorescência cimeira terminal, pluriflora. Botões florais obovados. Flores hermafroditas, pediceladas, vistosas. Cálice tubular a campanulado, anguloso, tubo 0,8–1,1 cm de compr., inflado, verde claro, glabro, lacínias lanceoladas a triangulares, partida menos da metade do comprimento, com tricomas simples ou glandulares nas margens. Corola púrpura, passando a alva na senescência, hipocrateriforme, tubo da corola 1,0–1,7 cm de compr., lobos arredondados, revestido por tricomas esparsos glandulares, diminutos. Fenologia: coletada com flores em fevereiro. Material examinado – BRASIL, Bahia: Vitória da Conquista, matinha da Universidade Estadual do Sudoeste da Bahia, 04/II/2020, Moura & Caires 21 (HUESBVC, HVC). Brunfelsia obovata é uma espécie endêmica do Brasil, está distribuída na região Nordeste (BA, PI), Centro-Oeste (DF, GO) e Sudeste (MG, RJ, SP), em áreas dos domínios fitogeográficos da Caatinga, Cerrado e Mata Atlântica (Flora do Brasil, 2020). Na Bahia, a espécie é pouco amostrada; há registros somente para as regiões oeste, sudoeste e sul do Estado (CRIA, 2020). Habita ambientes úmidos (Plowman, 1998; Ruszczyk; Nascimento, 1999). Na área em estudo, a espécie foi encontrada em borda de um fragmento florestal de mata-de-cipó. 7 de 33 Paubrasilia 2021;4:e0049 Moura & Caires Figura 2. Brunfelsia obovata Benth. a: Hábito; b–c: Face adaxial e abaxial da folha; d: Detalhe da flor; e: Botões florais; f: Detalhe do botão e cálice; g: Corola em perfil; h: Antera com deiscência longitudinal; i: Tricomas glandulares no tubo da corola; j: Tricomas glandulares na borda do cálice. igura 2. Brunfelsia obovata Benth. a: Hábito; b–c: Face adaxial e abaxial da folha; d: Detalhe da flor; e: Botões florais; f: Detalhe do b Antera com deiscência longitudinal; i: Tricomas glandulares no tubo da corola; j: Tricomas glandulares na borda do cálice. Paubrasilia 2021;4:e0049 8 de 33 Moura & Caires triangulares a lanceolados. Anteras amarelo-ouro, deiscência longitudinal. Estigma capitado ou lobado, ovário subgloboso. 2. Brunfelsia obovata Benth., Prodr. [A.P. de Candolle] 1846;10:199. Figura 2a–j. Baga 1,4 × 0,8 cm, ovoide, alongada, glabra, verde quando jovem, roxo- nigrescente na maturidade, cálice frutífero não envolvendo o fruto. Sementes não observadas. 3. Capsicum baccatum L., Mant. Pl. 1767;1:47. Figura 3a–g. 3. Capsicum baccatum L., Mant. Pl. 1767;1:47. Figura 3a–g. Arbusto 40 cm alt., ereto, inerme. Caules e ramos cilíndricos, pubescentes, revestidos por tricomas simples. Folhas simples, solitárias, discolores, membranáceas, lâmina 3,6–5,0 × 2,5–3,2 cm, elíptica a ovada, base oblíqua, ápice cuspidado a acuminado, margem inteira, vilosa, ambas as faces glabras a pubescentes, com tricomas simples; pecíolo 0,7–1,2 cm de compr., cilíndrico. Inflorescência cimeira axilar, extra-axilar. Botões florais não observados. Flores hermafroditas, pediceladas. Cálice com borda truncada, lobos 0,4 × 0,3 mm, pubescentes, com tricomas simples, com cinco lacínias filiformes a aciculares, pubescentes. Corola 0,4–0,6 cm diâm., rotada, branca com manchas esverdeadas, pubescente, lacínias triangulares a agudas. Anteras amarelas, deiscência longitudinal. Ovário oblongo, estigma bilobado. Baga 0,6 × 1,0 cm, lisa, subglobosa a alongada, epicarpo alaranjado a vermelho na maturidade, cálice frutífero não envolvendo o fruto. Sementes numerosas, reniformes. Fenologia: coletada com flores e frutos maduros em fevereiro. Fenologia: coletada com flores e frutos maduros em fevereiro. Material examinado – BRASIL, Bahia: Vitória da Conquista, próximo à comunidade Santa Marta, na margem do rio Verruga, 20/II/2020, Moura & Caires 23 (HUESBVC, HVC). Cestrum axillare é uma espécie que apresenta ampla distribuição no Brasil, havendo registros para as regiões Norte (AC, AM), Nordeste (AL, BA, CE, MA, PB, PE, RN, SE), Centro-Oeste (DF, GO, MS, MT), Sudeste (ES, MG, RJ, SP) e Sul (PR, SC), abrangendo os domínios fitogeográficos da Mata Atlântica, Cerrado, Pantanal, Caatinga e Amazônia (Flora do Brasil, 2020). A espécie ocorre em especial nas regiões Sudeste e Centro-Oeste, em regiões litorâneas do Nordeste, como na Bahia, em Sergipe, Alagoas e Pernambuco. Para a Bahia, há uma maior distribuição para o centro-norte, centro-leste, leste, sul e porções da região sudoeste do Estado (CRIA, 2020). Fenologia: coletada com flores e frutos em fevereiro. Material examinado – BRASIL, Bahia: Vitória da Conquista, curral próximo ao colégio CETEP, localizado no campus da Universidade Estadual do Sudoeste da Bahia, 20/II/2020, Moura & Caires 25 (HUESBVC, HVC). Material examinado – BRASIL, Bahia: Vitória da Conquista, curral próximo ao colégio CETEP, localizado no campus da Universidade Estadual do Sudoeste da Bahia, 20/II/2020, Moura & Caires 25 (HUESBVC, HVC). É considerada uma planta hepatotóxica, portanto, causa necrose hepática em espécies bovinas. 5. Datura stramonium L., Sp. Pl. 1753;1:179. Figura 5a–h. 5. Datura stramonium L., Sp. Pl. 1753;1:179. Figura 5a–h. 5. Datura stramonium L., Sp. Pl. 1753;1:179. Figura 5a h. Arbusto 45 cm alt., ereto, inerme. Caules e ramos cilíndricos a levemente sulcados, glabros a pubescentes, com tricomas simples. Folhas simples, alternas, discolores, membranáceas a subcoriáceas, lâmina 7,6–12,6 × 5,0–6,9 cm, oval a elíptica, ápice acuminado, base oblíqua, margem dentada, face adaxial pubescente, com tricomas simples, nervuras bem desenvolvidas, ambas as faces pubescentes quando jovens, com tricomas esparsos simples, glabras na maturidade, face adaxial verde escura, face abaxial verde claro; pecíolo 0,8–1,8 cm de compr., glabro. Inflorescência solitária, extra-axilar. Botões florais ovoides a oblongos. Flores hermafroditas, pediceladas. Cálice 2,7 × 1,3 cm, tubular, anguloso, verde claro, circunciso, lacínias triangulares. Corola branca, tubular, tubo 5,0–7,5 cm de compr., com seis apêndices filiformes. Anteras alvas a cremes, deiscência longitudinal. Estigma globoso, ovário 0,3–0,2 mm, subgloboso, espinescente. Cápsula 4,1 × 3,3 cm, espinescente, epicarpo verde quando jovem e pardo na maturidade, cálice frutífero não envolvendo o fruto. Sementes não observadas. 4. Cestrum axillare Vell., Fl. Flumin. 1829;101. Icone 1827;3: t. 6. Figura 4a–h. 4. Cestrum axillare Vell., Fl. Flumin. 1829;101. Icone 1827;3: t. 6. Figura 4a–h. Arvoreta, 2,30 m alt., inerme. Caules e ramos glabros a glabrescentes, com tricomas esparsos simples, pseudoestípulas elípticas presentes. Folhas simples, alternas, glabras, levemente discolores, papiráceas, lâmina 4,4–8,5 × 2,2–3,5 cm, elíptico- lanceolada ou oblonga, ápice agudo, base obtuso-acunheada, margem inteira a ondulada, ambas as faces glabras, face adaxial verde, face abaxial verde claro; pecíolo 0,4–1,2 cm de compr., cilíndrico, glabro. Inflorescência em cimeiras, plurifloras, axilares ou terminais, panículas, pubescentes, tricomas simples. Botões florais não observados. Flores hermafroditas, sésseis ou apresentando um pedicelo curto. Cálice 0,6 × 0,3 mm, tubuloso, pubescente, revestido por tricomas simples, lobos triangulares a dentados, hipsofilo 0,7 × 0,3 mm. Corola branco-arroxeada a creme-esverdeada, hipocrateriforme, tubo 1,7–2,5 cm de compr., glabrescente, lobos Arvoreta, 2,30 m alt., inerme. Caules e ramos glabros a glabrescentes, com tricomas esparsos simples, pseudoestípulas elípticas presentes. Folhas simples, alternas, glabras, levemente discolores, papiráceas, lâmina 4,4–8,5 × 2,2–3,5 cm, elíptico- lanceolada ou oblonga, ápice agudo, base obtuso-acunheada, margem inteira a ondulada, ambas as faces glabras, face adaxial verde, face abaxial verde claro; pecíolo 0,4–1,2 cm de compr., cilíndrico, glabro. Inflorescência em cimeiras, plurifloras, axilares ou terminais, panículas, pubescentes, tricomas simples. Botões florais não observados. Flores hermafroditas, sésseis ou apresentando um pedicelo curto. 2. Brunfelsia obovata Benth., Prodr. [A.P. de Candolle] 1846;10:199. Figura 2a–j. No estado da Bahia é frequentemente utilizada para sombreamento nas plantações de cacau. Coloniza margens de estradas, bordas de fragmentos em que haja cursos de água e restingas das regiões tropicais do país (Marinho et al., 2018). Na área em estudo, a espécie foi encontrada na margem do rio Verruga em solo úmido. Capsicum baccatum é uma espécie que possui a América do Sul como centro de dispersão, é amplamente cultivada na Argentina, Colômbia, Equador, Peru, Bolívia e Brasil (Soares et al., 2008). No Brasil, a espécie ocorre no Nordeste (BA), Sudeste (ES, MG, RJ, SP), Sul (PR, RS, SC) e está presente nos domínios fitogeográficos do Cerrado, Mata Atlântica e Pantanal (Flora do Brasil, 2020). Na Bahia, a espécie é pouco amostrada, com alguns registros somente para o centro-norte, centro-leste e litoral sul do Estado (CRIA, 2020). É facilmente encontrada em clareiras, bordas de mata e locais antropizados. A espécie apresenta ampla importância econômica, visto que é cultivada em diversos lugares do mundo como uma especiaria ou hortaliça (Carvalho et al., 2006). Na área em estudo, a espécie foi encontrada em uma área de pastagem, em local úmido. 5. Datura stramonium L., Sp. Pl. 1753;1:179. Figura 5a–h. Cálice 0,6 × 0,3 mm, tubuloso, pubescente, revestido por tricomas simples, lobos triangulares a dentados, hipsofilo 0,7 × 0,3 mm. Corola branco-arroxeada a creme-esverdeada, hipocrateriforme, tubo 1,7–2,5 cm de compr., glabrescente, lobos Fenologia: coletada com flores e frutos imaturos em fevereiro e agosto. Material examinado – BRASIL, Bahia: Vitória da Conquista, curral próximo ao colégio CETEP, 20/XIII/2019, Moura & Caires 8 (HUESBVC, HVC); próximo à comunidade Santa Marta, 20/II/2020, Moura & Caires 24 (HUESBVC, HVC). Material examinado – BRASIL, Bahia: Vitória da Conquista, curral próximo ao colégio CETEP, 20/XIII/2019, Moura & Caires 8 (HUESBVC, HVC); próximo à comunidade Santa Marta, 20/II/2020, Moura & Caires 24 (HUESBVC, HVC). Moura & Caires Paubrasilia 2021;4:e0049 9 de 33 Figura 3. Capsicum baccatum L. a: Hábito. b–c: Face adaxial e abaxial da folha; d: Margem foliar ciliada; e: Detalhe da flor; f: Cálice truncado com lacínias filiformes; g: Frutos. Paubrasilia 2021;4:e0049 Moura & Caires 9 de 33 igura 3. Capsicum baccatum L. a: Hábito. b–c: Face adaxial e abaxial da folha; d: Margem foliar ciliada; e: Detalhe da flor; f: Cálice F Paubrasilia 2021;4:e0049 Moura & Caires 10 de 33 a 4. Cestrum axillare Vell. a: Hábito; b–c: Face adaxial e abaxial da folha; d: Detalhe da corola; e: Inflorescência; f: hipsofilo g: pseudoestípulas; h: Frutos madu Figura 4. Cestrum axillare Vell. a: Hábito; b–c: Face adaxial e abaxial da folha; d: Detalhe da corola; e: Inflorescência; f: hipsofilo g: pseudoestípulas; h: Frutos maduros. igura 4. Cestrum axillare Vell. a: Hábito; b–c: Face adaxial e abaxial da folha; d: Detalhe da corola; e: Inflorescência; f: hipsofilo g: pse Paubrasilia 2021;4:e0049 Moura & Caires 11 de 33 5. Datura stramonium L. a: Hábito; b–c: Face adaxial e abaxial da folha; d: Detalhe da corola; e: Botão floral; f: Secção transversal do ovário; g: Tricomas s adaxial da folha; h: Cápsula espinescente. Figura 5. Datura stramonium L. a: Hábito; b–c: Face adaxial e abaxial da folha; d: Detalhe da corola; e: Botão floral; f: Secção trans da face adaxial da folha; h: Cápsula espinescente. Paubrasilia 2021;4:e0049 Moura & Caires 12 de 33 Na Bahia, a espécie ainda é pouca amostrada, sua distribuição concentra-se mais ao sul do Estado (CRIA, 2020). Datura stramonium, assim como as demais espécies do gênero, é nativa das Américas, sendo o México seu principal centro de diversidade (Luna-Cavazos; Bye, 2011). 7. Nicandra physalodes (L.) Gaertn., Fruct. Sem. Pl. 1791;2:237, t. 131, f. 2. Figura 7a–g. Arbusto 45 cm alt., ereto, inerme. Caules e ramos sulcados, glabros a pubescentes, com tricomas simples e glandulares. Folhas simples, discolores, membranáceas, lâmina 5,5–12,8 × 4,3–8,7 cm, ovada a elíptica, ápice acuminado, base atenuada, margem dentada, ambas as faces com tricomas simples ou glandulares; pecíolo 1,2–3,4 cm de compr., cilíndrico, sulcado, glabro a pubescente. Inflorescência solitária, axilar, pedunculada. Botões florais não observados. Flores hermafroditas, pediceladas, pétalas soldadas na maior parte do seu comprimento. Cálice 1,8 × 1,6 cm, campanulado, lobos auriculados, ápice cuspidado, livre na maior do seu comprimento. Corola lilás a alva, campanulada, tubo 0,8–1,6 cm de compr., lacínias ovais. Anteras amarelo-pálidas, deltoides, deiscência longitudinal. Estigma capitado, ovário cônico ovoide, tetralocular. Baga 1,7 × 1,6 cm, globosa, coberta pelo cálice acrescente, epicarpo verde quando jovem, pardo a marrom na maturidade. Sementes não observadas. 6. Iochroma arborescens (L.) J.M.H. Shaw, Plantsman 2018;17(3): 200. Figura 6a–f. 6. Iochroma arborescens (L.) J.M.H. Shaw, Plantsman 2018;17(3): 200. Figura 6a–f. Arvoreta 2 m alt., ereta, inerme. Caules e ramos cilíndricos, glabrescentes. Folhas simples, discolores, lâmina 5,0–14,0 × 2,9–7,3 cm, membranácea, ovada a lanceolada, ápice agudo, base atenuada, margem inteira a ondulada, face adaxial, revestida por tricomas simples, em especial nas nervuras foliares, face abaxial, com tricomas simples; pecíolo 1,0–2,8 cm de compr., cilíndrico, pubescentes. Inflorescência pluriflora, fasciculada a glomeruliforme, com tricomas simples. Botões florais não observados. Flores pediceladas com tricomas simples, hermafroditas. Cálice campanulado, tubo 0,4–0,6 mm de compr., glabrescente com tricomas simples, lacínias triangulares a lanceoladas. Corola 2,1 × 0,6 cm, campanulada, alva a creme com manchas esverdeadas, pétalas soldadas na maior parte do seu comprimento, lobos revolutos. Anteras amarelas, oblongas, deiscência longitudinal. Estigma capitado, ovário ovoide. Baga 1,0 × 0,9 cm, globosa, epicarpo glabro, verde quando jovem, alaranjado na maturidade, cálice frutífero não acrescente. Sementes numerosas, discoides. Fenologia: Coletada com flores e frutos em agosto e setembro. Material examinado – BRASIL, Bahia: Vitória da Conquista, curral próximo ao colégio CETEP, localizado no campus da Universidade Estadual do Sudoeste da Bahia, 20/VIII/2019, Moura & Caires 5 (HUESBVC, HVC); Bairro Primavera, 14/IX/2019 Moura & Caires 10 (HUESBVC, HVC). Material examinado – BRASIL, Bahia: Vitória da Conquista, curral próximo ao colégio CETEP, localizado no campus da Universidade Estadual do Sudoeste da Bahia, 20/VIII/2019, Moura & Caires 5 (HUESBVC, HVC); Bairro Primavera, 14/IX/2019 Moura & Caires 10 (HUESBVC, HVC). 5. Datura stramonium L., Sp. Pl. 1753;1:179. Figura 5a–h. Atualmente é considerada cosmopolita após sua introdução pelos europeus em diversas partes do mundo (Luna-Cavazos; Bye, 2011; Al-Snafi, 2017). No Brasil, possui ocorrências nas regiões Nordeste (AL, BA, CE, PB, PE, PI, RN, SE), Centro-Oeste (DF, GO), Sudeste (SP) e Sul (RS, SC), em domínios fitogeográficos da Caatinga, Cerrado e Mata Atlântica (Flora do Brasil, 2020). Na Bahia, a espécie é pouco amostrada, com alguns registros para as regiões leste, centro-leste e sudoeste do Estado. Possui grande plasticidade de adaptação ao ambiente e, portanto, é considerada uma espécie muito importante para restauração de ambientes degradados, tendo em vista que atrai muitos polinizadores e dispersores devido às atrativas flores e aos vistosos frutos (Verçoza et al., 2012). Estudos farmacológicos apontam a existência de princípios ativos anticancerígenos na planta (Carvalho et al., 2001; Verçoza et al., 2012). Na área em estudo a espécie foi encontrada em margens de estradas, em fragmentos antropizados e em local próximo a curso de água. A espécie é conhecida popularmente como figueira-do-inferno ou cálice-de-vênus, sendo considerada uma solanácea invasora de caráter tóxico, em razão das substâncias constituintes com efeitos alucinógenos e eufóricos (Soni et al., 2012). Entretanto, na medicina popular, pode ser utilizada no tratamento de enfermidades como úlceras, feridas e inflamações (Soni et al., 2012). No presente estudo a espécie foi encontrada em uma área de pastagem em local úmido e também próximo a curso de água. 7. Nicandra physalodes (L.) Gaertn., Fruct. Sem. Pl. 1791;2:237, t. 131, f. 2. Figura 7a–g. 7. Nicandra physalodes (L.) Gaertn., Fruct. Sem. Pl. 1791;2:237, t. 131, f. 2. Figura 7a–g. Nicandra physalodes é originária da América do Sul, nativa do Peru e conhecida popularmente como joá-de-capote ou lanterna-da-china (Wang et al., 2017). No Brasil, a espécie se distribui pelas regiões Norte (PA), Nordeste (AL, BA, CE, PB, PE, SE), Centro-Oeste (DF, GO, MS), Sudeste (ES, MG, RJ, SP) e Sul (PR, SC), em domínios fitogeográficos da Caatinga, Cerrado e Mata Atlântica (Flora do Brasil, 2020). Na Bahia, a espécie é pouco amostrada, com registros apenas em regiões do centro-norte, leste e sul do Estado. Fenologia: Coletada com flores e frutos maduros em fevereiro e dezembro. Material examinado – BRASIL, Bahia: Vitória da Conquista, Avenida Iolando Fonseca, próximo ao Samu, 6/XII/2019, Moura & Caires 18 (HUESBVC, HVC); próximo ao Residencial Palmira Bittencourt – Bairro Candeias, 13/II/2020, Moura & Caires 29 (HUESBVC, HVC). Material examinado – BRASIL, Bahia: Vitória da Conquista, Avenida Iolando Fonseca, próximo ao Samu, 6/XII/2019, Moura & Caires 18 (HUESBVC, HVC); próximo ao Residencial Palmira Bittencourt – Bairro Candeias, 13/II/2020, Moura & Caires 29 (HUESBVC, HVC). Trata-se de uma espécie que frequentemente coloniza locais férteis e úmidos, incluindo áreas agrícolas, onde causa sombreamento, prejudicando algumas culturas. Possui potencial medicinal e estudos da medicina tradicional chinesa apontam diversas propriedades, como sedativas e expectorantes (Silva; Agra, 2005; Wang et al., 2017). Na área em estudo, a espécie foi encontrada em pastagem e em terrenos baldios. Iochroma arborescens pode ser encontrada no sul do México, na América Central, nas Antilhas e na América do Sul, em especial em margens de fragmentos florestais e clareiras (Verçoza et al., 2012). No Brasil, possui ocorrências nas regiões Sul (RS, SC), Sudeste (RJ, SP, MG) e Nordeste (BA, CE, PE) (Flora do Brasil, 2020), sendo popularmente conhecida como fruta-de-sabiá (Verçoza et al., 2012). Paubrasilia 2021;4:e0049 Moura & Caires Moura & Caires 13 de 33 Figura 6. Iochroma arborescens (L.) J.M.H.Shaw. a: Hábito; b: Face adaxial da folha; c: Inflorescências; d: Detalhe da flor; e: Antera com deiscência longitudinal; f: Frutos maduros. H.Shaw. a: Hábito; b: Face adaxial da folha; c: Inflorescências; d: Detalhe da flor; e: Antera com deiscência longitudinal; f: Frutos Figura 6. Iochroma arborescens (L.) J.M.H.Shaw. a: Hábito; b: Face adaxial da folha; c: Inflorescências; d: Detalhe da flor; e: Antera mad ros Paubrasilia 2021;4:e0049 Moura & Caires 14 de 33 Figura 7. Nicandra physalodes (L.) Gaertn. 7. Nicandra physalodes (L.) Gaertn., Fruct. Sem. Pl. 1791;2:237, t. 131, f. 2. Figura 7a–g. a: Hábito; b: Corola em vista frontal; c: Detalhe do cálice frutífero acrescente; d: Frut longitudinal; f: Cálice frutífero; g: Fruto maduro. Figura 7. Nicandra physalodes (L.) Gaertn. a: Hábito; b: Corola em vista frontal; c: Detalhe do cálice frutífero acrescente; d: Fruto imaturo; e: Anteras com deiscência longitudinal; f: Cálice frutífero; g: Fruto maduro. Paubrasilia 2021;4:e0049 15 de 33 Moura & Caires 8. Nicotiana glauca Graham, Bot. Mag. 1828;55(2/2): t. 2837. Figura 8a–e. uniflora. Botões florais não observados. Flores hermafroditas, com pedicelo cilíndrico, pubescente. Cálice 0,5 × 0,4 mm, campanulado, sépalas soldadas na maior parte do seu comprimento. Corola rotáceo- campanulada, amarelo-pálida, tubo 0,8–0,9 mm de compr., com máculas marrons no interior do tubo, lacínias triangulares. Anteras azuis, deiscência longitudinal. Estigma capitado, ovário subgloboso. Baga 1,6 × 1,5 cm, globosa envolvida pelo cálice acrescente e inflado, epicarpo verde quando jovem. Sementes não observadas. Arvoreta 5 m alt., ereta, inerme. Caules e ramos cilíndricos, glabros. Folhas simples, concolores, membranáceas a subcoriáceas, lâmina 5,5–7,7 × 3,1–4,2 cm, elíptica a lanceolada, ápice cuspidado, base obtusa, margem inteira, ambas as faces glabras, verde cinéreo; pecíolo, 1,1–3,2 cm de compr. Inflorescências terminais, plurifloras, glabras. Botões florais não observados. Flores pediceladas, hermafroditas. Cálice 1,5 × 0,7cm, campanulado a tubuloso, glabro, lobos acuminados. Corola amarelo-esverdeada, tubulosa, constrita na região apical, tubo 2,6–3,3 × 0,6–0,9 cm, pubescente, revestida por tricomas simples na face externa, lobos agudos; anteras pardas a amarelas, deiscência longitudinal. Estigma bilobado, ovário oval. Cápsula 2,0 × 1,3 cm, elipsoide, glabra, verde quando jovem, marrom na maturidade, cálice envolvendo o fruto. Sementes marrons, numerosas, sub-reniformes. Fenologia: Coletada com flores e frutos imaturos em agosto. Material examinado – BRASIL, Bahia: Vitória da Conquista, curral próximo ao colégio CETEP localizado no campus da Universidade Estadual do Sudoeste da Bahia, 20/VIII/2019, Moura & Caires 6 (HUESBVC, HVC). Physalis angulata é uma espécie que ocorre abundantemente na América Central, América do Sul e em parte da América do Norte, como nos Estados Unidos (Soares et al., 2009). No Brasil, ocorre em quase todos os estados, exceto em Roraima, Tocantins e Sergipe, assim como ocorre em todos os domínios fitogeográficos, exceto no Pampa (Flora do Brasil, 2020). Para a Bahia, há uma maior distribuição nas regiões centro-leste, leste e litoral sul baiano (CRIA, 2020). É considerada uma erva daninha que habita preferencialmente ambientes antropizados, ruderais e terrenos baldios. Fenologia: Coletada com flores e frutos em agosto e novembro Material examinado – BRASIL, Bahia: Vitória da Conquista, margem da estrada da Barra, próximo ao anel viário da avenida Rosa Cruz, 08/VIII/2019, Moura & Caires 2 (HUESBVC, HVC); fundo do condomínio Alphaville, próx. à guarita da Universidade Estadual do Sudoeste da Bahia, 19/XI/2019, Moura & Caires 16 (HUESBVC, HVC). Nicotiana glauca é uma espécie nativa da América do Sul e encontra- se distribuída em zonas áridas e semiáridas do mundo, como na Arábia Saudita, Chile, Estados Unidos e México (Fabricante et al., 2015). A espécie pode ser encontrada ainda na Austrália, Croácia, Espanha, Namíbia e Portugal (Silva et al., 2007; Fabricante et al., 2015). No Brasil, a espécie distribui-se pelas regiões Nordeste (AL, BA, CE, PB, PE, PI, RN, SE), Centro-Oeste (DF, GO, MS), Sudeste (ES, MG, RJ, SP) e Sul (PR, RS, SC), em domínios fitogeográficos da Caatinga, Cerrado e Mata Atlântica (Flora do Brasil, 2020). Na Bahia, a espécie está em maior abundância nas regiões norte, centro-norte e sudoeste do Estado (CRIA, 2020). 10. Solanum agrarium Sendtn., Fl. Bras. [Martius] 1846;10:69. Figura 9a–h. 10. Solanum agrarium Sendtn., Fl. Bras. [Martius] 1846;10:69. Figura 9a–h. 10. Solanum agrarium Sendtn., Fl. Bras. [Martius] 1846;10:69. Figura 9a–h. Erva ou subarbusto, ereta, 20 cm alt., aculeada. Caules e ramos cilíndricos, pubescentes, com tricomas simples, estrelados sésseis ou glandulares, acúleos 0,2–0,8 mm de compr., aciculares, unidade simpodial difoliada. Folhas simples, alternas, discolores, aculeadas, acúleos cônicos, membranáceas a coriáceas, lâmina 2,8–5,9 × 1,6–3,9 cm, ovado-elíptica, ápice agudo, base sub-cordada, margem lobada, face adaxial pubescente, tomentosa, com tricomas simples, glandulares, face abaxial com tricomas estrelados, sésseis; pecíolo 0,9–2,5 cm de compr., cilíndrico. Inflorescência em cimeira, extra- axilar. Botões florais ovoides. Flores hermafroditas, pediceladas. Cálice 0,5 × 0,4 mm, campanulado, piloso, levemente aculeado, acúleos cônicos a aciculares, lobos triangulares a agudos. Corola 1,7 × 2,3 cm de diâm., estrelada, esverdeada, lobos triangulares, revolutos. Anteras verdes, lanceoladas, deiscência longitudinal. Ovário globoso. Baga 1,8 × 1,3 cm, globosa, não envolvida pelo cálice frutífero, epicarpo verde claro, pubescente. Sementes não observadas. É considerada uma planta invasora, podendo ser encontrada em ambientes úmidos, ruderais e bordas de estradas. Apresenta toxicidade tanto para animais quanto para humanos, em virtude da presença de princípios ativos, como o alcaloide anabasina (Fabricante et al., 2015). Indivíduos de Nicotiana glauca possuem grande habilidade de produzir e dispersar propágulos, podendo variar de 10.000–1.000.000 de propágulos por indivíduo (Fabricante et al., 2015). Na área em estudo, a espécie foi encontrada em margens de estradas e em terrenos baldios, sempre em pequenas populações. 7. Nicandra physalodes (L.) Gaertn., Fruct. Sem. Pl. 1791;2:237, t. 131, f. 2. Figura 7a–g. Possui propriedades anti-inflamatória, anti-diabética, além de outras, razão pela qual é amplamente utilizada na medicina popular (Silva; Agra, 2005). Na área em estudo, a espécie foi encontrada em uma pastagem, em local úmido. Fenologia: Coletada com flores e frutos em agosto e novembro. 9. Physalis angulata L., Sp. Pl. 1753;1:183. 9. Physalis angulata L., Sp. Pl. 1753;1:183. Fenologia: Coleta com flores e frutos imaturos em janeiro. Erva 24 cm alt., ereta, inerme. Caules e ramos angulosos, glabros a pubescentes, com tricomas esparsos, simples. Folhas alternas, simples, discolores, membranáceas, lâmina 4,5–8,8 × 3,5–7,1 cm, ovada a elíptica, ápice acuminado, base oblíqua, margem dentada ou levemente lobada, ambas as faces com tricomas glabrescentes; pecíolo 0,3–3,5 cm de compr., anguloso. Inflorescência axilar, Material examinado – BRASIL, Bahia: Vitória da Conquista, Avenida Eduardo Campos, 23/I/2020, Moura & Caires 20 (HUESBVC, HVC). 16 de 33 Paubrasilia 2021;4:e0049 Moura & Caires ; igura 8. Nicotiana glauca Graham. a: Hábito; b. Inflorescência; c: Detalhe da flor; d: Antera com deiscência longitudinal; e: Frutos maduros. Figura 8. Nicotiana glauca Graham. a: Hábito; b. Inflorescência; c: Detalhe da flor; d: Antera com deiscência longitudinal; e: Fru Paubrasilia 2021;4:e0049 Moura & Caires 17 de 33 : Hábito; b–c: Face adaxial e abaxial da folha; d–e: Detalhe da flor e anteras; f: Inflorescência; g: Tricoma séssil estrelado da face igura 9. Solanum agrarium Sendtn. a: Hábito; b–c: Face adaxial e abaxial da folha; d–e: Detalhe da flor e anteras; f: Inflorescência; g baxial da folha; h: Fruto. Figura 9. Solanum agrarium Sendtn. a: Hábito; b–c: Face adaxial e abaxial da folha; d–e: Detalhe da flor e anteras; f: Inflorescência; g: Tricoma séssil estrelado da face abaxial da folha; h: Fruto. Paubrasilia 2021;4:e0049 Moura & Caires 18 de 33 curto e longo estipitado; unidade simpodial difoliada. Folhas simples, discolores, coriáceas, lâmina 7,0–16,6 × 6,5–12,4 cm, ovadas a elípticas, ápice agudo, base cordiforme a oblíqua, margem lobada, com acúleos cônicos ao longo da nervura principal, ambas as faces velutinas, tomentosas, com tricomas porrecto estrelados, sésseis, curto e longo estipitados, face abaxial mais tomentosa, tricomas porrecto- estrelados; pecíolo 2,0–3,5 cm de compr., cilíndrico. Inflorescência simples, escorpioide, extra-axilar, densamente tomentosa. Botões florais oblongos. Flores hermafroditas, pediceladas. Cálice 3,2 × 2,9 cm, campanulado, oblongo, densamente tomentoso, com tricomas estrelados, aculeado, lobos largo-lanceolados. Corola 4,5–5,0 cm de diâm., púrpura, rotácea, partida menos da metade do seu comprimento, lobos triangulares a deltoides. Anteras amarelas, lanceoladas, pilosas, deiscência poricida. Estigma capitado, ovário subgloboso. Baga 5,0 × 4,7 cm, globosa, epicarpo castanho, velutino, tomentoso, com tricomas porrecto-estrelados, cálice frutífero não acrescente. Sementes numerosas, ovadas. Solanum agrarium é uma espécie que pode ser encontrada na Colômbia, Venezuela, Antilhas Holandesas e Caribe, em ambientes quentes e secos (Agra et al., 2009). 9. Physalis angulata L., Sp. Pl. 1753;1:183. No Brasil, S. agrarium ocorre nas regiões Norte (TO), Centro-Oeste (GO) e Sudeste (MG) e em todos os estados da região Nordeste (Flora do Brasil, 2020). Para a Bahia, a espécie distribui-se em partes do norte, centro-norte, centro-leste e sudoeste do Estado (CRIA, 2020). Habita áreas antropizadas, restingas e bordas de florestas. Conforme estudos de Matias et al. (2019), a espécie apresenta metabólitos eficientes na atividade medicinal, podendo agir como diurético e antisséptico, atuando como enzimas reguladoras. Na área estudada, o espécime foi encontrado em uma margem de estrada, como o único exemplar do local. 11. Solanum americanum Mill., Gard. Dict. 1768; ed. 8: 5. Figura 10a–f. 11. Solanum americanum Mill., Gard. Dict. 1768; ed. 8: 5. Figura 10a–f. 11. Solanum americanum Mill., Gard. Dict. 1768; ed. 8: 5. Figura 10a–f. Erva ereta, 40 cm alt., inerme. Caules e ramos cilíndricos, glabros a pubescentes, com tricomas simples; unidade simpodial difoliada. Folhas simples, concolores, membranáceas, lâmina 3,7–6,9 × 2,9–4,5 cm, oval a elíptica, ápice agudo, base atenuada, ambas as faces glabras a pubescentes, com tricomas diminutos, simples esparsos; pecíolo 0,3–1,4 cm de compr., cilíndrico. Inflorescência simples, lateral, umbeliforme. Botões florais globosos a elipsoides. Flores hermafroditas, pediceladas. Cálice campanulado, tubo 0,1–0,2 mm de compr., lobos diminutos, agudos. Corola 0,5 × 0,4 cm, branca, pubescente, estrelada, partida mais da metade do seu comprimento, lobos deltoides a lanceolados. Anteras amarelas, oblongas, deiscência poricida abrindo-se em fendas. Estigma globoso, ovário subgloboso, pubescente. Baga 0,5 × 0,5 mm, globosa, não envolvida pelo cálice frutífero, epicarpo glabro, verde quando jovem e negro na maturidade. Sementes não observadas. Fenologia: Coletada com flores em novembro e frutos imaturos em fevereiro. Material examinado – BRASIL, Bahia: Vitória da Conquista, estrada da Barra após a entrada do condomínio Alphaville, 19/XI/2019, Moura & Caires 14 (HUESBVC, HVC); próximo ao condomínio Vogue Alegro, 13/II/2020, Moura & Caires 26 (HUESBVC, HVC). Material examinado – BRASIL, Bahia: Vitória da Conquista, estrada da Barra após a entrada do condomínio Alphaville, 19/XI/2019, Moura & Caires 14 (HUESBVC, HVC); próximo ao condomínio Vogue Alegro, 13/II/2020, Moura & Caires 26 (HUESBVC, HVC). Solanum crinitum é uma espécie restrita aos países da América do Sul, sendo registrada na Bolívia, Brasil, Colômbia, Equador, Guianas, Peru e Venezuela (Giacomin; Gomes, 2018). Para o Brasil, a espécie já foi amostrada para quase todos os estados, com exceção da região Sul (SC, RS, PR), Centro-Oeste (MS) e Nordeste (AL, RN) (Giacomin; Gomes, 2018; Flora do Brasil, 2020). Para a Bahia, a espécie é bem amostrada para o sul, centro-oeste, centro-sul e uma porção do oeste do Estado (CRIA, 2020). Habita preferencialmente ambientes secos, áreas de cerrado, restingas e campos rupestres (Agra et al., 2009; Sampaio et al., 2019). Os frutos são considerados tóxicos devido à presença de alcaloides e flavonoides de atividade citotóxica (Araújo et al., 2010). Na área em estudo, a espécie foi em encontrada em um terreno aberto e na borda de mata-de-cipó em regeneração. Fenologia: coletada com flores e frutos maduros em agosto. Material examinado – BRASIL, Bahia: Vitória da Conquista, curral próximo ao colégio CETEP, localizado na Universidade Estadual do Sudoeste da Bahia, 20/VIII/2019, Moura & Caires 7 (HUESBVC, HVC). 11. Solanum americanum Mill., Gard. Dict. 1768; ed. 8: 5. Figura 10a–f. Material examinado – BRASIL, Bahia: Vitória da Conquista, curral próximo ao colégio CETEP, localizado na Universidade Estadual do Sudoeste da Bahia, 20/VIII/2019, Moura & Caires 7 (HUESBVC, HVC). Solanum americanum possui ampla distribuição mundial, ocorre em toda a América, desde os EUA até a Argentina e o Chile (Giacomin; Gomes, 2018). No Brasil, possui distribuição ampla, ocorrendo em toda extensão do território brasileiro, bem como em todos os domínios fitogeográficos (Flora do Brasil, 2020). Para a Bahia, a espécie é bem amostrada para o litoral sul, leste e centro-leste do Estado (CRIA, 2020). É uma espécie frequente em ambientes úmidos, sombreados, fragmentos antropizados e clareiras (Moreira; Bragança, 2011). É considerada uma das principais espécie invasoras nas culturas comerciais no Brasil (Parreira et al., 2010). Na área em estudo, a espécie foi encontrada em uma pastagem, em local úmido. 13. Solanum palinacanthum Dunal, Prodr. [A.P. de Candolle] 1852;13(1):245. Figura 12a–h. 13. Solanum palinacanthum Dunal, Prodr. [A.P. de Candolle] 1852;13(1):245. Figura 12a–h. Arbusto 30 cm alt., ereta, fortemente aculeada, acúleos 0,3–0,8 cm de compr., aciculares arroxeados. Caules e ramos cilíndricos, tomentosos, revestidos com tricomas simples, glandulares e estrelados; unidade simpodial difoliada. Folhas simples, discolores, aculeadas, acúleos aciculares ao longo das nervuras, 0,4–1,3 cm de compr., coriáceas, lâmina 4,2–6,4 × 3,8–5,2 cm, ovada a cordada, ápice agudo, base cordiforme, margem lobada, face adaxial pubescente, com tricomas simples, glandulares, face abaxial com tricomas estrelados sésseis, glandulares; pecíolo 1,3–2,7 cm de compr., cilíndrico. Inflorescência simples, extra-axilar. Botões florais ovoides a oblongos. Flores hermafroditas, pediceladas. Cálice 12. Solanum crinitum Lam., Tab. Encycl. 1794;2:20. Figura 11a–h. Arbusto ca. 2,0 m alt., ereto, fortemente aculeado, acúleos 1,0–2,0 cm de compr., aciculares a cônicos. Caules e ramos tomentosos, com indumento ferrugíneo, apresentando tricomas porrecto-estrelados, Moura & Caires Paubrasilia 2021;4:e0049 19 de 33 Paubrasilia 2021;4:e0049 Moura & Caires 20 de 33 Hábito; b–c: Face adaxial e abaxial da folha; d: Detalhe da flor; e: Flor em vista lateral; f: Detalhe do ramo; g: Detalhe do cálice; a 11. Solanum crinitum Lam. a: Hábito; b–c: Face adaxial e abaxial da folha; d: Detalhe da flor; e: Flor em vista lateral; f: Detalhe d igura 11. Solanum crinitum Lam. a: Hábito; b–c: Face adaxial e abaxial da folha; d: Detalhe da flor; e: Flor em vista lateral; f: Detal Tricomas. ra 11. Solanum crinitum Lam. 11. Solanum americanum Mill., Gard. Dict. 1768; ed. 8: 5. Figura 10a–f. a: Hábito; b–c: Face adaxial e abaxial da folha; d: Detalhe da flor; e: Flor em vista lateral; f: Detalhe do ramo; g: Detalhe do cá icomas. Paubrasilia 2021;4:e0049 Moura & Caires 21 de 33 a 12. Solanum palinacanthum Dunal. a: Hábito; b–c: Face adaxial e abaxial da folha; d: Detalhe da flor; e–f: Fruto imaturo e maturo; g: Tricoma estrelado perfície abaxial da folha; h: Tricomas glandulares simples do caule. igura 12. Solanum palinacanthum Dunal. a: Hábito; b–c: Face adaxial e abaxial da folha; d: Detalhe da flor; e–f: Fruto imaturo e m a superfície abaxial da folha; h: Tricomas glandulares simples do caule. nal. a: Hábito; b–c: Face adaxial e abaxial da folha; d: Detalhe da flor; e–f: Fruto imaturo e maturo; g: Tricoma estrelado-séssil comas glandulares simples do caule. Figura 12. Solanum palinacanthum Dunal. a: Hábito; b–c: Face adaxial e abaxial da folha; d: Detalhe da flor; e–f: Fruto imaturo e maturo; g: Tricoma estrelado-séssil da superfície abaxial da folha; h: Tricomas glandulares simples do caule. Paubrasilia 2021;4:e0049 Moura & Caires 22 de 33 Solanum paniculatum é uma espécie nativa da América do Sul, encontrada no Brasil, Paraguai e Argentina (Nee, 1999; Mendonça; Lopes, 2019). No Brasil, está amplamente distribuída, ocorrendo em todos os estados, exceto no Amazonas, Acre, Roraima, Amapá e Tocantins (Flora do Brasil, 2020). Para a Bahia, a espécie é bem amostrada, com exceção da região norte, oeste, parte da região sudoeste e extremo sul do Estado (CRIA, 2020). 0,8 × 0,7 cm, campanulado, lacínias com ápice oval a agudo, tricomas simples e glandulares na face externa. Corola 2,3–2,5 cm de diâm., lilás, estrelada, lacínias lanceoladas, revolutas. Anteras amarelas, oblongas a lanceoladas, deiscência poricida. Estigma capitado, ovário ovoide, pubescente. Baga 2,7 × 2,3 cm, inerme, globosa, epicarpo verde claro, amarelo na maturidade, glabrescente, cálice não envolvendo fruto. Sementes não observadas. 0,8 × 0,7 cm, campanulado, lacínias com ápice oval a agudo, tricomas simples e glandulares na face externa. Corola 2,3–2,5 cm de diâm., lilás, estrelada, lacínias lanceoladas, revolutas. Anteras amarelas, oblongas a lanceoladas, deiscência poricida. Estigma capitado, ovário ovoide, pubescente. Baga 2,7 × 2,3 cm, inerme, globosa, epicarpo verde claro, amarelo na maturidade, glabrescente, cálice não envolvendo fruto. Sementes não observadas. 15. Solanum rhytidoandrum Sendtn., Fl. Bras. [Martius] 1846;10:85. Figura 14a–g. 15. Solanum rhytidoandrum Sendtn., Fl. Bras. [Martius] 1846;10:85. Figura 14a–g. Arbusto 1,53 m alt., ereto, aculeado, acúleos 0,4–0,6 cm de compr., cônicos. Caules e ramos cilíndricos densamente tomentos, com tricomas porrecto-estrelados, pediculados e sésseis; unidade simpodial plurifoliada. Folhas simples, discolores, cartáceas, lâmina 6,5–8,2 × 2,9–6,5 cm, ovada a elíptica, ápice agudo, base oblíqua, margem levemente lobada, face adaxial verde-ferrugíneo, velutina, tomentosa, com tricomas porrecto-estrelados sésseis e pediculados, face abaxial verde cinéreo, velutina, tricomas porrecto estrelados, acúleos 0,2–0,4 mm de compr., esparsos na nervura principal; indumento ferrugíneo; pecíolo 0,8–1,9 cm de compr., tomentoso, aculeado. Inflorescências ramificadas, terminais, plurifloras, tomentosas. Botões florais ovoides. Flores hermafroditas, pediceladas. Cálice 0,5 × 0,4 mm, campanulado, tomentoso, tricomas porrecto-estrelados, lobos lanceolados, ápice agudo. Corola 2,0–2,7 cm de diâm., alva a creme, estrelada, lobos lanceolados. Anteras amarelas, lanceoladas, deiscência poricida. Estigma capitado, ovário subgloboso, revestido por tricomas glandulares e simples. Baga 1,4 × 1,2 cm, globosa, inerme, epicarpo piloso a pubescente, com tricomas glandulares e estrelados, cálice frutífero não envolvendo o fruto. Sementes não observadas. 11. Solanum americanum Mill., Gard. Dict. 1768; ed. 8: 5. Figura 10a–f. A espécie possui grande capacidade de adaptação a ambientes hostis e facilmente coloniza áreas abertas, logo, pode ser comumente encontrada em rodovias, bordas de estradas, clareiras e em terrenos baldios, o que lhe confere caráter ruderal (Assunção et al., 2006). Assim como outras espécies da família, S. paniculatum apresenta propriedades medicinais e, portanto, tem despertado grande interesse das indústrias farmacêuticas e em estudos farmacobotânicos (Terço; Lima, 2016). Na área em estudo, a espécie foi encontrada em margens de estradas, sempre em grandes concentrações. Fenologia: Coletada com flores e frutos maduros em fevereiro e novembro. Material examinado – BRASIL, Bahia: Vitória da Conquista, avenida José Pedral Sampaio, 06/XI/2019, Moura & Caires 13 (HUESBVC, HVC); próximo à guarita da Universidade Estadual do Sudoeste da Bahia, 04/II/2020, Moura & Caires 22 (HUESBVC, HVC). Solanum palinacanthum é uma espécie restrita à América do Sul, ocorrente no Brasil, Bolívia, Paraguai e Argentina. O Brasil possui uma distribuição ampla da espécie, com exceção da região Norte, onde ainda não há registros para os estados do Acre, Amazonas, Roraima e Amapá (Agra et al., 2009; Flora do Brasil, 2020). Para a Bahia, há maior abundância da espécie no centro-oeste, leste, sul do Estado (CRIA, 2020). Habita pastos, terrenos baldios, bordas de estradas, terrenos úmidos, bem como, áreas degradadas (Pereira et al., 2008). Na área em estudo, a espécie foi coletada em margem de estradas e em área aberta. 14. Solanum paniculatum L., Sp. Pl. 1762;1:267. Figura 13a–g. 14. Solanum paniculatum L., Sp. Pl. 1762;1:267. Figura 13a–g. Arbusto 3 m alt., ereto, aculeado, acúleos 0,2–0,5 cm, cônicos. Caules e ramos cilíndricos, tomentosos, velutinos, com tricomas porrecto- estrelados, sésseis ou pedicelados; unidade simpodial plurifoliada. Folhas simples, discolores, cartáceas a subcoriáceas, lâmina 12,3–14,8 × 3,8–3,2 cm, inerme ou aculeada, elíptica a lanceolada, ápice acuminado a agudo, base oblíqua, margem lobada, face adaxial pubescente a glabrescente, verde-escuro, face abaxial densamente tomentosa com tricomas estrelados, sésseis ou pediculados, indumento alvo; pecíolo 1,1–3,5 cm de compr., cilíndrico, tomentoso, velutino. Inflorescências terminais, ramificadas, plurifloras, com eixos densamente pilosos. Botões florais ovoides. Flores hermafroditas, pediceladas. Cálice 0,4 × 0,3 mm, campanulado, tomentoso, com tricomas estrelados, lobos acuminados. Corola 2,1–2,4 cm de diâm., alva a lilás, rotáceo-estrelada, partida menos da metade do seu comprimento. Anteras amarelas, lanceoladas, deiscência poricida. Estigma capitado, ovário subgloboso, pubescente. Baga 1,2 × 1,1 cm, globosa, epicarpo glabro, verde com manchas verdes escuras, marrom a nigrescente na maturidade, cálice frutífero não envolvendo o fruto. Sementes não observadas. Fenologia: Coletada com flores e frutos imaturos em fevereiro, setembro e novembro. Fenologia: Coletada com flores e frutos imaturos em fevereiro, setembro e novembro. Material examinado – BRASIL, Bahia: Vitória da Conquista, próximo ao condomínio Morada Jardim Guanabara, 17/IX/2019, Moura & Caires 12 (HUESBVC, HVC); fundo do condomínio Alphaville, próx. à guarita da Universidade Estadual do Sudoeste da Bahia, 19/XI/2019, Moura & Caires 17 (HUESBVC, HVC); lateral do muro do Alphaville 2, 13/II/2020, Moura & Caires 30 (HUESBVC, HVC); próximo ao Residencial Palmira Bittencourt, 13/II/2020, Moura & Caires 27 (HUESBVC, HVC). Material examinado – BRASIL, Bahia: Vitória da Conquista, próximo ao condomínio Morada Jardim Guanabara, 17/IX/2019, Moura & Caires 12 (HUESBVC, HVC); fundo do condomínio Alphaville, próx. à guarita da Universidade Estadual do Sudoeste da Bahia, 19/XI/2019, Moura & Caires 17 (HUESBVC, HVC); lateral do muro do Alphaville 2, 13/II/2020, Moura & Caires 30 (HUESBVC, HVC); próximo ao Residencial Palmira Bittencourt, 13/II/2020, Moura & Caires 27 (HUESBVC, HVC). Fenologia: Coletada com flores e frutos em agosto e setembro. Material examinado – BRASIL, Bahia: Vitória da Conquista, margem da estrada da Barra, entre o anel da Avenida Luís Eduardo e a BR, 08/VIII/2019, Moura & Caires 3 (HUESBVC, HVC); Avenida Luís Eduardo Magalhães, 17/IX/2019, Moura & Caires 28 (HUESBVC, HVC); próximo ao condomínio Vila Grécia, 17/IX/2019, Moura & Caires 11 (HUESBVC, HVC). Paubrasilia 2021;4:e0049 Moura & Caires 23 de 33 ; igura 13. Solanum paniculatum L. 14. Solanum paniculatum L., Sp. Pl. 1762;1:267. Figura 13a–g. a: Hábito; b: Face adaxial da folha; c–d: Detalhes da flor; e: Anteras com deiscência poricida; f: Tricoma estrelado da superfíc baxial da folha; g: Frutos imaturos. igura 13. Solanum paniculatum L. a: Hábito; b: Face adaxial da folha; c–d: Detalhes da flor; e: Anteras com deiscência poricida; f baxial da folha; g: Frutos imaturos. baxial da folha; g: Frutos imaturos. Paubrasilia 2021;4:e0049 Moura & Caires 24 de 33 Figura 14. Solanum rhytidoandrum Sendtn. a: Hábito; b–c: Face adaxial e abaxial da folha; d: Inflorescência e ramo piloso; e: Deta da superfície adaxial da folha; g: Fruto imaturo. Figura 14. Solanum rhytidoandrum Sendtn. a: Hábito; b–c: Face adaxial e abaxial da folha; d: Inflorescência e ramo piloso; e: Detalhe da flor; f: Tricoma estrelado-séssil da superfície adaxial da folha; g: Fruto imaturo. da superfície adaxial da folha; g: Fruto imaturo. Paubrasilia 2021;4:e0049 25 de 33 Moura & Caires Solanum rhytidoandrum é uma espécie nativa da América do Sul, bem representada na Bolívia, Paraguai e Brasil, em áreas do semiárido (Agra et al., 2009). No Brasil, a espécie é distribuída nas regiões Norte (AM, PA, RO, TO), Nordeste (BA, CE, MA, PB, PE, PI, RN) e Centro- Oeste (DF, GO, MS, MT), nos domínios fitogeográficos da Amazônia, Caatinga e Cerrado (Flora do Brasil, 2020). Na Bahia, a espécie ainda é pouco amostrada (CRIA, 2020) e é encontrada frequentemente em ambientes secos, de restingas, brejos ou em matas secundárias (Agra et al., 2009). Na área em estudo, a espécie foi encontrada em áreas de regeneração e terrenos baldios. 17. Solanum stipulaceum Willd. ex Roem. & Schult., Syst. Veg. 1819;4:662. Figura 16a–h. Arbusto 1,55 m alt., ereto, inerme. Caules e ramos cilíndricos, estriados, densamente pilosos, cobertos com tricomas estrelados sésseis; unidade simpodial plurifoliada. Folhas simples, discolores, membranáceas a subcoriáceas, lâmina 4,5–10,5 × 2,4–3,6 cm, lanceolada, ápice acuminado a agudo, base atenuada, margem inteira, face adaxial verde escuro, moderadamente pilosas, com tricomas estrelados, face abaxial velutina, com tricomas estrelados; pecíolo 0,9–2,6 cm de compr., cilíndrico, associado a uma pseudoestípula auriculada. Inflorescências ramificadas, escorpioides, plurifloras, com muitas flores concentradas no ápice do ramo. Flores hermafroditas, pediceladas. Cálice 0,6 × 0,5 mm, campanulado, densamente coberto por tricomas na face abaxial, sépalas parcialmente soldadas. Corola 0,9–1,2 cm de diâm., lilás, estrelada, lobos triangulares, com muitos tricomas na face abaxial. Anteras amarelas, oblongas, deiscência poricida. Estigma globoso, ovário subgloboso, glabro. 16. Solanum sisymbriifolium Lam., Tabl. Encycl. 1794;2:25. Figura 15a–g. Subarbusto 65 cm alt., ereto, fortemente aculeado, acúleos 0,5–0,8 mm de compr., aciculares. Caules e ramos cilíndricos, pubescentes, revestidos por tricomas simples e glandulares, raramente estrelados; unidade simpodial plurifoliada. Folhas discolores, pinatissectas, membranáceas, lâmina 5,2–16,4 × 3,5–10,2 cm, ovada a oblonga, ápice agudo, base assimétrica, face adaxial revestida por tricomas simples, glandulares, raramente estrelados, face abaxial pilosa, revestida por tricomas estrelados, acúleos aciculares presentes; pecíolo 1,4–3,9 cm de compr., cilíndrico. Inflorescências simples, extra-axilares, escorpioides, pubescentes. Botão floral elipsoide. Flores hermafroditas, pediceladas. Cálice 1,3 × 1,2 cm, campanulado, fortemente armado, piloso, com tricomas glandulares e simples, lacínias triangulares a lanceoladas. Corola 2,1–2,2 cm de diâm., branca, rotáceo-estrelada, partida menos da metade do seu comprimento, lobos triangulares. Anteras amarelas, lanceoladas, deiscência poricida. Estigma capitado, ovário globoso, glabro. Baga 1,8 × 1,7 cm, epicarpo verde quando jovem, vermelho a negro na maturidade, glabro, cálice frutífero envolvendo o fruto, fortemente armado. Sementes não observadas. Fenologia: Coletada com flores e frutos imaturos em agosto. Material examinado – BRASIL, Bahia: Vitória da Conquista, em frente ao Módulo Amélia Barreto da Universidade Estadual do Sudoeste da Bahia, 01/VIII/2019, Moura & Caires 1 (HUESBVC, HVC). Material examinado – BRASIL, Bahia: Vitória da Conquista, em frente ao Módulo Amélia Barreto da Universidade Estadual do Sudoeste da Bahia, 01/VIII/2019, Moura & Caires 1 (HUESBVC, HVC). Solanum stipulaceum é uma espécie nativa e endêmica do Brasil, ocorre no Nordeste (AL, BA, CE, PB, PE, PI, SE), Centro Oeste (GO) e Sudeste (MG) e é frequentemente distribuída em áreas de matas secas, bordas de estradas, áreas antropizadas, terrenos baldios bem como, regiões montanhosas, áreas de Savana, Savana Estépica e Floresta Ombrófila (Agra et al., 2009; Sampaio et al., 2019). Na Bahia, a espécie é bem amostrada para todo o Estado (CRIA, 2020). Na área em estudo, a espécie foi coletada em área urbana, sendo comum em todos os fragmentos antropizados do município. Fenologia: Coletada com flores e frutos maduros em janeiro. Fenologia: Coletada com flores e frutos maduros em janeiro. Material examinado – BRASIL, Bahia: Vitória da Conquista, Avenida Eduardo Campos, 23/I/2020, Moura & Caires 19 (HUESBVC, HVC). 14. Solanum paniculatum L., Sp. Pl. 1762;1:267. Figura 13a–g. Baga 1,3 × 1,2 cm, globosa, inerme, pubescente, epicarpo verde cinéreo quando imaturo, cálice frutífero não envolvendo o fruto, apresentando tricomas esparsos. Sementes numerosas, marrons, sub-reniformes. 16. Solanum sisymbriifolium Lam., Tabl. Encycl. 1794;2:25. Figura 15a–g. 18. Solanum thomasiifolium Sendtn., Fl. Bras. [Martius] 1846;10:74. Figura 17a–g. 18. Solanum thomasiifolium Sendtn., Fl. Bras. [Martius] 1846;10:74. Figura 17a–g. Solanum sisymbriifolium é uma espécie nativa das regiões secas da América do Sul e atualmente encontra-se bastante distribuída nas regiões tropicais e subtropicais (Sampaio et al., 2019). Apesar de ser nativa da América do Sul, a espécie já foi introduzida na América do Norte, em alguns países dos continentes Europeu, Africano, Asiático e também para a região da Australásia (Srivastava et al., 2015). No Brasil, distribui-se pela região Norte (AC, RO), Nordeste (BA), Centro-Oeste (DF, GO, MS, MT), Sudeste (ES, MG, RJ, SP) e Sul (PR, RS, SC), abrangendo todos os domínios fitogeográficos (Flora do Brasil, 2020). Na Bahia, a espécie ainda é pouco amostrada (CRIA, 2020). Solanum sisymbriifolium possui propriedades medicinais, sendo utilizadas as raízes como diurético, analgésico e contraceptivo (Srivastava et al., 2015). Para a área em estudo, a espécie foi encontrada em margem de estrada em área antropizada. Arbusto 1,60 m alt., ereto, aculeado, acúleos 0,6–1,0 cm de compr., cônicos, ferrugíneos. Caules e ramos cilíndricos, pubescentes, velutinos, indumento ferrugíneo, revestido por tricomas glandulares estrelados, sésseis; unidade simpodial difoliada. Folhas simples, solitárias, subcoriáceas, lâmina 2,5–4,5 × 2,3–3,4 cm, ovada a elíptica, ápice agudo, base cordada, margem inteira a lobada, ambas as faces tomentosas, velutinas, revestidas por tricomas glandulares estrelados sésseis, acúleos esparsos ao longo da nervura principal; pecíolo 0,6– 1,5 cm de compr., cilíndrico, tomentoso. Inflorescências simples, extra-axilares, plurifloras, tomentosas. Flores hermafroditas, pediceladas. Cálice 1,1 × 1,0 cm, campanulado, sépalas partidas mais da metade do seu comprimento, lobos triangulares, ápice acuminado, Moura & Caires Paubrasilia 2021;4:e0049 26 de 33 Figura 15. Solanum sisymbriifolium Lam. a: Hábito; b: Detalhe da lâmina foliar; c: Detalhe da flor; d: Inflorescência; e: Cálice frutífero armado; f: Fruto maduro; g: Tricomas da lâmina foliar. Paubrasilia 2021;4:e0049 Moura & Caires 26 de 33 Figura 15. Solanum sisymbriifolium Lam. a: Hábito; b: Detalhe da lâmina foliar; c: Detalhe da flor; d: Inflorescência; e: Cálice frutífero armado; f: Fruto maduro; g: Tricomas da lâmina foliar igura 15. Solanum sisymbriifolium Lam. a: Hábito; b: Detalhe da lâmina foliar; c: Detalhe da flor; d: Inflorescência; e: Cálice frutíf ricomas da lâmina foliar. Paubrasilia 2021;4:e0049 Moura & Caires 27 de 33 ; 16. Solanum stipulaceum Willd. ex Roem. & Schult. 19. Solanum viarum Dunal, Prodr. [A.P. de Candolle] 1852;13:240. Figura 18a–f. 19. Solanum viarum Dunal, Prodr. [A.P. de Candolle] 1852;13:240. Figura 18a–f. Arbusto 30 cm alt., ereto, aculeado, acúleos 0,3–1,2 cm de compr., aciculares esverdeados. Caules e ramos cilíndricos, pilosos, com tricomas simples e glandulares; unidade simpodial difoliada. Folhas simples, solitárias ou geminadas, pilosas, cartáceas, lâmina 4,5–7,4 × 3,9–6,6 cm, ovada, ápice agudo, base cordada, margem lobada, lobos agudos, acúleos, 0,5–1,5 cm de compr., face adaxial pubescente, revestida por tricomas simples e glandulares, face abaxial tomentosa, com tricomas simples e estrelados; pecíolo 3,2–4,5 cm de compr., cilíndrico. Inflorescência simples, extra-axilar ou lateral, pubescente. Botões florais elipsoides. Flores hermafroditas, pediceladas. Cálice 0,8–0,7 mm de compr., campanulado, sépalas soldadas até metade do seu comprimento, lobos lanceolados, tricomas simples e glandulares. Corola 2,0–2,5 cm de diâm., branca, estrelada, lobos lanceolados, revolutos; anteras brancas, lanceoladas, deiscência poricida. Estigma capitado, ovário subgloboso. Baga 3,5 × 3,4 cm, globosa, epicarpo glabrescente, verde quando jovem e amarelo quando maturo, cálice frutífero não acrescente. Sementes não observadas. Arbusto 30 cm alt., ereto, aculeado, acúleos 0,3–1,2 cm de compr., aciculares esverdeados. Caules e ramos cilíndricos, pilosos, com tricomas simples e glandulares; unidade simpodial difoliada. Folhas simples, solitárias ou geminadas, pilosas, cartáceas, lâmina 4,5–7,4 × 3,9–6,6 cm, ovada, ápice agudo, base cordada, margem lobada, lobos agudos, acúleos, 0,5–1,5 cm de compr., face adaxial pubescente, revestida por tricomas simples e glandulares, face abaxial tomentosa, com tricomas simples e estrelados; pecíolo 3,2–4,5 cm de compr., cilíndrico. Inflorescência simples, extra-axilar ou lateral, pubescente. Conceitualização: CSC. Curadoria de dados: JNM. Análise formal: JNM. Aquisição de financiamento: CSC. Investigação: JNM, CSC. Metodologia: JNM, CSC. Administração do projeto: CSC. Recursos: CSC. Supervisão: CSC. Redação – rascunho original: JNM. Redação – revisão e edição: JNM, CSC. 18. Solanum thomasiifolium Sendtn., Fl. Bras. [Martius] 1846;10:74. Figura 17a–g. Baga 1,2 × 1,1 cm, globosa, inerme, epicarpo verde claro com manchas quando imaturo, glabrescente, revestido por tricomas glandulares estrelados, cálice frutífero não envolvendo o fruto. Sementes não observadas. Fenologia: Coletada com flores e frutos imaturos em agosto. Material examinado – BRASIL, Bahia: Vitória da Conquista, margem da estrada lateral da Universidade Estadual do Sudoeste da Bahia, próximo à guarita da Matinha, 08/VIII/2019, Moura & Caires 4 (HUESBVC, HVC). 18. Solanum thomasiifolium Sendtn., Fl. Bras. [Martius] 1846;10:74. Figura 17a–g. a: Hábito; b: Face adaxial e abaxial da folha; c: Detalhe da flor; d: Detalhe do cálice e pétalas; e: In la; f: Anteras com deiscência poricida; g: Pseudoestípula auriculada; h: Fruto imaturo. Figura 16. Solanum stipulaceum Willd. ex Roem. & Schult. a: Hábito; b: Face adaxial e abaxial da folha; c: Detalhe da flor; d: Det da corola; f: Anteras com deiscência poricida; g: Pseudoestípula auriculada; h: Fruto imaturo. Figura 16. Solanum stipulaceum Willd. ex Roem. & Schult. a: Hábito; b: Face adaxial e abaxial da folha; c: Detalhe da flor; d: Detalhe do cálice e pétalas; e: Indumento da corola; f: Anteras com deiscência poricida; g: Pseudoestípula auriculada; h: Fruto imaturo. Paubrasilia 2021;4:e0049 Moura & Caires 28 de 33 Figura 17. Solanum thomasiifolium Sendtn. a: Hábito; b–c: Detalhes da flor; d: Inflorescência e botões florais; e: Tricoma estrela folha; f: Anteras com deiscência poricida; g: Frutos imaturos. Figura 17. Solanum thomasiifolium Sendtn. a: Hábito; b c: Detalhes da flor; d: Inflorescência e botões florais; e: Tricoma estrelado glandular da superfície abaxial da folha; f: Anteras com deiscência poricida; g: Frutos imaturos. Paubrasilia 2021;4:e0049 Moura & Caires 29 de 33 (PR, RS, SC), nos domínios Amazônia, Mata Atlântica, Cerrado e Pantanal (Sampaio, 2013; Flora do Brasil, 2020). Na Bahia, a espécie está concentrada nas regiões sul e leste (CRIA, 2020). Solanum viarum é considerada uma erva daninha e coloniza áreas de lavouras anuais, pastagens, terrenos baldios e bordas de estradas. Desenvolve-se preferencialmente em ambientes úmidos e arenosos e seus frutos, quando maduros, são tóxicos (Mendonça; Lopes, 2019). Na área em estudo, a espécie foi encontrada em uma pastagem, em local úmido. (PR, RS, SC), nos domínios Amazônia, Mata Atlântica, Cerrado e Pantanal (Sampaio, 2013; Flora do Brasil, 2020). Na Bahia, a espécie está concentrada nas regiões sul e leste (CRIA, 2020). Solanum viarum é considerada uma erva daninha e coloniza áreas de lavouras anuais, pastagens, terrenos baldios e bordas de estradas. Desenvolve-se preferencialmente em ambientes úmidos e arenosos e seus frutos, quando maduros, são tóxicos (Mendonça; Lopes, 2019). Na área em estudo, a espécie foi encontrada em uma pastagem, em local úmido. revestido por tricomas glandulares. Corola 1,7 × 2,0 cm de diâm., branca ou lilás, rotada, pétalas soldadas na maior parte do seu comprimento, lobos agudos; anteras amarelas, lanceoladas, deiscência poricida. Estigma capitado, ovário subgloboso, pubescente. Financiamento Fenologia: Coletada com flores e frutos maduros em agosto. A Fundação de Amparo à Pesquisa do Estado da Bahia (FAPESB) concedeu bolsa de Iniciação Científica à primeira autora. Material examinado – BRASIL, Bahia: Vitória da Conquista, curral próximo ao CETEP, localizado na Universidade Estadual do Sudoeste da Bahia, 20/VIII/2019, Moura & Caires 9 (HUESBVC, HVC). Material examinado – BRASIL, Bahia: Vitória da Conquista, curral próximo ao CETEP, localizado na Universidade Estadual do Sudoeste da Bahia, 20/VIII/2019, Moura & Caires 9 (HUESBVC, HVC). Agradecimentos À Dra. Greta Aline Dettke, pelo tratamento das pranchas. À Dra. Maria Rosa Vargas Zanatta, pela confecção dos mapas de distribuição geográfica. À Universidade Estadual do Sudoeste da Bahia, pelo apoio logístico; Ao Laboratório de Botânica, pelo espaço e pelos equipamentos necessários para o desenvolvimento da pesquisa; e à Fundação de Amparo à Pesquisa do Estado da Bahia (FAPESB), pela concessão da bolsa de Iniciação Científica à primeira autora. Conclusão A família Solanaceae apresentou-se com grande diversidade no presente estudo (nove gêneros e 19 espécies), evidenciando não só a riqueza da área estudada, mas também a qualidade do ambiente, onde seu comportamento como pioneira, observado em espécies subespontâneas e ruderais, confere a essas plantas um papel fundamental na reestruturação das comunidades vegetais. Observou- se que os gêneros e as espécies foram encontrados em diferentes fisionomias vegetais, sendo o gênero Solanum o mais abundante, colonizando todos os ambientes analisados. Algumas espécies estiveram presentes só em ambientes mais áridos, como S. sisymbriifolium, enquanto outras desenvolveram-se em locais mais úmidos, como S. americanum. Nossos resultados demonstram a necessidade de preservação das variadas comunidades vegetais, com a elaboração de ações conservacionistas, visto que estas abrigam diversas formas de vida que auxiliam no processo de estruturação ecológica dos fragmentos florestais. Solanum thomasiifolium é uma espécie endêmica do Brasil e distribui- se pela região Nordeste (AL, BA, SE), Centro-Oeste (GO) e Sudeste (ES, MG), em domínios fitogeográficos da Caatinga, Cerrado e Mata Atlântica. Nessas regiões, espalha-se por áreas secas da Caatinga, Cerrado e Campos Rupestres (Agra, 2007; Flora do Brasil, 2020). Na Bahia, há uma maior distribuição da espécie no centro-norte, centro- leste, parte do Sudoeste e no litoral baiano (CRIA, 2020). Para a área em estudo, a espécie foi encontrada em margem de estrada, em ambiente seco com mata em regeneração. A Fundação de Amparo à Pesquisa do Estado da Bahia (FAPESB) concedeu bolsa de Iniciação Científica à primeira autora. Referências Disponibilidade dos Dados Referências Agra MF. A new species of Solanum section Micracantha (Solanaceae) from Northeastern Brazil. Novon 1992;2(3):179‒ 181. doi:10.2307/3391542 Agra MF. A new species of Solanum subgenus Leptostemonum (Solanaceae) from Chapada Diamantina, Bahia, Brazil. Novon 1999a;9(3):292‒295. doi:10.2307/3391723 Os dados integrais analisados durante o estudo atual estão apresentados no corpo do manuscrito e os espécimes serão depositados nos herbários HUESBVC e HVC. Agra MF. A new species of Solanum section Micracantha (Solanaceae) from Northeastern Brazil. Novon 1992;2(3):179‒ 181. doi:10.2307/3391542 Contribuições de Autoria Solanum viarum é uma espécie nativa do Brasil e Argentina, frequentemente encontrada em toda a América do Sul, México, Honduras, Caribe, África, Índia, Nepal (Nee, 1991). No Brasil, distribui-se pela região Norte (AC, RO), Nordeste (AL, BA, PB), Centro-Oeste (DF, GO, MS, MT), Sudeste (ES, MG, RJ, SP) e Sul Paubrasilia 2021;4:e0049 30 de 33 Moura & Caires Figura 18. Solanum viarum Dunal. a: Hábito; b: Face adaxial da folha; c–d: Detalhe da flor e das anteras; e: Indumento das folhas; f: Frutos maduros. Figura 18. Solanum viarum Dunal. a: Hábito; b: Face adaxial da folha; c–d: Detalhe da flor e das anteras; e: Indumento das folhas; f: F olanum viarum Dunal. a: Hábito; b: Face adaxial da folha; c–d: Detalhe da flor e das anteras; e: Indumento das folhas; f: Frutos maduros. Figura 18. Solanum viarum Dunal. a: Hábito; b: Face adaxial da folha; c–d: Detalhe da flor e das anteras; e: Indumento das folhas; f: Frutos maduros. Disponibilidade dos Dados Os dados integrais analisados durante o estudo atual estão apresentados no corpo do manuscrito e os espécimes serão depositados nos herbários HUESBVC e HVC. Conformidade Ética Não se aplica. Referências Agra MF. A new species of Solanum section Micracantha (Solanaceae) from Northeastern Brazil. Novon 1992;2(3):179‒ 181. doi:10.2307/3391542 Agra MF. A new species of Solanum subgenus Leptostemonum (Solanaceae) from Chapada Diamantina, Bahia, Brazil. Novon 1999a;9(3):292‒295. doi:10.2307/3391723 Referências Conformidade Ética Não se aplica. 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Pharmacological properties of Datura stramonium L. as a potential medicine tree: an overview. Asian Pacific Journal of Tropical Biomedicine 2012;2(12):1002- 1008 doi:10 1016/S2221 1691(13)60014 3 (ABNT) MOURA, J.N.; CAIRES, C.S. A família Solanaceae Juss. no município de Vitória da Conquista, Bahia, Brasil. Paubrasilia, Porto Seguro, v. 4, e0049, 2021. DOI: 10.33447/paubrasilia.2021.e0049 (ABNT) MOURA, J.N.; CAIRES, C.S. A família Solanaceae Juss. no município de Vitória da Conquista, Bahia, Brasil. Paubrasilia, Porto Seguro, v. 4, e0049, 2021. DOI: 10.33447/paubrasilia.2021.e0049 Como citar este artigo How to cite this article: Como citar este artigo How to cite this article: Stommel JR, Bosland PW. Ornamental pepper. In: Anderson NO, editors. Flower breeding and genetics. Springer; 2007. p. 561‒569. doi:10.1007/978-1-4020-4428-1_21 Sendtner O. Solanaceae et Cestrinneae. In: Martius CFP von, editor. Flora Brasiliensis. v. 6, pt. 10. Vindobonae et Lipsiae: Weinheim, J. Cramer 1846. p. 1‒338. Superintendência de estudos econômicos e sociais da Bahia – SEI. Físico-ambientais – Mapas estaduais [internet]. [Salvador]: SEI; 1998 [acesso em 25 out 2020]. Disponível em: https://www.sei.ba.gov.br/ Silva KN, Agra MF. Estudo farmacobotânico comparativo entre Nicandra physalodes e Physalis angulata (Solanaceae). Revista Brasileira Silva KN, Agra MF. Estudo farmacobotânico comparativo entre Nicandra physalodes e Physalis angulata (Solanaceae). Revista Brasileira de Farmacognosia 2005;15(4):344‒351. doi:10.1590/S0102- 695X2005000400016 J p 1008. doi:10.1016/S2221-1691(13)60014-3 Srivastava S, Dvivedi A, Shukla RP. Solanum sisymbriifolium Lam. (Solanaceae): a new invasive undershrub of the old-fields of northeastern Uttar Pradesh. CheckList 2015;11(3):1643. doi:10.15560/11.3.1643 (Vancouver) Moura JN, Caires CS. A família Solanaceae Juss. no município de Vitória da Conquista, Bahia, Brasil. Paubrasilia 2021;4:e0049. doi:10.33447/paubrasilia.2021.e0049 Stehmann JR. Estudos taxonômicos na tribo Nicotianeae G.Don (Solanaceae): revisão de Petunia Jussieu, das espécies brasileiras de Calibrachos La Llave & Lexarza e o estabelecimento do novo gênero Petuniopsis Stehmann & Semir [tese]. Campinas, SP: Universidade Estadual de Campinas; 1999.
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Citizen Science, Education, and Learning: Challenges and Opportunities
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Citizen Science, Education, and Learning: Challenges and Opportunities Joseph Roche 1*, Laura Bell 1, Cecília Galvão 2, Yaela N. Golumbic 3, Laure Kloetzer 4, Nieke Knoben 5, Mari Laakso 6, Julia Lorke 7, Greg Mannion 8, Luciano Massetti 9, Alice Mauchline 10, Kai Pata 11, Andy Ruck 12, Pavel Taraba 13 and Silvia Winter 14 1 Trinity College Dublin, Dublin, Ireland, 2 University of Lisbon, Lisbon, Portugal, 3 University of Sydney, Sydney, NSW, Australia, 4 University of Neuchâtel, Neuchâtel, Switzerland, 5 Naturalis Biodiversity Centre, Leiden, Netherlands, 6 Ministry of Agriculture and Forestry, Helsinki, Finland, 7 Wissenschaft im Dialog, Bürger Schaffen Wissen, Berlin, Germany, 8 University of Stirling, Stirling, United Kingdom, 9 Institute of Bioeconomy, Italian National Research Council, Rome, Italy, 10 University of Reading, Reading, United Kingdom, 11 Tallinn University, Tallinn, Estonia, 12 University of the Highlands and Islands, Perth, United Kingdom, 13 Tomas Bata University in Zlín, Zlín, Czechia, 14 Vienna University of Natural Resources and Life Sciences, Vienna, Austria Citizen science is a growing field of research and practice, generating new knowledge and understanding through the collaboration of citizens in scientific research. As the field expands, it is becoming increasingly important to consider its potential to foster education and learning opportunities. Although progress has been made to support learning in citizen science projects, as well as to facilitate citizen science in formal and informal learning environments, challenges still arise. This paper identifies a number of dilemmas facing the field—from competing scientific goals and learning outcomes, differing underlying ontologies and epistemologies, diverging communication strategies, to clashing values around advocacy and activism. Although such challenges can become barriers to the successful integration of citizen science into mainstream education systems, they also serve as signposts for possible synergies and opportunities. One of the key emerging recommendations is to align educational learning outcomes with citizen science project goals at the planning stage of the project using co-creation approaches to ensure issues of accessibility and inclusivity are paramount throughout the design and implementation of every project. Only then can citizen science realise its true potential to empower citizens to take ownership of their own science education and learning. Keywords: learning environments, teachers, ontology and epistemology, activism, science communication, public engagement Edited by: Minela Kerla, Consultant, Sarajevo, Bosnia and Herzegovina Edited by: Minela Kerla, Consultant, Sarajevo, Bosnia and Herzegovina Edited by: Minela Kerla, Consultant, Sarajevo, Bosnia and Herzegovina Reviewed by: Johan Buitendag, University of Pretoria, South Africa Wendy Purcell, Harvard University, United States *Correspondence: Joseph Roche Joseph.Roche@tcd.ie Reviewed by: Johan Buitendag, University of Pretoria, South Africa Wendy Purcell, Harvard University, United States *Correspondence: Joseph Roche Reviewed by: Johan Buitendag, University of Pretoria, South Africa Wendy Purcell, Harvard University, United States Reviewed by: Johan Buitendag, University of Pretoria, South Africa Wendy Purcell, Harvard University, United States Specialty section: This article was submitted to Sociological Theory, a section of the journal Frontiers in Sociology Received: 03 October 2020 Accepted: 09 November 2020 Published: 02 December 2020 Citation: Roche J, Bell L, Galvão C, Golumbic YN, Kloetzer L, Knoben N, Laakso M, Lorke J, Mannion G, Massetti L, Mauchline A, Pata K, Ruck A, Taraba P and Winter S (2020) Citizen Science, Education, and Learning: Challenges and Opportunities. Front. Sociol. 5:613814. doi: 10.3389/fsoc.2020.613814 Specialty section: This article was submitted to Sociological Theory, a section of the journal Frontiers in Sociology Received: 03 October 2020 Accepted: 09 November 2020 Published: 02 December 2020 Specialty section: This article was submitted to Sociological Theory, a section of the journal Frontiers in Sociology Received: 03 October 2020 Accepted: 09 November 2020 Published: 02 December 2020 CONCEPTUAL ANALYSIS published: 02 December 2020 doi: 10.3389/fsoc.2020.613814 INTRODUCTION Roche J, Bell L, Galvão C, Golumbic YN, Kloetzer L, Knoben N, Laakso M, Lorke J, Mannion G, Massetti L, Mauchline A, Pata K, Ruck A, Taraba P and Winter S (2020) Citizen Science, Education, and Learning: Challenges and Opportunities. Front. Sociol. 5:613814. doi: 10.3389/fsoc.2020.613814 Citizen science has long been considered to hold vast potential in the field of science education and learning (Bonney et al., 2009a). It is also a rapidly growing field of research in its own right, with increasing prominence in areas such as astronomy, ecology, meteorology, and medicine (Lewandowski et al., 2017). As the term “citizen science” applies to science that involves people who are not professional scientists, it occupies a unique position in the scientific community. As well as being its own distinct field of enquiry (Jordan et al., 2015), it can also reach beyond individual scientific disciplines Citizen science has long been considered to hold vast potential in the field of science education and learning (Bonney et al., 2009a). It is also a rapidly growing field of research in its own right, with increasing prominence in areas such as astronomy, ecology, meteorology, and medicine (Lewandowski et al., 2017). As the term “citizen science” applies to science that involves people who are not professional scientists, it occupies a unique position in the scientific community. As well as being its own distinct field of enquiry (Jordan et al., 2015), it can also reach beyond individual scientific disciplines December 2020 | Volume 5 | Article 613814 Frontiers in Sociology | www.frontiersin.org Citizen Science, Education, and Learning Roche et al. to attract wider public participation in scientific research, leading to the overall advancement of scientific knowledge (Bonney et al., 2009b). Citizen science has ample capacity for transdisciplinarity and for integrating natural, physical, and health sciences with the humanities and social sciences (Pykett et al., 2020; Tauginiene et al., 2020). It is an excellent method of harnessing non-traditional data sources to tackle societal challenges and contribute to certain Sustainable Development Goals of the United Nations (Fritz et al., 2019; Fraisl et al., 2020). opportunities for strengthening the relationship between citizen science and education must be identified so that appropriate support can be offered and integration achieved. INTRODUCTION Many questions have been raised regarding the role of citizen science within science education (Bonney et al., 2016); even the term “citizen science,” and the individual component words of that term can have different meanings subject to context (Eitzel et al., 2017). A report prepared by the US National Academies of Sciences, Engineering, and Medicine set out to tackle these issues—not simply by discussing the “potential of citizen science to support science learning” but by endeavouring to “identify promising practices and programs that exemplify the promising practices, and lay out a research agenda that can fill gaps in the current understanding of how citizen science can support science learning and enhance science education” (National Academies of Sciences, 2018, p. 2). A number of associations have been established world-wide, with the aim of bringing together people who are involved in citizen science. The most distinguished of these are the Citizen Science Association (ostensibly a US-based association, but offering global membership), the European Citizen Science Association, and the Australian Citizen Science Association. Each of these relatively new associations have highlighted education and learning as critical issues for citizen science as an emerging professional field (Storksdieck et al., 2016; Roche and Davis, 2017a). Citizen science has the capacity to “develop connections between students’ everyday lives and science so that they will have tangible reasons for continuing with the lifelong learning of science” (Jenkins, 2011, p. 501). It can function as a means of engaging the public with science on the scale of individual experiments, creating a unique position of combining participation, monitoring, and social change (Doyle et al., 2019; Dawson et al., 2020). Citizen science also offers a route by which the tenets of responsible research and innovation (Owen et al., 2012) may be fulfilled, particularly by facilitating lesser- heard communities in having their voices heard in relation to scientific policy-making and governance. This is now of more importance than ever, as researchers and academic experts find society’s trust in their authority diminished (Roche and Davis, 2017b), while the COVID-19 pandemic has demonstrated the acute need for public trust to be strengthened (Henderson et al., 2020). Indeed, citizen science might offer a more pressing model for science in a post-pandemic world (Provenzi and Barello, 2020). Despite its growing importance, citizen science is rarely considered in terms of science education research (Kelemen- Finan et al., 2018). INTRODUCTION For the purposes of this paper, the term “science” is taken to encompass systematic and evidence-based investigations in the pursuit of new knowledge, while the term “education” is considered to be the acquisition of knowledge through learning. Learning can be self-directed, but often relies on the guidance of a teacher. The learning can take place in either formal or informal environments and the methods of teaching, or pedagogy, can be as varied as the settings themselves. While these definitions are not all-inclusive, they provide a starting point where science, education, and learning can be considered in relation to the emerging challenges and opportunities stemming from citizen science. In Europe, efforts are also underway to identify the challenges and opportunities that may arise when citizen science and education are brought together through project collaboration, networks, research, and practice. Alongside the European Commission’s “Science with and for Society” work programme (European Commission, 2017), and the European Citizen Science Association, a COST Action (CA15212) was also established in order “to promote creativity, scientific literacy, and innovation throughout Europe” through citizen science (COST, 2016). This COST Action included a working group entitled “Develop synergies with education” and, through a dedicated workshop, brought together researchers and practitioners with a range of different backgrounds and contexts for interpreting citizen science in relation to education and learning. The subsequent discussions that emerged from the working group led to this paper, which provides an international perspective on some of the main challenges and opportunities facing citizen science in education. While the diversity of the working group ensured that a broad selection of perspectives were considered, it was by no means exhaustive. There are undoubtedly other arising challenges and opportunities that have not yet been considered, and it is hoped that this paper will serve as a starting point for developing a comprehensive research agenda for supporting citizen science in education and learning. CHALLENGES FOR CITIZEN SCIENCE, EDUCATION, AND LEARNING Every person who participates in citizen science is also involved in a learning process (Bela et al., 2016), not just the acquisition of the skills necessary for participation in citizen science, but also a deeper understanding of scientific concepts and processes—historically referred to as “scientific literacy” (Miller, 1983). The development of scientific literacy in tandem with the contribution to genuine scientific outcomes has been a longstanding goal of the field (Brossard et al., 2005; Jordan et al., 2011; Saunders et al., 2018). Logistical tensions tend to arise between citizen science and education due to unavoidable constraints concerning time, space, staff, and other key resources. While training could help address a number of these issues, the Supporting Citizen Science in Education and Learning In order to ensure that citizen science lives up to its vast potential to extend beyond individual projects and disciplines, December 2020 | Volume 5 | Article 613814 Frontiers in Sociology | www.frontiersin.org 2 Citizen Science, Education, and Learning Roche et al. while generating valuable data for the project at large. Both of these projects offer web tools for downloading data, as well as instructions for data analysis to empower participants to perform their own analysis. The construction of materials, the maintenance of an interactive website, smartphone apps, and continuous email contact requires considerable resources, especially in terms of staffwith relevant experience in science and education. More recent projects like the School of Ants (Lucky et al., 2014), LandSense (Olteanu-Raimond et al., 2018), and eMammal (Schuttler et al., 2019) have mirrored the success of these large-scale schools projects, while national schools-based citizen science projects in the future are likely to tackle aspects of post-pandemic life (Eichler et al., 2020; Ugolini et al., 2020). Smaller contributory projects sometimes lack such infrastructure and resources and, consequently, participants are often only involved in data collection without gaining experience of the complete inquiry process (Jenkins et al., 2015). Zoellick et al. (2012) argue that a third party, for example, a university, is a necessary intermediary between scientists and educators in order to ensure that specific research and educational outcomes are ultimately achieved. Their proposed model for school-based research projects describes scientists’ and educators’ inputs, their interactions during the design and implementation phase, and separate outputs and outcomes for students and scientists. While this model addresses the tension between collaborating scientists, schools, teachers, and students, it could be further improved with the added consideration of student input alongside outcomes for the educators (Jenkins, 2006). Co-constructed citizen science projects, where students are actively involved in the scientific process are labour and resource intensive for scientists, students, and teachers, but are more likely to achieve the scientific and educational goals of the project (Gray et al., 2012). associated costs often present a barrier, especially in fields where participant goodwill and volunteer work are crucial (Lorke et al., 2019). Many citizen science projects have little flexibility in terms of timing and the allocation of resources, and navigating these issues will invariably remain challenging for many citizen science coordinators and programme managers. Supporting Citizen Science in Education and Learning Beyond logistics, the goal of citizen science—to bring about scientific progress—and the goal of education—to support learning—may not necessarily always align. Citizen science can be integrated into education in both formal and informal learning environments. Formal learning generally occurs in school, college, or university environments with clear learning objectives, whereas informal education can take place outside of the classroom or after school, often in public engagement spaces like museums, zoos, or aquariums (Eshach, 2007; National Research Council, 2009). Each environment raises unique challenges for practitioners. Challenges may also arise as a result of the different needs of the scientists, students, teachers, educators, researchers, and other actors involved; the issue of how information is communicated and shared; as well as from potential conflict between the capacity of citizen science for activism and the desire or obligation to reach specific learning objectives. Citizen Science and Education in Formal Learning Environments Specific learning objectives, background information, and lesson plans are generally utilised by educators to integrate citizen science projects into curricula in formal learning environments, particularly when teaching children and adolescents at primary and post-primary level (Bonney et al., 2009b, 2014). Consequently, project engagement becomes contingent on the educators themselves; as the students or learners may have been effectively volunteered to participate, rather than electing to do so, motivation and engagement may be lacking compared to other groups of citizen science participants. Therefore teachers, as the citizen science intermediaries in formal learning environments (Weinstein, 2012), play a crucial role in successfully integrating such projects into their classrooms and schools. That some teachers may lack confidence in their own general level of scientific content knowledge and scientific literacy can considerably impede this process—for example, issues of content knowledge could arise on projects that require teachers to explore outdoor environments where they cannot fulfil the perceived demand to be an expert (Kelemen-Finan and Dedova, 2014; Jenkins et al., 2015). The participation of schools can also be constrained by school curricula, timetables, or logistical issues. For those teachers and schools that are interested in engaging in citizen science projects, it may be difficult to navigate the rapidly growing number of initiatives available to them. Frontiers in Sociology | www.frontiersin.org Citizen Science and Education in Informal Learning Environments Informal education generally refers to the learning that takes places outside of classrooms and lecture theatres. Informal environments may sometimes be further subdivided into non- formal and semiformal categories (Werquin, 2007), but for the purpose of this paper, all learning environments outside of those involving schools, higher education, or universities, can be considered informal. Informal learning environments, such as science centres and museums, are critical to science education. Citizen science projects find a natural home in these domains due to a shared strong commitment to public engagement (Dickinson et al., 2012; Ballard et al., 2017). The impact that citizen science projects can have on education in these environments is affected by the same challenge that faces informal learning environments in general—finding the best way to support learners and facilitators (Stewart and Jordan, 2017). Tension may arise between designing projects that are “fun” for casual participants and ensuring that data generated is of sufficient quality. The use of “fun” activities can increase participation, create interest in a given research topic, and nurture a love of science—particularly in projects involving young people (Kountoupes and Oberhauser, 2008). However, Additional challenges stem from the type of classroom involvement that can be facilitated. Projects such as the Monarch Larva Monitoring Project (Kountoupes and Oberhauser, 2008) or Classroom FeederWatch (Bonney and Dhondt, 1997) are considered examples of best practice from the last two decades, where materials are provided for local school involvement, December 2020 | Volume 5 | Article 613814 3 Citizen Science, Education, and Learning Roche et al. aligned to the UNESCO Incheon Declaration and Framework for Action (UNESCO, 2015) which seeks to ensure inclusive, equitable, and quality education on a global scale. It encompasses three distinct dimensions: a citizen’s legal citizenship, socio- economic background, and socio-cultural background (Kalekin- Fishman et al., 2007). Legal citizenship enables an individual to channel their political agency, although, as highlighted by Eitzel et al. (2017), the definition of citizenship is complex and can be problematic in some contexts. Socio-economic power can create demand for education, transforming learning into a desirable consumer commodity and potentially creating resources that can supplement underfunded or overlooked government services. The socio-cultural dimension of active citizenship focuses on ethics, and seeks to foster cohesion, inclusion, and tolerance in the personal and public spheres. Citizen Science and Education in Informal Learning Environments Citizen science practice could be exercised as one means of educating active citizens; by empowering communities to advocate for their local environment through research, or by enabling citizens to gather evidence on, and articulate, pressing issues. The results of active citizenship, often shared with the wider public through social media, can even hasten the actions of decision-makers (Eitzel et al., 2017). there may be a trade-offregarding the time and resources necessary to make these activities engaging, and the efforts to serve the scientific and educational goals of the project. A report by the US Committee on Learning Science in Informal Environments in 2009 found that although tensions often arise between the “reasonable goals for learning science in informal environments” and the education “agenda,” it was deemed ‘unproductive to blindly adopt either purely academic goals or purely subjective learning goals’ in informal learning settings (Bell et al., 2009, p. 3). The learning that takes place in informal environments through citizen science projects can be difficult to capture. Initial efforts have been undertaken to find ways to evaluate the intended learning outcomes for the participants in these projects (Phillips et al., 2014, 2018), but Edwards (2014) has highlighted that the specific impact that citizen science can have on the lifelong learning of people outside the classroom has not yet been comprehensively explored. Likewise, while understanding social and cultural capital is critical to interpreting how people engage with informal science education institutions (Dawson, 2014), there has not yet been enough consideration given to how this capital affects participation in citizen science projects and the resulting issues of equity that may emerge (Birmingham, 2016). Citizen science has the same issues of inequity that are endemic throughout society, with innate barriers to participation for minorities and underserved communities (Soleri et al., 2016; Fiske et al., 2019). Science capital—a concept that explores how a person’s environment and social class can affect their involvement in science—could allow “inequalities in science participation” to be discovered more readily, which in turn could be used to promote “social justice within science education” (Archer et al., 2015, p. 943). Citizen Science and Education in Informal Learning Environments If citizen science is to fulfil its potential in improving equity of access to, and participation in, both science and science education in informal learning environments, then “the extent to which citizen science can build science capital and enable wider engagement with science-related issues [...] deserves further experimentation and investigation” (Edwards et al., 2018, p. 390). However, despite the benefits of potentially bolstering science education through active citizenship, tension may arise between the traditional role of the learner in some learning environments, acquiring pre-determined knowledge and values, and the process of learning continuously through active citizenship, which may result in social transformation. Educators may feel uncomfortable in sharing decision-making power with other participants in citizen-led activities and may feel uncertainty as to the value of that learning process (Mueller and Tippins, 2015). In citizen science activities, practitioners, and participants may not be able to retain their usual roles in some learning environments (Fazio and Karrow, 2015) and significant changes may need to be made in order to enable and facilitate social activism. Frontiers in Sociology | www.frontiersin.org Dissemination, Dialogue, and Participatory Communication Citizen science projects often aim not only to advance scientific knowledge, but to share it too. The manner in which communication takes place in these projects, and the effect it has on learning, must tread the line between outreach and engagement, and warrants a communication plan that not only connects with the right audiences but retains their interest over time (Veeckman et al., 2019). Projects tend to adopt either a two- way approach that emphasises participatory dialogue (McCallie et al., 2009; Haywood and Besley, 2013), or a one-way approach that focuses on outreach and dissemination. Two-way communication between citizens and scientists within projects leads to the sharing of ideas, information, and knowledge, while one-way dissemination to a wider audience can involve the communication of results, funding- specific public relations obligations, or participant recruitment (Tulloch et al., 2013; Groulx et al., 2017). While the two-way participatory approach is more time consuming, and can put additional pressure on project resources, it is more likely to foster collaborative work, relationship building, and learning (Mercer and Littleton, 2007). The tension between outreach and engagement is mirrored in the field of science communication with its models of deficit and dialogue (Trench, 2008; Lewenstein, 2015). Competing tensions in citizen science can also be considered through three stances in education suggested by Stetsenko’s (2008) acquisition, participation, and transformation—which are evident at each level of Haklay’s typology. In the first stance, “acquisition,” stakeholders see citizen science processes as being concerned with generating pre-existing, fixed, factual knowledge that is gained by individuals primarily through passive input. The second stance, “participation,” positions science and education practices as potentially being affected by other factors—such as location or culture—and necessitates an initiation process in order for participants to gain full access to the community. This stance places citizen scientists into a more participatory role, and educators and scientists are aware that citizen science often generates findings that are culturally located, generated, shared, mutable, and communicated over time. This stance may bring about tension from stakeholders who don’t wholly subscribe to the idea that findings are culturally embedded; however, “participation” provides access for novices, e.g., pupils, into the community of science practitioners. Whereas participatory engagement is a powerful way to support learning (Gleason and Von Gillern, 2018), one-way dissemination also has a valuable role in citizen science. Theoretical Perspectives on Citizen Science and Educational Practice This transformative stance embodies the fourth level of Haklay’s typology; presenting an ideal common ground for both education and citizen science, resolving potential onto-epistemological tensions, and generating synergy. Building on Arnstein’s concept, Haklay (2013) designed an adapted model for citizen science in which the fourth and final level of citizen participation enables all stakeholders— scientists, educators, facilitators, the public, education partner organisations, and policy makers—to collaborate. At this level, citizen science would emerge as a truly transformative practice that has the power to change and influence the world. In his typology, Haklay’s (2013) suggests that increasing the involvement and engagement of the public in citizen science will result in the empowerment of learners while significantly democratising citizen science input. As members of the public are empowered to engage more deeply with, and learn more about the scientific projects they are involved in, they are likely to move up the structure—from merely acting as sensors for science projects that are conducted elsewhere, to collaboratively shaping scientific endeavours from their inception, and participating in their analysis throughout. Theoretical Perspectives on Citizen Science and Educational Practice Citizen Science, Education, and Activism Arnstein (1969) pioneered the concept of citizen participation with her “Ladder of Citizen Participation,” which described the eight levels of citizen power, from non-participatory “manipulation” to “citizen control.” The role citizen science may play in activism and in advocacy—citizens intervening on behalf of, or representing, a socio-political goal (Letiecq and Anderson, 2017; Reis, 2020)—is a key consideration in its interactions with education and learning. From the perspective of civic society, citizen science should encourage individuals to take an active role in their communities—operationalizing active citizenship (Burls and Recknagel, 2013). This role of active citizenship aligns with Arnstein’s rising level of citizen participation and is especially pertinent in citizen science projects that focus on environmental activism and climate change— empowering people to take responsibility for the future of their environments (Baptista et al., 2018; Kythreotis et al., 2019; Dawson et al., 2020). The concept of active citizenship is closely Sc e ce a d duca o a ac ce Ontologies and epistemologies are theories surrounding the nature of being and knowing, or generating knowledge, and provide the assumptions which naturally underlie both educational practice and citizen science practice. Ontology and epistemology are often linked, because how the world is understood, and the phenomena that are available for study within it, are very much dependent on how people think they can come to know, and what they consider “valid” knowledge. Therefore, onto-epistemological differences, namely, tensions that arise from the disparate ways each person interprets the world, including the understanding of what phenomena can be studied, how it can be studied, and the conclusions that can subsequently be drawn, mean that the differences inherent between various citizen science fields and educational environments will result in disparate learning outcomes. As noted by Shirk et al. (2012), tension may be generated due to the often dissimilar interests of scientific and public stakeholder groups in the wider field of public participation December 2020 | Volume 5 | Article 613814 Frontiers in Sociology | www.frontiersin.org 4 Citizen Science, Education, and Learning Roche et al. in scientific research (PPSR), in which citizen science is intrinsic. Competing onto-epistemologies are likely contributory factors to the difficulties inherent in engaging various publics in scientific research, and the alignment of these competing constituents could facilitate greater synergy between citizen science and education. would evolve into a collaborative, co-creative approach. Frontiers in Sociology | www.frontiersin.org Recommendations for Finding Synergy processes, especially regarding societal challenges (Newman et al., 2012). Recommendations for Finding Synergy There has already been a great deal of work conducted with a view to establishing best practice principles for citizen science notably, the European Citizen Science Association’s “Ten Principles of Citizen Science” [European Citizen Science Association, 2015; and the subsequent characteristics of citizen science (European Citizen Science Association, 2020) which expand on the principles]. Assuming adherence to these principles, the following recommendations may create meaningful opportunities for citizen science in education and learning. In as much as onto-epistemological tensions may arise between citizen science and education, one-way dissemination may generate significant tension in a learning environment when science is positioned as the sole truth, and the scientific method the only way to produce reliable knowledge. A two- way participatory approach, by contrast, not only bridges the gap between science education and science communication but poses science as one of many types of knowledge, and the scientific method as one of a multitude of ways to describe the world (Baram-Tsabari and Osborne, 2015). This interplay between science and society is ever more critical in the era of fake news and misinformation (Scheufele and Krause, 2019). One of the most effective solutions to such tensions is to involve scientists in all aspects of the communication process in citizen science projects (Riesch and Potter, 2014). This has a positive effect on participant recruitment, retention, instruction, knowledge sharing, awareness raising, and increases the credibility and authority of the work taking place. However, some scientists may be hesitant to engage in efforts to communicate if they feel that they are not specifically trained to do so (Golumbic et al., 2017). Communication activities, such as public talks, interviews, or popular science articles can be time consuming, and some scientists may find participation uncomfortable (Van Vliet et al., 2014). An increasing number of research funding initiatives at both national and European levels require the inclusion of public engagement and communication strategies, thus increasing the pressure on scientists involved in citizen science projects to directly engage with public audiences. This may be particularly challenging for scientists if these activities are not supported by their institutions, or if their career progression is primarily evaluated on the quality of their publications in scientific journals (Kreiman and Maunsell, 2011). Recommendations for Finding Synergy (2013) demonstrated that such workshops could improve scientific literacy for workshop participants, assessed with context-specific measures. However, unique challenges are still likely to arise. Jordan et al. (2011) could not detect any increase in scientific literacy, and the potential failure of these training sessions was attributed to a lack of time for active learning, which must provide a provision for reflection, and allow participants to make mistakes (Gray et al., 2012; Jordan et al., 2015). To further embed citizen science in informal learning spaces, gamification is an effective tool in engaging participants, and in-game rewards can be carefully planned in order to reward focus on good quality data (Tippins and Jensen, 2012; Bowser et al., 2013; Morschheuser et al., 2019; Piper, 2020). Ensuring alignment between the onto-epistemological positions of the citizen science, education, and learning aspects of any project is a worthwhile endeavour. It is clear that the achievement of the educational goals of citizen science projects are contingent on those goals being taken into consideration at the design stage (Bonney et al., 2014). Following frameworks for measuring individual learning outcomes from participation in citizen science—such as Phillips et al. (2018)—would facilitate the alignment of learning outcomes and the underlying onto- epistemological stances. Additionally, building a co-creation component into citizen science projects from the outset would significantly increase the likelihood that both the educational and scientific goals of the project will be met (Gray et al., 2012). Such co-creation approaches should be considered obligatory, where possible, for every new project. Recommendations for Finding Synergy While not without its critics (Khazragui and Hudson, 2015; Watermeyer, 2016), the Research Excellence Framework in the UK is a notable example of a research evaluation process that gives consideration to the societal impact of research. meaningful opportunities for citizen science in education and learning. Professional development training workshops (Jeanpierre et al., 2005) facilitating citizen science in classrooms can be effective in overcoming some of the barriers that schools, teachers, and students may encounter while participating in citizen science projects (Eberbach and Crowley, 2009; Scheuch et al., 2018). Crall et al. (2013) demonstrated that such workshops could improve scientific literacy for workshop participants, assessed with context-specific measures. However, unique challenges are still likely to arise. Jordan et al. (2011) could not detect any increase in scientific literacy, and the potential failure of these training sessions was attributed to a lack of time for active learning, which must provide a provision for reflection, and allow participants to make mistakes (Gray et al., 2012; Jordan et al., 2015). To further embed citizen science in informal learning spaces, gamification is an effective tool in engaging participants, and in-game rewards can be carefully planned in order to reward focus on good quality data (Tippins and Jensen, 2012; Bowser et al., 2013; Morschheuser et al., 2019; Piper, 2020). Ensuring alignment between the onto-epistemological positions of the citizen science, education, and learning aspects of any project is a worthwhile endeavour. It is clear that the achievement of the educational goals of citizen science projects are contingent on those goals being taken into consideration at the design stage (Bonney et al., 2014). Following frameworks for measuring individual learning outcomes from participation in citizen science—such as Phillips et al. (2018)—would facilitate the alignment of learning outcomes and the underlying onto- epistemological stances. Additionally, building a co-creation component into citizen science projects from the outset would significantly increase the likelihood that both the educational and scientific goals of the project will be met (Gray et al., 2012). Such co-creation approaches should be considered obligatory, where possible, for every new project. h ll d d d Professional development training workshops (Jeanpierre et al., 2005) facilitating citizen science in classrooms can be effective in overcoming some of the barriers that schools, teachers, and students may encounter while participating in citizen science projects (Eberbach and Crowley, 2009; Scheuch et al., 2018). Crall et al. Dissemination, Dialogue, and Participatory Communication Communicating the mission and vision of a project outside of its immediate community can be one of the most important goals for project leaders (Kerzner, 2013). The way in which these values are communicated can vary, depending on the scientists, citizens, and policymakers involved. In particular, there is often a perceived disconnect between policymakers and other key stakeholders, such as citizens and scientists (Socientize Consortium, 2013). Using a Public Relations (PR) approach is a commonly employed method of bridging this gap (Scott, 2013), and involves implementing a strategic communications plan that can include public lectures, workshops, festivals, exhibits, tours, and open laboratories. To supplement these activities, a strategic PR plan for citizen science projects is often used to directly engage policymakers with demonstrations of the usefulness of the project and the need for new knowledge generation (Socientize Consortium, 2014). Although a common concern when employing a communication approach that focuses on PR is the potential tendency to overlook negative results and issues of uncertainty, which are part of the scientific process, if effective communication is adhered to between stakeholders, it can lead to citizen science projects enhancing public debate and citizen participation in decision-making Applying Stetsenko’s third stance, citizen science can become “transformative” when embedded in educational programming. This transformation could lead to change at individual, community-wide, and global levels if citizen science expands in scale and scope. The intrinsic risk of the transformative approach is that it can replace a system of knowledge with one that still does not appropriately recognise marginalised forms of knowledge (Leibowitz, 2017). An example of a transformative project could be “WeatherBlur” a co-created citizen science project bringing together, fishermen, students, teachers, and research scientists from island and coastal communities on the east coast of the US “to share, analyse, and interpret data about the local impact of climate change” (Kermish-Allen et al., 2019, p. 627). “Knowing” and acquiring knowledge are presented by Stetsenko as active and collective activities; thus citizen science Frontiers in Sociology | www.frontiersin.org December 2020 | Volume 5 | Article 613814 5 Citizen Science, Education, and Learning Roche et al. OPPORTUNITIES FOR CITIZEN SCIENCE, EDUCATION, AND LEARNING Challenges surrounding communication, dissemination, and dialogue may be addressed by increasing science communication training opportunities for scientists involved in citizen science, as well as for scientists in general. Collaboration between scientists and citizens with public relations and communication professionals could lead to more open strategies for communicating with different audiences and could generate clear alignment between both the dissemination and participatory modes of communication. Crucially, to ensure that scientists contribute not only to the scientific goals of citizen science projects, but also to the communication and educational aspects, public engagement Thoroughly exploring the obstacles that prevent the successful integration of citizen science practice into mainstream education systems is key to overcoming them. Recommendations based on the challenges that have been highlighted in this paper could help citizen science to fulfil its potential as a truly transformative social innovation for education and learning. This could, in turn, encourage citizen science practitioners and educators to take an adaptive and flexible position in the face of new and emerging societal challenges and a fluctuating political and economic landscape that continuously strains the relationship between science and society. December 2020 | Volume 5 | Article 613814 Frontiers in Sociology | www.frontiersin.org 6 Citizen Science, Education, and Learning Roche et al. should be recognised as scholarly work. This would be made possible through research institutions redefining public engagement as a metric to be evaluated in academic career progression, in a manner akin to research output (Smith et al., 2014). attending to learning and practicing science in ways that are more in tune with learners’ motivations, with local places, and in ways that are socio-culturally distributed among all participants, including scientists, teachers, students, community members, policymakers, and any other stakeholders (Mannion et al., 2013; Haywood et al., 2016). Taking a transformative stance on citizen science in education could be key to engendering a more vital role for science in the public sphere, generating responses to current and future eco-social problems (Dillon et al., 2016), and helping to achieve the UN Sustainable Development Goals (Fritz et al., 2019; Fraisl et al., 2020). The greater recognition of citizen science and activism in recent years may, in part, be due to a growing focus on equality, open access, and public participation caused by the current global political climate (Roche and Davis, 2017b; Hutter and Kriesi, 2019). OPPORTUNITIES FOR CITIZEN SCIENCE, EDUCATION, AND LEARNING Once public engagement is fully integrated into the missions of both research performing organisations and research funding organisations, social activism must be given due consideration as an inevitable counterpart to citizen science. As recommended by the National Academies of Sciences (2018), issues of equity and power should be given particular consideration at all stages of citizen science project design and implementation, in all settings. Citizen science is not merely a method of involving the public in scientific research but is also a way of empowering citizens to take ownership of their own science education and learning. The future of how citizen science will be integrated into education and learning will continue to be influenced by globally-accessible digital platforms. The newest of these, EU- Citizen.Science, is an online platform for citizen science in Europe that is being established with the support of a Horizon 2020 grant from the European Commission. This platform will not only make citizen science projects and data more readily accessible, but it will also act as a mutual learning space for sharing useful tools, guidelines, training, and best practice examples in several languages to help citizens, scientists, teachers, students, schools, and other stakeholders to determine how they can engage with local and international citizen science projects. Global initiatives such as these will be key to realising the education and learning potential of citizen science as a far- reaching social innovation. The Future of Citizen Science in Education and Learning Transformative approaches to education are becoming more widely accepted; within education, and in higher education specifically, there is significant interest in developing co- researcher partnerships (Healey et al., 2016). Such partnerships can lead to the co-design of curricula (Bovill, 2014) and the co-production of knowledge (McCulloch, 2009). A contributory approach necessitates a whole new learning paradigm requiring novel educational methods. The outdated metaphor of ‘students as consumers’ (Dearing, 1997; Palfreyman and Warner, 1998), which has a negative impact on student engagement and performance (Bunce et al., 2017), could be replaced by a citizen science partnership that supports educators and students, where knowledge is exchanged in both directions, and the students are active partners in their learning (Freeman et al., 2014) and in participating in authentic scientific research. AUTHOR CONTRIBUTIONS JR and SW were the lead authors and oversaw the completion of the writing. JR, LB, CG, YG, LK, NK, ML, JL, GM, LM, AM, KP, AR, PT, and SW contributed writing to individual subsections of the manuscript. All authors have read and agreed to the published version of the manuscript and were involved in the conceptual design of the manuscript. FUNDING This work would not have been possible without the support of the COST (European Cooperation in Science and Technology) Action CA15212 (Citizen Science to promote creativity, scientific literacy, and innovation throughout Europe) and the EU-Citizen Science project which received funding from the European Union’s Horizon 2020 Research and Innovation Programme under Grant Agreement No. 824580. This work would not have been possible without the support of the COST (European Cooperation in Science and Technology) Action CA15212 (Citizen Science to promote creativity, scientific literacy, and innovation throughout Europe) and the EU-Citizen Science project which received funding from the European Union’s Horizon 2020 Research and Innovation Programme under Grant Agreement No. 824580. Citizen science practitioners and programmes seeking links with schools may find that tapping into more transformative models of learner engagement is a starting point for enhanced participation. The adoption of a transformative onto- epistemological stance opens up much greater potential for synergy between citizen science and education. The outcomes of transformative citizen science will result in changes to what is known, how it is known, and to the individual, socio-cultural, and wider world. Mueller and Tippins (2012) rhetorically ask why citizen science programming in education generally aims to advance science literacy, when learners’ motivations are predominantly to care for what is often a local environment. Within this transformational framing, potential exists for Frontiers in Sociology | www.frontiersin.org REFERENCES Dawson, E. (2014). “Not designed for us”: how science museums and science centers socially exclude low-income, minority ethnic groups. Sci. Educ. 98, 981–1008. doi: 10.1002/sce.21133 Archer, L., Dawson, E., DeWitt, J., Seakins, A., and Wong, B. (2015). “Science capital”: a conceptual, methodological, and empirical argument for extending bourdieusian notions of capital beyond the arts. J. Res. Sci.Teach. 52, 922–948. doi: 10.1002/tea.21227 Dawson, T., Hambly, J., Kelley, A., Lees, W., and Miller, S. (2020). Coastal heritage, global climate change, public engagement, and citizen science. Proc. Natl. Acad. Sci. 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Evaluation of reporting quality of cohort studies using real-world data based on RECORD Ran Zhao  Institute of Information on Traditional Chinese Medicine, China Academy of Chinese Medical Sciences Wen Zhang  Institute of Basic Research in Clinical Medicine, China Academy of Chinese Medical Sciences Zedan Zhang  Traditional Chinese Medicine Data Center, China Academy of Chinese Medical Sciences Chang He  Institute of Information on Traditional Chinese Medicine, China Academy of Chinese Medical Sciences Rong Xu  Guang'anmeng Hospital, China Academy of Chinese Medical Sciences Xudong Tang  China Academy of Chinese Medical Sciences Bin Wang  (  wangbin135@139.com ) Institute of Information on Traditional Chinese Medicine, China Academy of Chinese Medical Sciences Research Article Keywords: RWD, RWE, cohort studies, reporting quality, RECORD Posted Date: January 27th, 2023 DOI: https://doi.org/10.21203/rs.3.rs-2407386/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License.   Read Full License Additional Declarations: No competing interests reported. Version of Record: A version of this preprint was published at BMC Medical Research Methodology on June 29th, 2023. See the published version at https://doi.org/10.1186/s12874-023-01960-2. Evaluation of reporting quality of cohort studies using real-world data based on RECORD Ran Zhao  Institute of Information on Traditional Chinese Medicine, China Academy of Chinese Medical Sciences Wen Zhang  Institute of Basic Research in Clinical Medicine, China Academy of Chinese Medical Sciences Zedan Zhang  Traditional Chinese Medicine Data Center, China Academy of Chinese Medical Sciences Chang He  Institute of Information on Traditional Chinese Medicine, China Academy of Chinese Medical Sciences Rong Xu  Guang'anmeng Hospital, China Academy of Chinese Medical Sciences Xudong Tang  China Academy of Chinese Medical Sciences Bin Wang  (  wangbin135@139.com ) Institute of Information on Traditional Chinese Medicine, China Academy of Chinese Medical Sciences Research Article Keywords: RWD, RWE, cohort studies, reporting quality, RECORD Posted Date: January 27th, 2023 DOI: https://doi.org/10.21203/rs.3.rs-2407386/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Additional Declarations: No competing interests reported. Version of Record: A version of this preprint was published at BMC Medical Research Methodology on June 29th, 2023. See the published version at https://doi.org/10.1186/s12874-023-01960-2. Evaluation of reporting quality of cohort studies using real-world data based on RECORD Ran Zhao  Institute of Information on Traditional Chinese Medicine, China Academy of Chinese Medical Sciences Wen Zhang  Institute of Basic Research in Clinical Medicine, China Academy of Chinese Medical Sciences Zedan Zhang  Traditional Chinese Medicine Data Center, China Academy of Chinese Medical Sciences Chang He  Institute of Information on Traditional Chinese Medicine, China Academy of Chinese Medical Sciences Rong Xu  Guang'anmeng Hospital, China Academy of Chinese Medical Sciences Xudong Tang  China Academy of Chinese Medical Sciences Bin Wang  (  wangbin135@139.com ) Institute of Information on Traditional Chinese Medicine, China Academy of Chinese Medical Sciences Research Article Keywords: RWD, RWE, cohort studies, reporting quality, RECORD Posted Date: January 27th, 2023 DOI: https://doi.org/10.21203/rs.3.rs-2407386/v1 Research Article Keywords: RWD, RWE, cohort studies, reporting quality, RECORD Posted Date: January 27th, 2023 DOI: https://doi.org/10.21203/rs.3.rs-2407386/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Posted Date: January 27th, 2023 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Additional Declarations: No competing interests reported. Additional Declarations: No competing interests reported. Version of Record: A version of this preprint was published at BMC Medical Research Methodology on June 29th, 2023. See the published version at https://doi.org/10.1186/s12874-023-01960-2. Page 1/19 Page 1/19 Abstract Objective: Real-world data (RWD) and real-world evidence (RWE) have been paid more and more attention in recent years. We aimed to evaluate the reporting quality of cohort studies using real-world data (RWD) published between 2013 to 2021 and analyze the possible factors. Methods: We conducted a comprehensive search in Medline and Embase through the OVID interface for cohort studies published from 2013 to 2021 on April 29, 2022. The evaluation was based on the REporting of studies Conducted using Observational Routinely-collected health Data (RECORD) statement. Pearson chi-square test or Fisher's exact test and Mann-Whitney U test were used to analyze the possible factors, including the release of RECORD, journal IFs, and article citations. Results: 187 articles were finally included. The mean±SD of the percentage of adequately reported items in the 187 articles was 44.7±14.3 with a range of 11.1% to 87%. Of 23 items, the adequate reporting rate of 10 items reached 50%, and the reporting rate of some vital items was inadequate. The reporting of only two items significantly improved after the release of RECORD and there was no significant improvement in the overall report quality. The journal IFs and citations were respectively related to 6 areas and significantly higher in high-reporting quality articles. Conclusion: The reporting of cohort studies using RWD was generally inadequate and has not improved in recent years. We encourage researchers to endorse relevant guidelines when utilizing RWD for research. 2.2. Search strategy We conducted a comprehensive search in Medline and Embase through the OVID interface for English language articles published between 2013 to 2021(Search on April 29, 2022). The indexing terms included study design, data sources of studies (e.g., “routinely collected data”, “health information system”, “electronic medical record”, “registry”), excluded publication types or article types (e.g., “review”, “protocol”, “meta-analysis”) and excluded outcomes. The search filters developed by Scottish Intercollegiate Guidelines Network [24] and the strategy developed by Lars G. Hemkens et al [19] were integrated into our search strategy (available in S1 File). 2.1. Eligibility of studies We selected cohort studies that aimed to compare the effectiveness and safety of the exposure factors and used real-world data, including prospective, retrospective, and both. For example, we excluded studies that classified populations by disease type [23]. Studies on morbidity, mortality, or hazard factors of diseases and economic benefits or medical expenses of exposure factors were excluded. We only considered the English language articles published between 2013 to 2021. For the wider applicability of research results, no more restrictions on research diseases or participants, exposure measures, and the settings of control groups. Introduction We concentrated on cohort studies that compared the effectiveness or safety of exposure factors, since cohort studies are helpful to provide evidence indicating causality, the strength of correlation between exposure factors and outcomes, and can usually produce highly generalizable results [20,21] while comparative research could complement or assess the evidence originated from randomized trials and inform decisions about health policy and clinical care [22]. The RECORD checklist was transformed into a series of questions for convenient and accurate evaluation. We aimed to evaluate the reporting quality of cohort studies using RWD published from 2013 to 2021 and analyze the possible factors of reporting quality. Comparative analyses were conducted to ascertain whether the report quality is related to the release of the RECORD statement, journal impact factors (IFs), and citations of individual articles. Introduction Real-world data is defined as the data relating to patient health status and/or the delivery of health care that is routinely collected from a variety of sources, such as patient registries, electronic medical records (EMRs), electronic health records (EHRs), insurance claims, and patient health records [1,2], by the US Food and Drug Administration [3]. Real-world evidence (RWE) generated by studies using RWD could be the complement of clinical trials for observing the effectiveness or safety of drugs, products, operations, or any other treatment measures and play an increasingly significant role in decision-making while it is more reflective of clinical practice compared to the evidence generated by randomized trials [4–7]. However, the reporting of studies using RWD currently exists with the problems of inadequate and lack of transparency, which will limit the reproducibility and replicability of studies and arouse concerns, doubts, and reductions of confidence in RWE [8–11]. Some reporting guidelines have been established to standardize studies reports and promote the quality of RWE [9,12–15]. The REporting of studies Conducted using Observational Routinely-collected health Data (RECORD) statement [12], which is an extension of the STrengthening the Reporting of OBservational studies in Epidemiology (STROBE) statement [16], was created to address the specific reporting issues of studies using routinely-collected data. Previous studies have shown the reporting of observational studies was usually insufficient whether based on RECORD or STROBE [17–19]. Page 2/19 According to the RECORD checklist, we evaluated the quality of reporting on specific aspects of studies using RWD, such as codes and algorithms, data linkage and cleaning, and discussion of peculiar limitations. We concentrated on cohort studies that compared the effectiveness or safety of exposure factors, since cohort studies are helpful to provide evidence indicating causality, the strength of correlation between exposure factors and outcomes, and can usually produce highly generalizable results [20,21] while comparative research could complement or assess the evidence originated from randomized trials and inform decisions about health policy and clinical care [22]. The RECORD checklist was transformed into a series of questions for convenient and accurate evaluation. According to the RECORD checklist, we evaluated the quality of reporting on specific aspects of studies using RWD, such as codes and algorithms, data linkage and cleaning, and discussion of peculiar limitations. 2.3. Screening and data extraction We imported search results into Note Express (Beijing Aegean Hailezhi Technology Co., Ltd., Beijing, China) for management, removing duplicated articles and screening. First, articles that did not conform to Page 3/19 the eligibility criteria were excluded by independently screening the titles and abstracts by two reviewers (R.Z. and W.Z.). Subsequently, we downloaded all the available full texts of preliminary included articles. Two reviewers (R.Z. and W.Z.) read each full-text article to screen out the articles that were ultimately included and record the reason for exclusion. Any discrepancies in the screening process were resolved via discussion or determined by a third author (one of B.W., Z.D.Z, C.H.). We extracted the characteristics of each qualified article including the year of publication, country of the corresponding author, type of disease, journal name, journal IFs in the year of publication, citations, type of therapy, and type of data source. To guarantee the accuracy of the journal names and journal impact factors, we identified the journals by DOI, PMID, or site link provided in the articles and searched the ISSN of journals on the Web of Science to obtain the journal impact factors in the year of publication. Article citations were accessed on Google Scholar. Two reviewers (two of R.Z., W.Z., Z.D.Z, and C.H.) completed the process of extraction and recorded data in Microsoft Excel (Microsoft Corporation, USA). Any discrepancies were resolved via consensus. 2.4. Evaluation Included articles were evaluated by the RECORD checklist, an extension of the STROBE checklist. Considering that some items of the RECORD checklist contain various aspects, we split and transformed the 13 items of the RECORD checklist into 23 questions that can be answered “yes”, “partly yes”, “no”, or “not applicable”. Reported as “yes” was considered adequate reporting while reported as “partly yes” or “no” was considered inadequate reporting. Some questions may not correspond to the RECORD checklist, but they are emphasized in the explanations of the RECORD statement and are necessary to be reported. For the split items, we renamed the different items with lowercase letters (e.g., R1.1 split to R1.1a, R1.1b). Five items appear to be inapplicable, in brief, three items (R1.3, R6.3, and R12.3) would be inapplicable if the study did not require database linkage and two items (R6.2 and R19.1e) would be inapplicable if the study did not report the codes or algorithms for population selection. We considered that other items should be reported in studies using RWD and therefore applied by default. The complete list of questions, descriptions, and examples can be obtained in S1 Table. We randomly selected 10 included articles and four reviewers (R.Z., W.Z., Z.D.Z, C.H.) preliminary evaluated them through the constructed 23 questions. Afterwards every reviewer proposed problems and suggestions generated in the preliminary evaluation for a panel discussion to eliminate all discrepancies and ameliorate the 23 questions. Finally, two independent reviewers (R.Z. and W.Z.) evaluated each of included articles and integrated the results in Microsoft Excel (Microsoft Corporation, USA), any discrepancies were resolved via discussion or determined by two other authors (Z.D.Z, C.H.). 2.5. Data analysis Page 4/19 We respectively performed horizontal and vertical calculations. For each item, the number of answers that were “yes”, “partly yes”, “no” and “not applicable” was calculated, as well as the percentage after removing the inapplicable items. For each article, the number of each type of answer was similarly calculated, as well as the rate of adequate reporting which was regarded as the overall quality of the individual articles. To compare the reporting quality before and after the release of RECORD (released on October 6, 2015), we conducted a before-and-after analysis, with articles published from 2013 to 2015 defined as Pre- RECORD articles and articles published from 2018 to 2021 defined as Post-RECORD articles. Considering the time required to publish the article and the dissemination of RECORD, a two-year interval was allowed. The median and interquartile range (IQR) were used to describe continuous variables. We compared the reporting of each item of Pre-RECORD articles and Post-RECORD articles using the Pearson chi-square test or Fisher's exact test and calculated odd ratios and 95% confidence intervals. Due to the non-normal distribution of the data, the Mann-Whitney U test was used to analyze the correlations of journal IFs and citations with reporting quality. We also set a 50% reporting rate as a threshold to compare the difference in impact factors and citations between articles with higher reporting quality and others reporting relatively lower. Articles without IF or citations would not be included in the analysis. A p-value less than 0.05 with two-sided was considered a statistically significant difference and all statistical analyses were performed on SPSS v26.0 (IBM Corp., Armonk, NY, USA). Table 1. characteristics of included studies 3.1. Screening results and characteristics of included studies A total of 5824 articles were identified, and 187 articles were finally included after the screening (Figure 1). The complete list of included articles is available in S2 File. The number of articles has been on the rise in recent years. Articles from the USA were at most (56,29.9%). Cardiovascular diseases (65,34.8%) and carcinoma/neoplasms (39, 20.9%) are the most studied diseases. The most common therapy is medication (144,77%). Most studies used a single database (121, 64.7%), with multicenter registries accounting for the majority (116, 62%). Details about the characteristics of included studies are shown in Table 1. Table 1. characteristics of included studies Page 5/19 Characteristics  Studies [n (%)] Characteristics  Studies [n (%)] Country   Publication Year   USA Germany Japan  Korea  China Denmark Spain Italy Other 56(29.9) 16(8.6) 14(7.5) 13(7) 11(5.9) 10(5.3) 9(4.8) 9(4.8) 59(31.6) 2013 2014 2015 2016 2017 2018 2019 2020 2021 8(4.3) 10(5.3) 15(8) 14(7.5) 24(12.8) 26(13.9) 29(15.5) 31(16.6) 30(16) Type of Diseases   Type of Therapies   Cardiovascular Disease Carcinoma/ Neoplasm Metabolic Disease Rheumatic Disease Nervous System Disease Skin Disease Liver Disease Other diseases 65(34.8) 39(20.9) 19(10.2) 14(7.5) 8(4.3) 7(3.7) 6(3.2) 29(15.5) Medication Surgical Procedure Chemoradiotherapy Other 144(77) 36(19.3) 5(2.7) 13(16) Type of Data Sources   Number of databases   Multicenter Registry Single center registry EMR/EHR Claims Other 116(62) 6(3.2) 34(18.2) 11(5.9) 29(15.5) 1 2 3 4 ≥5 121(64.7) 32(17.1) 13(7) 5(2.7) 16(8.6) Journal IF (n=176)   Citations (n=187)   Median (IQR) Range 3.54(2.4,5.55) 0.52-27.97 Median (IQR) Range 12(6,25) 0-548 Studies [n (%)] Characteristics  Studies [n (%)] IQR, interquartile range; EMR, electronic medical record; EHR, electronic health record; IF Country means the country of the corresponding author; therapies involve exposure group and control group; journal IF is in the publication year of the article. Country means the country of the corresponding author; therapies involve exposure group and control group; journal IF is in the publication year of the article. artile range; EMR, electronic medical record; EHR, electronic health record; IF, impact factor 3.2 Reporting quality of cohort studies using RWD The mean±SD of the percentage of adequately reported items in the 187 articles was 44.7±14.3 with a range of 11.1% to 87% (disregarding inapplicable items). 72 articles (38.5%) adequately reported 50% and above items, the evaluation details of each article can be obtained in S3 File. Out of 23 items in total, the adequate reporting rate of 10 items reached 50%, and the reporting rate of some vital items was inadequate. Of 66 (35.3%) studies involving database linkage, 33 (50%), 12 (18.2%), and 28(42.4%) studies adequately reported databases linkage in title or abstract (R1.3), flowcharts or other diagrams to display linkage (R6.3), and the detail of linkage (R12.3) respectively (Table 2). Of the 75 (40.1%) articles that adequately reported codes or algorithms of population selection (R6.1), only 21(28%), 19 (25.3%) articles adequately reported the validation methods (R6.2), and the limitations of codes or algorithms and the validation used to population selection (R19.1c) respectively (Table 2). Page 6/19 In addition, the reporting of some other items was also critically insufficient, such as codes of algorithms (R7.1a:28, 15%; R7.1b:43, 23%; R7.1c:23, 12.3%), data-cleaning methods (R12.2:65, 34.8%), discussion of change in eligibility of results over time (R19.1e:34, 18.2%), and availability of study protocol and raw study (R22.1a:44, 23.5%, R22.1b:38, 20.3%) (Table 2). Table 2. 3.2 Reporting quality of cohort studies using RWD Reporting of cohort studies using RWD Items Brief Descriptions Reporting [n (%)] Not Applicable Yes Partly Yes No Title and abstract R1.1a Type of data 182(97.3)   5(2.7)   R1.1b Name of databases 134(71.7) 2(1.1) 51(27.2)   R1.2a Geographic region 117(62.6)   70(37.4)   R1.2b Time frame 97(51.9)   90(48.1)   R1.3 Linkage between databases 33(50)   33(50) 121 Methods R6.1 Population selection methods 75(40.1) 112(59.9)     R6.2 Validation of codes/algorithms for population selection 21(28)   54(72) 112 R6.3 Graphical display of data linkage process 12(18.2)   54(81.8) 121 R7.1a Codes/algorithms of exposures 28(15) 2(1) 157(84)   R7.1b Codes/algorithms of outcomes 43(23) 34(18.2) 11(5.9)   R7.1c Codes/algorithms of confounders and effect modifiers 23(12.3) 7(3.7) 157(84)   R12.1 The extent of database accessed 153(81.8)   34(18.2)   R12.2 Data-cleaning methods 65(34.8)   122(65.2)   R12.3 Detail of data linkage 28(42.4) 28(42.4) 10(15.2) 121 Results R13.1 Detail of population selection 107(57.2) 19(10.2) 61(32.6)   Discussion R19.1a Inherent limitations 155(82.9)   32(17.1)   R19.1b Risk of misclassification 58(31)   129(69)   R19.1c Limitations of codes/algorithms and the validation 19(25.3)   56(74.7) 112 R19.1d Implications of the missing variables and missing data 118(63.1)   69(36.9)   R19.1e Eligibility of results over time 34(18.2)   153(81.8)   Other information R22.1a Study protocol availability 44(23.5)   143(76.5)   R22.1b Raw data availability 38(20.3)   149(79.7)   R22.1c Supplementary materials availability 100(53.5)   87(46.5) RWD, real-world data 3.3 Reporting before and after the release of RECORD Compared to Pre-RECORD period (33 articles), significant improvement in reporting quality for only two items in Post-RECORD period (116 articles): reporting of availability of raw data (R22.1b, p<0.001), and availability of other supplementary materials (R22.1c, OR: 3.2, 95%CI:1.43-7.16, p=0.004)(Table 3). There was no significant improvement in the overall report quality. Table 3. Reporting before and after the release of RECORD Page 7/19 Table 4. Correlations of journal IFs and citations with reporting quality Page 7/19 Items Reporting Studies(n) OR (95%CI) p-value Pre-RECORD Post-RECORD R1.1a Yes Partly Yes/No 32 1 115 1 3.59(0.22,59.06) 0.395 R1.1b Yes Partly Yes/No 26 7 84 32 0.71(0.28,1.79) 0.462 R1.2a Yes Partly Yes/No 18 15 77 39 1.65(0.75,3.61) 0.212 R1.2b Yes Partly Yes/No 16 17 64 52 1.31(0.6,2.84) 0.497 R1.3 Yes Partly Yes/No 6 10 26 16 2.71(0.83,8.89) 0.095 R6.1 Yes Partly Yes/No 14 19 51 65 1.07(0.49,2.33) 0.875 R6.2 Yes Partly Yes/No 6 8 12 39 0.41(0.12,1.42) 0.152 R6.3 Yes Partly Yes/No 3 13 9 33 1.18(0.28,5.07) 0.822 R7.1a Yes Partly Yes/No 4 29 23 93 1.79(0.57,5.61) 0.443 R7.1b Yes Partly Yes/No 8 25 30 86 1.09(0.44,2.68) 0.851 R7.1c Yes Partly Yes/No 4 29 14 102 1(0.3,3.26) 1 R12.1 Yes Partly Yes/No 27 6 103 13 1.76(0.61,5.06) 0.289 R12.2 Yes Partly Yes/No 11 22 42 74 1.14(0.5,2.57) 0.761 R12.3 Yes Partly Yes/No 5 11 22 20 2.42(0.72,8.18) 0.149 R13.1 Yes Partly Yes/No 19 14 71 45 1.16(0.53,2.55) 0.71 R19.1a Yes Partly Yes/No 27 6 96 20 1.07(0.39,2.92) 0.9 R19.1b Yes Partly Yes/No 11 22 33 83 0.8(0.35,1.82) 0.587 R19.1c Yes Partly Yes/No 6 8 12 39 0.41(0.12,1.42) 0.152 R19.1d Yes Partly Yes/No 25 8 71 45 0.51(0.21,1.22) 0.123 R19.1e Yes Partly Yes/No 8 25 21 95 0.69(0.27,1.74) 0.432 R22.1a Yes Partly Yes/No 9 24 28 88 0.85(0.35,2.04) 0.713 R22.1b Yes Partly Yes/No 0 33 36 80 -- <0.001 R22.1c Yes Partly Yes/No 12 21 75 41 3.2(1.43,7.16) 0.004 Total   -- -- 1.17(0.98,1.39) 0.082 Page 8/19 RECORD, REporting of studies Conducted using Observational Routinely-collected health Data Pre-RECORD refers to articles published in 2013-2015, Post-RECORD refers to articles published in 2018-2021. Pre-RECORD refers to articles published in 2013-2015, Post-RECORD refers to articles published in 2018-2021. 3.4 Correlations of journal IFs and citations with reporting quality The journal IFs were significantly higher for adequately reported articles in 6 items: population selection methods (R6.1: p=0.04), codes or algorithms of outcomes (R7.1b: p=0.014), the extent of database accessed (R12.1: p=0.004), eligibility of results over time (R19.1e: p=0.001), availability of raw data and supplementary materials (R22.1b: p<0.001; and R22.1c: p=0.002)(Table 4). The citations of articles were also significantly higher for adequately reported articles in 6 items: R6.1: p=0.035; R7.1a: p=0.02; R7.1c: p=0.001; R19.1a: p=0.01; R19.1d: p=0.001; R22.1b: p=0.015 (Table 4). In total, the journal IFs and citations were significantly higher for articles with advanced reporting rates (≥50%)(IFs: 4.2 versus 3.17, p=0.002; Citations: 16.5 versus 10, p=0.004)(Table 4). Discussion To our knowledge, this is the first time that the complete RECORD checklist was used to evaluate the reporting quality of studies using RWD, with the analysis of the change in reporting quality and its relationship to the journal IFs and citations. We found that only 72 (38.5%) articles adequately reported more than 50% of the items, and some vital items were very insufficiently reported. The overall reporting quality was poor. Some items were reported similarly to analogous studies, such as the codes and algorithms for population selection and their validation studies, as well as data cleaning (R6.1, R6.2, R12.2), while some items were reported worse than analogous studies, such as the codes and algorithms of exposures, outcomes, confounders, and the availability of supplementary materials (R7.1a, R7.1b, R22.1c) [19,25]. While most studies stated that there were inherent limitations when using RWD, there is insufficient discussion of the limitations in some specific areas. Data cleaning, data linkage, and disclosure of relevant information were also severely underreported. Previous studies have investigated changes in reporting quality before and after the release of other checklists, such as STROBE [26], CONSORT [27], etc. We used similar approaches to compare changes in reporting quality before and after the release of RECORD, and we found statistically significant increases in reporting quality for only two items which suggests years after the release of RECORD, the reporting quality of cohort studies has not improved in total. The journal IFs and citations were related to some areas and significantly higher in high-reporting quality articles. Our results were compatible with the study conducted by Pol CB van der et al that high-quality studies were cited more frequently [28]. Interestingly, we found that few high-quality articles had low journal IFs and few citations instead, and vice versa. However, our analysis was cursory because the effect of time was not removed, and the newly published articles may have fewer citations. RWE, on one hand, holds the potential to address important questions [29], on the other hand, there is some controversy due to the issues such as the quality of data sources or studies. The reporting quality issue of concern to this research, to some extent, influences the quality of RWE and its utility in decision- making. 3.4 Correlations of journal IFs and citations with reporting quality Page 9/19 Items Reporting Journal IFs Citations Studies(n) Median (IQR) p-value Studies(n) Median (IQR) p-value R1.1a Yes Partly Yes/No 171 5 3.58(2.39,5.55) 3.19(1.56,4.94) 0.49 182 5 12(6,26) 12(4.5,15) 0.458 R1.1b Yes Partly Yes/No 125 51 3.71(2.46,5.52) 3.39(2.28,6.24) 0.841 134 53 12 (7,25.25) 12(4,25.5) 0.493 R1.2a Yes Partly Yes/No 110 66 3.85(2.45,5.56) 3.3(2.35,5.54) 0.446 117 70 12(5.5,24.5) 12.5(7,26) 0.602 R1.2b Yes Partly Yes/No 88 88 3.42(2.3,5.17) 3.79(2.46,6.11) 0.376 97 90 10(4.5,24.5) 15(7.75,26.25) 0.113 R1.3 Yes Partly Yes/No 33 32 4.4(2.65,5.36) 4.65(3.3,8.62) 0.322 33 33 10(4.5,30.5) 17(7,39.5) 0.16 R6.1 Yes Partly Yes/No 70 106 4.11(2.65,5.99) 3.24(2.26,5.22) 0.04 75 112 16(7,32) 10.5(5,19.75) 0.035 R6.2 Yes Partly Yes/No 20 50 4.01(3.14,6.13) 4.15(2.38,5.99) 0.326 21 55 23(9,60.5) 15.5(6.75,27.25) 0.088 R6.3 Yes Partly Yes/No 12 53 4.53(3.3,5.66) 4.4(2.9,6.73) 0.906 12 54 16.5(4.25,36.5) 13.5(5.75,35) 0.874 R7.1a Yes Partly Yes/No 25 151 4.07(2.78,6.8) 3.4(2.32,5.48) 0.116 28 159 17(9.5,43.75) 11(6,23) 0.02 R7.1b Yes Partly Yes/No 40 136 4.15(3.23,6.45) 3.24(2.23,5.49) 0.014 43 144 15(7,41) 11.5(5.25,23) 0.129 R7.1c Yes Partly Yes/No 20 156 4.58(2.82,12.43) 3.42(2.35,5.42) 0.036 23 164 19(12,66) 10.5(5,23) 0.001 R12.1 Yes Partly Yes/No 145 31 3.94(2.55,5.71) 2.6(1.71,4.4) 0.004 153 34 12(6,27.5) 12.5(5.5,18.75) 0.534 R12.2 Yes Partly Yes/No 61 115 4.07(2.62,6.21) 3.28(2.32,5.1) 0.072 65 122 14(7,29.5) 11(5,23) 0.276 R12.3 Yes Partly Yes/No 28 37 5.16(3.61,8.46) 4.01(2.64,6.07) 0.086 28 38 15(4.25,51.5) 13(5,30) 0.559 R13.1 Yes Partly Yes/No 101 75 3.95(2.62,5.71) 3.19(2.14,5.49) 0.119 107 80 11(5,23) 15(6.25,26) 0.628 R19.1a Yes Partly Yes/No 145 31 3.75(2.46,5.59) 3.15(1.95,5.49) 0.133 155 32 13(7,27) 8.5(3.25,15,75) 0.01 R19.1b Yes Partly Yes/No 54 122 4.02(2.59,6.51) 3.47(2.34,5.22) 0.135 58 129 15(8,30) 10(5,23) 0.099 R19.1c Yes Partly Yes/No 19 51 4.07(3.06,5.57) 4.15(2.45,6.29) 0.751 19 56 27(9,41) 15.5(6,27.75) 0.09 R19.1d Yes Partly Yes/No 112 64 3.68(2.42,5.6) 3.39(2.33,5.42) 0.476 118 69 15(8,29.25) 9(3.5,18) 0.001 R19.1e Yes Partly Yes/No 30 146 2.53(1.7,3.77) 3.98(2.55,5.66) 0.001 34 153 10.5(7.75,20.75) 12(5,25.5) 0.855 R22.1a Yes Partly Yes/No 44 132 3.91(2.38,6.45) 3.47(2.41,5.42) 0.582 44 143 14.5(7,31.25) 11(6,25) 0.469 R22.1b Yes Partly Yes/No 36 140 4.64(2.78,6.49) 3.26(2.33,5.19) 0.022 38 149 8(2,18) 14(7,26) 0.015 R22.1c Yes Partly Yes/No 83 93 4.4(3.03,6.21) 2.65(1.85,4.43) <0.001 87 100 12(6.25,25.75) 12(6,25) 0.681 Total ≥ 50% 50% 65 111 4.2(2.97,6.02) 3 17(2 14 5 18) 0.002 72 115 16.5(8.25,36.5) 10(5 19) 0.004 Page 10/19 IFs, impact factors; IQR, interquartile range Journal IFs of 11 articles were unavailable and therefore not included in the analysis. Citations were obtained from Google Scholar on November 18. Discussion Databases may have issues with incomplete, inconsistent, and inaccurate coding, which makes it more challenging and complex to create data linkages and seriously impedes the reproducibility and replicability of studies, and therefore distinct reporting of codes or algorithms and their validation allows critical assessment by readers and benefits the generalization of study findings [30-32]. Linkage of databases can supplement and enrich data sources and transparent reporting can increase confidence in RWE [33,34]. Only 28 (42.4%) articles in the present research reported details such as the level or methods of linkage, yet this may still be insufficient, and some extended guidelines have been developed for more detailed issues of data linkage [34,35]. The disclosure of research-related information is also Page 11/19 Page 11/19 fundamental to improving the transparency of studies, especially the availability of raw data, which enables readers to assess the authenticity and reliability of the findings. We believe that establishing a high-quality analytical database with accuracy, completeness, consistency, and wide applicability is the core of acquiring reliable RWE [36], and the crucial elements involve codes or algorithms and their validations, quality of data linkage, data cleaning, and the establishment of data specifications. This necessitates that we concentrate on both the quality of the research process, including methodology and reporting quality, as well as the quality of the data sources, such as standardized data structures and rigorous data quality assessments [37,38]. In the meantime, health data not collected for specific purposes are generally not standardized, in contrast to the strictly conducted RCTs. It is almost impossible to balance all confounders and eliminate the impact of quality problems such as data errors and missing. However, we must still be comprehensively aware of the specific limitations of studies using such data and do everything possible to lessen their impact. Our research demonstrated the current reporting state of cohort studies using RWD in recent years. There are no restrictions on population and exposure measures, and results have wider applicability. According to our research, we can recognize that there are various issues with this type of study which may be caused by the inadequate dissemination and endorsement of pertinent guidelines, the incomplete methodology consensus, etc. However, we realized that full compliance with the RECORD guidelines is almost impossible in some circumstances because of the technical issues involved in data processing, local policy implications, etc. Funding This work was supported by the National Natural Science Foundation of China (Grant No. 82274685); the Scientific and Technological Innovation Project of China Academy of Chinese Medical Sciences (Grant No. CI2021A01009; the Scientific and Technological Innovation Project of China Academy of Chinese Medical Sciences (Grant No. CI2021A05312). Consent for publication Not applicable. Availability of data and materials The full data set is available on request from the corresponding author. Ethics approval and consent to participate Not applicable. Authors' contributions R Zhao and W Zhang: project development, search strategy development, data screening and extraction, evaluation, data analysis, manuscript writing. ZD Zhang and C He: data screening and extraction, evaluation, data rectification. R Xu: search strategy development, data analysis, manuscript writing. B Wang and XD Tang: project development, data rectification, manuscript writing. All authors revised the manuscript and approved the final version. The authors declare that they have no competing interests. The authors declare that they have no competing interests. Discussion But for real-world studies, RWD should be made realistic, standardized, and easy to handle from the inception of the study dataset development, otherwise such studies would create more risks of bias different from traditional research methods, which requires the involvement of policy makers, technical personnel, investigators, clinicians, epidemiologists, and methodologists. And at least, researchers can standardize data and research processes as much as possible, we anticipate that our research can promote the spreading of RECORD and suggest the possible direction for researchers to improve RWE quality. There are some limitations to this research. First, we only used the RECORD checklist to evaluate included articles, and other important aspects of observational studies mentioned in the STROBE checklist were not evaluated, such as details of study design, statistical methods, and reporting of results. Second, some items may not have undergone a strict enough evaluation, such as item R12.3 was deemed sufficient if the author described the level, techniques, and methods of data link or the method to evaluate its quality, which did not demand to be fully detailed. Third, our search strategy cannot retrieve studies that did not mention the “real world” in the paper, consequently, we may overlook many articles that met the criteria, but the final inclusion result was within the acceptable range. Finally, subjectivity may be an inevitable part of the research process. We did not analyze the differences between the results of different reviewers, but we tried to eliminate discrepancies by ensuring the independence of the review and conducting panel discussions. Page 12/19 To conclude, the reporting of cohort studies using RWD was generally inadequate and has not improved in recent years. Journal IFs and article citations were significantly related to the reporting of some areas. We encourage researchers to endorse relevant guidelines when utilizing RWD for research to maximize the value of RWD, obtain high-quality RWE, and prevent misleading clinical decisions. Acknowledgements Page 13/19 Page 13/19 Not applicable. References 1. 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Linking electronic health data in pharmacoepidemiology: Appropriateness and feasibility. Pharmacoepidemiol Drug Saf 2020;29:18–29. https://doi.org/10.1002/pds.4918. 34. Pratt NL, Mack CD, Meyer AM, Davis KJ, Hammill BG, Hampp C, et al. Data linkage in pharmacoepidemiology: A call for rigorous evaluation and reporting. Pharmacoepidemiol Drug Saf 2020;29:9–17. https://doi.org/10.1002/pds.4924. 34. Pratt NL, Mack CD, Meyer AM, Davis KJ, Hammill BG, Hampp C, et al. Data linkage in pharmacoepidemiology: A call for rigorous evaluation and reporting. Pharmacoepidemiol Drug Saf 2020;29:9–17. https://doi.org/10.1002/pds.4924. 35. Gilbert R, Lafferty R, Hagger-Johnson G, Harron K, Zhang L-C, Smith P, et al. GUILD: GUidance for Information about Linking Data sets. J Public Health Oxf Engl 2018;40:191–8. https://doi.org/10.1093/pubmed/fdx037. 35. Gilbert R, Lafferty R, Hagger-Johnson G, Harron K, Zhang L-C, Smith P, et al. GUILD: GUidance for Information about Linking Data sets. J Public Health Oxf Engl 2018;40:191–8. https://doi.org/10.1093/pubmed/fdx037. 36. Ehsani-Moghaddam B, Martin K, Queenan JA. Data quality in healthcare: A report of practical experience with the Canadian Primary Care Sentinel Surveillance Network data. Health Inf Manag J 2021;50:88–92. https://doi.org/10.1177/1833358319887743. 36. Ehsani-Moghaddam B, Martin K, Queenan JA. Data quality in healthcare: A report of practical experience with the Canadian Primary Care Sentinel Surveillance Network data. Health Inf Manag J 2021;50:88–92. https://doi.org/10.1177/1833358319887743. 37. Reps JM, Schuemie MJ, Suchard MA, Ryan PB, Rijnbeek PR. Design and implementation of a standardized framework to generate and evaluate patient-level prediction models using 37. Reps JM, Schuemie MJ, Suchard MA, Ryan PB, Rijnbeek PR. Design and implementation of a standardized framework to generate and evaluate patient-level prediction models using Page 16/19 Page 16/19 Figure 1 Flow diagram. Figures Page 17/19 This is a list of supplementary files associated with this preprint. Click to download. This is a list of supplementary files associated with this preprint. Click to download. Page 18/19 PRISMA2020abstractchecklist.docx PRISMA2020checklist.docx S1Table.TransformedRECORDchecklist..docx S1File.Searchstrategy..docx S2File.Listofincludedarticles..docx S3File.Detailsofevaluationresults..xlsx Page 19/19
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Communication, the centrosome and the immunological synapse
Philosophical transactions - Royal Society. Biological sciences
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Review Cite this article: Stinchcombe JC, Griffiths GM. 2014 Communication, the centrosome and the immunological synapse. Phil. Trans. R. Soc. B 369: 20130463. 1. Directed cell-to-cell interaction in the immune system The mammalian immune system is composed of a variety of cell types each specialized to combat a particular area of infection and disease. CD4 T cells, for example, act as ‘helper’ cells. On activation during an immune response, CD4 T cells release cytokines which can enhance the response of other immune cells. By contrast, cytolytic immune cells such as cytotoxic T-lymphocytes (CTL) and natural killer (NK) cells release lytic proteins which induce apoptosis and kill unwanted tumour or virally infected cells. To prevent aberrant killing, lytic pro- teins are stored within the immune cell in specialized ‘secretory’ lysosomes [1] (also called secretory or cytolytic granules) and only release their content on encountering an unhealthy cell. http://dx.doi.org/10.1098/rstb.2013.0463 One contribution of 18 to a Theme Issue ‘The centrosome renaissance’. One contribution of 18 to a Theme Issue ‘The centrosome renaissance’. rstb.royalsocietypublishing.org Recent findings on the behaviour of the centrosome at the immunological synapse suggest a critical role for centrosome polarization in controlling the communication between immune cells required to generate an effective immune response. The features observed at the immunological synapse show parallels to centrosome (basal body) polarization seen in cilia and fla- gella, and the cellular communication that is now known to occur at all of these sites. Jane C. Stinchcombe and Gillian M. Griffiths Jane C. Stinchcombe and Gillian M. Griffiths Cambridge Institute for Medical Research, University of Cambridge, Cambridge CB2 OXY, UK Subject Areas: cellular biology The function of these and many other immune cells depends on intimate inter- actions between the immune cell and a second cell, usually termed the ‘antigen- presenting cell (APC)’ or ‘target’ (figure 1a(iii)). The nature of these interactions varies with respect to frequency, length and stability between different cells of the immune system. For example, cytokine production by CD4 T cells is often acti- vated by interaction with dendritic cells. These interactions are stable, lasting for hours, and result in sustained cytokine production. Other interactions, for example those of CTL or NK cells with infected or tumour cells are fast, transient and sequential, and result in destruction of the unwanted ‘target’. Despite differences in duration, stability and outcome, most interactions share important structural and functional features and involve receptor-triggered recognition (for example by T-cell receptor (TCR) in T cells) accompanied by dramatic changes in protein organization and cell morphology. The membrane proteins across the cell : cell contact site reorganize to form a distinct topological rearrangement termed the ‘immunological synapse’ comprising membrane domains known as ‘supramole- cular activation clusters’ (SMAC) (figures 1b and 2a). Synapse structures were first identified in CD4 T cells interacting with APC, in which a peripheral ring of T-cell integrin proteins (pSMAC) was found to surround a central (cSMAC) region that accumulated TCR and other signalling proteins [2,3]. An outer distal (dSMAC) ring, rich in actin and actin-associated proteins, has also been identified. Similar basic structures have since been identified at the contact site between many immune cells and their APCs although there are cell type-specific differences. For example, synapses formed between CTL and unhealthy target cells contain an additional central ‘secretory’ domain. This appears opposite a space between the two cells termed the secretory cleft into which lytic proteins are released [4] (figure 2a). It is thought that this sort of membrane organization allows cell-to- cell communication including exchange of signals, material and information between the two cells. Morphological changes involve polarization of the Cambridge Institute for Medical Research, University of Cambridge, Cambridge CB2 OXY, UK Cambridge Institute for Medical Research, University of Cambridge, Cambridge CB2 OXY, UK Keywords: target synapse a) (i) (i) (ii) (iii) (ii) (iii) (iv) (b) target target 2 target 1 target target rstb.royalsocietypublishing.org Phil. Trans. R. Soc. B 369: 20130463 2 2 (b) (a) a) (i) (ii) (i) (i) rstb.royalsocietypublishing.org (ii) (iii) (iv) target (iv) (iv) target 2 target 1 (iii) target (iii) Figure 1. Properties of immune cell centrosomes interacting with target cells. (a) Centrosome positioning in T cells. In rounded naive or sedentary T lymphocytes (i), the centrosome (red) is close to the nucleus (dark orange) and microtubules (black) radiate from the cell centre towards the membrane. Centrosomes are located in the uropod at the back of the cell in migrating lymphocytes (ii), and rapidly polarize to the contact site synapse on interaction with a target (grey, target) (iii). On contact with further targets (iv), the centrosome retracts from the first target (1) and polarizes towards the second (2). Straight arrow in (ii) shows the direction of cell movement during migration. Curved arrow in (iv) shows repolarization of the centrosome on contact with a second target. (b) Centrosome polarization, secretion and signalling at different stages of immune cell interaction with targets. Low power cartoon (left) and corresponding medium power diagram (right) showing the different stages of immune cell (blue, cartoon; main cell, diagram) interaction with a target (grey, target, cartoon). On meeting a target (i), the immune cell centrosome (red, cartoon; brown, diagram) orientates towards the target and moves towards the contact site, reorganizing the microtubule network (black/ grey lines) and microtubule-associated organelles, including secretory vesicles (yellow) and cytolytic secretory granules (orange). Signalling pathways are activated on cell : cell contact (red arrows). Tight centrosome polarization to the membrane (ii) aligns microtubules along the contact site and allows secretory organelles to access secretory sites at the synapse (pale yellow, diagram). Retraction of the centrosome (iii) removes the microtubule network and associated organelles from the contact site and prevents further secretion, terminating the functional response. Arrows indicate direction of centrosome movement. Membrane domains (or ‘supra- molecular activation clusters’ (SMAC)) of the immunological synapse are indicated by colour bars in the diagram and show the integrin-containing peripheral ring (pSMAC, green) surrounding a TCR- and signalling protein-rich central domain (cSMAC, red) and a secretory domain (yellow) which, in lytic cells, forms beside the cSMAC and opposite an intracellular cleft (pale yellow, ii) (see also figure 2a). Keywords: ‘polarization’ phase during which the centrosome moves from the uropod round the nucleus to face the target, and a second slower ‘docking’ phase as it moves up to the synapse at the con- tact site [8]. Only the initial phase seems to occur when TCR signalling is compromised [9]. Live cell imaging has shown that the centrosome can oscillate back and forth at the mem- brane and may retract and abort completely, moving back into the cell body or polarizing to a different target [10]. At the end of the interaction the centrosome either repolarizes to a second target (figure 1a(iv)), moves back to its migratory position behind the nucleus in the uropod (figure 1a(ii)) or, in static cells, sits next to the nucleus (figure 1a(i)). Full retraction of the centrosome back into the cell body from the cell surface may be important in terminating the response at or before cell separation (see §7 and figure 1b). immune cell, in particular, orientation of the cytoskeleton towards the target cell [5–7] (figure 1a(iii)). Key to many of these changes, both at the membrane and within the cell, is polarization of the centrosome to the immunological synapse. Keywords: & 2014 The Authors. Published by the Royal Society under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0/, which permits unrestricted use, provided the original author and source are credited. target synapse (a) (i) (i) (ii) (iii) (ii) (iii) (iv) (b) target target 2 target 1 target target Figure 1. Properties of immune cell centrosomes interacting with target cells. (a) Centrosome positioning in T cells. In rounded naive or sedentary T lymphocytes (i), the centrosome (red) is close to the nucleus (dark orange) and microtubules (black) radiate from the cell centre towards the membrane. Centrosomes are located in the uropod at the back of the cell in migrating lymphocytes (ii), and rapidly polarize to the contact site synapse on interaction with a target (grey, target) (iii). On contact with further targets (iv), the centrosome retracts from the first target (1) and polarizes towards the second (2). Straight arrow in (ii) shows the direction of cell movement during migration. Curved arrow in (iv) shows repolarization of the centrosome on contact with a second target. (b) Centrosome polarization, secretion and signalling at different stages of immune cell interaction with targets. Low power cartoon (left) and corresponding medium power diagram (right) showing the different stages of immune cell (blue, cartoon; main cell, diagram) interaction with a target (grey, target, cartoon). On meeting a target (i), the immune cell centrosome (red, cartoon; brown, diagram) orientates towards the target and moves towards the contact site, reorganizing the microtubule network (black/ grey lines) and microtubule-associated organelles, including secretory vesicles (yellow) and cytolytic secretory granules (orange). Signalling pathways are activated on cell : cell contact (red arrows). Tight centrosome polarization to the membrane (ii) aligns microtubules along the contact site and allows secretory organelles to access secretory sites at the synapse (pale yellow, diagram). Retraction of the centrosome (iii) removes the microtubule network and associated organelles from the contact site and prevents further secretion, terminating the functional response. Arrows indicate direction of centrosome movement. Membrane domains (or ‘supra- molecular activation clusters’ (SMAC)) of the immunological synapse are indicated by colour bars in the diagram and show the integrin-containing peripheral ring (pSMAC, green) surrounding a TCR- and signalling protein-rich central domain (cSMAC, red) and a secretory domain (yellow) which, in lytic cells, forms beside the cSMAC and opposite an intracellular cleft (pale yellow, ii) (see also figure 2a). 2. The polarized centrosome 2 1 ciliated cell 3 2 1 4 5 (b) (b) ciliated cell ciliated cell Figure 2. Morphological and functional similarities between the immunological synapse and cilia. Side views of the immunological synapse in CTL (a) and cilia region of a ciliated epithelial cell (b), showing centrosomes (brown), microtubules (black, grey lines) and associated organelles organized under the membrane. (a) Morphological features of the synapse include organization of the membrane into distinct ‘supramolecular activation cluster’ (SMAC) domains in which actin is present in a distal ring (dSMAC, blue) surrounding a peripheral ring of integrins (pSMAC, green) enclosing both a central (c)SMAC (red) containing TCR, and a secretory domain (yellow) opposite an intracellular cleft (pale yellow). The centrosome (brown) contactsthe membrane between the cSMAC and secretory domain. Microtubule-associated organelles including the Golgi complex (light green) and biosynthetic vesicles (dark green), early and recycling endocytic compartments (blue), late endosomes and multivesicular bodies (grey/ beige), constitutive secretory organelles (yellow) and regulated secretory lysosomes/lytic granules (orange) are present in the cytoplasm around the centrosome beneath the membrane. Signalling pathways (black numbers) include ion or protein channels (1) or membrane receptors (2,3). Membrane receptors can be activated at the cSMAC (2) or the target cell (3), by ligands (4–6) either present on opposing membranes (4), associated with released lysosomal vesicular content [1] (5), or released as free soluble proteins (6). Membrane trafficking pathways (grey numbers/arrows) include endocytic recycling (light blue, 1), degradative (grey, 2), biosynthetic (green, 3) and secretory (yellow/orange, 4,5) pathways. Secretory vesicles (yellow, 4) and cytolytic secretory organelles (orange, 5) are delivered to secretory sites at the cleft (pale yellow). (b) Morphological features of ciliated epithelia include (right hand side) separation of the apical surface (black membrane) from the basal (red membrane) by adherens junctions (sludge green) and (centre) organization of the surface membrane into distinct domains at and around the cilium, including the endocytic (tur- quoise) and secretory (yellow) regions of the cilia pocket at the cilium base, and the transition zone (green), inversin compartment (dark pink) and distal regions (light pink) of the cilium. The basal body (brown) sits at, and anchors, the base of the cilium. Microtubule-associated organelles including the Golgi complex (light green) and biosynthetic vesicles (dark green), endocytic recycling compartments (blue), late endosomes and multivesicular bodies (grey/brown), and constitutive (yellow) and regu- lated (orange) secretory organelles are all present around or near the centrosome at the cilia base. 2. The polarized centrosome (a) Morphological features of the synapse include organization of the membrane into distinct ‘supramolecular activation cluster’ (SMAC) domains in which actin is present in a distal ring (dSMAC, blue) surrounding a peripheral ring of integrins (pSMAC, green) enclosing both a central (c)SMAC (red) containing TCR, and a secretory domain (yellow) opposite an intracellular cleft (pale yellow). The centrosome (brown) contactsthe membrane between the cSMAC and secretory domain. Microtubule-associated organelles including the Golgi complex (light green) and biosynthetic vesicles (dark green), early and recycling endocytic compartments (blue), late endosomes and multivesicular bodies (grey/ beige), constitutive secretory organelles (yellow) and regulated secretory lysosomes/lytic granules (orange) are present in the cytoplasm around the centrosome beneath the membrane. Signalling pathways (black numbers) include ion or protein channels (1) or membrane receptors (2,3). Membrane receptors can be activated at the cSMAC (2) or the target cell (3), by ligands (4–6) either present on opposing membranes (4), associated with released lysosomal vesicular content [1] (5), or released as free soluble proteins (6). Membrane trafficking pathways (grey numbers/arrows) include endocytic recycling (light blue, 1), degradative (grey, 2), biosynthetic (green, 3) and secretory (yellow/orange, 4,5) pathways. Secretory vesicles (yellow, 4) and cytolytic secretory organelles (orange, 5) are delivered to secretory sites at the cleft (pale yellow). (b) Morphological features of ciliated epithelia include (right hand side) separation of the apical surface (black membrane) from the basal (red membrane) by adherens junctions (sludge green) and (centre) organization of the surface membrane into distinct domains at and around the cilium, including the endocytic (tur- quoise) and secretory (yellow) regions of the cilia pocket at the cilium base, and the transition zone (green), inversin compartment (dark pink) and distal regions (light pink) of the cilium. The basal body (brown) sits at, and anchors, the base of the cilium. Microtubule-associated organelles including the Golgi complex (light green) and biosynthetic vesicles (dark green), endocytic recycling compartments (blue), late endosomes and multivesicular bodies (grey/brown), and constitutive (yellow) and regu- lated (orange) secretory organelles are all present around or near the centrosome at the cilia base. Signalling pathways (black numbers) include activation by extracellular ligands of ion or protein channels (1) or membrane receptors at the cilium membrane (2). Membrane trafficking pathways (grey numbers and arrows) include endocytic recycling (light blue, 1), degradative (dark blue/grey/beige, 2), biosynthetic (green, 3), and secretory (constitutive (yellow, 4) and regulated (orange, 5)) pathways. 2. The polarized centrosome Centrosome positioning varies in T cells (figure 1a). In migratory T cells, the centrosome is in the uropod, slightly dis- tant from the nucleus (e.g. figure 1a(ii)). This contrasts with most migratory cells which move with the centrosome in front of the nucleus. Upon contact with a target, the centrosome begins to polarize towards the synapse (figure 1a(iii)). Recent studies suggest that polarization may be biphasic, with a fast initial 3 4 2 1 5 5 2 1 target lytic cell ciliated cell 6 6 3 4 2 1 5 3 2 1 4 5 (a) (b) ological and functional similarities between the immunological synapse and cilia. Side views of the immunological synapse in CTL (a) and cell (b), showing centrosomes (brown), microtubules (black, grey lines) and associated organelles organized under the membrane. (a napse include organization of the membrane into distinct ‘supramolecular activation cluster’ (SMAC) domains in which actin is present ounding a peripheral ring of integrins (pSMAC, green) enclosing both a central (c)SMAC (red) containing TCR, and a secretory domain (y ft (pale yellow). The centrosome (brown) contactsthe membrane between the cSMAC and secretory domain. Microtubule-associated orga (light green) and biosynthetic vesicles (dark green), early and recycling endocytic compartments (blue), late endosomes and multivesicu e secretory organelles (yellow) and regulated secretory lysosomes/lytic granules (orange) are present in the cytoplasm around the cent gnalling pathways (black numbers) include ion or protein channels (1) or membrane receptors (2,3). Membrane receptors can be activat ell (3), by ligands (4–6) either present on opposing membranes (4), associated with released lysosomal vesicular content [1] (5), or 6) M b t ffi ki th ( b / ) i l d d ti li (li ht bl 1) d d ti ( 2) bi th ti 3 4 2 1 5 5 target lytic cell 6 6 3 4 2 1 5 (a) (a) 2 1 lytic cell ciliated cell 3 2 1 3 2 1 4 5 (b) Figure 2. Morphological and functional similarities between the immunological synapse and cilia. Side views of the immunological synapse in CTL (a) and cilia region of a ciliated epithelial cell (b), showing centrosomes (brown), microtubules (black, grey lines) and associated organelles organized under the membrane. 2. The polarized centrosome Signalling pathways (black numbers) include activation by extracellular ligands of ion or protein channels (1) or membrane receptors at the cilium membrane (2). Membrane trafficking pathways (grey numbers and arrows) include endocytic recycling (light blue, 1), degradative (dark blue/grey/beige, 2), biosynthetic (green, 3), and secretory (constitutive (yellow, 4) and regulated (orange, 5)) pathways. The movement of the centrosome across almost the entire length of the migrating T cell is far more dramatic than that seen in other cell types where the centrosome may only polar- ize from one side of the nucleus to the other [11]. Very tight polarization of the centrosome to the plasma membrane at the synapse was first identified in studies on CTL and sub- sequently observed by electron microscopy (EM) for other lytic cells [12–15]. A time course of EM images suggests that the tight polarization process may take longer in CD4 T cells and only appears in samples fixed after 2–4 h of interaction, the time of peak cytokine secretion [14]. been shown to polarize with the centrosome [23]. It was pro- posed that this might be due to loss of actin clearance from the site of release as cdc42 is required for actin remodelling at secretory sites. 4 rstb.royalsocietypublishing.org 3. The centrosome as the master regulator The importance of centrosome movement towards the contact site for immune cell function was first recognized in early studies showing that centrosome polarization towards the point of membrane contact occurred during target killing [5–7]. The centrosome was subsequently found to move right up to the contact site and associate with the membrane at a specific point between the region of TCR clustering and the sites of secretory granule docking and secretion [12,13]. These observations demonstrated a role for centrosome polarization in granule delivery to the secretory cleft since this reorganizes the microtubule network to lie parallel to the membrane under the secretory sites, thus directing and delivering the gran- ules to the secretory sites during minus-end directed movement along the polarized microtubules [12,13] (figure 1b(i–ii)). Evidence is now emerging that suggests that, like the synapse (figure 2a), the ciliary sheath and the plasma mem- brane surrounding the polarized centrosome of cilia and flagella (called basal body) is organized into distinct functional domains [27] (figure 2b). The transition zone acts as a selective barrier between the cell body and the cilium [27–29], while sig- nalling, signal transduction and protein release occur within the cilium itself [30–33]. Thus, like the synapse of immune cells, the membrane surrounding polarized basal bodies at sites of cilia formation also contains distinct domains devoted to adhesion, signalling and secretion. Other insights into centrosome polarization to the synapse have come from studies on B cells in which the centrosome was ablated [16,17]. B cells also secrete lysosomal content on interaction with APCs, and proteases released from the lyso- somes are involved in extracting antigens from the target cell membranes. B cells lacking centrosomes showed no reor- ganization of their microtubule cytoskeleton, no movement of microtubule-organizing centre (MTOC)-associated organelles towards the target and no release of lysosomal content. Further- more, lysosomes showed no directed movement either towards the contact site or the site of the ablated centrosome. Thus, the centrosome itself is required for polarized secretion in B cells. 4. Morphological similarities between centrosome polarization sites at the immunological synapse and cilia Tight centrosome polarization right up to the membrane is unusual but is also seen in cells with cilia and flagella. Although cilia are much more stable than the transient synapses formed by immune cells, there are many morpho- logical similarities between these structures (summarized in figure 2). Intriguingly, primary cilia and flagella are often embedded within invaginations of the cell membrane, termed the cilia/flagella pocket [24,25]. This pocket bears similarities to the secretory clefts which form opposite polar- ized centrosomes of immune synapses [4]. Curiously, the membrane of immune cells can form membrane bumps and protrusions that project into these clefts above the centrosome [13,26]. Although these may appear to parallel cilia structures they lack axonemes and their significance is unclear. 5. Molecular and functional similarities between cilia and the synapse Centrosome polarization directs many events at the synapse. MTOC-associated organelles including the Golgi complex, which processes newly synthesized proteins, and endocytic recycling compartments, responsible for downregu- lation of membrane proteins including TCR and associated proteins LAT and Lck [18–20], are focused at the immunologi- cal synapse when the centrosome is at the membrane (figure 2a). This means that centrosome polarization can effectively control communication at the synapse, with signalling, secretion and recycling effectively activated (‘switched on’) when the centrosome is in contact with the plasma membrane (figure 1b(ii)) and ‘switched off’ when retracted (figure 1b(i,iii)). This seems likely to be particularly relevant for control- ling secretion from cytolytic cells because mutations which allow centrosome polarization close to, but not at, the plasma membrane, prevent secretion [9]. Several proteins required for ciliogenesis have also been shown to be involved in formation of the immunological synapse. Cor- rect positioning of the basal body during ciliogenesis is known to involve polarity proteins including dishevelled (Dvl), Par3, Par6 and atypical protein kinase C proteins (aPKCs) [34,35]. Many of these have now been shown to be involved at different stages in T-cell polarity, with Par6 and the aPKC isoform aPKC-z involved during T-cell migration and detection of target cells [36], while Par3, Scribble and Discs large (Dlg) show polarized front-back and lateral distributions during interactions of T cells with APCs [37,38], and aPKC-z enriches with Par3 at the synapse [39]. Of particular interest, ‘cilia’ proteins have now been shown to be present in events at the immunological synapse. The intraflagellar transport (IFT) components, IFT88 and IFT20, first identified as part of the complex transporting material through cilia and flagella (reviewed in [40]), have been impli- cated in TCR recycling at the synapse [41]. Roles for IFT proteins outside the cilium in ciliated cells had previously been described. For example, IFT20 is involved in vesicular transport between the Golgi complex and the base of the grow- ing/mature cilium [42] and in some ciliated cell types, such as Centrosome polarization may not be required for secretion of all cytokines from CD4 T cells [21]. A recent study suggested cytokine release from CD4 T cells can occur in the absence of centrosome polarization [22]. 6. Polarized movement of centrosomes to and from the cell surface in immune and cilliated cells y y g y p Signal transduction is now recognized as a major function of primary cilia, with recent reports suggesting that primary cilia are specialized calcium-signalling organelles [50]. Sev- eral signalling pathways have been identified in cilia including Hedgehog (Hh), Wnt, Notch, mTOR and receptor tyrosine kinase pathways (reviewed in [30–33]). A primary role for cilia in signalling is in cell-to-cell communication and information exchange. Thus, signalling in cilia parallels that in immune cells, where signalling is involved in, and results from, interaction of immune cells with targets. Cilia project from the cell surface allowing them to transduce sig- nals between the extracellular environment and the cell body. Components of the signalling pathways (including membrane receptors, signal transducers (including ion chan- nels) and effector proteins) are localized to the cilia membrane which allows them to detect and respond to extra- cellular ligands and/or changes in the external environment [30–33] (figure 2b). Signal reception and transduction may be molecular, via detection of chemical changes (e.g. for uni- cellular organisms) or binding of ligands to receptors on the cilia membrane (e.g. binding of Hh to Patched (Ptch) [51–53]) or at the cilium base (e.g. Wnt binding to Frizzled) and activate downstream signalling pathways within the cilium and/or the cell body. Alternatively, extracellular signals may be mechanical, mediated by bending forces on the cilia membrane which activate calcium fluxes and are driven, for example, by fluid flow in the surrounding milieu [54,55] (e.g. for renal tubule cells of the kidney). In addition, cilia generate signals which act locally or at a distance, for example by cleavage of peptide domains (e.g. proteolytic processing of polycystin-1 in the kidney duct) or shedding of membrane [49] from the cilia surface. The details of the mechanics controlling centrosome move- ment in immune cells are gradually emerging from a number of different studies. IQGAP1 forms part of a complex of proteins linking the plus ends of microtubules to the actin cortex [61,62]. Our early investigations suggested that IQGAP might link the movement of the centrosome to the reorganiz- ation of actin at the synapse [12]. This was supported by the finding that dynein associated with the outer ring of actin at the synapse, suggesting dynein might play a role in pulling the centrosome towards the synapse [10]. 5. Molecular and functional similarities between cilia and the synapse Silencing of cdc42 at the site of secretion did not affect centrosome polarization but did prevent release of IL2 and IFN-g, two cytokines that have ciliated retinal cells, appears exclusively localized to Golgi- associated vesicle transport pathways [43]. IFT88, although primarily involved in transport within the cilium, is also found associated with basal bodies at early stages of cilio- genesis [43] and is required for correct positioning and orientation of developing cilia within the apical membranes of epithelia [44]. The observations in immune cells were the first demonstration of a role for ciliary proteins in cells that do not make cilia. The involvement of IFT88 in immune cells is particularly intriguing given the role of the centrosome in positioning organelles at both the synapse and cilium. IFT20 has since been identified in other non-ciliated cells such as reti- nal neurons, where several IFT proteins localize to the dendrites [45]. Recently, the ciliary membrane protein inver- sin/nephrocystin-2 was shown to be required for the polarity and directional migration of fibroblasts [46]. Thus, there is increasing evidence of ciliary proteins playing more general roles in pathways outside the cilium (see also [47]). controlling Hh signalling pathways (reviewed, for example in [51–53]). Binding of extracellular Hh to Ptch on the cilia surface causes translocation of Ptch out of the cilium and movement of the transmembrane protein Smoothened (Smo) into it. Reloca- tion of Smo to the primary cilium is required for translocation of Gli pathway activators to the nucleus and initiation of Gli-mediated transcription of target genes. It has been known for some time that Hh signalling is required during T cell devel- opment in the thymus [56]. What has only recently emerged is that TCR signalling at the immunological synapse triggers Hh signalling within T cells [57]. The Hh pathway plays an impor- tant functional role in CTL since cells in which Smo is deleted show impaired target cell killing. How does this happen? It takes 4–5 days for CTL to be generated from naive T cells, and TCR signalling is required to trigger small, naive T cells to produce the cytolytic secretory granules and dynamic cytos- keleton required for delivery of these secretory granules to the synapse. Although granules develop normally after Smo del- etion, Rac1, required for actin remodelling and centrosome polarization [58–60], fails to upregulate. rstb.royalsocietypublishing.org Phil. Trans. R. Soc. B 369: 20130463 5 5 rstb.royalsocietypublishing.org Phil. Trans. R. Soc. B 369: 20130463 rstb.royalsocietypublishing.org rg Phil. Trans. R. Soc. B 369: 20130463 As at the immunological synapse, secretion can also be focused at cilia (figure 2b). Unicellular paramecia release their specialized storage organelles (trichocysts) at distinct exocytic secretory domains at the base of cilia [48]. Membrane vesicles have been shown to be released from the cilium of Chlamydomo- nas [49]. These ‘ectosomes’ contain proteases required for cell wall breakdown during reproduction and provide a remarkable parallel to the release of lytic and lysosomal proteases from secretory lysosomes at the immunological synapse. 5. Molecular and functional similarities between cilia and the synapse In this way, Hh signal- ling triggered by TCR at the synapse controls the ability of the centrosome to polarize to the synapse which is required for CTL to destroy target cells. 7. Centrosome retraction At the end of interaction of an immune cell with a target, the centrosome retracts back from the surface into the cell body (figure 1b(iii)) and the domain organization of the synapse membrane proteins disperses as the two cells separate. Retrac- tion of the basal body is also seen during downregulation of cilia and flagella in response to changes in the extracellular environment, for example tissue damage, release of stress or changes in nutritional supply. For immune cells, retraction allows repolarization to further targets or return to a migratory phase (figure 1a(ii, iv)), while for ciliated cells it permits re-entry into the cell cycle and cell division. In the unicellulate Chlamydo- monas, flagella are resorbed under stress conditions by severing between the basal body and transition zone by the microtubule ATPase, Katanin p60 [71–73], which is thought to liberate basal bodies to return to the cell body for mitosis. In mammalian cells, control is regulated by displacement of TTBK2 and MARK4 kinases and rebinding of CP110, Cep97, Kif24 and Trichoplein (reviewed in [74]). Little is known about the mechanisms gov- erning retraction of the centrosome from the synapse since it is not yet known if or how the centrosome ‘docks’ at the plasma membrane. What is clear is that in immune cells, as in cilia, tight association with the plasma membrane appears to be able to regulate function at the immunological synapse. The absence of cilia in immune cells is also unusual because there is a correlation between the presence of a centrosome and the capacity to form cilia or flagella, and most cell types which lack flagella or cilia also lack centrioles. For example, unciliated myotubes lose their centrosomes during differentiation [79], while cilia are not observed on cells/species with alternative microtubule-organizing structures, for example Dictyostelium and Saccharomyces cerevisiae [78]. A few specialized cell types do appear to retain polarized centrosomes in the absence of cilia. Differentiated enterocytes show permanently polar- ized centrosomes at the apical surface but lose their cilia as embryogenesis and tissue differentiation progress [80]. The centrosome remains at the surface associated with a vestigial cilium remnant but the adult tissue entirely lacks mature cilia, suggesting downregulation or loss of cilia mechanisms with development and differentiation. Other specialized tissues have functionally modified cilia or flagella, differen- tiated for particular roles by accentuation or loss of specific cilia components. 6. Polarized movement of centrosomes to and from the cell surface in immune and cilliated cells More recently, the centrosomal protein CK1d has been shown to be involved since its depletion leads to loss of centrosome polarization [63]. Intriguingly, CK1d phosphorylates the micro- tubule plus-end binding protein EB1, an activity that seems to be required for centrosome translocation, leading to the propo- sal that Ck1d plays a role in centrosome polarization in T cells by increasing microtubule growth speeds. The most recent observations on centrosome polarization also implicate micro- tubule dynamics in bringing the centrosome forward to the synapse [8]. This study suggested that the ‘pioneer’ microtubules seen in migrating cells [64] might also be present and tethered at the synapse and shrink asthe centrosome moves forward, raising thepossibilitythat centrosomepolarizationoccurs byaprocess of end-on capture-shrinkage. A growing body of evidence suggests formins are also involved in the mechanisms and direction of centrosome movement (reviewed in [65]). Formins are Rho-family GTPase effectors which act as linear actin nucleators and have roles including stabilization of microtubules. TCR acti- vation induces the formins INF2, DIA1 and FMNL1 to form an array of stable, detyrosinated, glutamated (Glu) microtu- bules around the centrosome. It is thought that these disrupt the steady-state organization of the microtubule Hh pathways are involved in regulating a number of devel- opmental processes. Many key steps involved in Hh signalling occur within the primary cilium, and movement and exchange of proteins between the cilia and the cell body is critical for mother centriole in front of the daughter and associated via the distal appendages of the mother centriole (figure 2b). On docking, distal end microtubules elongate from the mother centriole to form the axoneme of the cilium, and the basal body is anchored at the membrane, forming a stable structure. By contrast, immune cell centrosomes remain associated with the synapse only transiently. As CTL can kill targets rapidly (within 5 min of interaction) while movingbetween multiple tar- gets [75], the time of centrosome interaction with the plasma membrane must be brief. To date, there is noevidence of ciliafor- mation upon polarization of centrosomes to synapses [76,77]. It is not yet known whether this is because they lack components required for ciliaformation, ciliogenesis is regulated or inhibited, or simply because centrosome association with the plasma membrane is too transient to allow ciliogenesis to be initiated. network and that this results in pushing forces which drive and direct the centrosome forwards towards the target APC. rstb.royalsocietypublishing.org Phil. Trans. R. Soc. B 369: 20130463 6 6 rstb.royalsocietypublishing.org Phil. Trans. R. Soc. B 369: 20130463 6 Taken together, these data support the idea that actin and microtubule dynamics at the synapse play a role in generating the forces to pull the centrosome up to the synapse. Less is known about movement of the centriole to the surface during ciliogenesis. Early studies on primary cilia formation by Sorokin [66–68] suggested mother centrioles docked with membrane vesicles while still in the cell body. These vesicles coalesced to form vacuoles and the whole structure relocated to the surface. Axoneme extension and primary cilia growth was thought to initiate while the centrosome was still within the body and/or during transit to the surface. In other cells and tissues, centro- somes may interact directly with the plasma membrane at the cell surface, with transport of the centrosome mediated by forces generated by the cytoskeleton, possibly as has been pro- posed for immune cells (reviewed in [69,70]). It is not clear if the apparent differences in membrane association site reflect mechanistic differences between distinct cell or cilia types, for example primary cilia versus cilia on terminally differentiated cells, or whether different components of a single common pathway have been identified in distinct systems. Comparisons between centrosome behaviour during ciliogenesis and the mechanisms reported for immune cells may help elucidate the processes involved in both pathways in the future. org Phil. Trans. R. Soc. B 369: 20130463 7. Centrosome retraction Transient centrosome polarization in haemo- poietic cells could therefore be seen as another form of extreme cilia specialization, in this case without production of the cilium, and/or of downregulation of ciliogenesis as a result of the fully differentiated state. The identification of ‘ciliogen- esis’ proteins such as IFT and Hh [41,57] in immune cells supports this idea and suggests that at least some mechanistic proteins are conserved within immune cells and required for their function. Further studies to determine whether additional ‘ciliogenesis’ or ‘ciliary’ proteins are present and/or play roles in immune cell function, and vice versa, should shed more 9. Origins and evolutionary significance of centrosome polarization in immune cells Cilia and flagella are found on cells across phyla and are of particular functional importance to unicellulates. Further- more, although not all cells and tissues require cilia or flagella for their steady-state ‘normal’ function, most can pro- duce primary cilia under particular conditions, for example nutritional or environmental stress. Thus, they are evolution- arily primitive and widely conserved [78]. The fact that immune cells show centrosome polarization and share simi- larities with cilia on polarization but do not appear to make cilia is therefore particularly striking. This has led to the pro- posal that haemopoietic cells might exploit mechanisms used for primary ciliogenesis to orientate cell polarity, trafficking pathways, signalling processes and information towards target cells on target cell encounter [12,26]. 6. 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Molecular Mechanisms of Kidney Injury and Repair
International journal of molecular sciences
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  g p * Correspondence: asola@idibell.cat Citation: Rayego-Mateos, S.; Marquez-Expósito, L.; Rodrigues-Diez, R.; Sanz, A.B.; Guiteras, R.; Doladé, N.; Rubio-Soto, I.; Manonelles, A.; Codina, S.; Ortiz, A.; et al. Molecular Mechanisms of Kidney Injury and Repair. Int. J. Mol. Sci. 2022, 23, 1542. https://doi.org/ 10.3390/ijms23031542 Citation: Rayego-Mateos, S.; Marquez-Expósito, L.; Rodrigues-Diez, R.; Sanz, A.B.; Guiteras, R.; Doladé, N.; Rubio-Soto, I.; Manonelles, A.; Codina, S.; Ortiz, A.; et al. Molecular Mechanisms of Kidney Injury and Repair. Int. J. Mol. Sci. 2022, 23, 1542. https://doi.org/ 10.3390/ijms23031542 † These authors contributed equally to this work. Abstract: Chronic kidney disease (CKD) will become the fifth global cause of death by 2040, thus emphasizing the need to better understand the molecular mechanisms of damage and regeneration in the kidney. CKD predisposes to acute kidney injury (AKI) which, in turn, promotes CKD progression. This implies that CKD or the AKI-to-CKD transition are associated with dysfunctional kidney repair mechanisms. Current therapeutic options slow CKD progression but fail to treat or accelerate recovery from AKI and are unable to promote kidney regeneration. Unraveling the cellular and molecular mechanisms involved in kidney injury and repair, including the failure of this process, may provide novel biomarkers and therapeutic tools. We now review the contribution of different molecular and cellular events to the AKI-to-CKD transition, focusing on the role of macrophages in kidney injury, the different forms of regulated cell death and necroinflammation, cellular senescence and the senescence-associated secretory phenotype (SAPS), polyploidization, and podocyte injury and activation of parietal epithelial cells. Next, we discuss key contributors to repair of kidney injury and opportunities for their therapeutic manipulation, with a focus on resident renal progenitor cells, stem cells and their reparative secretome, certain macrophage subphenotypes within the M2 phenotype and senescent cell clearance. Received: 8 January 2022 Accepted: 26 January 2022 Published: 28 January 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Keywords: acute kidney injury; chronic kidney disease; regeneration; kidney; molecular mechanisms; cell death; treatment 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/). Review Molecular Mechanisms of Kidney Injury and Repair Sandra Rayego-Mateos 1,2 , Laura Marquez-Expósito 1,2 , Raquel Rodrigues-Diez 3,4 , Ana B. Sanz 2,5, Roser Guiteras 6, Nuria Doladé 7, Irene Rubio-Soto 1,2, Anna Manonelles 2,8 , Sergi Codina 8 , Alberto Ortiz 2,5 , Josep M. Cruzado 2,6,8, Marta Ruiz-Ortega 1,2,† and Anna Sola 2,6,*,† Sandra Rayego-Mateos 1,2 , Laura Marquez-Expósito 1,2 , Raquel Rodrigues-Diez 3,4 , Ana B. Sanz 2,5, Roser Guiteras 6, Nuria Doladé 7, Irene Rubio-Soto 1,2, Anna Manonelles 2,8 , Sergi Codina 8 , Alberto Ortiz 2,5 , Josep M. Cruzado 2,6,8, Marta Ruiz-Ortega 1,2,† and Anna Sola 2,6,*,† 1 Cellular Biology in Renal Diseases Laboratory, IIS-Fundación Jiménez Díaz, Universidad Autónoma Madrid, 28040 Madrid, Spain; srayego@fjd.es (S.R.-M.); laura.marqueze@quironsalud.es (L.M.-E.); 1 Cellular Biology in Renal Diseases Laboratory, IIS-Fundación Jiménez Díaz, Universidad Autónoma Madrid 28040 Madrid, Spain; srayego@fjd.es (S.R.-M.); laura.marqueze@quironsalud.es (L.M.-E.); p y g j q irene.rubios@quironsalud.es (I.R.-S.); Mruizo@fjd.es (M.R.-O.) 2 Departament of Systems Biology, Facultad de Medicina, Universidad de Alcalá, 28805 Alcalá de Henares, Spain; ASANZB@fjd.es (A.B.S.); amanonelles@bellvitgehospital.cat (A.M.); aortiz@fjd.es (A.O.); jmcruzado@bellvitgehospital.cat (J.M.C.) j ( ) j g p (J ) 3 Department of Pharmacology, Facultad de Medicina, Research Institute Hospital La Paz (IdiPAZ), U i id d A ó d M d id 28029 M d id S i l d i @ j ( ) j g p (J ) 3 Department of Pharmacology, Facultad de Medicina, Research Institute Hospital La Paz Universidad Autónoma de Madrid, 28029 Madrid, Spain; raquel.rodrigues@uam.es Department of Pharmacology, Facultad de Medicina, Research Institute Hospital La Pa Universidad Autónoma de Madrid, 28029 Madrid, Spain; raquel.rodrigues@uam.es Universidad Autónoma de Madrid, 28029 Madrid, Spain; raquel.rodrigues@uam.es 4 Centro de Investigación Biomédica en Red enfermedades Cardiovasculares (CIBERCV), Av. Monforte de Lemos, 28029 Madrid, Spain 4 Centro de Investigación Biomédica en Red enfermedades Cardiovasculares (CIBERCV), 5 Division of Nephrology and Hypertension, IIS-Fundación Jiménez Díaz, Universidad Autónoma Madrid, 28040 Madrid, Spain p 6 Department of Nephrology and Transplant Group, Institut d’Investigació Biomédica de Bellvitge (IDIBELL) 6 Department of Nephrology and Transplant Group, Institut d’Investigació Biomédica de Bellvitge (ID Hospitalet de Llobregat 08907 Barcelona Spain; r guiteras@gmail com Hospitalet de Llobregat, 08907 Barcelona, Spain; r.guiteras@gmail.com 7 Vascular and Renal Translational Research Group, Institut de Recerca Biomèdica de Lleida IRBLleida, 25198 Lleida, Spain; nuriadolade@gmail.com p g 8 Department of Nephrology, Hospital Universitari Bellvitge, 08907 Barcelona, Spain; scodina@bellvitgehospital.cat p g 8 Department of Nephrology, Hospital Universitari Bellvitge, 08907 Barcelona, Spain; International Journal of Molecular Sciences International Journal of Molecular Sciences International Journal of Molecular Sciences International Journal of Molecular Sciences International Journal of Molecular Sciences 1. Introduction Chronic kidney disease (CKD) is a growing disease burden, predicted to become the fifth global cause of death by 2040 [1]. The causes of CKD can be genetic, metabolic, immunological and vascular, among others, with metabolic (i.e., diabetes) and vascular https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2022, 23, 1542. https://doi.org/10.3390/ijms23031542 Int. J. Mol. Sci. 2022, 23, 1542 2 of 20 causes being the most common [2,3]. Therefore, different mediators and molecular mech- anisms participate in the progression of kidney damage (Figure 1) [2,3]. However, CKD is associated with an increased risk of CKD progression, death and acute kidney injury (AKI), which is independent from the cause of CKD. AKI may also have different causes, classically classified as prerenal (decreased perfusion), renal (parenchyma cell injury) and post-renal (urinary tract obstruction). Renal causes include untreated and persistent ob- struction or decreased perfusion, as well as tubular, glomerular and interstitial injury. A suboptimal array of early diagnostic biomarkers and of effective treatments for AKI or CKD contributes to the increasing global impact of kidney disease. Therefore, there is an urgent unmet medical need to further understand the mechanisms of kidney damage and repair to develop novel biomarkers and therapies that improve the outcomes of kidney diseases. Figure 1. Mechanisms of kidney injury and repair. Figure 1. Mechanisms of kidney injury and repair. There is a well-established link between AKI and CKD. CKD predisposes to AKI and AKI, especially repeat or severe AKI episodes, which in turn promote CKD progression [2,3]. Beyond the severity or persistence of AKI, there are no good biomarkers that predict the AKI-to-CKD transition and may guide the enrollment of early AKI patients in clinical trials aimed at preventing this transition. Thus, even potentially reversible causes of AKI, such as obstruction, may evolve into CKD if obstruction is not relieved within a certain time frame, as has been well documented experimentally [4]. Failure of serum creatinine to decrease is currently the best early indicator of the AKI-to-CKD transition, but it is still a late event that does not allow the initiating of preventive strategies when AKI is diagnosed. This raises the issue of defective kidney repair following AKI and/or a negative impact of prior CKD on kidney repair. The adult kidney displays repair mechanisms after AKI but has limited cell turnover. 2.1. Macrophages in Kidney Injury Inflammatory cell recruitment is an early event in AKI. Neutrophils are first recruited and favor the immediate recruitment of macrophages. These recruited neutrophils have a short life [7], whereas recruited macrophages and resident macrophages can persist for long periods in glomeruli or the tubulointerstitium. Macrophages have a critical role in wound healing and promote regeneration by bridging the initial inflammation and later tissue regeneration and repair phases. However, persistent or long-lasting macrophage presence in tissues may extend the damage phase, which finally leads to a failure of tubular repair, resulting in maladaptive kidney repair and contributing to the AKI-to-CKD transition [8–10]. Macrophages have a great plasticity to adapt to the environment, and their role in damage and/or repair is phenotype-dependent [11–13]. Thus, activation or deactivation of macrophages by different stimuli may determine the balance of healing versus injury. M1 macrophages have been proposed as inflammatory cells triggering kidney damage through pro-inflammatory cytokine secretion, including IL-6, TNFα and IL-1β [14,15] while M2 macrophages are involved in kidney tissue repair [16]. Different macrophage types coexist in the different disease phases of inflammation, repair/regeneration and fibrosis, but the M1/M2 ratio changes over time [12]. Pro-inflammatory macrophages have a key role in the onset of murine kidney disease through the specific transmembrane pattern recognition receptor Mincle [17], the M1 polar- ization regulator KLF4 [18] and/or the release of the specific macrophage inflammatory factors HMGB1, IL-1β and TNFα [19–22]. Moreover, damaged tubular epithelial cells communicate with and recruit M1 macrophages through miR-23a-enriched exosomes [23] or damage-associated molecular patterns (DAMPs) [24], promoting tubulointerstitial in- flammation. Thus, M1 depletion protects against kidney ischemia-reperfusion injury (IRI) in vivo [12], while the transference of M1 macrophages increases kidney injury [24]. M2 macrophages are classically considered anti-inflammatory cells. However, M2 macrophages are also involved in kidney fibrosis in mice [13,25], and in patients with type-2 diabetes there was a positive correlation between kidney CD163+ (M2) macrophages and fibrosis [26]. Hence, the study of the role of renal resident and/or infiltrating monocytes, macrophages and dendritic cells is complicated by the overlapping phenotypic heterogeneity, phenotypic transitions, surface marker expression and functional diversity in the kidney during health and injury [27]. Independent of the exact phenotype, however, a common feature is their close association with kidney fibrosis [28]. The AKI-to-CKD progression is more likely in the elderly. 2. Molecular and Cellular Events Contributing to the AKI-to-CKD Transition Key contributors to kidney injury and to the AKI-to-CKD transition include macrophages, the different forms of regulated cell death and necroinflammation, cellular senescence and SAPS, polyploidization, and podocyte injury and activation of parietal epithelial cells. 1. Introduction The precise mechanisms of kidney repair remain to be fully understood as they are related to the relative contributions of specialized renal progenitor resident cells or the division of de-differentiated mature cells. In addition, the molecular footprints of AKI and biological processes associated with unsuccessful attempted repair can become a threat to the organ. Int. J. Mol. Sci. 2022, 23, 1542 3 of 20 3 of 20 The acute phase of AKI, characterized by cell death, is followed by a recovery phase, where there is an activation of protective and regenerative mechanisms in surviving cells to restore epithelial cell properties and functions [3,5]. However, failed resolution may result in partial epithelial–mesenchymal transition (EMT), cell senescence, G2/M cell- cycle-arrest (CCA), activation of fibroblasts and immune cells and epigenetic modifications that perpetuate inflammation and release a profibrogenic secretome, contributing to the progressive decline of kidney function and fibrosis [3,5,6] (Figure 1). In this review, we examine key molecular and cellular mechanisms involved in both maladaptive responses and the endogenous repair mechanisms in the kidney, which offer an opportunity for the design of new therapeutic approaches. 2.2. Regulated Cell Death Renal tubular cell death is a key process during AKI. Indeed, tubular necrosis and loss of brush border showed the best correlation with renal dysfunction in biopsies of AKI patients [30,31]. The classic paradigm is that after an initial wave of tubular cell death, surviving tubular cells’ dedifferentiation and self-duplication potentially overshoot, and a second wave of cell death adjusts the final cell numbers [32]. First, apoptosis was described as the main form of cell death involved in AKI [33]. However, in the last decade, there is increasing evidence for a key contribution of regulated necrosis, such as necroptosis or ferroptosis, to cell death during AKI [34,35]. Apoptosis and necrosis are interconnected, and inhibition of one could activate the other [36,37]. However, the impact on inflammation and further tissue injury clearly differs between apoptosis and necrosis. Ferroptosis and necroptosis may occur synchronously, such as in kidney IRI [38,39], or sequentially, such as in folic acid nephropathy [37,40]. Necroptosis and ferroptosis also contribute to oxalate nephropathy and necroptosis is involved in cisplatin-induced AKI [41–43]. p p y p p Unlike cells dying from apoptosis, which display cell surface “eat-me” signals and are rapidly engulfed by macrophages or adjacent healthy cells, cells dying from regu- lated necrosis release DAMPs and alarmins that amplify tissue injury, in a process termed necroinflammation that interferes with kidney repair [44]. DAMPs and alarmins activate the innate immune system, triggering a stronger inflammatory response that alters the tubular microenvironment, promoting a profibrotic phenotype in tubular cells [45–47] and recruiting profibrotic macrophages [48,49]. Necroinflammation also induces an amplifica- tion loop of inflammation-related tubular cell death, leading to maladaptive repair [46]. In folic acid nephropathy, an initial ferroptosis wave triggers TWEAK-dependent necroin- flammation followed by a necroptosis wave that leads to persistence of AKI [37,40,50]. In this regard, necroptotic proteins RIPK3 or MLKL mediate early kidney injury after IRI and amplify injury through necroinflammation, leading to kidney fibrosis in long-term IRI [51]. RIPK3 also promoted fibrosis in a 28-day folic acid nephropathy model, suggesting a role of this protein in AKI–CKD transition; however, it is not clear whether this effect of RIPK3 is dependent on the necroptosis pathway [52], as bone marrow-derived RIPK3 mediates kidney inflammation in AKI independently from necroptosis [53]. 2.2. Regulated Cell Death Overall, the different forms of regulated necrosis contribute to kidney injury and may potentially drive the AKI-to-CKD transition, but they may also link to kidney repair through the recruitment of macrophages and the role of apoptosis to clear excess or damaged cells, including immune cells and fibroblasts. 2.1. Macrophages in Kidney Injury This may represent the exis- tence of subclinical pre-existent CKD and also underlying maladaptive repair mechanisms related to aging kidneys. In a recent study in aging mice, defective macrophage-M2 polar- Int. J. Mol. Sci. 2022, 23, 1542 4 of 20 ization contributed to persistent chronic inflammation after IRI, which played an important role in the progression of fibrosis and the decreasing of GFR. Interestingly, defective CSF-1 signaling mediated persistent M1-mediated inflammation and impaired macrophage M2 polarization [29]. 2.3. Cellular Senescence Cellular senescence may be triggered by different stimuli, such as oncogene induction, DNA damage, telomere shortening, inflammation, reactive oxygen species production and toxins [54]. In the kidney, cellular senescence has been described in different states of kidney repair and regeneration, including early AKI, the AKI-to-CKD transition, CKD progression and transplant rejection [55,56]. There are several senescence-related mechanisms (Figure 2). One of the most studied is the DNA-Damage-Response (DDR) pathway, which is activated by cellular DNA damage, as a protective process. DDR activation provokes cell cycle arrest. Initial steps include the phosphorylation of the histone H2AX (γH2AX), which is used as a DDR marker, and acti- vates the inhibitors of the cyclin-dependent-kinases (CDKs), p16ink4a and p21cip1, via p53 induction [55,57–59]. The final step in this process is the inhibition of CDK-mediated phos- phorylation of the retinoblastoma tumor suppressor (Rb). Then, Rb hypo-phosphorylation prevents cell cycle progression, blocking cellular proliferation [55]. After DNA repair, cells Int. J. Mol. Sci. 2022, 23, 1542 5 of 20 5 of 20 abandon this growth arrest state and re-enter the cell cycle [60]. However, in patholog- ical conditions, DDR and CCA are permanent, thus provoking cellular senescence [61]. Senescent cells accumulate in kidney disease and the activation of senescence mechanisms in tubular cells leads to the failure of regeneration following AKI or the AKI-to-CKD transition [29,62]. Proximal tubular cells in CCA suffer profound changes in intracellular signaling, provoking phenotypic alterations characterized by an aberrant and detrimental secretome termed the senescence-associated secretory phenotype (SASP) [59,63]. Glomeru- lar cells, such as podocytes and endothelial cells, may undergo senescence, leading to SASP, excessive ECM accumulation (Figure 2) and glomerulosclerosis [6]. Figure 2. Principal hallmarks of senescent cells. Many different stimuli may cause a DNA damage in the cell and activate the DNA-Damage Response (DDR), which may produce the expression of the Cell-Cycle Arrest (CCA) molecules. Prolonged activation of DDR and CCA proteins produce cellular senescence and the release of the Senescent-Associated Secretory Phenotype (SASP), enriched in different proinflammatory, profibrotic and growth factors that are regulated by NF-κB pathway activation and may cause activation of cellular senescence in a paracrine manner, inflammaging and fibrosis in the kidney, and other tissues. Figure 2. Principal hallmarks of senescent cells. Many different stimuli may cause a DNA damage in the cell and activate the DNA-Damage Response (DDR), which may produce the expression of the Cell-Cycle Arrest (CCA) molecules. 2.3. Cellular Senescence Prolonged activation of DDR and CCA proteins produce cellular senescence and the release of the Senescent-Associated Secretory Phenotype (SASP), enriched in different proinflammatory, profibrotic and growth factors that are regulated by NF-κB pathway activation and may cause activation of cellular senescence in a paracrine manner, inflammaging and fibrosis in the kidney, and other tissues. Cells of the immune system can also become senescent. The concept of immunose- nescence refers to altered immune cells that play a deleterious role promoting tissue inflammation, termed inflammaging [64,65]. In murine folic acid-induced AKI, aged kid- neys were predisposed to more severe injury and inflammation due to baseline senescence and inflammaging, when compared to young kidneys [56]. 2.4. The Senescence-Associated Secretory Phenotype (SAPS) The SASP is a key feature of senescent cells, enriched in pro-inflammatory cytokines, growth factors and other compounds. By releasing these factors, senescent cells can exert deleterious pro-inflammatory and pro-fibrotic actions [54,63,66]. The proinflammatory fac- tors found in the SASP include IL-6, IFN-γ, IL1β, TNF-α or MCP-1 [57]. As discussed above, transient inflammation participates in kidney repair, whereas persistent inflammation con- tributes to kidney injury progression and kidney failure [67–69]. MCP-1/CCL2 levels correlate with loss of kidney function and this chemokine was proposed as a biomarker of kidney fibrosis [70]. MCP-1/CCL2 could be a biomarker of kidney fibrosis and function decline [71,72]. SASP pro-inflammatory cytokines activate the nuclear factor-kappa B (NF-κB) signaling pathway [73,74], one of the most important drivers of inflammation and Int. J. Mol. Sci. 2022, 23, 1542 6 of 20 loss of nephroprotective factors, such as Klotho [75]. NF-κB is also activated in senescent cells, being the main regulator of SASP [57]. g g In age-related diseases, including CKD, the NLRP3-inflammasome is activated and contributes to inflammaging [76]. The NLRP3-inflammasome activates caspase-1 to pro- duce SASP components and other cytokines such as IL-1α, IL-1β and IL-18 [77–79]. In preclinical studies, overexpression of proinflammatory SASP genes was associated with increased senescence-related mechanisms and kidney injury following toxic or IRI AKI [56,80,81]. SASP components also include profibrotic factors, such as transforming growth factor- β1 (TGF-β1), cellular communication network factor 2 (CCN2/CTGF) and PAI-1 [66]. TGF-β1 is considered a master promoter of kidney fibrosis through the activation of canon- ical pathways, as well as the phosphorylation and activation of Smad2/3 proteins [82]. TGF-β1 activates fibroblasts and myofibroblasts, increases collagen production and ac- cumulation and regulates the expression of tissue inhibitors of MMPs. The transition of bone marrow-derived macrophages into collagen-producing myofibroblasts contributes to the accumulation of ECM proteins in the kidney. An elegant study using chimeric mice and in vitro studies clearly demonstrated the role of TGF-β/Smad3 signaling in this phenotype transition [83]. CCN2 is a matricellular ECM protein that participates in di- verse biological processes, including cell proliferation and migration, ECM remodeling and kidney fibrosis [3,6,84,85]. A key paradigm in fibrosis was to consider CCN2 a key downstream mediator of pro-fibrotic factors, including TGF-β1 and Angiotensin II [86,87]. CCN2 is widely used as a fibrotic marker in kidney tissue. 2.4. The Senescence-Associated Secretory Phenotype (SAPS) CCN2 promotes partial EMT in tubular cells and ECM accumulation in cultured renal cells, and is considered a therapeutic target in kidney fibrosis [6]. SASP pro-fibrotic factors also upregulate ECM components in kidney parenchymal cells and promote partial EMT in tubular cells, contributing to the AKI-to-CKD transition or CKD progression, leading to kidney fibrosis [3,88]. The SASP also negatively regulates cell growth, migration, and the differentiation of adjacent, non-senescent cells in a paracrine-manner. Thus, senescent cells may spread the maladaptive phenotype to neighboring cells, a process known as secondary senescence [66] that promotes the progression of kidney injury, inflammation and fibrosis [55,89,90]. 2.5. Polyploidization: A Question of Size; Samson or Delilah Tissue size is controlled by two biological strategies. One is through cell number, where cell proliferation directly affects tissue size and scaling, and the other is through cell size, where the DNA contents may increase through polyploidy. During repair, polyploid cells will occupy a large tissue space that might have been freed up by loss of other cells. This strategy would compensate cell loss and promote wound repair. Therefore, polyploidy plays a role in tissue repair and organ regeneration [91,92]. Cardiomyocytes and hepatocytes may undergo both proliferation and polyploidiza- tion. Direct observation of labelled cells during regeneration after partial hepatectomy disclosed that hepatocytes initially increase in size through ploidy, while cell proliferation contributes to hepatocyte regeneration only if most of the liver is removed [93–95]. p y g y However, polyploidization may impair tissue repair in some organs or circumstances. Thus, following completion of heart regeneration, polyploid cells are cleared by apoptosis and replaced by dividing cells [92]. In the case of the kidney, recently, Lazzeri et al. [96] described that PTC also becomes polyploidy after some insults, but the question is whether these alternative cell cycles contribute to repair or to a secondary event of damage. On the one hand, the researchers demonstrated that, as well as in the liver, the majority of tubular epithelial cells entering the cell cycle after AKI undergo hypertrophy, which would maintain (on a limited basis) the renal function, but would not promote repair. Potentially, this cell cycle arrest of tubular cells after acute damage could provide important protection by preventing the division of potentially damaged cells [97] but, at the same time, prolonged cell cycle detection may Int. J. Mol. Sci. 2022, 23, 1542 7 of 20 promote a pro-fibrotic and senescent phenotype by amplifying the secondary events of damage and promoting the AKI-to-CKD link. g p g Polyploidization of podocytes is a well-known feature [98]. Adult podocytes character- istically cannot complete cytokinesis although they can undergo a full cell cycle including mitosis, with a consequent increase in DNA content without cell division that restores a normal podocyte number after injury [99,100]. The increase in size is deleterious, as podocytes sit outside the glomerular capillaries and are exposed to the hydrostatic pressure that drives glomerular filtration and pushes them into the urinary space. 2.5. Polyploidization: A Question of Size; Samson or Delilah During CKD progression, loss of nephrons leads to a compensatory increase in the size of remnant glomeruli as well as to compensatory single nephron hyperfiltration, i.e., an increase in hydrostatic pressure. The combination of relative podocytopenia and increased pressures facilitates the loss of enlarged podocytes [101]. In diabetic nephropathy, the increase in glomerular size and resulting relative podocytopenia is an early phenomenon, which is also well described in Fabry nephropathy [102]. Abnormalities of podocyte mitosis, the increase in mitotic cell cycle proteins despite the lack of proliferation in vitro and also podocyte polyploidy in vivo were observed in experimental membranous nephropathy [103,104]. However, no interventional studies have yet targeted podocyte polyploidy to slow the transition from acute podocyte injury to CKD or CKD progression. 2.6. Podocyte Damage and Activation of Parietal Epithelial Cells Physiological stress or pathological stimuli cause podocyte injury and modify podocytes phenotypes. This response may be maladaptive, characterized by modifications in biological processes, including cellular metabolism, dysregulation and loss of integrity [105]. Mechan- ical stress induced by increased trans-capillary pressure can damage the podocyte, causing cell hypertrophy and cytoskeletal dysregulation [106]. As discussed above, glomerular hyperfiltration induces shear stress in podocyte foot processes and body surfaces that are magnified for large podocytes [107]. g g p y During glomerular injury, mediators such as inflammatory cytokines (e.g., TWEAK, MIF, TGF-β1, MCP-1 and IL-1β), complement, angiotensin II and metabolites (e.g., high glucose concentrations, lyso-Gb3 and 3,4-DGE) cause podocyte injury and contribute to podocyte loss, activating intracellular pathways such as oxidative stress and Notch signaling as well as adaptive responses such as HSP27/HSPB1expression [108–115]. Indeed, podocytes are also targets, in at least certain forms, of AKI, such as pigment-induced-AKI or IRI, considered so far as mainly driven by tubular cell injury [116,117]. Oxidative stress causes podocyte injury in AKI, namely diabetic or hypertensive nephropathy, triggering Rac-1 GTPase signaling, FOXO3a activation, endoplasmic reticu- lum stress and mitochondrial damage, among others [118–126]. Glomerular PECs are involved in fibrosis, leading to crescents in rapid progressive glomerulonephritis/crescentic glomerulonephritis (RPGN/CGN) such as ANCA-associated glomerulonephritis, and to focal and segmental glomerulosclerosis (FSGS) [127–130]. Dur- ing crescent formation, activated PECs invade the glomerular urinary space, irreversibly distorting the glomerular structure and obstructing the urinary space [131–133]. PEC “activation” implies a phenotype change that promotes their abnormal proliferation and their tuft-oriented migration towards the urinary space of the glomerulus. Activated PECs have an immature progenitor-like phenotype and a higher capacity for hyperplastic expansion (CD44+/cytokeratin-) and associate with inflammatory cells, mainly mono- cytes/macrophages CD68+) (Figure 3). CD44, CD9 and the glucocorticoid receptor contribute to PEC hyperplasia and activa- tion. CD44-deficient mice had milder glomerular cell proliferation, crescent formation and collapsing FSGS [134]. In murine CGN and FSGS, the PEC-specific gene blockade of Cd9 preserved glomerular injury through reduced expression of CD44 and β1 integrin as well as the migration of PECs into the glomerular tuft [135]. Surprisingly, given that CGN is treated in humans with glucocorticoids, glucocorticoid receptor inhibitors protected against murine CGN by decreasing cellular crescent formation and PEC proliferation/migration, Int. J. Mol. Sci. 2022, 23, 1542 8 of 20 8 of 20 uncovering the direct effects of glucocorticoids on PEC in addition to their well-known immune-suppressive actions [136]. 3. Recovery and Repair Strategies Key contributors to the repair of kidney injury include resident renal progenitor cells, stem cells and their reparative secretome, certain macrophage subphenotypes within the M2 phenotype and senescent cell clearance. 2.6. Podocyte Damage and Activation of Parietal Epithelial Cells Additionally, PECs are, together with podocytes, a source of glomerular heparin-binding epidermal growth factor-like growth factor (HB-EGF) during CGN. HB-EGF activation of the EGF receptor modifies podocyte phenotypes and favors CGN [137]. This points to a podocyte–PEC interaction; indeed, PECs are present in the neighborhood of injured podocytes [105,138–140], supporting the hypothesis that injured podocytes release soluble mediators that induce PEC activation and migration towards the inside of the glomerulus [141]. Figure 3. Glomerulonephritis characterized by a fibrous structure formation (crescent) in renal cor puscle. Glomerular PECs (cytokeratin+) are the main actors, infiltrating cells such as macrophages (CD68+) in crescent formation during rapid progressive glomerulonephritis/crescentic glomeru lonephritis (RPGN/CGN). Activated PECs (CD44+/cytokeratin-) invade the glomerular urinary space and obstruct the urinary space. PEC “activation” implies a phenotypic change that promotes their abnormal proliferation and their migration towards the urinary space of renal corpuscles. Figure 3 Glomerulonephritis characterized by a fibrous structure formation (crescent) in rena Figure 3. Glomerulonephritis characterized by a fibrous structure formation (crescent) in renal cor- puscle. Glomerular PECs (cytokeratin+) are the main actors, infiltrating cells such as macrophages (CD68+) in crescent formation during rapid progressive glomerulonephritis/crescentic glomeru- lonephritis (RPGN/CGN). Activated PECs (CD44+/cytokeratin-) invade the glomerular urinary space and obstruct the urinary space. PEC “activation” implies a phenotypic change that promotes their abnormal proliferation and their migration towards the urinary space of renal corpuscles. 3.1. Resident Renal Progenitor Cells Both circulating bone marrow stem cells and renal resident stem cells from different locations are thought to contribute to kidney repair and regeneration [142–144]. Renal Int. J. Mol. Sci. 2022, 23, 1542 9 of 20 9 of 20 progenitor cells, such as CD133+ CD24+ cells, have the ability to self-renew and to differen- tiate into different kidney cell types as well as to adipogenic, osteogenic and chondrogenic lineages [145,146]. g ACE inhibitors [147], retinoids [148] or corticosteroids [149] increased podocyte num- bers after depletion in the absence of proliferation, suggesting another cell source for novel podocytes. A bone marrow origin of circulating progenitors for podocytes is not clearly substantiated in experimental and human disease [150]. Podocytes and PECs originate from the same pool of mesenchymal cells and PECs could return to a fetal developmental reprogramming and become podocytes [151]. Indeed, in the glomerular vascular pole, a small population of PECs (“glomerular epithelial transitional cells”) co-express both podocyte and PEC proteins. Depending on the specific proteins expressed, these possible progenitor cells will be catalogued as “ectopic podocytes” or “parietal podocytes” [129,152]. Cultured CD133+ CD24+ PECs obtained from the tubular pole of the Bowman’s capsule need retinoids for normal survival and function [153], but have a multi-progenitor potential and can be differentiated into podocytes or tubular cells [146,154,155]. These findings are in line with observations that PECs could be precursor cells of podocytes in some renal diseases such as diabetic nephropathy (DN) or FSGS [156–162]. In experimental FSGS, PECs began to express podocyte proteins, and acquired podocyte phenotypes such as a decreased Notch signaling [163]. The chemokine stromal-derived factor (SFD/CXCL12) blockade increased renal progenitor differentiation towards a podocyte phenotype [164]. Migration of PECs to the glomerulus to replace senescent podocytes would be beneficial, but aberrant repair by PECs may be to contribute to podocyte damage and crescent formations, as detailed above [165–167]. Hence, is necessary to elucidate which secreted molecules from damaged podocytes recruit PECs to repopulate the glomerulus as opposed to signals that promote crescent formation. CD133+ CD24+ CD106+ renal progenitor cells from proximal tubules exhibit a higher rate of proliferation, self-renewal and differentiation than CD133+ CD24+ CD106- from Bowman’s capsule, despite sharing some markers such as vimentin and cytokeratin-7 [168], and also improve kidney function after kidney injury [154,168,169]. Lineage tracing identi- fied a Sox9+ progenitor cell population in a proximal tubule involved in AKI recovery [170]. 3.1. Resident Renal Progenitor Cells Other progenitor cells from tubules, especially from proximal tubules, express CD90, PAX-2 and CD44 but not CD133 [145,154,171], and are capable of differentiation into tubular epithelial cells and the mesodermal and chondrogenic lineages [145]. Tubular progenitor cells are more resistant to death and undergo mitosis to replace cells lost during injury [96]. 3.2. Stem Cells and the Reparative Secretome Kidney injury results in a local microenvironment that recruits and activates progeni- tor cells and cell-dependent tissue repair [144]. The regenerative potential of pluripotent embryonic (ESCs) or inducible (iPSCs) stem cells and mesenchymal stem cells (MSCs) is under study to repair experimental AKI and CKD [172,173]. MSC from different origins, including bone-marrow, the umbilical cord, amniotic fluid and adipose tissue [173,174], and renal progenitor cells (NPC) derived from human iPSCs [175,176] are renoprotective in experimental AKI and CKD. The renoprotective effects observed after stem cells’ transplan- tation depend on paracrine effects rather than on the integration of cells into host damage tissue [172,173]. These paracrine effects are mediated by the reparative secretome, i.e., by the release of growth factors, chemokines, cytokines and extracellular vesicles (EVs) that shuttle mRNA or miRNA to stimulate host cells’ regeneration, decrease inflammation and retard fibrosis [177–180]. Small clinical trials have reported promising proof-of-concept results regarding the safety and clinical feasibility of MSC-based therapies in AKI [181,182], diabetic nephropa- thy [183], atherosclerotic renovascular disease [184], lupus nephritis [185] and kidney transplant recipients [186–188]. MSC-based therapies need to overcome several issues, including a consistent source of cells with a stable phenotype, the optimal timing, dose and routes of administration and risks such as microvasculature collapse, rejection or tu- Int. J. Mol. Sci. 2022, 23, 1542 10 of 20 10 of 20 morigenicity [189]. In this regard, MSC-derived EVs represent an alternative to MSC-based therapies that overcome most of the problems associated with MSC transplantation but retain the beneficial effects [190]. Transplantation of embryonic [191] and MSC-derived [189] EVs was beneficial in preclinical AKI and CKD [189]. MSC-derived EVs accumulated in injury sites [192], where they induced proximal tubular cell proliferation and dedifferentiation and angiogenesis. Moreover, EVs prevented renal cell apoptosis and necrosis and inhibited oxidative stress, inflammation, EMT and fibrosis [189]. The regenerative and renoprotective effects of MSC- derived EVs are meditated by their cargo in proteins, mRNAs and microRNAs. Therefore, the identification and characterization of key factors, together with the selection of best cell types for each pathological situation and the definition of the optimal protocols for EV production and isolation before clinical translation is mandatory. Nevertheless, the first clinical trial employing umbilical cord–blood–MSC-derived EVs in CKD category G3–G4 patients safely improved kidney function and decreased inflammation [193]. 3.3. Macrophages in Repair Macrophages present a great phenotype plasticity modulated by specific factors such as the cytokine environment. The classical macrophage phenotypes are M1 and M2 macrophages, characterized by pro- or anti-inflammatory functions, respectively [194,195]. Several beneficial functions have been assigned to M2 macrophages, such as resolution of inflammation and tissue remodeling [67,196]. However, three M2 sub-phenotypes are recognized: M2a promotes tissue repair in response to IL-4 and/or IL-13; M2b triggers host defense; and M2c is anti-inflammatory [10,197]. y M2-phenotype-like macrophages participate in the later time points of kidney injury, during active tubular cell proliferation and kidney repair [12,198]. They contribute to epithe- lial regeneration after kidney IRI, the clearance of intraluminal debris and the attenuation of kidney inflammation [199–201]. Interestingly, Klotho induced M2 macrophage polar- ization and suppressed kidney inflammation caused by indoxyl sulfate-M1 macrophage activation in vitro [202], and the blockade of M2 polarization promoted the AKI-to-CKD transition [203]. Loss of Klotho is an early event in AKI and CKD that may be driven by albuminuria and NF-kB activation [74,204,205]. The inability of macrophages to switch between the M1 and M2 phenotypes could underlie the sustained inflammation and fi- brosis developing after IRI [8]. Thus, the macrophage phenotype plays a pivotal role during kidney repair and the therapeutic use of M2 macrophages should ensure that the M2 phenotype is stable and difficult to switch in vivo. In this regard, lipocalin-2 (Lcn- 2) is a potent modulator of macrophage polarization that stabilizes the M2 macrophage phenotype [206,207]. Hepatocyte growth factor (HGF) gene therapy increased the number of bone marrow- derived M2 macrophages and induced kidney repair in diabetic kidneys [208]. In a further step, cell therapy with M2 macrophages that had been stabilized with Lcn-2 effectively reduced kidney fibrosis in murine kidney disease, probably by the modulation of the renal inflammatory milieu [209,210]. Lcn-2 derived from macrophages was a major player in rescuing epithelial CCA, suggesting that Lcn-2 is a key pro-proliferative factor to rebuild damaged epithelia following injury [211]. g p g j y Hence, different phenotypes of macrophages exert diverse effects in kidney disease, offering the opportunity to therapeutically regulate macrophage activation, polarization and phenotype [212,213]. Author Contributions: Conceptualization, A.S. and M.R.-O.; Investigation, S.R.-M., L.M.-E., R.R.-D., A.B.S., R.G., N.D., I.R.-S., A.M., S.C., A.O., J.M.C., M.R.-O. and A.S.; Resources A.S., M.R.-O., A.O. and J.M.C.; Writing—Original Draft Preparation: S.R.-M., L.M.-E., R.R.-D.,. A.B.S., R.G., N.D., I.R.-S., A.O., J.M.C., M.R.-O. and A.S.; Writing-Review & Editing, S.R.-M., L.M.-E., R.R.-D., A.B.S., R.G., N.D., I.R.-S., A.M., S.C., A.O., J.M.C., M.R.-O. and A.S.; Funding Acquisition A.S., M.R.-O., A.O. and J.M.C. All authors have read and agreed to the published version of the manuscript. 3.4. Senescent Cell Clearance: Senotherapies As the expansion of senescent cells promotes CKD progression, therapeutic approaches have aimed at suppressing senescent cells or their consequences. Such senotherapies include the removal of senescent cells with senolytics, SASP suppression with senostatics, or boosting blockers of cellular senescence triggers [214]. This section is focused on senolytics. Int. J. Mol. Sci. 2022, 23, 1542 11 of 20 11 of 20 Senescent cells are resistant to apoptosis, as they undergo a switch from anti-survival to pro-survival signals, in which the B-cell lymphoma-2 (BCL-2) family of proteins plays a key role [57]. The BCL-2 family of proteins has prosurvival (e.g., BCL-2 and BCL-XL) and proapoptotic members (e.g., BAX) [33,215,216]. The BH3 domain is a key component of proapoptotic BCL-2 members and BH3-mimetics, such as ABT-737, are under study to treat malignancy by promoting cell death [217]. The BH3-mimetic Navitoclax (ABT-263) promotes the depletion of senescent human and murine renal epithelial cells [218,219]. In irradiated tubular cells, Navitoclax induced senescent cell apoptosis in a dose-dependent manner. In unilateral murine kidney IRI, Navitoclax decreased kidney injury and p21 expression and increased proliferation [219]. However, Navitoclax is undergoing clinical trials for cancer (NCT03366103) but not for CKD. Flavonoids have been explored to deplete senescent cells in kidney injury. The com- bination of Dasatinib, a tyrosine kinase inhibitor which promotes apoptosis, and the pro-apoptotic natural flavonoid Quercetin has been tested in vitro and in vivo [220,221]. In murine kidney IRI, Dasatinib + Quercetin increased apoptotic cells but decreased senescent cells’ markers and collagen accumulation, thus decreasing kidney injury [221]. In a pilot study in nine patients with diabetic kidney disease, Dasatinib + Quercetin for 3 days (a “hit- and-run” therapeutic approach) reduced adipose tissue senescent cells and macrophages at 11 days [222]. A larger (n = 30 patients with CKD) clinical trial is exploring the impact of a 3-day course on the proportion of senescent cells in skin, fat, and/or blood (NCT02848131). These early results are encouraging, but senescent kidney cells were not assessed and the concept of the depletion of senescent kidney cells by senolytics remains untested in humans. 4. AKI/CKD-to-Cancer Transition CKD patients have long been known to be at increased risk of kidney cancer. However, only recently has research focused on the contribution of AKI as an initial trigger or an aggravating factor in the chain of molecular and cellular events leading from kidney injury to kidney cancer. Thus, triggers of DNA damage may promote the expansion of pre- malignant and malignant cell clones. 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Huen, S.C.; Cantley, L.G. 5. Conclusions In conclusion, a better understanding of the drivers of the AKI-to-CKD transition and of the contribution to kidney repair of resident renal progenitor cells, stem cells and their reparative secretome, M2 macrophage subphenotypes within the M2 phenotype and senescent cell clearance may help identify novel biomarkers that guide therapy initiation and response to therapy as well as novel therapeutic approaches that not only prevent the AKI-to-CKD transition but also promote kidney regeneration following AKI or CKD. Author Contributions: Conceptualization, A.S. and M.R.-O.; Investigation, S.R.-M., L.M.-E., R.R.-D., A.B.S., R.G., N.D., I.R.-S., A.M., S.C., A.O., J.M.C., M.R.-O. and A.S.; Resources A.S., M.R.-O., A.O. and J.M.C.; Writing—Original Draft Preparation: S.R.-M., L.M.-E., R.R.-D.,. A.B.S., R.G., N.D., I.R.-S., A.O., J.M.C., M.R.-O. and A.S.; Writing-Review & Editing, S.R.-M., L.M.-E., R.R.-D., A.B.S., R.G., N.D., I.R.-S., A.M., S.C., A.O., J.M.C., M.R.-O. and A.S.; Funding Acquisition A.S., M.R.-O., A.O. and J.M.C. All authors have read and agreed to the published version of the manuscript. Int. J. Mol. Sci. 2022, 23, 1542 12 of 20 12 of 20 Funding: Instituto de Salud Carlos III (ISCIII) FEDER funds via the RICORS program: RICORS2040 (RD21/0005), FIS/ISCIII/FEDER (PI17/01411, PI17/00119, PI18/00910, PI18/01366, PI19/00588, PI19/00815, DTS18/00032, PI20/00140), ERA-PerMed-JTC2018 (KIDNEY ATTACK AC18/00064 and PERSTIGAN AC18/00071, ISCIII-RETIC REDinREN RD016/0009), Sociedad Española de Nefrología, FRIAT, Comunidad de Madrid en Biomedicina B2017/BMD-3686 CIFRA2-CM and NOVELREN-CM: (B2017/BMD-3751 to M.R.-O). “Convocatoria Dinamización Europa Investigación 2019” MINECO (EIN2019-103294 to M.R.-O and S.R.-M); Juan de la Cierva incorporacion grant: IJC2018-035187-I to S.R-M, Innovation pro-gramme under the Marie Skłodowska-Curie grant of the European Union’s Horizon 2020 (IMProve-PD ID: 812699) to M.R.-O. Acknowledgments: A.O. has received grants from Sanofiand consultancy or speaker fees or travel support from Advicciene, Astellas, Astrazeneca, Amicus, Amgen, Fresenius Medical Care, GSK, Bayer, Sanofi-Genzyme, Menarini, Kyowa Kirin, Alexion, Idorsia, Chiesi, Otsuka, Novo-Nordisk and Vifor Fresenius Medical Care Renal Pharma and is Director of the Catedra Mundipharma-UAM of diabetic kidney disease and the Catedra Astrazeneca-UAM of chronic kidney disease and electrolytes. Conflicts of Interest: The authors declare no conflict of interest. References Macrophages in Renal Injury and Repair. Annu. Rev. Physiol. 2017, 79, 449–469. [CrossRef] [PubMed] 12. Lee, S.; Huen, S.; Nishio, H.; Nishio, S.; Lee, H.K.; Choi, B.-S.; Ruhrberg, C.; Cantley, L.G. Distinct Macrophage Phenotypes Contribute to Kidney Injury and Repair J Am Soc Nephrol 2011 22 317–326 [CrossRef] [PubMed] 11. Huen, S.C.; Cantley, L.G. Macrophages in Renal Injury and Repair. Annu. Rev. Physiol. 2017, 79, 449–469. 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Biochem. 2017, 42, 1945–1960. [CrossRef] 210. Guiteras, R.; Sola, A.; Flaquer, M.; Manonelles, A.; Hotter, G.; Cruzado, J.M. Exploring macrophage cell therapy on Diabetic Kidney Disease. J. Cell. Mol. Med. 2019, 23, 841–851. [CrossRef] y 211. References Cao, Q.; Wang, Y.; Zheng, D.; Sun, Y.; Wang, C.; Wang, X.M.; Lee, V.W.S.; Wang, Y.; Zheng, G.; Tan, T.K.; et al. Failed renoprotection by alternatively activated bone marrow macrophages is due to a proliferation-dependent phenotype switch in vivo. Kidney Int. 2014, 85, 794–806. [CrossRef] [PubMed] 195. Sica, A.; Mantovani, A. Macrophage plasticity and polarization: In vivo veritas. J. Clin. Investig. 2012, 122, 787–795. [CrossRef] [PubMed] 196. Wang, Y.; Harris, D.C.H. Macrophages in Renal Disease. J. Am. Soc. Nephrol. 2011, 22, 21–27. [CrossRef 197. Mosser, D.M.; Edwards, J.P. Exploring the full spectrum of macrophage activation. Nat. Rev. Immunol. 2008, 8, 958–969. [CrossRef] 198. Baek, J.-H.; Zeng, R.; Weinmann-Menke, J.; Valerius, M.T.; Wada, Y.; Ajay, A.K.; Colonna, M.; Kelley, V.R. IL-34 mediates acute kidney injury and worsens subsequent chronic kidney disease. J. Clin. Investig. 2015, 125, 3198–3214. [CrossRef] 197. Mosser, D.M.; Edwards, J.P. Exploring the full spectrum of macrophage activation. Nat. Rev. Immunol. 20 198. Baek, J.-H.; Zeng, R.; Weinmann-Menke, J.; Valerius, M.T.; Wada, Y.; Ajay, A.K.; Colonna, M.; Kelley, V.R. IL-34 mediates acute kidney injury and worsens subsequent chronic kidney disease. J. Clin. Investig. 2015, 125, 3198–3214. [CrossRef] 199. Lin, S.-L.; Li, B.; Rao, S.; Yeo, E.-J.; Hudson, T.E.; Nowlin, B.T.; Pei, H.; Chen, L.; Zheng, J.J.; Carroll, T.J.; et al. Macrophage Wnt7b is critical for kidney repair and regeneration. Proc. Natl. Acad. Sci. USA 2010, 107, 4194–4199. [CrossRef] 199. Lin, S. L.; Li, B.; Rao, S.; Yeo, E. J.; Hudson, T.E.; Nowlin, B.T.; Pei, H.; Chen, L.; Zheng, J.J.; Carroll, T.J.; et al. Macrophage Wnt7b is critical for kidney repair and regeneration. Proc. Natl. Acad. Sci. USA 2010, 107, 4194–4199. [CrossRef] J g J J J p kidney repair and regeneration. Proc. Natl. Acad. Sci. USA 2010, 107, 4194–4199. [CrossRef] 200. Arai, S.; Kitada, K.; Yamazaki, T.; Takai, R.; Zhang, X.; Tsugawa, Y.; Sugisawa, R.; Matsumoto, A.; Mori, M.; Yoshihara, Y.; et al. Apoptosis inhibitor of macrophage protein enhances intraluminal debris clearance and ameliorates acute kidney injury in mice. Nat. Med. 2016, 22, 183–193. [CrossRef] 20 of 20 Int. J. Mol. Sci. 2022, 23, 1542 201. Sola, A.; Weigert, A.; Jung, M.; Vinuesa, E.; Brecht, K.; Weis, N.; Brüne, B.; Borregaard, N.; Hotter, G. Sphingosine-1-phosphate signalling induces the production of Lcn-2 by macrophages to promote kidney regeneration. J. Pathol. 2011, 225, 597–608. [CrossRef] [PubMed] [ ] [ ] 202. Lv, J.; Chen, J.; Wang, M.; Yan, F. References [CrossRef] [PubMed] , J ; , y g y J , , [ ] 221. Li, C.; Shen, Y.; Huang, L.; Liu, C.; Wang, J. Senolytic therapy ameliorates renal fibrosis postacute kidney injury by alleviating renal senescence. FASEB J. 2021, 35, e21229. [CrossRef] [PubMed] J 222. Hickson, L.J.; Langhi Prata, L.G.P.; Bobart, S.A.; Evans, T.K.; Giorgadze, N.; Hashmi, S.K.; Herrmann, S.M.; Jensen, M.D.; Jia, Q.; Jordan, K.L.; et al. Senolytics decrease senescent cells in humans: Preliminary report from a clinical trial of Dasatinib plus Quercetin in individuals with diabetic kidney disease. EBioMedicine 2019, 47, 446–456. [CrossRef] [PubMed] 223. Peired, A.J.; Lazzeri, E.; Guzzi, F.; Anders, H.-J.; Romagnani, P. From kidney injury to kidney cancer. Kidney Int. 2021, 100, 55–66. [CrossRef] [PubMed]
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Ligand sensitized fluorescence of uranyl in its fluoride complex by picolinic acid : An application for trace fluoride ion determination
Zenodo (CERN European Organization for Nuclear Research)
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Satendra Kumar, S. Maji Gay en* and K. Sankaran Analytical Chemistry and Spectroscopy Section, Chemistry Group, Indira Gandhi Centre for Atomic Research, Kalpakkam-603 102, Tamilnadu, India d Spectroscopy Section, Chemistry Group, Indira Gandhi Centre for Atomic Research, E-mail : siuli@igcar.gov. in Abstract : Picolinic acid has been used to form complex with uranyl fluoride to enhance the fluorescence intensity by about two times. Linearity in fluorescence intensity of uranyl-fluoride-picolinatt complex was obtained in the range of fluoride concentration of 40-400 ppb. The quenching effect of ions like CI-, N03 -, Soi-, Col- and Fe2+ on the • fluorescence intensity of uranyl-fluoride and uranyl-fluoride-picolina~ complexes have been studied. Stern-Volmer quenching constants for these quenchers were found to be about five times less in uranyl-fluoride-picolinate complex as compared to uranyl-fluoride complex. Fe2+ ion is found to have severe quenching effects on the fluorescence intensity of both the complexes of uranyl. This method can be used for determination of trace amount of fluoride in water samples with the tolerance limits of N03- (20 ppm), Sol- (60 ppm), Col- (100 ppm) and CI- (2 ppm). Keywords : Fluorescence, uranyl, fluoride, picolinic acid. Keywords : Fluorescence, uranyl, fluoride, picolinic acid. Introduction uranyl fluoride picolinate (U-F-PA) complex for the esti- mation of F- concentration in water. Quenching effects due to the presence of different ions on the fluorescence have also been studied. Determination of fluoride ion (F-) concentration is very important in various fields such as environmental, health sciences etc. 1. Fluoride has some beneficial effects to human body such as dental caries, if it is present within some specific limit, but it shows toxic effects if it is present in large concentration2. It has been known that fluorosis is caused by elevated intake of fluoride over prolonged period of time and mostly drinking water is the major contributor for fluoride intake. Ion selective electrode based methods of fluoride ion determination in materials like drinking water, fruit juice, tooth paste etc. are often found in literature3•4. .J. Indian Chern. Soc., Vol. 90, November 2013, pp. 2075-2078 .J. Indian Chern. Soc., Vol. 90, November 2013, pp. 2075-2078 .J. Indian Chern. Soc., Vol. 90, November 2013, pp. 2075-2078 Ligand sensitized fluorescence of uranyl in its fluoride complex by picolinic acid : An application for trace fluoride ion determination Satendra Kumar, S. Maji Gay en* and K. Sankaran Linear range and detection limit : Linear range and detection limit : The fluorescence intensity of U-F-PA complex was 7000 6000 1000 • 0 50 1 00 150 200 250 300 350 400 450 500 Auorlde ion cone In ppb Fig. 4. Calibration curve for fluorescence intensity of U-F-PA com- plex in water; U- 5 ppm, PA- 3 x to-4 M, pH 4.5. 7000 6000 1000 • 0 50 1 00 150 200 250 300 350 400 450 50 Auorlde ion cone In ppb Fig. 2. Emission spectra of (a) U; (b) U-F; (c) U-F-PA, U- 5 ppm ; F - 1 ppm; PA - 3 x 10-4M. Fluorescence of U-F complex : Fig. 1a and 1b shows the excitation spectra of uncomplexed uranium (without F-) and U-F complex in water respeaively. These spectra were recorded by fix- ing the emission wavelength at 515 nm. The concentra- tion ofF- in U-F complex was I ppm. The excitation spectra (Fig. 1a and 1b) are similar indicating that the absorbing specie is uranyl in both the cases. Fluores- cence emission spectra for l! and U-F are recorded with excitation at 255 nm and are shown in Fig. 2a and 2b, respectively. In the presence of F-, emission from U dis- plays well resolved spectral lines (Fig. 2b) with peak positions at 495, 515, 542 and 568 nm and the fluores- cence intensity is enhanced. Similar observations of fluo- rescence enhancement has been reported in the litera- ture6. The fluoride ions prevent the water molecules near- ing the U ion there by reducing the quenching effects and resulting in an enhancement in fluorescence inten- sity. Uranyl ion (Uol+; in the text it will be presented as U) is known to make strong complexes with fluoride ion and these uranyl fluoride (U-F) complexes can be charac- terized by fluorescence spectroscopy as they show fluo- rescence in aqueous medium5-7. U rany I ion is often found to exhibit fluorescence enhancement when complexed with suitable organic ligands8•9. The fluorescence enhancement is due to efficient energy transfer from the triplet level of ligand to the lowest lying emitting level of metal ion, a phenomenon known as ligand sensitized fluorescence8- 11. In this study, picolinic acid (PA) has been used as ligand for enhancing the fluorescence of U-F complex. In this paper, we made use of the enhanced fluorescence of 2075 J. Indian Chern. Soc., Vol. 90, November 2013 18000 ...-----------------, 18000 14000 ~ 12000 2! c: 10000 ~ ~ 8000 u.. 6000 4000 3.0 3.5 4.0 4.5 5.0 [pH] Fig. 3. The variation of fluorescence intensity of U-F-PA with pH of the solution; U- 5 ppm, F- 1 ppm, PA- 3 x 10-4M. 18000 ...-----------------, 18000 14000 ~ 12000 2! c: 10000 ~ ~ 8000 u.. 6000 4000 3.0 3.5 4.0 4.5 5.0 [pH] Fig. 3. The variation of fluorescence intensity of U-F-PA with pH of the solution; U- 5 ppm, F- 1 ppm, PA- 3 x 10-4M. Fluorescence of U-F complex : 200 220 240 260 280 300 320 340 380 380 400 420 Excitation wavelength (nm) Fig I Excitation spectra of (a) U; (b) U-F ; (c) U-F-PA U - 5 Excitation wavelength (nm) Fig. 3. The variation of fluorescence intensity of U-F-PA with pH of the solution; U- 5 ppm, F- 1 ppm, PA- 3 x 10-4M. Fig. I. Excitation spectra of (a) U; (b) U-F ; (c) U-F-PA, U - 5 ppm ; F - 1 ppm; PA - 3 x 10-4 M. plex is different from that of U-F case and it is likely that PA which is absorbing light. The emission spectrum shown in Fig. 2c, over the range 460-580 nm, recorded using excitation at 272 nm, displays well resolved features as displayed by U-F complex. We have also carried out experiments in which only U and PA solutions are mixed (without the fluoride). We have not observed any en- hancement in the uranium fluorescence. It is clear from the emission spectra (Fig. 2b and 2c) that PA enhances the fluorescence of U-F by about two times. These obser- vations strongly indicate a complex formation between U-F and PA. 460 460 500 520 540 580 580 Emission wavelenglh(nm) Fig. 2. Emission spectra of (a) U; (b) U-F; (c) U-F-PA, U- 5 ppm ; F - 1 ppm; PA - 3 x 10-4M. 460 460 500 520 540 580 580 Emission wavelenglh(nm) Fig 2 Emission spectra of (a) U; (b) U F; (c) U F PA U 5 Fluorescence of U-F-PA complex: At the outset, the fluorescence of U was recorded as a function of PA concentration and pH of the solution. Maximum fluorescence intensity was obtained for pH 4.5 (Fig. 3) and acid concentration of 3 x 10-4 M of PA. This acid concentration and pH was used throughout this study. The excitation and emission spectrum of U-F in the presence of PA, at pH 4.5, is shown in Fig. lc and 2c, respectively. The excitation maximum for U-F-PA (Fig. lc) occurs at 272 nm, which is different from exci- tation maximum for U and U-F of 255 nm (Fig. la and lb). This indicates that the absorber in the U-F-PA com- Auorlde ion cone In ppb Fig. 4. Calibration curve for fluorescence intensity of U-F-PA com- plex in water; U- 5 ppm, PA- 3 x to-4 M, pH 4.5. 2076 Kumar et at. : Ligand sensitized fluorescence of uranyl in its fluoride complex etc. Kumar et at. : Ligand sensitized fluorescence of uranyl in its fluoride complex etc. found to increase linearly with p- concentration which makes it useful for the trace determination of fluoride ion. Linearity in fluorescence intensity of this U-F-PA complex was obtained in the range of fluoride ion con- centration of 40-400 ppb which is shown in Fig. 4. The detection limit calculated using the criterion of 3cr of the blank is 12 ppb. The relative standard deviation for 5 replicate analysis of the sample containing 40 ppb of fluo- ride ion is -8%. blank is 12 ppb. The relative standard deviation for 5 replicate analysis of the sample containing 40 ppb of fluo- ride ion is -8%. Effect of ions : Effect of ions : complex was obtained in the range of fluoride ion con centration of 40-400 ppb which is shown in Fig. 4. The detection limit calculated using the criterion of 3cr of the 0.7 0.6 ~ -•- U-F 0.5 0.4 -o- U-F-PA .l~ ..!.... 0.3 ~ 0.2 0.1 0.0 200 400 600 800 1000 1200 [SO,'"] in ppm 0.7 0.6 / (c) 0.5 -•-U-F 0.4 -D-U·F-PA ~ 0.3 I • 0.2 v 0.1 c 0.0 -<).1 2 6 10 12 14 16 18 20 [NO,"] in ppm 6 ... I 4 ~ 0 0 Effect of ions : In order to apply this method for p- determination in water samples, it is necessary to see the effects of pres- 1.0 ..,---------------------, (b) ' ~ 0.4 0.8 0.6 / . ~ /~•-U-F v -0-UHA 0.2 0.0 -50 50 100 150 200 250 300 350 400 [C0 12"] in ppm 1.6 ~ 0.8 ~ 1.2 I ,~/ ! ~c -•-U-F v -0-U-F-PA 0.4 0.0 0 8 10 12 14 16 18 20 [Cllln ppm (e) -•-U-F -0-U-F-PA 3 4 6 {F e'"Jin ppm Fig. S. Stem-Volmer plots for U-F and U-F-PA complexes with different ions: (a) so42-; (b) col-; (c) N03-; (d) CI-; (e) Fe 2+; U- 5 ppm; F- 1 ppm; PA- 3 x 10-4 M for all cases. In order to apply this method for p- determination in water samples, it is necessary to see the effects of pres- 0.7 0.6 ~ -•- U-F 0.5 0.4 -o- U-F-PA .l~ ..!.... 0.3 ~ 0.2 0.1 0.0 200 400 600 800 1000 1200 [SO,'"] in ppm 1.0 ..,---------------------, (b) ' ~ 0.4 0.8 0.6 / . ~ /~•-U-F v -0-UHA 0.2 0.0 -50 50 100 150 200 250 300 350 400 [C0 12"] in ppm 0.7 0.6 ~ -•- U-F 0.5 0.4 -o- U-F-PA .l~ ..!.... 0.3 ~ 0.2 0.1 0.0 200 400 600 800 1000 1200 [SO,'"] in ppm 0.7 0.6 / (c) 0.5 -•-U-F 0.4 -D-U·F-PA ~ 0.3 I • 0.2 v 0.1 c 0.0 -<).1 2 6 10 12 14 16 18 20 [NO,"] in ppm 1.0 ..,---------------------, (b) ' ~ 0.4 0.8 0.6 / . ~ /~•-U-F v -0-UHA 0.2 0.0 -50 50 100 150 200 250 300 350 400 [C0 12"] in ppm 1.6 ~ 0.8 ~ 1.2 I ,~/ ! Effect of ions : ~c -•-U-F v -0-U-F-PA 0.4 0.0 0 8 10 12 14 16 18 20 [Cllln ppm 0.0 200 400 600 800 1000 1200 [SO,'"] in ppm 0.7 0.6 / (c) 0.5 -•-U-F 0.4 -D-U·F-PA ~ 0.3 I • 0.2 v 0.1 c 0.0 -<).1 2 6 10 12 14 16 18 20 [NO,"] in ppm 0.0 -50 50 100 150 200 250 300 350 400 [C0 12"] in ppm 1.6 ~ 0.8 ~ 1.2 I ,~/ ! ~c -•-U-F v -0-U-F-PA 0.4 0.0 0 8 10 12 14 16 18 20 [Cllln ppm 0.7 0.6 / (c) 0.5 -•-U-F 0.4 -D-U·F-PA ~ 0.3 I • 0.2 v 0.1 c 0.0 -<).1 2 6 10 12 14 16 18 20 [NO,"] in ppm 1.6 ~ 0.8 ~ 1.2 I ,~/ ! ~c -•-U-F v -0-U-F-PA 0.4 0.0 0 8 10 12 14 16 18 20 [Cllln ppm [NO,"] in ppm [Cllln ppm 6 ... I 4 ~ 0 0 (e) -•-U-F -0-U-F-PA 3 4 6 {F e'"Jin ppm Fig S Stem-Volmer plots for U-F and U-F-PA complexes with different ions: (a) so42 ; (b) col-; (c) N03-; (d) CI-; (e) Fe 2+; U 6 ... I 4 ~ 0 0 (e) -•-U-F -0-U-F-PA 3 4 6 {F e'"Jin ppm 6 ... I 4 ~ 0 0 (e) -•-U-F -0-U-F-PA 3 4 6 {F e'"Jin ppm Fig. S. Stem-Volmer plots for U-F and U-F-PA complexes with different ions: (a) so42-; (b) col-; (c) N03-; (d) CI-; (e) Fe 2+; U- 5 ppm; F- 1 ppm; PA- 3 x 10-4 M for all cases. 2077 JICS-22 J. Indian Chern. Soc., Vol. 90, November 2013 J. Indian Chern. Soc., Vol. 90, November 2013 maximum and uniform transmittance ( > 85%) above 400 nm, was placed in front of the emission monochromator to reduce the scatter of the incident beam entering the emission monochromator. Spectra were recorded at room temperature with a 90° collection geometry. All spectra had been corrected for instrument response. ence of other ions which are conunonly found in water, on the fluorescence intensity of U-F-PA complex. Differ- ent ions such as Cl-, N03-, soi-, Col- and Fe2+ were used for this study and found to quench the fluorescence intensity. References I. C. R. Nagendra Rao, "Proceedings of the Third Interna- tional Conference on Environment and Health", Chennai, India, 15-17 December, 2003. 2. Jenny Abanto Alvarez, Karla Mayra P. C. Rezende, Susana Maria Salazar Marocho, Fabiana B. T. Alves, Paula Celiberti and Ana Lidia Ciamponi, Med. Oral Patol. Oral. Cir. Bucal., 2009, 14, 103. 3. Serife Tokalioglu, Senol Kartal and Ugur Sahin, Turk. J. Chern., 2004, 28, 203. 4 . Ana B. Descalzo, Diego Jimenez, Jamal El Haskouri, Daniel Beltran, Pedro Amor6s, M. Dolores Marcos and Ramon Martinez-Mariezand Juan Soto, Chern. Commun., 2002, 562. 5. Zsolt Fazekas, Tomoo Yamamura and Hiroshi Tomiyasu, Journal of Alloys and Compounds, 1998, 756, 271. In summary picolinic acid was found to enhance the fluorescence of uranyl fluoride complex. Also fluores- cence quenching by other ions was observed to be less in case of U-F-PA as compared to U-F compl~o;x, which could lead the fluoride ion determination in trace levels. 6. James V. Beitz and Clatyon W. Williams, Journal of Alloys and Compounds, 1997,250,375. 7. By Akira Kirishima, Takaumi Kimura, Osamu Tochiyama and Zenko Yoshida, Radiochim. Acta, 2004, 92, 889. 8. S. Maji and K. S. Viswanathan, Journal of Luminescence, 2009, 129, 1242. 9. S. Maji and K. S. Viswanathan, Journal of Luminescence, 2011, 131, 1848. Effect of ions : Quenching behaviour of different ions can be obtained from Stern-Volmer equation12 : !0 I I = 1 + kD [Q] Standard solutions of uranium was prepared by dis- solving the required amount of uranium oxide in distilled HC104. Stock solution of fluoride ion was prepared by dissolving the required amount of sodium fluoride in water. All the ions like CI-, N03-, Soi-, CO/- were preapared from their highly pure sodium salt by dissolving in dis- tilled water. Stock solution of picolinic acid (Sigma Aldrich) was prepared by dissolving the required amount of the reagent in water. All chemicals were used as pur- chased from the supplier without any further purifica- tion. De-ionized water (18 MQ) obtained with a Milli-Q (Millipore) system was used in preparing all solutions. where !0 and I are the fluorescence intensity of the complexe without quencher and with quencher respec- tively, kD represents the Stern-Volmer constant for a par- ticular ion, and [Q] is the concentration of quencher. In Fig. 5, [(10/J)- 1] is plotted against the quencher concen- tration. Stern-Volmer plots for these ions are not linear which indicates the presence of both static and collision type of quenching operating in this system12. so42- and Col- ions (Fig. Sa and Sb) have less quenching effect as compared to No3- and Cl- which are found to have dras- tic quenching effect even at low concentrations as shown in Fig. Sd and Se respectively. The fluorescence intensity is decreased by about 25% at 500 ppm of sol-. The same effect is seen for co32- when the concentration is 250 ppm. The quenching effect of ions on the fluores- cence of U-F in absence of PAis also studied and plotted in the same Fig. 5. It is clear from these plots that the quenching effects of these ions, except Fe2+, are more severe in case of U-F than the case of U-F-PA. Fe2+ was found to have severe quenching effect in both the com- plexes as can be seen from Fig. 5e. Tolerance limits of different ions, which caused the maximum reduction in fluorescence intensity of U-F-PA by 10% were calcu- lated and found to be N03- (20 ppm), Sol- (60 ppm), co32- (100 ppm) and Cl- (2 ppm) for the determination of 200 ppb of F-. Experimental All fluorescence spectra were recorded using an Edinburgh FLS920 spectrofluorimeter, with a 450 W xenon lamp source. Solutions were taken in a 10 mm path length fused silica cell. The band-pass for the excita- tion and emission monochromators was set at 2 nm each. A long-wavelength pass filter (UV-39, Shimadzu), with a 10. J.-H. Yang, G.-Y. Zhu and B. Wu, Anal. Chim. Acta, 1987, 198, 287. 11 L. M. Perry and J. D. Winefordner, Anal. Chim. Acta, 1990, 237' 273. 12. Joseph R. Lakowicz, "Principle of Fluorescence Spec- troscopy", Springer, 3rd ed. 2078
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Dietary Zinc and Fibre Source can Influence the Mineral and Antioxidant Status of Piglets
Animals
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10,729
Received: 19 June 2019; Accepted: 24 July 2019; Published: 29 July 2019 Simple Summary: The post-weaning period is one of the most critical phases in a pig’s life, when trace mineral requirements increase because of the inadequate absorptive capacity of the intestine and reduced feed intake. Mineral bioavailability can be increased by using more available feed additives or the reduction of dietary antinutrients, such as phytates or fibres, in pig nutrition. Therefore, our study was focused on improving the absorption of trace elements, mainly zinc, from the small intestine of piglets using more efficient dietary sources, such as an organic form of zinc and potato fibre. Abstract: The study investigated the effect of dietary zinc glycine chelate and potato fibre on the absorption and utilisation of Zn, Cu, Fe, and Mn; the activity of Zn-containing enzymes (superoxide dismutase, SOD; alkaline phosphatase, ALP); and zinc transporter concentrations (metalothionein1, MT1; zinc transporter1, ZnT1) in tissues, with a special emphasis on the small intestine. Twenty-four barrows (Danbred × Duroc) were randomly allotted to four diets (supplemented with 10 g/kg of crude fibre and 120 mg Zn/kg) that consisted of cellulose and either zinc sulphate (C) or zinc glycinate (ZnGly), or contained potato fibre supplemented with ZnSO4 (PF) or ZnGly (PF + ZnGly). Feeding PF can influence the Zn absorption in the small intestine due to reduced zinc transporters MT1 and ZnT1 in the jejunum. The activity of antioxidant enzyme SOD and liver ZnT1, and duodenal iron concentrations were increased in the PF treatments. Dietary ZnGly did not significantly influence the Zn distribution, but it may alter the absorption of Fe and Mn. Given the elevated content of thiol groups and the Zn/Cu ratio in plasma, as well as the altered SOD activity and MT content in the tissues, we can conclude that feeding PF and ZnGly can influence the mineral and antioxidant status of growing piglets. However, further research is needed in order to elucidate the effect of both dietary sources on the transport systems of other minerals in enterocytes. Keywords: zinc glycine chelate; potato fibre; pigs; trace elements; zinc transporters Dietary Zinc and Fibre Source can Influence the Mineral and Antioxidant Status of Piglets 1 Institute of Animal Physiology, Centre of Biosciences of the Slovak Academy of Sciences, Soltesovej 4-6, 04001 Kosice, Slovakia 04001 Kosice, Slovakia 2 The Kielanowski Institute of Animal Physiology and Nutrition, Polish Academy of Sciences, Instytucka 3, 05-110 Jabłonna, Poland 2 The Kielanowski Institute of Animal Physiology and Nutrition, Polish Academy of Sciences, Instytuck 05-110 Jabłonna, Poland * Correspondence: gresakl@saske.sk; Tel.: + 421-55-792-2970 * Correspondence: gresakl@saske.sk; Tel.: + 421-55-792-2970 animals animals animals www.mdpi.com/journal/animals animals animals 1. Introduction Trace mineral requirements increase in young piglets, mainly after weaning. Upon weaning, the absorptive capacity of a piglet’s intestine is reduced due to several principal factors, such as changes in the villus structure, an inadequate ability to secrete acid and digestive enzyme levels [1]. Therefore, the diets of early weaning pigs should provide a higher content of trace minerals and mainly highly digestive feed components, while taking their limited feed intake into consideration. Moreover, the immunity or resistance to disease of piglets at this time are additional problems. Zinc, as the second most abundant essential trace element, performs multifarious physiological roles and, as Animals 2019, 9, 497; doi:10.3390/ani9080497 www.mdpi.com/journal/animals 2 of 16 Animals 2019, 9, 497 a component of many metalloenzymes, is involved in almost every metabolic pathway of the body [2]. Zinc deficiency may result in a reduced feed intake and growth of pigs, parakeratosis, alopecia, thymus atrophy and impaired immune functions [3]. Zinc recommendations for piglets (<11–25 kg body weight) are 140–150 mg Zn/kg in a complete diet and this decreases with increasing body weight [4]. It is well-known that the composition of feedstuffs and the ratio of dietary nutrients are important factors that affect mineral absorption from the gastrointestinal tract (GIT) of growing piglets in order to maintain or improve their body’s mineral status. Inorganic zinc sulphate widely used for the supplementation of animal feedstuffs is rapidly dissociated to free ions during digestion in the GIT and may interact with other dietary molecules, resulting in mineral losses prior to absorption in the small intestine [5]. For this reason, the majority of dietary zinc is excreted in animal faeces, and after soil fertilisation with manure, the accumulation of zinc is constantly increasing in the soil [6]. Therefore, it is necessary to improve the efficiency and absorption of trace elements by feed composition in order to decrease Zn supplementation in animal nutrition with respect to a substantial reduction of environmental pollution from animal husbandry. Using mineral amino acid chelates as feed additives in pig nutrition could increase mineral bioavailability and enhance mineral tissue storage. The stable structures of mineral chelates prevent undesirable interactions with feed compounds in the digestive tract, and using active transport pathways via metallotransporters across enterocytes contributes to better mineral absorption and utilisation in the animal body [5,7,8]. 1. Introduction Moreover, the elimination of mineral interference with some dietary nutrients, such as phytates or fibres, could improve the mineral efficacy. Therefore, the present study focused on improving the absorption of zinc (Zn) and other trace elements such as iron (Fe), copper (Cu), and manganese (Mn), from the small intestine of piglets using more efficient dietary sources, namely zinc glycine chelate and potato fibre. The main objective of our study was to investigate the effect of dietary zinc glycinate and potato fibre on trace element absorption and tissue distribution (Zn, Cu, Fe, and Mn) and the activity of Zn-containing enzymes in plasma and various tissues, with a special emphasis on the small intestine of piglets. Since zinc transporter 1 (ZnT1, SLC301A) and metallothionein 1 (MT1) are responsible for the amount of Zn released into the portal circulation for systemic distribution, we investigated the concentrations of these Zn transporters in the small intestine segments of piglets. Our study could elucidate the distribution of Zn in intestine cells and other tissues after feeding with different Zn and fibre sources. 2. Materials and Methods All procedures were done in accordance with European Community guidelines (EU Directive 2010/63/EU for animal experiments), and the experimental protocol was approved by the Local Animal Experimentation Ethics Committee (resolution number WAW2_21/2016, Warsaw University of Life Sciences-SGGW, Warsaw, Poland) and Polish Law on Animal Protection. 2.1. Experimental Design, Animals and Diets 2.1. Experimental Design, Animals and Diets A total of twenty-four Danbred × Duroc barrows 40 days old (initial body weight 10.8 ± 0.8 kg) were allocated to one of four dietary treatments, with six replicates per treatment in a completely randomized design. During the feeding period, which lasted for 4 weeks, the piglets were given a cereal-based diet supplemented with 10 g/kg crude fibre and 120 mg Zn/kg from two fibre and zinc sources. The experimental diets consisted of cellulose (Lonocel, Cargill Poland Ltd., Kiszkowo, Poland; 637 g/kg crude fibre) as the fibre source and zinc sulphate (C; ZnSO4.H2O, reagent grade, Sigma-Aldrich, USA) or zinc chelate of glycine hydrate (ZnGly; Glycinoplex-Zn, Phytobiotics Futterzusatzstoffe GmbH, Eltville, Germany), and potato fibre (Potex, Lyckeby Starch, Kristianstad, Sweden; 197 g/kg crude fibre) with ZnSO4 (PF) or zinc glycinate (PF + ZnGly). The diets were isonitrogenous, isoenergetic and prepared to contain a total of 150 mg Zn/kg of complete diet and did not exceed 50 g/kg of total crude fibre content. The diet composition of the basal cereal-based diet, containing either cellulose (for C and 3 of 16 Animals 2019, 9, 497 ZnGly treatments) or potato fibre (for PF and PF + ZnGly treatments) and a zinc-free vitamin-mineral mix (DSM Nutritional Products Ltd., Mszczonów, Poland), are shown in Table 1. ZnGly treatments) or potato fibre (for PF and PF + ZnGly treatments) and a zinc-free vitamin-mineral mix (DSM Nutritional Products Ltd., Mszczonów, Poland), are shown in Table 1. Piglets were housed individually in pens and kept in a 12 h light/12 h dark regimen. The housing temperature was maintained at 25 ◦C. Throughout the experiment, the animals had free access to feed and fresh potable water. Body weight and feed intake were measured weekly. Table 1. Ingredients and chemical composition of the cereal-based diets (g/kg). Table 1. Ingredients and chemical composition of the cereal-based diets (g/kg). 2.1. Experimental Design, Animals and Diets Ingredients, g/kg C PF Barley 200 200 Wheat 450 450 Soybean meal 170 170 Yellow lupin 60 60 Rapeseed oil 25.5 25.5 Corn starch 33 – Cellulose 17 – Potato fibre – 50 Mineral-vitamin mix 1 4 4 Monocalcium phosphate 7 7 Calcium carbonate 19 19 Sodium chloride 3 3 L-Lysine 6 6 DL-Methionine 2 2 L-Threonine 3 3 L-Tryptophan 0.5 0.5 Analysed Composition 2, g/kg C PF Dry matter 889 890 Crude ash 47 49 Crude protein 182 183 Crude fat 42 41 Crude fibre 41 40 Sugars 41 39 Starch 378 379 Calcium 9.6 9.5 Total phosphorus 5.1 5.1 Phytic phosphorus 1.9 1.7 aNDFom 108 112 ADFom 55 56 Lignin (sa) 11 10 Cellulose 43 47 Hemicellulose 53 55 Zinc (mg/kg) 142 141 Manganese (mg/kg) 99 94 Copper (mg/kg) 125 132 Iron (mg/kg) 315 300 Gross energy (MJ/kg) 16.8 16.6 diets containing cellulose fibre, PF:the diets containing potato fibre. 1 Provided per kg of diet: 1 n A, 1835 IU vitamin D3, 138 mg vitamin E, 4.59 vitamin K3, 2.94 mg vitamin B1, 7.34 mg vitamin B2, n B6, 44.0 µg vitamin B12, 36.7 mg nicotinic acid, 16.0 mg calcium D-pantothenate, 1.47 mg folic acid 228 mg Fe, 73.4 mg Mn, 156 mg Cu, 631 µg I, 411 µg Se. 2 The chemical composition of the cerea nd mineral content in the feed samples were analysed according to Official Methods of Analysis of ational (AOAC) procedures [9]. C: the diets containing cellulose fibre, PF:the diets containing potato fibre. 1 Provided per kg of diet: 14680 IU vitamin A, 1835 IU vitamin D3, 138 mg vitamin E, 4.59 vitamin K3, 2.94 mg vitamin B1, 7.34 mg vitamin B2, 4.40 mg vitamin B6, 44.0 µg vitamin B12, 36.7 mg nicotinic acid, 16.0 mg calcium D-pantothenate, 1.47 mg folic acid, 147 µg biotin, 228 mg Fe, 73.4 mg Mn, 156 mg Cu, 631 µg I, 411 µg Se. 2 The chemical composition of the cereal-based diets and mineral content in the feed samples were analysed according to Official Methods of Analysis of AOAC International (AOAC) procedures [9]. 2.3. Analytical Methods The diets were analysed for dry matter, crude protein, crude ash, crude fat, crude fibre, acid detergent fibre expressed exclusive of residual ash (ADFom), starch and phosphorus according to AOAC procedures [9], with values of 934.01, 984.13, 942.05, 920.39, 962.09, 973.18, 996.11, and 965.17, respectively. Lignin (sa) content was determined by the direct sulphuric acid method, as described by Robertson and Van Soest [10]. Neutral detergent fibre (aNDFom) was analysed with a heat stable amylase and expressed exclusive of residual ash, according to Mertens [11]. Hemicellulose content was calculated as the difference between aNDFom and ADFom, whereas cellulose was the difference between ADFom and lignin (sa). Phytic phosphorus was analysed after phytate precipitation with FeCl3 and subsequent colorimetric measurement of phosphorus according to AOAC [9] procedure 965.17. Sugar content was analysed using the anthrone method [12] after protein precipitation with Carrez solutions (K4Fe(CN)6·3H2O and Zn(CH3COO)2·2H2O). The Zn, Cu, Fe, Ca and Mn concentrations in the diets, plasma and tissues were analysed using a double-beam atomic absorption spectrophotometer (AA-7000 Series, Shimadzu Co., Kyoto, Japan) with a graphite furnace (GFA-7000, Shimadzu Co., Kyoto, Japan). The microwave-assisted digestion method using closed pressure vessels in an MWS 4 Speedwave device (Berghof Co., Germany) was used for the decomposition of all samples in two replicates. All samples, except blood, were wet acid-digested with a concentrated nitric acid and hydrogen peroxide mixture. Plasma Mn concentration and Cu content in muscle were determined using a graphite furnace atomic absorption spectrophotometer with deuterium background correction and pyrolytic-coated graphite tubes [13]. The certificate reference materials of bovine liver BCR-185R, bovine muscle ERM-BB184 and pig kidney ERM-BB186 from the Institute for Reference Materials and Measurements (IRMM, Geel, Belgium), bone ash SRM 1400 (NIST, National Institute of Standards and Technology, Gaithersburg, MD, USA) and ClinCheck control of lyophilized human plasma (Recipe, Munich, Germany) were included in each analysis to verify the instrument accuracy. Mineral concentrations in all tissue samples were expressed as mg/kg of DM, except bone (mg/kg of bone ash weight) and plasma (µg/L, mg/L). The total superoxide dismutase activity (SOD) was assayed by a modified spectrophotometric method, based on the ability of the enzyme to inhibit pyrogallol autoxidation by 50% [14]. Tissue samples were homogenized in ice-cold Tris and sucrose buffer (10 mM Tris, 0.25 M sucrose, pH 7.4). 2.2. Data Collection At the end of the feeding period (28 days), the piglets were stunned by electric shock and exsanguinated, followed by tissue collection. Blood samples were collected in heparinised tubes from each piglet and were centrifuged at 3350 g for 10 min. Tissue samples were collected from the liver, 4 of 16 Animals 2019, 9, 497 pancreas, left kidney cortex, the skeletal muscles m. longissimus dorsi, small intestinal segments and the metacarpal III and IV bones. Sampling was done at identical locations of each relevant tissue. Pieces 2–3 cm long of cut intestine segment samples were washed with saline solution and immediately frozen in liquid nitrogen for alkaline phosphatase (ALP) analysis. For the analyses of the mineral and transporter content or enzyme activity, excised intestinal sections of duodenum, jejunum and ileum were thoroughly rinsed with saline solution, and the intestinal mucosa of each region was then scraped offwith sterile glass slides on ice. All tissues and plasma samples were stored at −80 ◦C until further analysis. 2.3. Analytical Methods The homogenates were centrifuged at 10,000× g for 30 min at 4 ◦C, and the supernatants were used for the analysis. The specific Cu/Zn SOD inhibition by potassium cyanide allows the determination of enzyme activity under the same conditions. Enzyme activity is expressed as units of enzyme per mg of tissue protein. The protein concentration was measured using the spectrophotometric method [15]. MT1 and ZnT1 tissue content were determined with the colorimetric method using commercial ELISA kits (Porcine Zn-MT ELISA kit, NeoScientific, London, UK; SLC30A10 MyBioSource San Diego, CA, USA; respectively). The analysis of intestinal alkaline phosphatase activity (ALP) in the intestinal sections was performed using a modified simultaneous azo coupling method [16]. The histochemically stained slides were visualised using an Olympus microscope (BX 51) and a digital compact camera (Olympus DP 50) connected to a host computer. Image analysis was performed in the Ellipse program (ViDiTo, Slovakia), where the grey level of each pixel was given a value in the 0–255 range. A Vickers 5 of 16 Animals 2019, 9, 497 M85a microdensitometer was used to define the standard density values of enzyme activities at a wavelength of 520 nm, which was required for calibration. A special semi-interactive algorithm was used to find relevant pixels along the villus length, and the density of such pixels was measured [17]. The quantification of enzyme activity (pixel intensities) was carried out along the villus length in a whole section of at least four duodenal and jejunal slides, and the mean values recorded referred to one animal. The enzymatic activity of plasma ALP was evaluated photometrically in a kinetic way using a commercial kit (ALP MEG 500, Erba Lachema, Czech Republic). For photometrical determination of plasma albumin, an assay kit (ALB 500, Erba Lachema, Czech Republic) was used. The amount of thiobarbituric acid reactive substance (TBARS) was measured in blood plasma according to the method of Rice-Evans et al. [18]. The total concentration of thiol groups (TSH) was performed spectrophotometrically at 412 nm, as described by Ellman [19]. Briefly, aliquots of intestine mucosa (0.5 g) were homogenized in buffer (Tris 50 mM, pH 7.4) and subsequently centrifuged at 3000× g for 20 min at 4 ◦C. To determine non-protein thiols (NPSH), the protein fraction was precipitated with 10% trichloroacetic acid, followed by centrifugation, and the supernatant was used for analysis. 2.3. Analytical Methods The colorimetric assay was carried out using Ellman’s reagent and 0.1 M phosphate buffer (pH 7.4). A standard curve using glutathione was constructed in order to calculate the thiol content in the plasma and tissue samples. The pro-oxidant-antioxidant balance (PAB) in blood serum was analysed according to Koliakos and Hamidi Alamdari [20]. Briefly, 10 µL of serum was mixed in a well of a 96-well microplate with 200 µL of solution containing 3,3’,5,5’ tetramethylbenzidine (TMB), TMB cation, and 5 U of horseradish peroxidase in 0.05 mM phosphate-citrate buffer, pH 5.0. After 12 min of incubation, 100 µL of 2 M HCl was added and incubated for 45 min at room temperature in the dark. The absorbance was measured at 450 nm, with a reference wavelength of 620 nm, using a Maxmat PL multidiagnostic platform (Erba Diagnostics France SARL, Montpellier, France). The balance values were calculated from a standard curve prepared using 1 mM H2O2, as a representative of the oxidants, and 6 mM uric acid, as a representative of the antioxidants. The standard solutions were mixed in varying proportions, from 100% of 6 mM uric acid to 100% of 1 mM H2O2. The results were expressed in arbitrary Hamidi-Koliakos (HK). The HK unit is the percentage of H2O2 in the standard solution multiplied by 6. Therefore, greater HK values indicate higher oxidant concentrations in a sample. 2.4. Statistical Analysis Treatment effects on mineral tissue concentrations, enzyme activities and Zn transporter content were analysed according to a completely randomised 2 × 2 factorial design, with a factorial arrangement of treatments using two-way ANOVA, followed by a Bonferroni post-hoc test for pair-wise comparisons, where appropriate. The statistical model included the treatment effects (fibre and zinc source) and their interaction. When the interaction was significant, Fisher’s Least Significant Difference test (Fisher’s LSD) was applied post hoc to determine significant differences among the means. Pearson correlation was used to describe the relationships between the data sets. All statistical analyses were performed by the GraphPad Prism statistical software (GraphPad Prism version 8.1.2., GraphPad Software, San Diego, CA, USA). Differences between the mean values of the different dietary treatments were considered statistically significant at p < 0.05. Data are expressed as means and pooled standard errors of the mean (SEM). 3. Results The concentrations of Zn transporters MT1 and ZnT1 in the liver and small intestine sections of piglets are expressed in µg per gram of wet tissue (Table 2). The concentrations of both MT1 and ZnT1 in jejunum and liver tissues were affected by the fibre source, with significantly increased levels of jejunum in pigs from the dietary treatments containing cellulose fibre (p < 0.01 for MT1; p < 0.001 for ZnT1), although ZnT1 levels were elevated in the liver of piglets fed both PF diets (p < 0.01). Feeding 6 of 16 Animals 2019, 9, 497 diets with ZnGly increased the ileal ZnT1 content compared with feeding ZnSO4 (p < 0.05). The content of total and non-protein sulfhydryl groups in the liver and intestinal tissues (expressed as µmol/g tissue) was not affected by the dietary treatment (Table 2). Table 2. The tissue concentration of the metallothein 1 (MT1), zinc transporter 1 (ZnT1), and total and non-protein-bound thiols (TSH and NPSH, respectively) in piglets fed diets differing in zinc and fibre source. Table 2. The tissue concentration of the metallothein 1 (MT1), zinc transporter 1 (ZnT1), and total and non-protein-bound thiols (TSH and NPSH, respectively) in piglets fed diets differing in zinc and fibre source. Table 2. The tissue concentration of the metallothein 1 (MT1), zinc transporter 1 (ZnT1), and total and non-protein-bound thiols (TSH and NPSH, respectively) in piglets fed diets differing in zinc and fibre source. Table 2. The tissue concentration of the metallothein 1 (MT1), zinc transporter 1 (ZnT1), and total and non-protein-bound thiols (TSH and NPSH, respectively) in piglets fed diets differing in zinc and fibre source. 3. Results Dietary Treatment SEM Main Effects Interaction C ZnGly PF PF + ZnGly Zn Fibre Zn × Fibre MT1, µg/g tissue Liver 13.1 11.8 11.9 11.5 0.415 0.32 0.41 0.60 Duodenum 12.9 12.5 12.7 14.7 0.451 0.37 0.27 0.19 Jejunum 20.5 ab 21.7 b 16.7 a 19.4 a 0.302 0.36 0.007 0.17 Ileum 19.4 19.3 18.9 19.0 0.321 0.97 0.57 0.86 ZnT1, µg/g tissue Liver 16.7 a 16.7 a 20.0 ab 22.6 b 0.856 0.32 0.003 0.34 Duodenum 23.9 27.6 23.7 24.4 0.860 0.21 0.31 0.40 Jejunum 26.2 ab 30.1 b 23.9 a 23.1 a 0.684 0.20 0.001 0.06 Ileum 15.9 20.2 18.9 20.3 0.656 0.02 0.20 0.22 TSH, µmol/g tissue Jejunum 5.57 4.89 5.48 5.61 0.140 0.25 0.19 0.10 Ileum 5.62 5.48 5.55 6.12 0.131 0.42 0.29 0.19 NPSH, µmol/g tissue Jejunum 2.35 1.95 1.78 1.93 0.094 0.49 0.11 0.14 Ileum 1.96 1.89 1.88 2.17 0.068 0.42 0.45 0.19 C: cellulose, ZnGly: zinc chelate of glycine hydrate, PF: potato fibre. a,b Means within lines with different superscript letters are significantly different (p < 0.05) using by Bonferroni’s post hoc test; Bold values denote statistical significance at the p < 0.05. Regardless of the dietary source, the highest Zn concentration was observed in duodenal and ileal mucosa (85.5 ± 1.2 mg Zn/kg DM; 81.9 ± 1.22 mg Zn/kg DM, respectively) compared to Zn jejunal content (67.3 ± 0.66 mg Zn/kg DM, p < 0.0001). An increased ZnT1 content was observed in the duodenal and jejunal mucosa of all piglets (24.9 ± 0.74, 25.3 ± 0.71µg/g, respectively) in comparison with ZnT1 in the ileum (18.8 ± 0.57, p < 0.0001). A low MT1 content was recorded in the duodenum (13.2 ± 0.39 µg/g, p < 0.001), followed by an increased value in the ileum (18.4 ± 0.65 µg/g, p < 0.01), and the greatest MT1 content was in the jejunum (20.4 ± 0.24 µg/g, p < 0.01). Mineral concentrations of Zn, Cu, Fe and Mn in piglet liver, kidney, pancreas, bone, muscle, duodenum, jejunum and ileum tissues are expressed in mg/kg of tissue dry matter (Table 3). Intake of the diets containing potato fibre increased Zn concentrations in the ileal mucosa (p < 0.05). A Zn × fibre source interaction was observed for the Zn concentration in jejunal mucosa, with increased Zn levels in the control treatment supplemented with cellulose and ZnSO4 compared to the ZnGly or PF treatment (p < 0.05). 3. Results The Zn concentration in the other tissues and plasma of the piglets was not affected by the dietary treatment (Tables 3 and 4). 7 of 16 Animals 2019, 9, 497 Table 3. Tissue Zn, Cu, Fe and Mn concentrations of piglets fed diets differing in zinc and fibre source. Mineral Concentration Dietary Treatment SEM Main Effects Interaction C ZnGly PF PF + ZnGly Zn Fibre Zn × Fibre Zinc, mg/kg DM Liver 425 413 420 392 20.1 0.64 0.76 0.85 Kidney 169 153 150 143 4.60 0.14 0.22 0.60 Pancreas 168 152 160 147 9.51 0.48 0.75 0.93 Bone 1 293 287 283 290 3.59 0.96 0.64 0.37 Muscle 43.9 40.3 43.4 43.0 1.37 0.50 0.71 0.57 Duodenum 85.9 85.6 82.9 84.7 1.27 0.79 0.47 0.69 Jejunum 70.1 a 65.5 b 65.7 b 67.8 ab 0.76 0.37 0.45 0.03 Ileum 77.2 79.7 84.5 82.1 1.13 0.99 0.03 0.25 Copper, mg/kg DM Liver 90.3 79.3 133.8 92.9 8.21 0.12 0.08 0.36 Kidney 39.3 46.2 41.8 44.6 2.78 0.42 0.95 0.74 Pancreas 2.10 2.10 2.46 2.54 0.173 0.95 0.27 0.89 Bone 1 2.67 2.56 2.55 2.27 0.106 0.42 0.38 0.71 Muscle 2.53 2.39 2.62 2.76 0.163 0.99 0.52 0.69 Duodenum 57.3 54.3 75.4 51.6 4.90 0.43 0.18 0.30 Jejunum 12.8 13.5 12.3 10.5 0.515 0.61 0.09 0.22 Ileum 8.47 7.99 7.88 7.13 0.307 0.34 0.26 0.83 Iron, mg/kg DM Liver 354 342 389 415 15.3 0.83 0.08 0.85 Kidney 140 155 136 152 3.60 0.04 0.62 0.91 Pancreas 105 99.0 110 102 3.50 0.21 0.51 0.87 Bone 1 167 177 172 175 4.98 0.54 0.92 0.75 Muscle 20.7 22.3 20.8 21.3 0.563 0.36 0.68 0.65 Duodenum 566 719 1329 1187 106 0.50 0.01 0.74 Jejunum 36.3 34.9 35.7 38.9 1.16 0.48 0.20 0.09 Ileum 41.0 36.0 41.8 35.4 1.13 0.01 0.97 0.75 Manganese, mg/kg DM Liver 9.92 10.2 9.86 9.51 0.201 0.91 0.39 0.48 Kidney 6.63 ab 6.52 ab 6.20 a 6.94 b 0.119 0.18 0.92 0.05 Pancreas 5.06 4.57 4.88 4.57 0.115 0.11 0.72 0.71 Muscle 0.45 0.52 0.52 0.59 0.020 0.09 0.08 0.90 Duodenum 9.95 10.3 8.95 9.54 0.465 0.64 0.38 0.88 Jejunum 4.47 4.98 4.04 4.88 0.156 0.03 0.39 0.60 Ileum 3.50 3.58 3.50 3.40 0.128 0.98 0.73 0.75 C: cellulose, ZnGly: zinc chelate of glycine hydrate, PF: potato fibr. e e s e s g c y e e (p 0 05) s g y s e s e s S g c e e ce ( S ) pos oc mineral concentrations, mg/kg ash weight; Bold values denote statistical significance at the p < 0.05. 3. Results The effect of the Zn source was observed in the plasma albumin content, with increased values in the ZnGly treatments (p < 0.057). The concentrations of MT1, TBARS and PAB in plasma were not affected by the dietary treatment (Table 4). Table 4. Plasma concentration of Zn, Cu, Fe and Mn; total- and non-protein-bound thiols (TSH and NPSH, respectively); albumin (Alb); metalothionein 1 (MT); thiobarbituric acid reactive substances (TBARS); prooxidat-antioxidant balance (PAB); and plasma activity of alkaline phosphatase (ALP) in piglets fed diets differing in zinc and fibre source. Plasma Dietary Treatment SEM Main Effects Interaction C ZnGly PF PF + ZnGly Zn Fibre Zn × Fibre Zn, mg/L 0.842 0.877 0.836 0.963 0.025 0.18 0.50 0.44 Cu, mg/L 1.77 a 1.90 a 1.74 a 1.47 b 0.039 0.10 0.001 0.001 Zn/Cu ratio 0.476 a 0.460 a 0.478 a 0.658 b 0.022 0.01 0.002 0.003 Fe, mg/L 2.79 2.42 2.28 2.53 0.089 0.77 0.36 0.16 Mn, µg/L 5.28 4.93 5.22 5.43 0.220 0.88 0.65 0.57 ALP, U/L 548 a 415 ab 318 b 392 ab 27.97 0.54 0.02 0.04 Alb, g/L 30.5 32.2 31.7 37.4 1.014 0.06 0.09 0.28 MT, µg/L 13.9 14.1 14.4 13.9 0.110 0.53 0.52 0.15 TSH, mmol/L 0.346 A 0.344 A 0.251 B 0.360 A 0.014 0.06 0.13 0.03 NPSH, mmol/L 0.024 A 0.020 A 0.018 B 0.020 A 0.001 0.05 0.04 0.04 TBARS, µmol/L 0.41 0.53 0.45 0.41 0.026 0.36 0.43 0.14 PAB, HK unit 63.3 65.9 81.7 72.9 5.262 0.78 0.26 0.61 C: cellulose, ZnGly: zinc chelate of glycine hydrate, PF: potato fibre. a,b Means within lines with different superscript letters are significantly different (p < 0.05) using by Bonferroni’s post hoc test. A,B Means within lines with different superscript letters are significantly different (p < 0.05) using Fisher’s Least Significant Difference (LSD) post hoc test; Bold values denote statistical significance at the p < 0.05. C: cellulose, ZnGly: zinc chelate of glycine hydrate, PF: potato fibre. a,b Means within lines with different superscript letters are significantly different (p < 0.05) using by Bonferroni’s post hoc test. A,B Means within lines with different superscript letters are significantly different (p < 0.05) using Fisher’s Least Significant Difference (LSD) post hoc test; Bold values denote statistical significance at the p < 0.05. 3. Results a,b Means within lines with different superscript letters are significantly different (p < 0.05) using by Fisher’s Least Significant Difference (LSD) post hoc test. 1 Bone mineral concentrations, mg/kg ash weight; Bold values denote statistical significance at the p < 0.05. Table 3. Tissue Zn, Cu, Fe and Mn concentrations of piglets fed diets differing in zinc and fibre source. Intake of the diets supplemented with ZnGly increased Fe concentrations in the kidney (p < 0.05), but the Fe levels in the ileal mucosa were decreased in the treatments (p < 0.01). A nearly two-fold increase in Fe concentrations was determined in the duodenal mucosa of both PF treatments (p < 0.01). The liver tissue of piglets fed the PF diets showed a tendency towards increased Fe and Cu concentrations (both minerals, p = 0.08). Supplementation with the organic Zn source increased Mn concentrations in the jejunal mucosa of piglets (p < 0.05). A Zn × fibre source interaction was recorded for the Mn concentration in the kidney, with the highest Mn levels in the PF + ZnGly treatment (p < 0.05). Other tissue mineral concentrations were not changed by the dietary treatment (Table 3). The effect of fibre source was observed in the plasma Cu concentration and plasma ALP activity (p < 0.001, p < 0.05; respectively); however, a Zn × fibre interaction was also found (Table 4). Increased plasma ALP was found in the control treatment compared to the PF treatments (p < 0.05). Intake of the PF diets decreased Cu levels in the plasma, with the lowest concentration in the PF + ZnGly (p < 0.001) combination compared to all other treatments. However, plasma Zn levels were unaffected, and decreased plasma Cu levels mainly in the PF + ZnGly treatment resulted in an elevated Zn/Cu ratio, with the highest significant value in this treatment. Moreover, the Zn/Cu ratio was affected by 8 of 16 Animals 2019, 9, 497 both the zinc and fibre source (both p < 0.01), and a Zn × fibre interaction was also recorded (p < 0.01). The effect of both dietary sources, as well as a Zn × fibre interaction, was recorded in the plasma levels of non-protein thiols, with lower values of both total and non-protein thiols in the PF treatment (p < 0.05). Correlation Analyses A positive correlation was recorded between Zn concentrations and ALP activity in the liver (r = 0.506, p = 0.012), and plasma ALP and liver ALP activity (r = 0.619, p = 0.002). Cu content in the liver positively correlated with Cu/Zn SOD activity in the tissue (r = 0.446, p = 0.043). Relationships between Zn transporters, trace elements and other parameters in animal tissues are presented in Table 6. Correlation analyses showed a significant negative correlation between ZnT1 and MT1 levels in the duodenum (r = −0.417; p = 0.04), while jejunal ZnT1 positively correlated with MT1 in the jejunum (r = 0.454, p = 0.03). MT1 levels in the duodenum negatively correlated with Cu content in the segment of the small intestine (r = −0.517, p = 0.011), while a positive correlation was determined between Cu content and MT levels in the jejunum (r = 0.410, p < 0.05). No significant correlation was found between the zinc transporters (MT1, ZnT1) and Cu content in the ileum. Correlation analyses showed a positive correlation between Zn tissue concentrations, with the correlation coefficients (r) between kidney Zn and Zn concentrations in the liver, pancreas, jejunum and Cu content in the kidney at 0.577 (p = 0.003), 0.405 (p < 0.05), 0.486 (p = 0.02), and 0.472 (p = 0.03), respectively. Zn duodenal content positively correlated with Cu content in the duodenum (r = 0.52, p = 0.011), and Zn concentration in the jejunum and ileum (r = 0.43, p = 0.036; r = 0.446, and p = 0.029, respectively). Ileal Zn content strongly positively correlated with Cu and Mn levels in the ileum (r = 0.625, p = 0.002; r = 0.573, and p = 0.003, respectively). Cu content in the duodenum positively correlated with duodenal Fe content (r = 0.621, Table 6. Relationships between Zn transporters, trace elements and other parameters in tissues of piglets fed diets differing in zinc and fibre source. Table 6. Relationships between Zn transporters, trace elements and other parameters in tissues of piglets fed diets differing in zinc and fibre source. piglets fed diets differing in zinc and fibre source. Pearson Correlation Correlation Coefficient r p Value MT1 duodenum vs. ZnT1duodenum Cu duodenum −0.417 −0.517 0.043 0.001 MT1 jejunum vs. ZnT1 jejunum Cu jejunum +0.454 +0.410 0.029 0.046 Zn duodenum vs. Correlation Analyses Relationships between Zn transporters, trace elements and other parameters in animal tissues are presented in Table 6. Correlation analyses showed a significant negative correlation between ZnT1 and MT1 levels in the duodenum (r = −0.417; p = 0.04), while jejunal ZnT1 positively correlated with MT1 in the jejunum (r = 0.454, p = 0.03). MT1 levels in the duodenum negatively correlated with Cu content in the segment of the small intestine (r = −0.517, p = 0.011), while a positive correlation was determined between Cu content and MT levels in the jejunum (r = 0.410, p < 0.05). No significant correlation was found between the zinc transporters (MT1, ZnT1) and Cu content in the ileum. Correlation analyses showed a positive correlation between Zn tissue concentrations, with the correlation coefficients (r) between kidney Zn and Zn concentrations in the liver, pancreas, jejunum and Cu content in the kidney at 0.577 (p = 0.003), 0.405 (p < 0.05), 0.486 (p = 0.02), and 0.472 (p = 0.03), respectively. Zn duodenal content positively correlated with Cu content in the duodenum (r = 0.52, p = 0.011), and Zn concentration in the jejunum and ileum (r = 0.43, p = 0.036; r = 0.446, and p = 0.029, respectively). Ileal Zn content strongly positively correlated with Cu and Mn levels in the ileum (r = 0.625, p = 0.002; r = 0.573, and p = 0.003, respectively). Cu content in the duodenum positively correlated with duodenal Fe content (r = 0.621, p = 0.002). Mn concentration in the kidney positively correlated with Fe kidney content (r = 0.424, p = 0.039). Zn concentrations in the pancreas correlated positively with Zn content in the liver (r = 0.551, p = 0.005), in each segment of the small intestine (duodenum r = 0.562, p = 0.004; jejunum r = 0.629, p = 0.001; ileum r = 0.499, p = 0.013) and in Zn bone (r = 0.416, p < 0.05). A negative correlation was recorded between Zn muscle content and Zn concentrations in bone (r = −0.658, p < 0.001), and also in the Zn pancreas, duodenum and ileum (r = −0.474, p = 0.022; r = −0.447, p = 0.032; r = −0.474, p = 0.022, respectively). 3. Results a,b Means within lines with different superscript letters are significantly different (p < 0.05) using Fisher’s Least Significant Difference (LSD) post hoc test. 1 The enzyme activity is given as density values (pixel intensities) in duodenal and jejunal enterocytes at a wavelength of 520 nm; Bold values denote statistical significance at the p < 0.05 3. Results Increased activity of total SOD and Cu/Zn SOD was only recorded in the liver tissue of both PF treatments (p < 0.05), while a Zn × fibre source interaction for enzyme activity was determined in the duodenum (Table 5). The highest duodenal activity of total SOD was found in the ZnGly treatment compared to the control and PF + ZnGly treatment (p < 0.05). The activity of both SODs did not change in the kidney, pancreas and jejunum, nor did ALP activity in the duodenum, jejunum or liver tissue (data not shown). Table 5. Activity of total superoxide dismutase (SOD), Cu/Zn superoxide dismutase (Cu/Zn SOD) and alkaline phosphatase (ALP) in tissues of piglets fed diets differing in zinc and fibre source. Enzyme Activity Dietary Treatment SEM Main Effects Interaction C ZnGly PF PF + ZnGly Zn Fibre Zn × Fibre Total SOD, U/mg protein Liver 24.1 23.5 28.9 25.9 0.782 0.27 0.05 0.25 Kidney 7.67 7.91 6.60 8.84 0.428 0.16 0.93 0.25 Pancreas 8.70 6.24 7.94 7.40 0.612 0.25 0.88 0.45 Duodenum 9.93 a 13.5 b 12.7 ab 9.61 a 0.636 0.85 0.64 0.01 Jejunum 5.72 7.39 7.13 7.32 0.376 0.24 0.40 0.35 Cu/Zn SOD, U/mg protein Liver 21.6 21.6 26.3 22.9 0.843 0.25 0.03 0.46 Kidney 6.49 6.58 5.50 7.49 0.385 0.19 0.96 0.23 Pancreas 5.73 4.12 4.73 4.20 0.468 0.28 0.64 0.58 Duodenum 5.65 8.13 7.22 5.89 0.457 0.67 0.88 0.02 Jejunum 3.44 4.77 3.92 4.49 0.268 0.09 0.86 0.49 Table 5. Activity of total superoxide dismutase (SOD), Cu/Zn superoxide dismutase (Cu/Zn SOD) and alkaline phosphatase (ALP) in tissues of piglets fed diets differing in zinc and fibre source. 9 of 16 Animals 2019, 9, 497 Table 5. Cont. Enzyme Activity Dietary Treatment SEM Main Effects Interaction C ZnGly PF PF + ZnGly Zn Fibre Zn × Fibre ALP 1 Duodenum 14.7 14.0 13.6 15.2 0.353 0.36 0.93 0.06 Jejunum 12.2 12.0 12.3 12.8 0.253 0.80 0.44 0.49 C: cellulose, ZnGly: zinc chelate of glycine hydrate, PF: potato fibre. a,b Means within lines with different superscript letters are significantly different (p < 0.05) using Fisher’s Least Significant Difference (LSD) post hoc test. 1 The enzyme activity is given as density values (pixel intensities) in duodenal and jejunal enterocytes at a wavelength of 520 nm; Bold values denote statistical significance at the p < 0.05 C: cellulose, ZnGly: zinc chelate of glycine hydrate, PF: potato fibre. 4. Discussion The zinc active absorption efficiency depends on a number of proteins involved in intestinal Zn metabolism, such as the divalent metal transporter 1 (DMT1), zinc transporter ZIP4, MT1 and ZnT1 [21]. Regulation of Zn absorption or uptake is achieved by the transporter ZIP4, situated in the apical membrane of intestinal epithelial cells, which transports Zn ions into the cytoplasm, though it has been suggested that Zn absorption from ZnGly only partly depends on ZIP4 [22]. The divalent metal transporter DMT1, which is the principal iron transporter with a lower affinity to uptake other divalent metals into enterocytes, is also in the apical membrane; however, zinc regulates DMT1 expression and function [23]. The zinc transporter ZnT1, which decreases intracellular zinc levels due to the efflux of Zn from GIT cells into circulation, is localised at the opposite basolateral membrane of enterocytes, and in this way correlates with the intracellular free Zn2+ content. Therefore, ZnT1 is considered to be an intracellular zinc status indicator [21]. An intracellular zinc-binding protein, metallothionein MT1, serves as a dynamic Zn donor and acceptor and an exchangeable Zn pool and demonstrates the tissue distribution of endogenous zinc [24]. MT plays a primary role in Zn homeostasis and the scavenging of reactive oxygen species [21,24]. Given the literature data suggesting that mRNA expression of DMT1 and ZIP4 is susceptible to a low Zn intake, while ZnT1 and MT1 act as key regulators of Zn homeostasis under normal or high Zn conditions [25], and that the diverse impact of ZnGly on the gene expression of zinc absorption-related transporters has been indicated [22], we focused on the transporters ZnT1 and MT1 in the small intestine segments of piglets responsible for the amount of Zn released into portal circulation for systemic distribution. Zinc, like other trace elements bound in chelates or organic complexes, should be more stable, and this should prevent Zn ions from chelating by phytates or other feed compounds in the GIT, while the better absorption of organically bound Zn is explained by the use of other transport systems [26–28]. Organic sources of zinc may make more Zn available to the organism than inorganic zinc supplements, partially due to the stable cyclic structure that makes Zn more easily usable by transcription factor MTF-1, and so MT gene expression could be regulated more effectively [29]. Correlation Analyses Zn jejunum Zn ileum Cu duodenum +0.430 +0.446 +0.520 0.036 0.029 0.011 Cu duodenum vs. Fe duodenum +0.621 0.002 10 of 16 Animals 2019, 9, 497 Table 6. Cont. Pearson Correlation Correlation Coefficient r p Value Zn ileum vs. Cu ileum Mn ileum +0.625 +0.573 0.002 0.003 Zn kidney vs. Zn liver Zn pancreas Zn jejunum Cu kidney +0.577 +0.405 +0.486 +0.472 0.003 0.050 0.016 0.031 Mn kidney vs. Fe kidney +0.424 0.039 Zn liver vs. ALP liver +0.506 0.012 Cu liver vs. Cu/Zn SOD liver +0.446 0.043 Zn pancreas vs. Zn liver Zn duodenum Zn jejunum Zn ileum Zn bone +0.551 +0.562 +0.629 +0.499 +0.416 0.005 0.004 0.001 0.013 0.049 Zn muscle vs. Zn duodenum Zn ileum Zn pancreas Zn bone −0.447 −0.474 −0.474 −0.658 0.032 0.022 0.022 0.001 Zn plasma vs. TSH plasma ALB plasma +0.507 +0.478 0.019 0.024 Correlation Coefficient r 4. Discussion The upregulation of MT1 and ZnT1 mRNA expression was recorded on IPEC-J2 cells treated with different Zn sources, 11 of 16 Animals 2019, 9, 497 11 of 16 especially in the ZnGly-added treatment [22]. Similarly, organic dietary Zn sources increased gene expression of the zinc transporter ZnT1 in the jejunum of broiler chickens and the apparent Zn retention in piglets [27,30]. In our study, the feeding of both ZnGly diets elevated ZnT1 levels in the ileal mucosa, whereas MT levels and Zn concentration were not affected by the Zn source in any intestinal sections. However, the interaction of a challenge by Zn and also a fibre source was noted for the Zn concentration in the jejunal mucosa. The highest Zn content was noted in the jejunum of pigs fed the diet supplemented with cellulose and ZnSO4 compared to the ZnGly or PF diets. Our results indicate that ZnGly did not affect active Zn transport in the duodenum and jejunum, but it seems that it can alter Zn efflux from ileum into circulation without evidence of influencing zinc tissue deposition compared to ZnSO4. Both insoluble and soluble dietary fibres might have unfavourable effects on mineral absorption because of their mineral-binding properties. Cellulose is poorly soluble and fermentable, and the physical entrapment of minerals intrinsic to cellulose due to the formation of insoluble forms and/or large complexes results in decreasing mineral bioavailability [31]. On the other hand, the enhancing effect of soluble/fermentable fibre sources could be related to fermentation products that may increase passive absorption, the lowering of pH or the organic acid production that may have absorption enhancing properties [32]. The fermentation of soluble fibres in the colon often produces short chain fatty acids that can trigger the increased proliferation of epithelial cells, an increased absorptive surface area and hence absorption of some minerals, although this absorption enhancing property does not apply to all minerals and depends on the specific characteristics of the soluble fibre concerned [33]. Based on the mentioned properties of soluble fibres and our previous results indicating a positive effect of dietary potato fibre on mineral digestibility in pigs [34,35], the increased absorption of trace minerals, including zinc from the GIT of piglets, and the better mineral utilisation in tissues, were intended in the PF treatments. The intake of the PF diets decreased the MT1 and ZnT1 concentration in the jejunum of piglets in our study. 4. Discussion Intestinal MT regulates Zn absorption and can inhibit dietary Zn uptake by its sequestering capacity [36,37]. Cu has been shown to have a higher affinity than Zn for MT, presumably displacing Zn from MT and reducing Cu absorption [36]. The increased MT levels in the jejunum of piglets fed the cellulose-containing diets depressed the intracellular Zn and Cu concentrations by their sequestration inside the enterocytes bound to MT resulted in an apparent decreased Zn digestibility [38] and decreased Cu concentration in the liver. A tendency towards an increased liver Cu concentration in the PF treatments could result in the higher activity of Cu/Zn-dependent SOD in liver tissue. Liver SOD activity is sensitive to changes in Cu status and the increase in SOD activity is associated with an increased Cu content. Cu may not be safely bound in liver and acts as a pro-oxidant, leading to an increase in Cu/Zn superoxide dismutase activity in the tissue [39–41]. Moreover, a positive correlation between Cu/Zn SOD activity and Cu concentration in liver tissue was recorded in our study. Regardless of the dietary source of Zn and fibre, the highest contents of ZnT1 and MT1 were observed in the jejunum of piglets, and the Zn concentration decreased significantly in this intestinal section. Higher Zn concentrations were found in the duodenum, followed by an increased ZnT1 content and inversely correlated MT1. A kinetic study on intestinal zinc transport using an intestinal membrane isolated from small intestinal segments of rat indicated the lowest Zn transport rate in the jejunum, while the maximum Zn transport rate in the duodenum, which was not correlated with the mRNA expression of the Zn transporters ZnT1 and ZIP4, but showed an inverse correlation with MTs [42]. The authors suggested that the increased expression of MTs results in a low transport rate of zinc, and the increased relative expression of ZnT1, MT1 and MT2 was only reported in the jejunum compared to other segments of a rat’s small intestine. Our findings suggest a higher Zn absorption rate in the duodenum of piglets, while the lower Zn absorption in the jejunum was probably caused by the intracellular zinc-binding capacity of MTs. The high Zn content in the ileum might be caused by a non-saturable diffusive process, which is a different transport mechanism than the carrier-mediated absorption used in the duodenal and jejunal segments. 4. Discussion Similar results were described in small intestinal segments of broilers, where Zn absorption from organic sources was obviously greater than from Animals 2019, 9, 497 12 of 16 ZnSO4 [43]; however, the intake of ZnGly in our study only increased Zn efflux from the ileum by an increased ZnT1 content. The correlation analyses regarding Zn tissue concentration showed a strong positive correlation between Zn concentrations in the pancreas and each small intestinal segment, which confirms the important role of the pancreas in the endogenous excretion and reutilisation of zinc, as well as for Zn homeostasis. Zn, Cu and Fe are chemically similar, so the interaction among the elements appears to be tissue-specific and a positive correlation has been found between total Zn and Cu concentrations, mainly in liver and kidney tissues [40,41]. We found a positive correlation between Zn and Cu content in the kidney and ileum. In addition, ileal Zn content was positively correlated with Mn levels and duodenal Cu content was positively correlated with Fe levels in the intestinal segment. The positive correlation between Zn and Cu, Mn, and also Cu and Fe in intestinal tissues may indicate an intracellular trapping of the minerals caused by the impairment of efflux mechanisms. Low intracellular available Cu bound to MT could decrease the efflux of Fe and Mn during their transport out of the enterocytes through the Cu-dependent protein hephaestin that co-operates with their common transporter, ferroportin [44]. Unfortunately, we did not focus on other metallotransporters, so our results cannot clearly indicate any significant competition among the minerals that share the same transporter systems at the level of membrane transport. The effect of the Zn source was found in the increased kidney and reduced ileal Fe content, and the strong positive correlation between liver Fe and ZnT1 contents, mainly in the ZnGly treatments. Intake of the PF diets increased Fe concentrations in the duodenum, which could have led to better Fe utilisation by the liver tissue in the PF treatments. On the other hand, increased duodenal Fe could be caused by impaired Fe efflux out of the eneterocytes. Anyway, further investigation is needed to fully understand the effect of different Zn and fibre sources on Fe transporter mechanisms in enterocytes. 4. Discussion Our results showed a correlation between plasma Zn levels and total thiols or plasma albumin, with the highest values in the plasma of the piglets fed the diet enriched with ZnGly and PF. Plasma thiols were comprised of protein-bound and non-protein sulfhydryl groups, which contribute to the antioxidant capacity of plasma [45]. The most abundant plasma protein is albumin, which is the principal zinc carrier in plasma. The albumin molecule has 17 disulfide bridges and a single free cysteine residue, and given its structure, it is also a powerful radical scavenger in plasma and provides >50% of the total antioxidant of normal plasma [46]. It has been shown that during oxidative stress, plasma Zn levels and the plasma albumin-bound fraction of Zn are decreased, while plasma Cu levels are elevated [47]. Given the highest decrease of plasma Cu levels in the PF + ZnGly treatment, an elevated Zn/Cu ratio was recorded, whereas plasma Zn concentrations were similar in all piglets in our study. Low plasma Cu levels and low Cu/Zn ratios have been noted after excessive Zn treatments due to depressed Cu absorption and increased Cu sequestration in the mucosal cell bound to MT [48–50]. Nevertheless, Cu absorption is depressed when Zn is given in high excess over Cu [51], and many changes of the Zn/Cu imbalance could be used as a better indicator of metabolic disturbance than Zn or Cu status alone [52]. The zinc-related enzyme ALP is important in the detoxification of pathogenic bacteria endotoxins and enzyme alterations and can affect the absorptive and secretory ability of the small intestine. During the post-weaning phase, the downregulation of intestinal ALP has been suggested due to inflammatory processes and microbiota imbalance of the intestine of piglets [53]. Suitable feed supplementation can increase intestinal ALP expression, together with modulation of the gastrointestinal microbiota of pigs, and reduce intestinal inflammation or prevent diarrhoea. Although intestinal and liver ALP activities were unchanged by dietary treatments in our study, we noted the altered ALP activity in plasma that positively correlated with liver ALP activity. A positive correlation between Zn content and ALP activity in the liver indicates the importance of Zn in ALP enzymatic activity. However, total serum ALP originates from various tissues, mainly from the liver and bone, as well as from the intestine, spleen or kidney, but the bone-specific ALP isoenzyme predominates in the serum of young growing animals. 4. Discussion It has been shown that Zn influences bone formation and mineralisation, and also Animals 2019, 9, 497 13 of 16 stimulates ALP activity in osteoblast cells [54]. A recent study showed that the partial replacing of dietary inorganic minerals, including Zn with glycine chelates, increased immature collagen in the bone tissue of pheasants [55]. We can only speculate whether the variability of total plasma ALP could imply different bone formation in our piglets because of unchanged ALP levels in the liver or intestine; however, we did not measure any parameters related to bone formation or mineralisation. 5. Conclusions Thanks go to Renata Gerocova and Peter Jerga for their excellent assistance in data collection. Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. 5. Conclusions In conclusion, the present study showed that the dietary intake of potato fibre can influence the absorption of zinc and other trace elements from the small intestine due to affecting the Zn-transporter levels in the jejunum of piglets and increased zinc content in the ileum, which is the main site of non-saturable transport pathways. Feeding the potato fibre diets increased the ZnT1 content in the liver, while the content of both zinc transporters ZnT1 and MT1 was decreased in the jejunum. Similarly, dietary PF altered the activity of the Zn-containing enzymes in the liver SOD and plasma ALP. The substitution of dietary zinc glycine chelate in place of the commonly used zinc sulphate did not significantly influence the Zn distribution, but it may alter the absorption and utilisation of iron and manganese. Both dietary sources affected Fe concentrations in the small intestine and kidney tissues, and elevated the content of total thiols and the Zn/Cu ratio in plasma. We can conclude that feeding potato fibre and Zn glycinate can influence the mineral and antioxidant status of growing piglets, but further research is needed in order to elucidate the effect of both dietary sources on the transport systems of other minerals in enterocytes. Author Contributions: Conceptualization, M.B., M.T., K.C., and L.G.; data curation, M.B., L.G., M.H., A.T., and Z.S.; formal analysis, L.G.; funding acquisition, K.C.; investigation, M.B., A.T., M.T., K.C., and L.G.; methodology, M.B., M.T. and K.C.; project administration, M.B., M.T.; resources, M.B. and A.T.; validation, M.B. and L.G.; visualization, L.G.; writing—original draft preparation, L.G.; writing—review and editing, K.C. and L.G. Author Contributions: Conceptualization, M.B., M.T., K.C., and L.G.; data curation, M.B., L.G., M.H., A.T., and Z.S.; formal analysis, L.G.; funding acquisition, K.C.; investigation, M.B., A.T., M.T., K.C., and L.G.; methodology, M.B., M.T. and K.C.; project administration, M.B., M.T.; resources, M.B. and A.T.; validation, M.B. and L.G.; visualization, L.G.; writing—original draft preparation, L.G.; writing—review and editing, K.C. and L.G. Funding: This work was supported by the project ITMS 26220120066 of the Research and Development Operational Programme funded by the ERDF, by the Slovak Research and Development Support Agency APVV, grant number 17-0297 and by the Slovak Grant Agency VEGA, grant number 2/0069/17. This study is based upon work from COST Action FA1401 (PiGutNet), supported by COST (European Cooperation in Science and Technology). Acknowledgments: We thank David Lee McLean for providing language support that greatly improved the manuscript. References 1. Thacker, P. 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Learning Automata with Side-Effects
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Learning Automata with Side-Effects Gerco Van Heerdt, Matteo Sammartino, Alexandra Silva To cite this version: Gerco Van Heerdt, Matteo Sammartino, Alexandra Silva. Learning Automata with Side-Effects. 15th International Workshop on Coalgebraic Methods in Computer Science (CMCS), Apr 2020, Dublin, Ireland. pp.68-89, ￿10.1007/978-3-030-57201-3_5￿. ￿hal-03232354￿ Learning Automata with Side-Effects Gerco Van Heerdt, Matteo Sammartino, Alexandra Silva To cite this version: Gerco Van Heerdt, Matteo Sammartino, Alexandra Silva. Learning Automata with Side-Effects. 15th International Workshop on Coalgebraic Methods in Computer Science (CMCS), Apr 2020, Dublin, Ireland. pp.68-89, ￿10.1007/978-3-030-57201-3_5￿. ￿hal-03232354￿ Distributed under a Creative Commons Attribution 4.0 International License ⋆This work was partially supported by the ERC Starting Grant ProFoundNet (grant code 679127), a Leverhulme Prize (PLP–2016–129), and the EPSRC Standard Grant CLeVer (EP/S028641/1). Gerco van Heerdt1 (B), Matteo Sammartino1,2 , and Alexandra Silva1 1 University College London, United Kingdom {gerco.heerdt,alexandra.silva}@ucl.ac.uk 2 Royal Holloway, University of London, United Kingdom matteo.sammartino@rhul.ac.uk Abstract. Automata learning has been successfully applied in the verifi- cation of hardware and software. The size of the automaton model learned is a bottleneck for scalability, and hence optimizations that enable learning of compact representations are important. This paper exploits monads, both as a mathematical structure and a programming construct, to design and prove correct a wide class of such optimizations. Monads enable the development of a new learning algorithm and correctness proofs, building upon a general framework for automata learning based on category the- ory. The new algorithm is parametric on a monad, which provides a rich algebraic structure to capture non-determinism and other side-effects. We show that this allows us to uniformly capture existing algorithms, develop new ones, and add optimizations. Keywords: automata · learning · side-effects · monads · algebras. HAL Id: hal-03232354 https://inria.hal.science/hal-03232354v1 Submitted on 21 May 2021 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License Van Heerdt et al. 2 In this paper, we show how monads can be used to add optimizations to learning algorithms in order to obtain compact representations. We will use as playground for our approach the well known L⋆algorithm [2], which learns a minimal deterministic finite automaton (DFA) accepting a regular language by interacting with a teacher, i.e., an oracle that can reply to specific queries about the target language. Monads allow us to take an abstract approach, in which category theory is used to devise an optimized learning algorithm and a generic correctness proof for a broad class of compact models. The inspiration for this work is quite concrete: it is a well-known fact that non-deterministic finite automata (NFAs) can be much smaller than deterministic ones for a regular language. The subtle point is that given a regular language, there is a canonical deterministic automaton accepting it—the minimal one—but there might be many “minimal” non-deterministic automata accepting the same language. This raises a challenge for learning algorithms: which non-deterministic automaton should the algorithm learn? To overcome this, Bollig et al. [11] developed a version of Angluin’s L⋆algorithm, which they called NL⋆, in which they use a particular class of NFAs, Residual Finite State Automata (RFSAs), which do admit minimal canonical representatives. Though NL⋆indeed is a first step in incorporating a more compact representation of regular languages, there are several questions that remain to be addressed. We tackle them in this paper. q p p DFAs and NFAs are formally connected by the subset construction. Under- pinning this construction is the rich algebraic structure of languages and of the state space of the DFA obtained by determinizing an NFA. The state space of a determinized DFA—consisting of subsets of the state space of the original NFA—has a join-semilattice structure. Moreover, this structure is preserved in language acceptance: if there are subsets U and V , then the language of U ∪V is the union of the languages of the first two. Formally, the function that assigns to each state its language is a join-semilattice map, since languages themselves are just sets of words and have a lattice structure. And languages are even richer: they have the structure of complete atomic Boolean algebras. This leads to several questions: Can we exploit this structure and have even more compact representations? 1 Introduction The increasing complexity of software and hardware systems calls for new scalable methods to design, verify, and continuously improve systems. Black-box inference methods aim at building models of running systems by observing their response to certain queries. This reverse engineering process is very amenable for automation and allows for fine-tuning the precision of the model depending on the properties of interest, which is important for scalability. One of the most successful instances of black-box inference is automata learn- ing, which has been used in various verification tasks, ranging from finding bugs in implementations of network protocols [15] to rejuvenating legacy software [29]. Vaandrager [30] has written a comprehensive overview of the widespread use of automata learning in verification. A limitation in automata learning is that the models of real systems can become too large to be handled by tools. This demands compositional methods and techniques that enable compact representation of behaviors. ⋆This work was partially supported by the ERC Starting Grant ProFoundNet (grant code 679127), a Leverhulme Prize (PLP–2016–129), and the EPSRC Standard Grant CLeVer (EP/S028641/1). Van Heerdt et al. What if we slightly change the setting and look at weighted languages over a semiring, which have the structure of a semimodule (or vector space, if the semiring is a field)? The latter question is strongly motivated by the widespread use of weighted languages and corresponding weighted finite automata (WFAs) in verification, from the formal verification of quantitative properties [13,17,25], to probabilistic model-checking [5], to the verification of on-line algorithms [1]. Our key insight is that the algebraic structures mentioned above are in fact algebras for a monad T. In the case of join-semilattices this is the powerset monad, and in the case of vector spaces it is the free vector space monad. These monads can be used to define a notion of T-automaton, with states having the structure of an algebra for the monad T, which generalizes non-determinism as a side-effect. From T-automata we can derive a compact, equivalent version by Learning Automata with Side-Effects 3 taking as states a set of generators and transferring the algebraic structure of the original state space to the transition structure of the automaton. This general perspective enables us to generalize L⋆to a new algorithm L⋆ T , which learns compact automata featuring non-determinism and other side-effects captured by a monad. Moreover, L⋆ T incorporates further optimizations arising from the monadic representation, which lead to more scalable algorithms. We start by giving an overview of our approach, which also states our main contributions in greater detail and ends with a road map of the rest of the paper. 2 Overview and Contributions The state space of the hypothesis 4 Van Heerdt et al. Van Heerdt et al. 4 Van Heerdt et al. 1 S, E ←{ε} 2 repeat 3 while the table is not closed or not consistent 4 if the table is not closed 5 find t ∈S, a ∈A such that rowb(t)(a) ̸= rowt(s) for all s ∈S 6 S ←S ∪{ta} 7 if the table is not consistent 8 find s1, s2 ∈S, a ∈A, and e ∈E such that rowt(s1) = rowt(s2) and rowb(s1)(a)(e) ̸= rowb(s2)(a)(e) 9 E ←E ∪{ae} 10 Construct the hypothesis H and submit it to the teacher 11 if the teacher replies no, with a counterexample z 12 S ←S ∪prefixes(z) 13 until the teacher replies yes 14 return H if the table is not closed { } Construct the hypothesis H and submit it to the teacher 11 if the teacher replies no, with a counterexample z 12 S ←S ∪prefixes(z) if the teacher replies no, with a counterexample z 13 until the teacher replies yes 14 return H 2 Overview and Contributions In this section, we explain the original L⋆algorithm and discuss the challenges in adapting the algorithm to learn automata with side-effects, illustrating them through a concrete example—NFAs. We then highlight our main contributions. L⋆algorithm. This algorithm learns the minimal DFA accepting a language L ⊆A∗over a finite alphabet A. It assumes the existence of a minimally adequate teacher, which is an oracle that can answer two types of queries: 1. Membership queries: given a word w ∈A∗, does w belong to L? and 2. Equivalence queries: given a hypothesis DFA H, does H accept L? If not, the teacher returns a coun- terexample, i.e., a word incorrectly classified by H. The algorithm incrementally builds an observation table made of two parts: a top part, with rows ranging over a finite set S ⊆A∗; and a bottom part, with rows ranging over S · A (· is pointwise concatenation). Columns range over a finite E ⊆A∗. For each u ∈S ∪S · A and v ∈E, the corresponding cell in the table contains 1 if and only if uv ∈L. Intuitively, each row u contains enough information to fully identify the Myhill–Nerode equivalence class of u with respect to an approximation of the target language—rows with the same content are considered members of the same equivalence class. Cells are filled in using membership queries. As an example, and to set notation, consider the table below over A = {a, b}. It shows that L contains the word aa and does not contain the words ε (the empty word), a, b, ba, aaa, and baa. E ε a aa S  ε 0 0 1 S · A  a 0 1 0 b 0 0 0 rowt : S →2E rowt(u)(v) = 1 ⇐⇒ uv ∈L rowb : S →(2E)A rowb(u)(a)(v) = 1 ⇐⇒uav ∈L  We use functions rowt and rowb to describe the top and bottom parts of the table, respectively. Notice that S and S · A may intersect. For conciseness, when tables are depicted, elements in the intersection are only shown in the top part. A key idea of the algorithm is to construct a hypothesis DFA from the different rows in the table. The construction is the same as that of the minimal DFA from the Myhill–Nerode equivalence, and exploits the correspondence between table rows and Myhill–Nerode equivalence classes. Fig. 1: L⋆algorithm. DFA is given by the set H = {rowt(s) | s ∈S}. Note that there may be multiple rows with the same content, but they result in a single state, as they all belong to the same Myhill–Nerode equivalence class. The initial state is rowt(ε), and we use the ε column to determine whether a state is accepting: rowt(s) is accepting whenever rowt(s)(ε) = 1. The transition function is defined as rowt(s) a−→rowb(s)(a). (Notice that the continuation is drawn from the bottom part of the table). For the hypothesis automaton to be well-defined, ε must be in S and E, and the table must satisfy two properties: – Closedness states that each transition actually leads to a state of the hypothesis. That is, the table is closed if for all t ∈S and a ∈A there is s ∈S such that rowt(s) = rowb(t)(a). ( ) ( )( ) – Consistency states that there is no ambiguity in determining the transitions. That is, the table is consistent if for all s1, s2 ∈S such that rowt(s1) = rowt(s2) we have rowb(s1) = rowb(s2). The algorithm updates the sets S and E to satisfy these properties, constructs a hypothesis, submits it in an equivalence query, and, when given a counterexample, refines the hypothesis. This process continues until the hypothesis is correct. The algorithm is shown in Fig. 1. Example Run. We now run the algorithm with the target language L = {w ∈ {a}∗| |w| ̸= 1}. The minimal DFA accepting L is M = a a a (1) (1) Initially, S = E = {ε}. We build the observation table given in Fig. 2a. This table is not closed, because the row with label a, having 0 in the only column, Learning Automata with Side-Effects ε ε 1 a 0 (a) ε ε 1 a 0 aa 1 (b) a a (c) ε ε 1 a 0 aa 1 aaa 1 aaaa 1 (d) ε a ε 1 0 a 0 1 aa 1 1 aaa 1 1 aaaa 1 1 (e) Fig. 2: Example run of L⋆on L = {w ∈{a}∗| |w| ̸= 1}. Fig. 2: Example run of L⋆on L = {w ∈{a}∗| |w| ̸= 1}. does not appear in the top part of the table: the only row ε has 1. To fix this, we add the word a to the set S. Now the table (Fig. Fig. 1: L⋆algorithm. The crucial observation underlying our approach is that the language semantics of an NFA is defined in terms of its determinization, i.e., the DFA obtained by taking sets of states of the NFA as state space. In other words, this DFA is defined over an algebraic structure induced by the powerset, namely a (complete) join semilattice (JSL) whose join operation is set union. This automaton model does admit minimal representatives, which leads to the key idea for our algorithm: learning NFAs as automata over JSLs. In order to do so, we use an extended table where rows have a JSL structure, defined as follows. The join of two rows is given by an element-wise or, and the bottom element is the row containing only zeroes. More precisely, the new table consists of the two functions row♯ t : P(S) →2E row♯ b : P(S) →(2E)A given by row♯ t(U) = W{rowt(s) | s ∈U} and row♯ b(U)(a) = W{rowb(s)(a) | s ∈U}. Formally, these functions are JSL homomorphisms, and they induce the following general definitions: – The table is closed if for all U ⊆S, a ∈A there is U ′ ⊆S such that row♯ t(U ′) = row♯ b(U)(a). – The table is consistent if for all U1, U2 ⊆S s.t. row♯ t(U1) = row♯ t(U2) we have row♯ b(U1) = row♯ b(U2). – The table is closed if for all U ⊆S, a ∈A there is U ′ ⊆S such that row♯ t(U ′) = row♯ b(U)(a). ♯ ♯ We remark that our algorithm does not actually store the whole extended table, which can be quite large. It only needs to store the original table over S because all other rows in P(S) are freely generated and can be computed as needed, with no additional membership queries. The only lines in Fig. 1 that need to be adjusted are lines 5 and 8, where closedness and consistency are replaced with the new notions given above. Moreover, H is now built from the extended table. Optimizations. In this paper we also present two optimizations to our algorithm. For the first one, note that the state space of the hypothesis constructed by the algorithm can be very large since it encodes the entire algebraic structure. Fig. 1: L⋆algorithm. 2b) is closed and consistent, so we construct the hypothesis that is shown in Fig. 2c and pose an equivalence query. The teacher replies no and informs us that the word aaa should have been accepted. L⋆handles a counterexample by adding all its prefixes to the set S. We only have to add aa and aaa in this case. The next table (Fig. 2d) is closed, but not consistent: the rows ε and aa both have value 1, but their extensions a and aaa differ. To fix this, we prepend the continuation a to the column ε on which they differ and add a · ε = a to E. This distinguishes rowt(ε) from rowt(aa), as seen in the next table in Fig. 2e. The table is now closed and consistent, and the new hypothesis automaton is precisely the correct one M. As mentioned, the hypothesis construction approximates the theoretical construction of the minimal DFA, which is unique up to isomorphism. That is, for S = E = A∗the relation that identifies words of S having the same value in rowt is precisely the Myhill–Nerode’s right congruence. Learning non-deterministic automata. As is well known, NFAs can be smaller than the minimal DFA for a given language. For example, the language L above is accepted by the NFA N = a a a (2) N = a a a (2) (2) a which is smaller than the minimal DFA M. Though in this example, which we chose for simplicity, the state reduction is not massive, it is known that in general NFAs can be exponentially smaller than the minimal DFA [24]. This reduction of the state space is enabled by a side-effect—non-determinism, in this case. Learning NFAs can lead to a substantial gain in space complexity, but it is challenging. The main difficulty is that NFAs do not have a canonical minimal representative: there may be several non-isomorphic state-minimal NFAs accept- ing the same language, which poses problems for the development of the learning algorithm. To overcome this, Bollig et al. [11] proposed to use a particular class of NFAs, namely RFSAs, which do admit minimal canonical representatives. Van Heerdt et al. 6 However, their ad-hoc solution for NFAs does not extend to other automata, such as weighted or alternating. In this paper we present a solution that works for any side-effect, specified as a monad. Fig. 1: L⋆algorithm. We show that we can extract a minimal set of generators from the table and compute a succinct hypothesis in the form of an automaton with side-effects, without any algebraic structure. For JSLs, this consists in only taking rows that are not the join of other rows, i.e., the join-irreducibles. By applying this optimization to this specific case, we essentially recover the learning algorithm of Bollig et al. [11]. The second optimization is a generalization of the optimized counterexample handling method of Rivest and Schapire [28], originally intended for L⋆and DFAs. It consists in processing counterexamples by adding a single suffix of the counterexample to E, instead of adding all prefixes of the counterexample to S. This can avoid the algorithm posing a large number of membership queries. Learning Automata with Side-Effects 7 ε ε 1 a 0 (a) a a (b) (c) ε ε 1 a 0 aa 1 aaa 1 (d) ε a ε 1 0 a 0 1 aa 1 1 aaa 1 1 (e) Fig. 3: Example run of the L⋆adaptation for NFAs on L = {w ∈{a}∗| |w| ̸= 1}. ε ε 1 a 0 (a) a a (b) (c) ε ε 1 a 0 aa 1 aaa 1 (d) ε a ε 1 0 a 0 1 aa 1 1 aaa 1 1 (e) Fig. 3: Example run of the L⋆adaptation for NFAs on L = {w ∈{a}∗| |w| ̸= 1}. ε ε 1 a 0 (a) a a (b) (b) (c) Fig. 3: Example run of the L⋆adaptation for NFAs on L = {w ∈{a}∗| |w| ̸= 1}. Fig. 3: Example run of the L⋆adaptation for NFAs on L = {w ∈{a}∗| |w| ̸= 1}. Example Revisited. We now run the new algorithm on the language L = {w ∈ {a}∗| |w| ̸= 1} considered earlier. Starting from S = E = {ε}, the observation table (Fig. 3a) is immediately closed and consistent. (It is closed because we have row♯ t({a}) = row♯ t(∅).) This gives the JSL hypothesis shown in Fig. 3b, which leads to an NFA hypothesis having a single state that is initial, accepting, and has no transitions (Fig. 3c). The hypothesis is incorrect, and the teacher may supply us with counterexample aa. Adding prefixes a and aa to S leads to the table in Fig. 3d. Fig. 1: L⋆algorithm. The table is again closed, but not consistent: row♯ t({a}) = row♯ t(∅), but row♯ b({a})(a) = row♯ t({aa}) ̸= row♯ t(∅) = row♯ b(∅)(a). Thus, we add a to E. The resulting table (Fig. 3e) is closed and consistent. We note that row aa is the union of other rows: row♯ t({aa}) = row♯ t({ε, a}) (i.e., it is not a join-irreducible), and therefore can be ignored when building the succinct hypothesis. This hypothesis has two states, ε and a, and indeed it is the correct one N. Contributions and road map of the paper. After some preliminary notions in Section 3, we present the main contributions: Contributions and road map of the paper. After some preliminary notions in Section 3, we present the main contributions: Contributions and road map of the paper. After some preliminary notions in Section 3, we present the main contributions: – In Section 4, we develop a general algorithm L⋆ T , which generalizes the NFA one presented in Section 2 to an arbitrary monad T capturing side-effects, and we provide a general correctness proof for our algorithm. e a general correctness proof for our algorithm – In Section 5, we describe the first optimization and prove its cor – In Section 6 we describe the second optimization. We also show how it can be combined with the one of Section 5, and how it can lead to a further small optimization, where the consistency check on the table is dropped. – Finally, in Section 7 we show how L⋆ T can be applied to several automata models, highlighting further case-specific optimizations when available. 3 Preliminaries In this section we define a notion of T-automaton, a generalization of non- deterministic finite automata parametric in a monad T. We assume familiarity with basic notions of category theory: functors (in the category Set of sets and functions) and natural transformations. Side-effects can be conveniently captured as a monad. A monad T = (T, η, µ) is a triple consisting of an endofunctor T on Set and two natural transformations: Van Heerdt et al. 8 8 a unit η: Id ⇒T and a multiplication µ: T 2 ⇒T, which satisfy the compatibility laws µ ◦ηT = idT = µ ◦Tη and µ ◦µT = µ ◦Tµ. a unit η: Id ⇒T and a multiplication µ: T 2 ⇒T, which satisfy the compatibility laws µ ◦ηT = idT = µ ◦Tη and µ ◦µT = µ ◦Tµ. Example 1 (Monads). An example of a monad is the triple (P, {−}, S), where P denotes the powerset functor associating a collection of subsets to a set, {−} is the singleton operation, and S is just union of sets. Another example is the triple (V (−), e, m), where V (X) is the free semimodule (over a semiring S) over X, namely {ϕ | ϕ: X →S having finite support}. The support of a function ϕ: X →S is the set of x ∈X such that ϕ(x) ̸= 0. Then e: X →V (X) is the characteristic function for each x ∈X, and m: V (V (X)) →V (X) is defined for ϕ ∈V (V (X)) and x ∈X as m(ϕ)(x) = P ψ∈V (X) ϕ(ψ) × ψ(x). Given a monad T, a T-algebra is a pair (X, h) consisting of a carrier set X and a function h: TX →X such that h ◦µX = h ◦Th and h ◦ηX = idX. A T-homomorphism between two T-algebras (X, h) and (Y, k) is a function f : X →Y such that f ◦h = k ◦Tf. The abstract notion of T-algebra instantiates to expected notions, as illustrated in the following example. Example 2 (Algebras for a monad). The P-algebras are the (complete) join- semilattices, and their homomorphisms are join-preserving functions. If S is a field, V -algebras are vector spaces, and their homomorphisms are linear maps. We will often refer to a T-algebra (X, h) as X if h is understood or if its specific definition is irrelevant. 3 Preliminaries Given a set X, (TX, µX) is a T-algebra called the free T-algebra on X. One can build algebras pointwise for some operations. For instance, if Y is a set and (X, x) a T-algebra, then we have a T-algebra (XY , f), where f : T(XY ) →XY is given by f(W)(y) = (x ◦T(evy))(W) and evy : XY →X by evy(g) = g(y). If U and V are T-algebras and f : U →V is a T-algebra homomorphism, then the image img(f) of f is a T-algebra, with the T-algebra structure inherited from V . The following proposition connects algebra homomorphisms from the free T-algebra on a set U to an algebra V with functions U →V . We will make use of this later in the section. Proposition 3. Given a set U and a T-algebra (V, v), there is a bijective corre- spondence between T-algebra homomorphisms TU →V and functions U →V : for a T-algebra homomorphism f : TU →V , define f † = f ◦η: U →V ; for a function g: U →V , define g♯= v ◦Tg: TU →V . Then g♯is a T-algebra homomorphism called the free T-extension of g, and we have f †♯= f and g♯† = g. We now have all the ingredients to define our notion of automaton with side- effects and their language semantics. We fix a monad (T, η, µ) with T preserving finite sets, as well as a T-algebra O that models outputs of automata. Definition 4 (T-automaton). A T-automaton is a quadruple (Q, δ: Q → QA, out: Q →O, init ∈Q), where Q is a T-algebra, the transition map δ and output map out are T-algebra homomorphisms, and init is the initial state. Definition 4 (T-automaton). A T-automaton is a quadruple (Q, δ: Q → QA, out: Q →O, init ∈Q), where Q is a T-algebra, the transition map δ and output map out are T-algebra homomorphisms, and init is the initial state. Example 5. DFAs are Id-automata when O = 2 = {0, 1} is used to distinguish accepting from rejecting states. For the more general case of O being any set, DFAs generalize into Moore automata. 9 Learning Automata with Side-Effects Example 6. Recall that P-algebras are JSLs, and their homomorphisms are join- preserving functions. 3 Preliminaries In a P-automaton, Q is equipped with a join operation, and QA is a join-semilattice with pointwise join: (f ∨g)(a) = f(a) ∨g(a) for a ∈A. Since the automaton maps preserve joins, we have, in particular, δ(q1 ∨q2)(a) = δ(q1)(a) ∨δ(q2)(a). One can represent an NFA over a set of states S as a P- automaton by taking Q = (P(S), S) and O = 2, the Boolean join-semilattice with the or operation as its join. Let init ⊆S be the set of initial states and out: P(Q) →2 and δ: P(S) →P(S)A the respective extensions (Proposition 3) of the NFA’s output and transition functions. The resulting P-automaton is precisely the determinized version of the NFA. More generally, an automaton with side-effects given by a monad T always represents a T-automaton with a free state space. Proposition 7. A T-automaton of the form ((TX, µX), δ, out, init), for any set X, is completely defined by the set X with the element init ∈TX and functions δ† : X →(TX)A out† : X →O. We call such a T-automaton a succinct automaton, which we sometimes identify with the representation (X, δ†, out†, init). These automata are closely related to the ones studied in [18]. A (generalized) language is a function L: A∗→O. For every T-automaton we have an observability and a reachability map, telling respectively which state is reached by reading a given word and which language each state recognizes. Definition 8 (Reachability/Observability Maps). The reachability map of a T-automaton A is a function rA : A∗→Q inductively defined as: rA(ε) = init and rA(ua) = δ(rA(u))(a). The observability map of A is a function oA : Q → OA∗given by: oA(q)(ε) = out(q) and oA(q)(av) = oA(δ(q)(a))(v). The language accepted by A is the map LA = oA(init) = outA ◦rA : A∗→O. Example 9. For an NFA A represented as a P-automaton, as seen in Example 6, oA(q) is the language of q in the traditional sense. Note that q, in general, is a set of states: oA(q) takes the union of languages of singleton states. The set LA is the language accepted by the initial states, i.e., the language of the NFA. The reachability map rA(u) returns the set of states reached via all paths reading u. Van Heerdt et al. In the following, we will also make use of the minimal Moore automaton accepting L. Although this always exists—by instantiating Definition 10 with T = Id—it need not be finite. The following property says that finiteness of Moore automata and of T-automata accepting the same language are related. Proposition 11. The minimal Moore automaton accepting L is finite if and only if the minimal T-automaton accepting L is finite. 4 A General Algorithm In this section we introduce our extension of L⋆to learn automata with side-effects. The algorithm is parametric in the notion of side-effect, represented as the monad T, and is therefore called L⋆ T . We fix a language L: A∗→O that is to be learned, and we assume that there is a finite T-automaton accepting L. This assumption generalizes the requirement of L⋆that L is regular (i.e., accepted by a specific class of T-automata, see Example 5). An observation table consists of a pair of functions rowt : S →OE rowb : S →(OE)A rowb : S →(OE)A rowt : S →OE given by rowt(s)(e) = L(se) and rowb(s)(a)(e) = L(sae), where S, E ⊆A∗are finite sets with ε ∈S ∩E. For O = 2, we recover exactly the L⋆observation table. The key idea for L⋆ T is defining closedness and consistency over the free T-extensions of those functions. given by rowt(s)(e) = L(se) and rowb(s)(a)(e) = L(sae), where S, E ⊆A∗are finite sets with ε ∈S ∩E. For O = 2, we recover exactly the L⋆observation table. The key idea for L⋆ T is defining closedness and consistency over the free T-extensions of those functions. Definition 12 (Closedness and Consistency). The table is closed if for all U ∈T(S) and a ∈A there exists a U ′ ∈T(S) such that row♯ t(U ′) = row♯ b(U)(a). The table is consistent if for all U1, U2 ∈T(S) such that row♯ t(U1) = row♯ t(U2) we have row♯ b(U1) = row♯ b(U2). For closedness, we do not need to check all elements of T(S) × A against elements of T(S), but only those of S × A, thanks to the following result. Lemma 13. If for all s ∈S and a ∈A there is U ∈T(S) such that row♯ t(U) = rowb(s)(a), then the table is closed. Example 14. For NFAs represented as P-automata, the properties are as presented in Section 2. Recall that for T = P and O = 2, the Boolean join-semilattice, row♯ t and row♯ b describe a table where rows are labeled by subsets of S. Then we have, for instance, row♯ t({s1, s2})(e) = rowt(s1)(e) ∨rowt(s2)(e), i.e., row♯ t({s1, s2})(e) = 1 if and only if L(s1e) = 1 or L(s2e) = 1. 3 Preliminaries Given a language L: A∗→O, there exists a (unique) minimal T-automaton ML accepting L, which is minimal in the number of states. Its existence follows from general facts. See for example [19]. Definition 10 (Minimal T-Automaton for L). Let tL : A∗→OA∗be the function giving the residual languages of L, namely tL(u) = λv.L(uv). The minimal T-automaton ML accepting L has state space M = img(t♯ L), initial state init = tL(ε), and T-algebra homomorphisms out: M →O and δ: M →M A given by out(t♯ L(U)) = L(U) and δ(t♯ L(U))(a)(v) = t♯ L(U)(av). Definition 10 (Minimal T-Automaton for L). Let tL : A∗→OA∗be the function giving the residual languages of L, namely tL(u) = λv.L(uv). The minimal T-automaton ML accepting L has state space M = img(t♯ L), initial state init = tL(ε), and T-algebra homomorphisms out: M →O and δ: M →M A given by out(t♯ L(U)) = L(U) and δ(t♯ L(U))(a)(v) = t♯ L(U)(av). 10 4 A General Algorithm Closedness amounts to check whether each row in the bottom part of the table is the join of a set of rows in the top part. Consistency amounts to check whether, for all sets of rows U1, U2 ⊆S in the top part of the table whose joins are equal, the joins of rows U1 · {a} and U2 · {a} in the bottom part are also equal, for all a ∈A. Learning Automata with Side-Effects 11 1 S, E ←{ε} 2 repeat 3 while the table is not closed or not consistent 4 if the table is not closed 5 find s ∈S, a ∈A such that rowb(s)(a) ̸= row♯ t(U) for all U ∈T(S) 6 S ←S ∪{sa} 7 if the table is not consistent 8 find U1, U2 ∈T(S), a ∈A, and e ∈E such that row♯ t(U1) = row♯ t(U2) and row♯ b(U1)(a)(e) ̸= row♯ b(U2)(a)(e) 9 E ←E ∪{ae} 10 Construct the hypothesis H and submit it to the teacher 11 if the teacher replies no, with a counterexample z 12 S ←S ∪prefixes(z) 13 until the teacher replies yes 14 return H find s ∈S, a ∈A such that rowb(s)(a) ̸= row♯ t(U) for all U ∈T(S) S ←S ∪{sa} { } Construct the hypothesis H and submit it to the teacher if the teacher replies no, with a counterexample z ( ) 13 until the teacher replies yes 14 return H 3 Take the language {ap}, for some p ∈N and a singleton alphabet {a}. Its residual languages are ∅and {ai} for all 0 ≤i ≤p, thus the minimal DFA accepting the language has p + 2 states. However, the residual languages w.r.t. sets of words are all the subsets of {ε, a, aa, . . . , ap}—hence, the minimal T-automaton has 2p+1 states. Fig. 4: Adaptation of L⋆for T-automata. If z ∈A∗is such that LH(z) ̸= L(z) and prefixes(z) ⊆S, then there are a prefix ua of z, with u ∈A∗and a ∈A, and U ∈T(S) such that rowt(u) = row♯ t(U) and rowb(u)(a) ̸= row♯ b(U)(a). Now, note that by increasing S or E, the hypothesis state space H never decreases in size. Moreover, for S = A∗and E = A∗, row♯ t = t♯ L. Therefore, since H and M are defined as the images of row♯ t and t♯ L, respectively, the size of H is bounded by that of M. Since H increases while the algorithm loops, the algorithm must terminate and is thus correct. Note that the learning algorithm of Bollig et al. does not terminate using this counterexample processing method [10, Appendix F]. This is due to their notion of consistency being weaker than ours: we have shown that progress is guaranteed because a consistency defect, in our sense, is created using this method. Query complexity. The complexity of automata learning algorithms is usually measured in terms of the number of both membership and equivalence queries asked, as it is common to assume that computations within the algorithm are insignificant compared to evaluating the system under analysis in applications. The cost of answering queries themselves is not considered, as it depends on the implementation of the teacher, which the algorithm abstracts from. The table is a T-algebra homomorphism, so membership queries for rows labeled in S are enough to determine all other rows. We measure the query complexities in terms of the number of states n of the minimal Moore automaton, the number of states t of the minimal T-automaton, the size k of the alphabet, and the length m of the longest counterexample. Note that t cannot be smaller than n, but it can be much bigger. For example, when T = P, t may be in O(2n).3 ( ) The maximum number of closedness defects fixed by the algorithm is n, as a closedness defect for the setting with algebraic structure is also a closedness defect for the setting without that structure. The maximum number of consistency defects fixed by the algorithm is t, as fixing a consistency defect distinguishes two rows that were previously identified. Since counterexamples lead to consistency defects, this also means that the algorithm will not pose more than t equivalence queries. Fig. 4: Adaptation of L⋆for T-automata. Fig. 4: Adaptation of L⋆for T-automata. If closedness and consistency hold, we can define a hypothesis T-automaton H, with state space H = img(row♯ t), init = rowt(ε), and output and transitions out: H →O out(row♯ t(U)) = row♯ t(U)(ε) δ: H →HA δ(row♯ t(U)) = row♯ b(U). out: H →O out(row♯ t(U)) = row♯ t(U)(ε) δ: H →HA δ(row♯ t(U)) = row♯ b(U). out: H →O δ: H →HA The correctness of this definition follows from the abstract treatment of [21], instantiated to the category of T-algebras and their homomorphisms. The correctness of this definition follows from the abstract treatment of [21], instantiated to the category of T-algebras and their homomorphisms. We can now give algorithm L⋆ T . Similarly to the example in Section 2, we only have to adjust lines 5 and 8 in Fig. 1. The resulting algorithm is shown in Fig. 4. Correctness. Correctness for L⋆ T amounts to proving that, for any target language L, the algorithm terminates returning the minimal T-automaton ML accepting L. As in the original L⋆algorithm, we only need to prove that the algorithm terminates, that is, that only finitely many hypotheses are produced. Correctness follows from termination, since line 13 causes the algorithm to terminate only if the hypothesis automaton coincides with ML. In order to show termination, we argue that the state space H of the hypothesis increases while the algorithm loops, and that H cannot be larger than M, the state space of ML. In fact, when a closedness defect is resolved (line 6), a row that was not previously found in the image of row♯ t : T(S) →OE is added, so the set H grows larger. When a consistency defect is resolved (line 9), two previously equal rows become distinguished, which also increases the size of H. As for counterexamples, adding their prefixes to S (line 11) creates a consis- tency defect, which will be fixed during the next iteration, causing H to increase. This is due to the following result, which says that the counterexample z has a Van Heerdt et al. 12 prefix that violates consistency. Note that the hypothesis H in the statement below is the hypothesis obtained before adding the prefixes of z to S. Proposition 15. 4 Here and hereafter we assume that T(S′) ⊆T(S), and more generally that T preserves inclusion maps. To eliminate this assumption, one could take the inclusion map f : S′ ,→S and write row♯ t(T(f)(U)) instead of row♯ t(U). Fig. 4: Adaptation of L⋆for T-automata. A word is added to S when fixing a closedness defect, and O(m) words are added to S when processing a counterexample. The number of rows that we need to fill using queries is therefore in O(tmk). The number of columns added to the table is given by the number of times a consistency defect is fixed and thus in O(t). Altogether, the number of membership queries is in O(t2mk). 13 Learning Automata with Side-Effects 5 Succinct Hypotheses We now describe the first of two optimizations, which is enabled by the use of monads. Our algorithm produces hypotheses that can be quite large, as their state space is the image of row♯ t, which has the whole set T(S) as its domain. For instance, when T = P, T(S) is exponentially larger than S. We show how we can compute succinct hypotheses, whose state space is given by a subset of S. We start by defining sets of generators for the table. Definition 16. A set S′ ⊆S is a set of generators for the table whenever for all s ∈S there is U ∈T(S′) such that rowt(s) = row♯ t(U).4 Intuitively, U is the decomposition of s into a “combination” of generators. When T = P, S′ generates the table whenever each row can be obtained as the join of a set of rows labeled by S′. Explicitly: for all s ∈S there is {s1, . . . , sn} ⊆S′ such that rowt(s) = row♯ t({s1, . . . , sn}) = rowt(s1) ∨· · · ∨rowt(sn). Recall that H, with state space H, is the hypothesis automaton for the table. The existence of generators S′ allows us to compute a T-automaton with state space T(S′) equivalent to H. We call this the succinct hypothesis, although T(S′) may be larger than H. Proposition 7 tells us that the succinct hypothesis can be represented as an automaton with side-effects in T that has S′ as its state space. This results in a lower space complexity when storing the hypothesis. We now show how the succinct hypothesis is computed. Observe that, if generators S′ exist, row♯ t factors through the restriction of itself to T(S′). Denote this latter function d rowt ♯. Since we have T(S′) ⊆T(S), the image of d rowt ♯ coincides with img(row♯ t) = H, and therefore the surjection restricting d rowt ♯to its image has the form e: T(S′) →H. Any right inverse i: H →T(S′) of the function e (that is, e ◦i = idH, but whereas e is a T-algebra homomorphism, i need not be one) yields a succinct hypothesis as follows. Definition 17 (Succinct Hypothesis). The succinct hypothesis is the T- automaton S = (T(S′), δ, out, init) given by init = i(rowt(ε)) and out† : S′ →O out†(s) = rowt(s)(ε) δ† : S′ →T(S′)A δ†(s)(a) = i(rowb(s)(a)). return S′ To determine whether U as in the above algorithm exists, one can always naively enumerate all possibilities, using that T preserves finite sets. This is what we call the basic algorithm. For specific algebraic structures, one may find more efficient methods, as we show in the following example. Example 20. Consider the powerset monad T = P. We now exemplify two ways of computing succinct hypotheses, which are inspired by canonical RFSAs [16]. The basic idea is to start from a deterministic automaton and to remove states that are equivalent to a set of other states. The algorithm given in Proposition 19 computes a minimal S′ that only contains labels of rows that are not the join of other rows. (In case two rows are equal, only one of their labels is kept.) In other words, as mentioned in Section 2, S′ contains labels of join-irreducible rows. To concretize the algorithm efficiently, we use a method introduced by Bollig et al. [11], which essentially exploits the natural order on the JSL of table rows. In contrast to the basic exponential algorithm, this results in a polynomial one.5 Bollig et al. determine whether a row is a join of other rows by comparing the row just to the join of rows below it. Like them, we make use of this also to compute right inverses of e, for which we will formalize the order. The function e: P(S′) →H tells us which sets of rows are equivalent to a single state in H. We show two right inverses H →P(S′) for it. The first one, i1(h) = {s ∈S′ | rowt(s) ≤h}, stems from the construction of the canonical RFSA of a language [16]. Here we use the order a ≤b ⇐⇒a ∨b = b induced by the JSL structure. The resulting construction of a succinct hypothesis was first used by Bollig et al. [11]. This succinct hypothesis has a “maximal” transition function, meaning that no more transitions can be added without changing the language of the automaton. The second inverse is i2(h) = {s ∈S′ | rowt(s) ≤h and for all s′ ∈S′ s.t. rowt(s) ≤rowt(s′) ≤h we have rowt(s) = rowt(s′)}, resulting in a more economical transition function, where some redundancies are removed. This corresponds to the simplified canonical RFSA [16]. Example 21. Consider T = P, and recall the table in Fig. 3e. 5 When we refer to computational complexities, as opposed to query complexities, they are in terms of the sizes of S, E, and A. 5 Succinct Hypotheses out† : S′ →O out†(s) = rowt(s)(ε) δ† : S′ →T(S′)A δ†(s)(a) = i(rowb(s)(a)). This definition is inspired by that of a scoop, due to Arbib and Manes [4]. This definition is inspired by that of a scoop, due to Arbib and Manes [4]. Proposition 18. Any succinct hypothesis of H accepts the language of H. We now give a simple procedure to compute a minimal set of generators, that is, a set S′ such that no proper subset is a set of generators. This generalizes a procedure defined by Angluin et al. [3] for non-deterministic, universal, and alternating automata. 4 Here and hereafter we assume that T(S′) ⊆T(S), and more generally that T preserves inclusion maps. To eliminate this assumption, one could take the inclusion map f : S′ ,→S and write row♯ t(T(f)(U)) instead of row♯ t(U). 14 Van Heerdt et al. Proposition 19. The following algorithm returns a minimal set of generators for the table: p for the table: S′ ←S while there are s ∈S′ and U ∈T(S′ \ {s}) s.t. row♯ t(U) = rowt(s) S′ ←S′ \ {s} return S′ S′ ←S while there are s ∈S′ and U ∈T(S′ \ {s}) s.t. row♯ t(U) = rowt(s) S′ ←S′ \ {s} return S′ When S′ = S, the right inverse given by i1 yields the succinct hypothesis shown below. 5 When we refer to computational complexities, as opposed to query complexities, they are in terms of the sizes of S, E, and A. 15 Learning Automata with Side-Effects a a a a a a a Note that i1(rowt(aa)) = {ε, a, aa}. Taking i2 instead, the succinct hypothesis is just the DFA (1) because i2(rowt(aa)) = {aa}. Rather than constructing a succinct hypothesis directly, our algorithm first reduces the set S′. In this case, we have rowt(aa) = row♯ t({ε, a}), so we remove aa from S′. Now i1 and i2 coincide and produce the NFA (2). Minimizing the set S′ in this setting essentially comes down to determining what Bollig et al. [11] call the prime rows of the table. Remark 22. The algorithm in Proposition 19 implicitly assumes an order in which elements of S are checked. Although the algorithm is correct for any such order, different orders may give results that differ in size. 6 Optimized Counterexample Handling The second optimization we give generalizes the counterexample processing method due to Rivest and Schapire [28], which improves the worst case complexity of the number of membership queries needed in L⋆. Maler and Pnueli [26] proposed to add all suffixes of the counterexample to the set E instead of adding all prefixes to the set S. This eliminates the need for consistency checks in the deterministic setting. The method by Rivest and Schapire finds a single suffix of the counterexample and adds it to E. This suffix is chosen in such a way that it either distinguishes two existing rows or creates a closedness defect, both of which imply that the hypothesis automaton will grow. The main idea is finding the distinguishing suffix via the hypothesis automaton H. Given u ∈A∗, let qu be the state in H reached by reading u, i.e., qu = rH(u). For each q ∈H, we pick any Uq ∈T(S) that yields q according to the table, i.e., such that row♯ t(Uq) = q. Then for a counterexample z we have that the residual language w.r.t. Uqz does not “agree” with the residual language w.r.t. z. The above intuition can be formalized as follows. Let R: A∗→OA∗be given by R(u) = t♯ L(Uqu) for all u ∈A∗, the residual language computation. We have the following technical lemma, saying that a counterexample z distinguishes the residual languages tL(z) and R(z). Lemma 23. If z ∈A∗is such that LH(z) ̸= L(z), then tL(z)(ε) ̸= R(z)(ε). Lemma 23. If z ∈A∗is such that LH(z) ̸= L(z), then tL(z)(ε) ̸= R(z)(ε). Lemma 23. If z ∈A∗is such that LH(z) ̸= L(z), then tL(z)(ε) ̸= R(z)(ε). We assume that Uqε = η(ε). For a counterexample z, we then have R(ε)(z) = tL(ε)(z) ̸= R(z)(ε). While reading z, the hypothesis automaton passes a sequence of states qu0, qu1,qu2,. . . ,qun, where u0 = ϵ, un = z, and ui+1 = uia for some a ∈A is a prefix of z. If z were correctly classified by H, all residuals R(ui) would classify the remaining suffix v of z, i.e., such that z = uiv, in the same way. However, the previous lemma tells us that, for a counterexample z, this is not case, meaning that for some suffix v we have R(ua)(v) ̸= R(u)(av). In short, this inequality is discovered along a transition in the path to z. Van Heerdt et al. 16 Corollary 24. If z ∈A∗is such that LH(z) ̸= L(z), then there are u, v ∈A∗ and a ∈A such that uav = z and R(ua)(v) ̸= R(u)(av). Corollary 24. If z ∈A∗is such that LH(z) ̸= L(z), then there are u, v ∈A∗ and a ∈A such that uav = z and R(ua)(v) ̸= R(u)(av). To find such a decomposition efficiently, Rivest and Schapire use a binary search algorithm. We conclude with the following result that turns the above property into the elimination of a closedness witness. That is, given a coun- terexample z and the resulting decomposition uav from the above corollary, we show that, while currently row♯ t(Uqua) = row♯ b(Uqu)(a), after adding v to E we have row♯ t(Uqua)(v) ̸= row♯ b(Uqu)(a)(v). (To see that the latter follows from the proposition below, note that for all U ∈T(S) and e ∈E, row♯ t(U)(e) = t♯ L(U)(e) and for each a′ ∈A, row♯ b(U)(a′)(e) = t♯ L(U)(a′e).) The inequality means that either we have a closedness defect, or there still exists some U ∈T(S) such that row♯ t(U) = row♯ b(Uqu)(a). In this case, the rows row♯ t(U) and row♯ t(Uqua) have become distinguished by adding v, which means that the size of H has increased. A closedness defect also increases the size of H, so in any case we make progress. Proposition 25. Lemma 23. If z ∈A∗is such that LH(z) ̸= L(z), then tL(z)(ε) ̸= R(z)(ε). Again, we measure the membership and equivalence query complexities in terms of the number of states n of the minimal Moore automaton, the number of states t of the minimal T-automaton, the size k of the alphabet, and the length m of the longest counterexample. A counterexample now gives an additional column instead of a set of rows, and we have seen that this leads to either a closedness defect or to two rows being distinguished. Thus, the number of equivalence queries is still at most t, and the number of columns is still in O(t). However, the number of rows that we need to fill using membership queries is now in O(nk). This means that a total of O(tnk) membership queries is needed to fill the table. Apart from filling the table, we also need queries to analyze counterexamples. The binary search algorithm mentioned after Corollary 24 requires for each counterexample O(log m) computations of R(x)(y) for varying words x and y. Let r be the maximum number of queries required for a single such computation. Note that for u, v ∈A∗, and letting α: TO →O be the algebra structure on O, we have R(u)(v) = α(T(evv ◦tL)(Uqu)) for the original definition of R and R(u)(v) = α(T(evv ◦tL)(r† S(u))) in the succinct hypothesis case. Since the restricted map T(evv ◦tL): TS →TO is completely determined by evv ◦tL : S →O, r is at most |S|, which is bounded by n in this optimized algorithm. For some examples (see for instance the writer automata in Section 7), we even have r = 1. The overall membership query complexity is O(tnk + tr log m). Dropping Consistency. We described the counterexample processing method based around Proposition 25 in terms of the succinct hypothesis S rather than the actual hypothesis H by showing that R can be defined using S. Since the definition of the succinct hypothesis does not rely on the property of consistency to be well-defined, this means we could drop the consistency check from the algorithm altogether. We can still measure progress in terms of the size of the set H, but it will not be the state space of an actual hypothesis during intermediate stages. This observation also explains why Bollig et al. [11] are able to use a weaker notion of consistency in their algorithm. Lemma 23. If z ∈A∗is such that LH(z) ̸= L(z), then tL(z)(ε) ̸= R(z)(ε). If z ∈A∗is such that LH(z) ̸= L(z), then there are u, v ∈A∗ and a ∈A such that row♯ t(Uqua) = row♯ b(Uqu)(a) and t♯ L(Uqua)(v) ̸= t♯ L(Uqu)(av). We now show how to combine this optimized counterexample processing method with the succinct hypothesis optimization from Section 5. Recall that the succinct hypothesis S is based on a right inverse i: H →T(S′) of e: T(S′) →H. Choosing such an i is equivalent to choosing Uq for each q ∈H. We then redefine R using the reachability map of the succinct hypothesis. Specifically, R(u) = t♯ L(rS(u)) for all u ∈A∗. L Unfortunately, there is one complication. We assumed earlier that Uqε = η(ε), or more specifically R(ε)(z) = L(z). This now may be impossible because we do not even necessarily have ε ∈S′. We show next that if this equality does not hold, then there are two rows that we can distinguish by adding z to E. Thus, after testing whether R(ε)(z) = L(z), we either add z to E (if the test fails) or proceed with the original method. Proposition 26. If z ∈A∗is such that R(ε)(z) ̸= L(z), then row♯ t(initS) = rowt(ε) and t♯ L(initS)(z) ̸= tL(ε)(z). Proposition 26. If z ∈A∗is such that R(ε)(z) ̸= L(z), then row♯ t(initS) = rowt(ε) and t♯ L(initS)(z) ̸= tL(ε)(z). To see that the original method still works, we prove the analogue of Proposi- tion 25 for the new definition of R. Proposition 27. If z ∈A∗is such that LS(z) ̸= L(z) and R(ε)(z) = L(z), then there are u, v ∈A∗and a ∈A such that row♯ t(r† S(ua)) = row♯ b(r† S(u))(a) and t♯ L(r† S(ua))(v) ̸= t♯ L(r† S(u))(av). Example 28. Recall the succinct hypothesis S from Fig. 3c for the table in Fig. 2a. Note that S′ = S cannot be further reduced. The hypothesis is based on the right inverse i: H →P(S) of e: P(S) →H given by i(rowt(ε)) = {ε} and i(row♯ t(∅)) = ∅. This is the only possible right inverse because e is bijective. For the prefixes of the counterexample aa we have rS(ε) = {ε} and rS(a) = rS(aa) = ∅. Note that t♯ L({ε})(aa) = 1 while tL(∅)(a) = tL(∅)(ε) = 0. Thus, R(ε)(aa) ̸= R(a)(a). Adding a to E would indeed create a closedness defect. 17 Learning Automata with Side-Effects Query complexity. Lemma 23. If z ∈A∗is such that LH(z) ̸= L(z), then tL(z)(ε) ̸= R(z)(ε). Interestingly, they exploit the canonicity of their choice of succinct hypotheses to arrive at a polynomial membership query complexity that does not involve the factor t. 7 Examples In this section we list several examples that can be seen as T-automata and hence learned via an instance of L⋆ T . We remark that, since our algorithm operates on finite structures (recall that T preserves finite sets), for each automaton type one can obtain a basic, correct-by-construction instance of L⋆ T for free, by plugging the concrete definition of the monad into the abstract algorithm. However, we note that this is not how L⋆ T is intended to be used in a real-world context; it should be Van Heerdt et al. 18 seen as an abstract specification of the operations each concrete implementation needs to perform, or, in other words, as a template for real implementations. For each instance below, we discuss whether certain operations admit a more efficient implementation than the basic one, based on the specific algebraic structure induced by the monad. Due to our general treatment, the optimizations of Sections 5 and 6 apply to all of these instances. Non-deterministic automata. As discussed before, non-deterministic automata are P-automata with a free state space, provided that O = 2 is equipped with the “or” operation as its P-algebra structure. We also mentioned that, as Bollig et al. [11] showed, there is a polynomial time algorithm to check whether a given row is the join of other rows. This gives an efficient method for handling closedness straight away. Moreover, as shown in Example 20, it allows for an efficient construction of the succinct hypothesis. Unfortunately, checking for consistency defects seems to require a number of computations exponential in the number of rows. However, as explained at the end of Section 6, we can in fact drop consistency altogether. Universal automata. Just like non-deterministic automata, universal automata can be seen as P-automata with a free state space. The difference is that the P-algebra structure on O = 2 is dual: it is given by the “and” rather than the “or” operation. Universal automata accept a word when all paths reading that word are accepting. One can dualize the optimized specific algorithms for the case of non-deterministic automata. This is precisely what Angluin et al. [3] have done. Partial automata. Consider the maybe monad Maybe(X) = 1 + X, with natural transformations having components ηX : X →1 + X and µX : 1 + 1 + X →1 + X defined in the standard way. 7 Examples Partial automata with states X can be represented as Maybe-automata with state space Maybe(X) = 1+X, where there is an additional sink state, and output algebra O = Maybe(1) = 1 + 1. Here the left value is for rejecting states, including the sink one. The transition map δ: 1 + X →(1 + X)A represents an undefined transition as one going to the sink state. The algorithm L⋆ Maybe is mostly like L⋆, except that implicitly the table has an additional row with zeroes in every column. Since the monad only adds a single element to each set, there is no need to optimize the basic algorithm for this specific case. Weighted automata. Recall from Section 3 the free semimodule monad V , sending a set X to the free semimodule over a finite semiring S. Weighted automata over a set of states X can be represented as V -automata whose state space is the semimodule V (X), the output function out: V (X) →S assigns a weight to each state, and the transition map δ: V (X) →V (X)A sends each state and each input symbol to a linear combination of states. The obvious semimodule structure on S extends to a pointwise structure on the potential rows of the table. The basic algorithm loops over all linear combinations of rows to check closedness and over all pairs of combinations of rows to check consistency, making them extremely expensive operations. If S is a field, a row can be decomposed into a linear 19 Learning Automata with Side-Effects combination of other rows in polynomial time using standard techniques from linear algebra. As a result, there are efficient procedures for checking closedness and constructing succinct hypotheses. It was shown by Van Heerdt et al. [21] that consistency in this setting is equivalent to closedness of the transpose of the table. This trick is due to Bergadano and Varricchio [7], who first studied learning of weighted automata. Alternating automata. We use the characterization of alternating automata due to Bertrand and Rot [9]. Recall that, given a partially ordered set (P, ≤), an upset is a subset U of P such that, if x ∈U and x ≤y, then y ∈U. Given Q ⊆P, we write ↑Q for the upward closure of Q, that is the smallest upset of P containing Q. 7 Examples We consider the monad A that maps a set X to the set of all upsets of P(X). Its unit is given by ηX(x) =↑{{x}} and its multiplication by µX(U) = {V ⊆X | ∃W ∈U ∀Y ∈W ∃Z∈Y Z ⊆V }. Algebras for the monad A are completely distributive lattices [27]. The sets of sets in A(X) can be seen as DNF formulae over elements of X, where the outer powerset is disjunctive and the inner one is conjunctive. Accordingly, we define an algebra structure β : A(2) →2 on the output set 2 by letting β(U) = 1 if {1} ∈U, 0 otherwise. Alternating automata with states X can be represented as A-automata with state space A(X), output map out: A(X) →2, and transition map δ: A(X) →A(X)A, sending each state to a DNF formula over X. The only difference with the usual definition of alternating automata is that A(X) is not the full set PP(X), which is not a monad [23]. However, for each formula in PP(X) there is an equivalent one in A(X). An adaptation of L⋆for alternating automata was introduced by Angluin et al. [3] and further investigated by Berndt et al. [8]. The former found that given a row r ∈2E and a set of rows X ⊆2E, r is equal to a DNF combination of rows from X (where logical operators are applied component-wise) if and only if it is equal to the combination defined by Y = {{x ∈X | x(e) = 1} | e ∈E ∧r(e) = 1}. We can reuse this idea to efficiently find closedness defects and to construct the hypothesis. Even though the monad A formally requires the use of DNF formulae representing upsets, in the actual implementation we can use smaller formulae, e.g., Y above instead of its upward closure. In fact, it is easy to check that DNF combinations of rows are invariant under upward closure. Similar as before, we do not know of an efficient way to ensure consistency, but we could drop it. Writer automata. The examples considered so far involve existing classes of automata. To further demonstrate the generality of our approach, we introduce a new (as far as we know) type of automaton, which we call writer automaton. Van Heerdt et al. The algebras for this monad are sets Q equipped with an M-action. One may take the output object to be the set M with the monoid multiplication as its action. Writer-automata with a free state space can be represented as deterministic automata that have an element of M associated with each transition. The semantics is as expected: M-elements multiply along paths and finally multiply with the output of the last state to produce the actual output. The basic learning algorithm has polynomial time complexity. To determine whether a given row is a combination of rows in the table, i.e., whether it is given by a monoid value applied to one of the rows in the table, one simply tries all of these values. This allows us to check for closedness, to minimize the generators, and to construct the succinct hypothesis, in polynominal time. Consistency involves comparing all ways of applying monoid values to rows and, for each comparison, at most |A| further comparisons between one-letter extensions. The total number of comparisons is clearly polynomial in |M|, |S|, and |A|. 7 Examples The writer monad Writer(X) = M × X for a finite monoid M has a unit ηX : X →M × X given by adding the unit e of the monoid, ηX(x) = (e, x), and a multiplication µX : M × M × X →M × X given by performing the monoid multiplication, µX(m1, m2, x) = (m1m2, x). In Haskell, the writer monad is used for such tasks as collecting successive log messages, where the monoid is given by the set of sets or lists of possible messages and the multiplication adds a message. Writer automata. The examples considered so far involve existing classes of automata. To further demonstrate the generality of our approach, we introduce a new (as far as we know) type of automaton, which we call writer automaton. ( ) y The writer monad Writer(X) = M × X for a finite monoid M has a unit ηX : X →M × X given by adding the unit e of the monoid, ηX(x) = (e, x), and a multiplication µX : M × M × X →M × X given by performing the monoid multiplication, µX(m1, m2, x) = (m1m2, x). In Haskell, the writer monad is used for such tasks as collecting successive log messages, where the monoid is given by the set of sets or lists of possible messages and the multiplication adds a message. 20 8 Conclusion We have presented L⋆ T , a general adaptation of L⋆that uses monads to learn an automaton with algebraic structure, as well as a method for finding a succinct equivalent based on its generators. Furthermore, we adapted the optimized counterexample handling method of Rivest and Schapire [28] to this setting and discussed instantiations to non-deterministic, universal, partial, weighted, alternating, and writer automata. Related Work. This paper builds on and extends the theoretical toolkit of Van Heerdt et al. [21,19], who are developing a categorical automata learning framework (CALF) in which learning algorithms can be understood and developed in a structured way. An adaptation of L⋆that produces NFAs was first developed by Bollig et al. [11]. Their algorithm learns a special subclass of NFAs consisting of RFSAs, which were introduced by Denis et al. [16]. Angluin et al. [3] unified algorithms for NFAs, universal automata, and alternating automata, the latter of which was further improved by Berndt et al. [8]. We are able to provide a more general framework, which encompasses and goes beyond those classes of automata. Moreover, we study optimized counterexample handling, which [3,11,8] do not consider. The algorithm for weighted automata over an arbitrary field was studied in a category theoretical context by Jacobs and Silva [22] and elaborated on by Van Heerdt et al. [21]. The algorithm itself was introduced by Bergadano and Varricchio [7]. The theory of succinct automata used for our hypotheses is based on the work of Arbib and Manes [4], revamped to more recent category theory. Future Work. Whereas our general algorithm effortlessly instantiates to monads that preserve finite sets, a major challenge lies in investigating monads that do not enjoy this property. The algorithm for weighted automata generalizes to an infinite field [7,22,21] and even a principal ideal domain [20]. However, for an Learning Automata with Side-Effects 21 infinite semiring in general we cannot guarantee termination, which is because a finitely generated semimodule may have an infinite chain of strict submodules [20]. Intuitively, this means that while fixing closedness defects increases the size of the hypothesis state space semimodule, an infinite number of steps may be needed to resolve all closedness defects. In future work we would like to characterize more precisely for which semirings we can learn, and ideally formulate this characterization on the monad level. 8 Conclusion As a result of the correspondence between learning and conformance test- ing [6,21], it should be possible to include in our framework the W-method [14], which is often used in case studies deploying L⋆(e.g. [12,15]). We defer a thorough investigation of conformance testing to future work. References 1. Benjamin Aminof, Orna Kupferman, and Robby Lampert. Reasoning about online algorithms with weighted automata. ACM Trans. Algorithms, 6(2):28:1–28:36, 2010. 2. Dana Angluin. Learning regular sets from queries and counterexamples. Inform. Comput., 75:87–106, 1987. 3. Dana Angluin, Sarah Eisenstat, and Dana Fisman. Learning regular languages via alternating automata. In IJCAI, pages 3308–3314, 2015. 4. Michael A. Arbib and Ernest G. Manes. Fuzzy machines in a category. Bulletin of the AMS, 13:169–210, 1975. 5. Christel Baier, Marcus Gr¨oßer, and Frank Ciesinski. Model checking linear-time properties of probabilistic systems. In Handbook of Weighted automata, 2009. 6. Therese Berg, Olga Grinchtein, Bengt Jonsson, Martin Leucker, Harald Raffelt, and Bernhard Steffen. On the correspondence between conformance testing and regular inference. In FASE, volume 3442, pages 175–189, 2005. 7. Francesco Bergadano and Stefano Varricchio. Learning behaviors of automata from multiplicity and equivalence queries. SIAM Journal on Computing, 25:1268–1280, 1996. 8. Sebastian Berndt, Maciej Li´skiewicz, Matthias Lutter, and R¨udiger Reischuk. Learning residual alternating automata. In AAAI, pages 1749–1755, 2017. 9. Meven Bertrand and Jurriaan Rot. Coalgebraic determinization of alternating automata. arXiv preprint arXiv:1804.02546, 2018. 10. Benedikt Bollig, Peter Habermehl, Carsten Kern, and Martin Leucker. Angluin-style learning of nfa (research report lsv-08-28). Technical report, ENS Cachan, 2008. ( ) 11. Benedikt Bollig, Peter Habermehl, Carsten Kern, and Martin Leucker. Angluin-style learning of NFA. In IJCAI, volume 9, pages 1004–1009, 2009. 12. Georg Chalupar, Stefan Peherstorfer, Erik Poll, and Joeri de Ruiter. Automated reverse engineering using Lego R ⃝. In WOOT, 2014. 13. Krishnendu Chatterjee, Laurent Doyen, and Thomas A. Henzinger. Quantitative languages. In CSL, pages 385–400, 2008. 14. Tsun S. Chow. Testing software design modeled by finite-state machines. IEEE Trans. Software Eng., 4:178–187, 1978. 15. Joeri de Ruiter and Erik Poll. Protocol state fuzzing of TLS implementations. In USENIX Security, pages 193–206, 2015. 16. Fran¸cois Denis, Aur´elien Lemay, and Alain Terlutte. Residual finite state automata. Fundamenta Informaticae, 51:339–368, 2002. Van Heerdt et al. 22 17. Manfred Droste and Paul Gastin. Weighted automata and weighted logics. In ICALP, pages 513–525, 2005. 18. Sergey Goncharov, Stefan Milius, and Alexandra Silva. Towards a coalgebraic chomsky hierarchy. In TCS, pages 265–280. Springer, 2014. 19. Gerco van Heerdt. An abstract automata learning framework. Master’s thesis, Radboud University Nijmegen, 2016. 20. Gerco van Heerdt, Clemens Kupke, Jurriaan Rot, and Alexandra Silva. Learning weighted automata over principal ideal domains. arXiv preprint arXiv:1911.04404, 2019. 21. Gerco van Heerdt, Matteo Sammartino, and Alexandra Silva. References CALF: categorical automata learning framework. In CSL, pages 29:1–29:24, 2017. 22. Bart Jacobs and Alexandra Silva. Automata learning: A categorical perspective. In Horizons of the Mind, volume 8464, pages 384–406, 2014. 23. Bartek Klin and Julian Salamanca. Iterated covariant powerset is not a monad. 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Мелихов В.Н. Федеральное государственное унитарное предприятие «Всероссийский научно- исследовательский геологический институт имени А.П.Карпинского» (ФГУП «ВСЕГЕИ»), Санкт-Петербург, Россия, vsegei@vsegei.ru РЕСУРСНЫЙ ПОТЕНЦИАЛ И ПЕРСПЕКТИВЫ РАЗВЕДКИ ТРАНСГРАНИЧНЫХ НЕФТЕГАЗОНОСНЫХ БАССЕЙНОВ ЮГО-ВОСТОЧНОГО ТУРКМЕНИСТАНА, ЮЖНЫХ РЕГИОНОВ УЗБЕКИСТАНА И ТАДЖИКИСТАНА, СЕВЕРНОГО АФГАНИСТАНА И СЕВЕРО-ВОСТОЧНОГО ИРАНА 1 1 ISSN 2070-5379 Neftegasovaâ geologiâ. Teoriâ i practika (RUS) URL: http://www.ngtp.ru 1 УДК 553.98.04(575.1+575.3/.4)+55+581.3 РЕСУРСНЫЙ ПОТЕНЦИАЛ И ПЕРСПЕКТИВЫ РАЗВЕДКИ ТРАНСГРАНИЧНЫХ НЕФТЕГАЗОНОСНЫХ БАССЕЙНОВ ЮГО-ВОСТОЧНОГО ТУРКМЕНИСТАНА, ЮЖНЫХ РЕГИОНОВ УЗБЕКИСТАНА И ТАДЖИКИСТАНА, СЕВЕРНОГО АФГАНИСТАНА И СЕВЕРО-ВОСТОЧНОГО ИРАНА Производится оценка ресурсов газа и нефти, результатов и перспектив разведки трансграничных нефтегазоносных бассейнов Юго-Восточного Туркменистана и сопредельных регионов Узбекистана, Таджикистана, Афганистана и Ирана. Рекомендуется разведка на нефть южных бортовых зон Мургабской впадины. Ключевые слова: нефтегазоносный бассейн, ресурсы нефти и газа, месторождение, перспективы разведки, юг Центральной Азии. В пределы рассматриваемой территории попадают юго-восточные части Амударьинского (Туркменистан, Узбекистан, Афганистан) и Предкопетдагского (Туркменистан, Иран) бассейнов, Верхнеамударьинский нефтегазоносный бассейн (НГБ) (Афганистан, Таджикистан, Узбекистан), Калаиморско-Каларинский газонефтеносный бассейн (ГНБ) (Туркменистан, Афганистан), перспективный НГБ Тирпуль (Афганистан, Иран) и новый Кучано-Мешхедский ГНБ (Иран). В статье анализируются и сопоставляются ресурсно-геологические данные по названным трансграничным бассейнам, выявляются особенности их нефтегазоносности и распределения ресурсов углеводородов (УВ), крайне важные для развития нефтегазового комплекса стран юга Центральной Азии и Мира. Эти вопросы слабо освещены в специальной литературе [Амурский и др., 1976; Самсонов и др., 1989; Халылов и др., 1992; Соловьёв, Кузьминов, Салина, 1996; Жмуд, Мелихов, 2000; Мелихов, Сибирёв, 2003; Аверков, 2006; Мелихов, 2009; В Афганистане обнаружено..., 2010; Asia Times..., 2012; Емельянов, 2012; Прогнозно-минерагеническая..., 2012]. Информация по результатам разведки и оценке ресурсов УВ Верхнеамударьинского бассейна излагается раздельно по таджикскому, узбекскому и афганскому национальным секторам. В афганском разделе анализируется также юго-восточная часть Амударьинского бассейна и перспективные НГБ Западного и Южного Афганистана, трансграничные с Ираном и Пакистаном. В туркменском разделе приводятся новые данные о ресурсном ISSN 2070-5379 Neftegasovaâ geologiâ. Teoriâ i practika (RUS) URL: http://www.ngtp.ru SN 2070-5379 Neftegasovaâ geologiâ. Teoriâ i practika (RUS) URL: http://www.ngtp.ru ISSN 2070-5379 Neftegasovaâ geologiâ. Teoriâ i practika (RUS) URL: http://www.ngtp.ru 2 потенциале УВ Юго-Восточного Туркменистана и о перспективах разведки на нефть приграничной с Афганистаном территории Амударьинского и Калаиморско-Каларинского бассейнов. В иранском разделе приведены известные и новые данные по бассейнам Северо- Восточного Ирана. 2 потенциале УВ Юго-Восточного Туркменистана и о перспективах разведки на нефть приграничной с Афганистаном территории Амударьинского и Калаиморско-Каларинского бассейнов. В иранском разделе приведены известные и новые данные по бассейнам Северо- Восточного Ирана. Схема тектонических элементов и продуктивности трансграничных НГБ юга Центральной Азии представлена на рис. 1. ______________________________________________________________________________ © Нефтегазовая геология. Теория и практика. – 2013. -Т.8. - №1. - http://www.ngtp.ru/rub/6/6 Верхнеамударьинский нефтегазоносный бассейн 1 – границы крупных и средних тектонических элементов; 2 – отдельные антиклинальные структуры, перспективные на нефть и газ; 3 – внебассейновые складчатые сооружения, сложенные породами мезозоя, палеозоя и протерозоя; 4 – граница Амударьинского ГНМБ; 5 – границы ГНБ и НГБ в составе Амударьинского мегабассейна и вне его; 6 – индексация и перечень газонефтеносных и нефтегазоносных бассейнов (I - Амударьинский ГНБ; II - Предкопетдагский ГНБ; III - Калаиморско-Каларинский ГНБ; I+II+III - Амударьинский ГНМБ; IV - Верхнеамударьинский НГБ; V - Тирпульский ПНГБ; VI - Кучано-Мешхедский ГНБ); 7-9 – месторождения УВ, индексация основных и значительных месторождений: 7 – газовые, газоконденсатные, 8 – нефтегазовые, нефтегазоконденсатные, газонефтяные, 9 – нефтяные; 10 – притоки и проявления нефти и газа; 11 – государственные границы. р 1 – границы крупных и средних тектонических элементов; 2 – отдельные антиклинальные структуры, перспективные на нефть и газ; 3 – внебассейновые складчатые сооружения, сложенные породами мезозоя, палеозоя и протерозоя; 4 – граница Амударьинского ГНМБ; 5 – границы ГНБ и НГБ в составе Амударьинского мегабассейна и вне его; 6 – индексация и перечень газонефтеносных и нефтегазоносных бассейнов (I - Амударьинский ГНБ; II - Предкопетдагский ГНБ; III - Калаиморско-Каларинский ГНБ; I+II+III - Амударьинский ГНМБ; IV - Верхнеамударьинский НГБ; V - Тирпульский ПНГБ; VI - Кучано-Мешхедский ГНБ); 7-9 – месторождения УВ, индексация основных и значительных месторождений: 7 – газовые, газоконденсатные, 8 – нефтегазовые, нефтегазоконденсатные, газонефтяные, 9 – нефтяные; 10 – притоки и проявления нефти и газа; 11 – государственные границы. государственные границы. Перечень индексированных месторождений УВ, их фазовый состав (Н, ГН, НГ, НГК, ГК, Г) и крупность (крупное – 30-100 млн. т н.э., крупнейшее – 100-500 млн. т н.э.; уникальное - >500 млн. т н.э.): у р р ц Перечень индексированных месторождений УВ, их фазовый состав (Н, ГН, НГ, НГК, ГК, Г) и крупность (крупное – 30-100 млн. т н.э., крупнейшее – 100-500 млн. т н.э.; уникальное - >500 млн. т н э ): Амударьинский ГНБ. 1 – Кандым ГК – крупнейшее; 2 – Самантепе ГК – крупное; 3 – Хаузак- Денгизкуль-Шады ГК – крупнейшее; 4 – Чендыр ГК; 5 – Караулбазар-Сарыташ НГ; 6 – Юж. Кемачи НГ - крупное; 7 – Умид НГК; 8 – Уртабулак ГК – крупнейшее; 9 – Зеварды ГК – крупнейшее; 10 – Памук ГК – крупное; 11 – Алан ГК – крупнейшее; 12 – Култак ГК – крупное; 13 – Кокдумалак НГК – крупнейшее; 14 – Янгуи ГК; 15 – Карим НГ; 16 – Юж. Верхнеамударьинский нефтегазоносный бассейн Для Верхнеамударьинского бассейна, более известного как Афгано-Таджикский, используется историческое название, отражающее его генетическое родство и идентичность платформенного юрско-палеогенового развития с Амударьинским бассейном. В новейшее время Верхнеамударьинский НГБ отделился от Амударьинского бассейна и развивался как межгорный со сложной складчато- надвиговой тектоникой. Верхнеамударьинская впадина составлена Байсунским, Сурхандарьинским, Вахшским и Кулябским прогибами и разобщающими их зонами поднятий (ЗП) – Шерабадской, Кафирниганской и Обигармской. В юго-восточной части впадины (Афганистан) Вахшский и Кулябский прогибы сливаются. Южная часть наиболее крупного Сурхандарьинского прогиба известна в Афганистане как прогиб Мазари-Шариф. Южная афганская часть Верхнеамударьинского бассейна, так же как и юго-восточная афганская часть Амударьинского бассейна, детально исследовались советскими и афганскими геологами [по материалам ВНИГНИ, 1978; Браташ и др., 1970; Северный Афганистан..., 1978;Самсонов и др., 1989; Ахмедзянов, 2005]. В последние десять лет эта территория стала объектом дистанционных и полевых исследований Геологической службы США (ГС США) [Аверков, 2006; В Афганистане обнаружено..., 2010; Емельянов, 2012]. Северная узбекско-таджикская часть Верхнеамударьинского НГБ традиционно изучалась российскими, узбекскими и таджикскими специалистами, в последние годы к ним присоединились специалисты нефтегазовых компаний, работающих в Таджикистане и Узбекистане [Абидов и др., 1987; Карта нефтегазогеологического..., 1989; Аверков, 2006; Абидов, 2009; Гулев, 2009; В Афганистане обнаружено..., 2010; Абдуллаев, Нурматов, 2012; Канадская компания..., 2012; Asia Times..., 2012]. Основополагающими аналитико- картографическими материалами по общей и нефтегазовой геологии Афганистана и сопредельных регионов Туркменистана, Узбекистана и Таджикистана остаются советские отчёты, публикации и карты. _____________________________________________________________________________ азовая геология. Теория и практика. – 2013. -Т.8. - №1. - http://www.ngtp.ru/rub/6/6_2013.pd ISSN 2070-5379 Neftegasovaâ geologiâ. Teoriâ i practika (RUS) URL: http://www.ngtp.ru SSN 2070-5379 Neftegasovaâ geologiâ. Teoriâ i practika (RUS) URL: http://www.ngtp.ru Рис. 1. Схема тектонических элементов и продуктивности трансграничных нефтегазоносных бассейнов Юго-Восточного Туркменистана, Западного и Южного Узбекистана, Юго-Западного Таджикистана, Северного и Западного Афганистана, Северо-Восточного Ирана 3 Рис. 1. Схема тектонических элементов и продуктивности трансграничных нефтегазоносных бассейнов Юго-Восточного Туркменистана, Западного и Южного Узбекистана, Юго-Западного Таджикистана, Северного и Западного Афганистана, Северо-Восточного Ирана _______________________________________________________________________________________ © Нефтегазовая геология. Теория и практика. – 2013. -Т.8. - №1. - http://www.ngtp.ru/rub/6/6_2013.pdf ISSN 2070-5379 Neftegasovaâ geologiâ. Teoriâ i practika (RUS) URL: http://www.ngtp.ru ISSN 2070-5379 Neftegasovaâ geologiâ. Teoriâ i practika (RUS) URL: http://www.ngtp.ru 4 Условные обозначения к рис. 1. Условные обозначения к рис. 1. Верхнеамударьинский нефтегазоносный бассейн Мубарек ГК; 17 – Карактай Г; 18 – Бешкент ГК; 19 – Сев. Нишан НГК; 20 – Гирсан ГК; 21 – Шуртан ГК – уникальное; 22 – Сев. Шуртан НГК; 23 – Пачкамар ГК; 24 – Адамташ ГК – крупное; 25 – Гумбулак ГК – крупное; 26 – Юж. Тандырча ГК - крупное; 27 – Оджа (Аккайры) ГК – крупное; 28 – Аккумулям ГК; 29 – Сакар ГК; 30 – Келяка ГК; 31 – Багаджа ГК - крупное; 32 – Малай-Чартак ГК – крупнейшее; 33 – Елан Г; 34 – Байрамали ГК – крупное; 35 – Сейраб ГК – крупное; 36 – Учаджи ГК – крупное; 37 – Вост. Учаджи ГК; 38 – Елкуи ГК – крупное; 39 – Бешкызыл ГК – крупное; 40 – Зап. Шатлык ГК – крупнейшее; 41 – Вост. Шатлык ГК – крупнейшее; 42 – Даулетабад – Донмез ГК - уникальное; 43 – Галкыныш (Минара-Иолотань- Юж. Иолотань – Осман – Джурджи) ГК – уникальное; 43а – Юж. Иолотань Н (межсолевые доломиты); 44 – Яшлар (Зап. Яшлар – Вост. Яшлар - Молодёжная – Каритли) ГК – уникальное; 45 – Шахмолла ГК; 46 – Дервезекем ГК; 47 – Гуррукбил ГК – крупное; 48 – Карабиль Г – крупное; 49 – Ходжагугирдоб (Тек-Тек) НГ; 50 – Алигул Н; 51 – Джангаликолон ГК; 52 – Джума Г; 53 – Башикурд Г; 54 – Шакарак Г; 55 – Джаркудук Г - крупное; 56 – Етымдаг ГК – крупное; 57 – Ходжагугердаг ГК – крупное; 58 – Ходжабулан НГ; 59 – Ангот; 60 – Акдарья Н; 61 – Кашкари Н; 62 – Базарками Н; 63 – Замрадсай Н. -Каларинский ГНБ. 1 – Моргуновское ГК; 2 – Ислим ГК; 3 – Карачоп ГК; 4 – Тореших шор ГК. Предкопетдагский ГНБ. 1 – Гараджаовлак ГК – крупнейшее; 2 – Теджен Г – крупное; 3 – Хангирен ГК – крупнейшее; 4 – Гонбадли ГК – крупное. Предкопетдагский ГНБ. 1 – Гараджаовлак ГК – крупнейшее; 2 – Теджен Г – крупное; 3 – Хангирен ГК – крупнейшее; 4 – Гонбадли ГК – крупное. Верхнеамударьинский НГБ. 1 – Гаджак ГК – крупное; 2 – Миршади Н; 3 – Юж. Верхнеамударьинский НГБ. 1 – Гаджак ГК – крупное; 2 – Миршади Н; 3 – Юж. Миршади Н; 4 – Каштар Н; 5 – Ляльмикар ГН; 6 – Джалаир Н; 7 – Хаудаг Н; 8 – Учкызыл Н; 9 – Кокайты Н; 10 – Актау Н; 11 – Досманага Н; 12 – Джейранхона Н; 13 – Корсаглы Н; 14 – Амударья Н; 15 – Кызылтумшук НГК; 16 – Акбашадыр Н; 17 – Кичикбель Н; 18 – Бештентяк НГК; 19 – Сульдузы Н; 20 – Узунохор Н; 21 – Юж. Пушион Н; 22 – Ходжасартис НГ; 23 – Шаамбары Г; 24 – Комсомольское Г; 25 – Андыген Г; 26 – Сев. Курганча Н. Верхнеамударьинский нефтегазоносный бассейн Миршади Н; 4 – Каштар Н; 5 – Ляльмикар ГН; 6 – Джалаир Н; 7 – Хаудаг Н; 8 – Учкызыл Н; 9 – Кокайты Н; 10 – Актау Н; 11 – Досманага Н; 12 – Джейранхона Н; 13 – Корсаглы Н; 14 – Амударья Н; 15 – Кызылтумшук НГК; 16 – Акбашадыр Н; 17 – Кичикбель Н; 18 – Бештентяк НГК; 19 – Сульдузы Н; 20 – Узунохор Н; 21 – Юж. Пушион Н; 22 – Ходжасартис НГ; 23 – Шаамбары Г; 24 – Комсомольское Г; 25 – Андыген Г; 26 – Сев. Курганча Н. ISSN 2070-5379 Neftegasovaâ geologiâ. Teoriâ i practika (RUS) URL: http://www.ngtp.ru N 2070-5379 Neftegasovaâ geologiâ. Teoriâ i practika (RUS) URL: http://www.ngtp.ru 5 Естественно, за 20-летний постсоветский период наработаны и новые геолого- геофизические материалы [Халылов и др., 1992; Соловьёв, Кузьминов, Салина, 1996; Давыдов, 1997; Одеков, Пашаев, 2001; Одеков и др., 2001; Мелихов, Сибирёв, 2003; Афгано- Таджикская и Мургабская..., 2007; Крылов, Кучеря, 2008; Абидов, 2009; Мелихов, 2009; Абдуллаев, Нурматов, 2012, Прогнозно-минерагеническая..., 2012]. Узбекско-Таджикская часть Верхнеамударьинского нефтегазоносного бассейна Базовые советские оценки строения, объектов геологоразведочных работ (ГРР) и ресурсов УВ по узбекско-таджикской части Верхнеамударьинского бассейна произведены К.Н. Кравченко и др. (ВНИГНИ, 1989), Н.А. Крыловым и др. (ИГИРГИ), узбекскими и таджикскими геологами (ИГИРНИГМ и др.). Актуализированное определение основных плеев – направлений ГРР и самые последние оценки ресурсов УВ рассматриваемого региона, его составных прогибов и нефтегазоносных комплексов (палеоген, мел, подсолевой карбонатный комплекс верхней юры) осуществлены Н.А. Крыловым, М.С. Кучерей (ВНИИГАЗ, 2008). Основными направлениями ГРР на нефть и газ Верхнеамударьинского бассейна являются: - карбонатно-терригенный палеоген-сенон [палеоген-верхний мел]: а) в прогибах бассейна; б) в поднадвиговых структурах на бортах Вахшского прогиба и в Кафирниганской зоне поднятий; - терригенно-карбонатный сеноман - нижний мел [нижний мел – надсолевой титон] всех прогибов бассейна и, возможно, отдельных структур в зонах поднятий; - подсолевой карбонатный комплекс верхней юры [верхнего келловея-оксфорда] в пределах всего Верхнеамударьинского бассейна; - терригенный комплекс средней-нижней юры, считающийся потенциально газонефтеносным на всей территории бассейна, однако, ввиду запредельно больших глубин залегания, не являющийся первостепенным для разведки. Согласно Н.А. Крылову, М.С. Кучеря [Крылов, Кучеря, 2008] неразведанные ресурсы нефти палеогена таджикско-узбекской части Верхнеамударьинского бассейна достигают 160 млн. т. Извлекаемые перспективные ресурсы УВ (преимущественно нефти) категории С3 составляют 57,5 млн. т н.э., распределяясь по прогибам бассейна следующим образом: Сурхандарьинский прогиб – 51,8 млн. т н.э.; Вахшский прогиб – 2,2 млн. т н.э.; Кулябский прогиб – 2,5 млн. т н.э. Средние запасы потенциальных палеогеновых месторождений нефти региона предполагаются небольшими – в пределах 1,5-5 млн. т. _______________________________________________________________________________________ © Нефтегазовая геология. Теория и практика. – 2013. -Т.8. - №1. - http://www.ngtp.ru/rub/6/6_2013.pdf ISSN 2070-5379 Neftegasovaâ geologiâ. Teoriâ i practika (RUS) URL: http://www.ngtp.ru ______________________________________________________________________________ © Нефтегазовая геология. Теория и практика. – 2013. -Т.8. - №1. - http://www.ngtp.ru/rub/6/6 ISSN 2070-5379 Neftegasovaâ geologiâ. Teoriâ i practika (RUS) URL: http://www.ngtp.ru 6 Неразведанные ресурсы нефти и газа меловых отложений рассматриваемого региона достигают 160,2 млн. т н.э., в том числе 121,1 млн. т н.э. в Сурхандарьинском прогибе, 16,1 млн. т н.э. в Вахшском прогибе и 23 млн. т н.э. в Кулябском прогибе. Суммарные неразведанные ресурсы УВ надсолевых мел-палеогеновых отложений таджикско-узбекской части Верхнеамударьинского бассейна составляют 320 млн. т н.э. Они, вероятно, являются более значимыми (до 400 млн. т н.э.), поскольку не учитывают ресурсы УВ поднадвиговых структур на бортах Вахшского прогиба и в Кафирниганской зоне поднятий [Крылов, Кучеря, 2008]. Неразведанные ресурсы газа глубокопогруженного (4-7 км и более) подсолевого карбонатного комплекса региона из-за крайне слабой изученности структуры, состава и литофациальной зональности комплекса недостаточно обоснованы и произвольно прогнозируются в диапазоне 1-3 трлн. м3. Практически неизученными остаются подсолевые антиклинальные структуры, которые по генезису и морфологии не могут быть интерполированы от многочисленных известных надсолевых поднятий. Примерами подсолевых объектов являются окраинно-бассейновые, минимально погруженные структуры – газовые месторождения – крупное месторождение Гаджак с запасами около 100 млрд. м3 (Байсунский прогиб, глубина подсолевых карбонатов 3,3-4 км) и мелкие месторождения Душанбинского прогиба (глубина карбонатов 1,7-2,7 км). Таджикская часть Верхнеамударьинского нефтегазоносного бассейна Ресурсы УВ Таджикистана представлялись в 1988 г. следующим образом: газ – 863 млрд. м3; нефть – 113 млн. т. Из этих ресурсов УВ 80,8% принадлежат Верхнеамударьинскому бассейну, остальные 19,2% - Ферганскому бассейну. В 2011 г Главное геологическое управление при СМ Таджикистана обнародовало прогнозную оценку недр страны – более 3 трлн. м3 газа и около 200 млн. т нефти. В 2003 г. правительство Таджикистана и «Газпром» заключили долгосрочное (до 2028 г.) соглашение о стратегическом сотрудничестве в области разведки и добычи газа и нефти и газификации Таджикистана. В 2006 г. «Газпрому» в лице дочерней компании «Газпром Зарубежнефтегаз» (Gazprom International) выдали лицензию на проведение геологического изучения площадей Саргазон и Ренган с прогнозными ресурсами газа соответственно 30 млрд. м3 и 35 млрд. м3. В 2008 г Gazprom International и правительство Таджикистана подписали соглашение об общих принципах проведения геологического изучения недр на 4 площадях (Саргазон, Ренган, Сарыкамыш, Шохамбары), обладающих суммарными прогнозными ресурсами газа в пределах 80-160 млрд. м3. ISSN 2070-5379 Neftegasovaâ geologiâ. Teoriâ i practika (RUS) URL: http://www.ngtp.ru ISSN 2070-5379 Neftegasovaâ geologiâ. Teoriâ i practika (RUS) URL: http://www.ngtp.ru ISSN 2070-5379 Neftegasovaâ geologiâ. Teoriâ i practika (RUS) URL: http://www.ngtp.ru 7 Все разведочные площади Gazprom International расположены на севере Верхнеамударьинского НГБ [Гулев, 2009], в то время как плотность подсолевых ресурсов газа и нефти прогнозируется максимальной на юге бассейна. Впрочем, такая локализация основных ресурсов УВ Верхнеамударьинского бассейна является сугубо предварительной ввиду крайне слабой изученности подсолевых карбонатов, как потенциально основной продуктивной толщи. В 2009 г. на площади Саргазон в ходе первого этапа сейсморазведки 2D была получена первая геологическая информация о высокоперспективных на газ глубокозалегающих подсолевых горизонтах юры. Оказалось, что подсолевой структуры, выявленной по недостаточно качественным сейсмическим материалам 1980-х гг., не существует. Однако здесь были выделены новые интересные объекты, нуждающиеся в доизучении сейсморазведкой 2D-3D. В 2009-2010 г.г. в ходе сейсморазведки 2D-3D на площади Сарыкамыш обнаружены новые целевые горизонты, которые могут содержать природный газ (подсолевые отложения верхней юры) и нефть (палеоген). Здесь подготовлена к бурению структура Шахринав, содержащая следующие перспективные ресурсы УВ: свободный газ – 18 млрд. м3; нефть – 17 млн. т (7 млн. т извлекаемых); растворённый газ – 2 млрд. м3. В 2011 г. на структуре Шахринав заложена самая глубокая в Таджикистане и во всей Средней Азии поисковая скв. 1 проектной глубиной 6300 м. В настоящее время её забой достиг 5500 м. Эта скважина будет иметь принципиальное значение для оценки нефтегазоносности глубоких горизонтов северной части Верхнеамударьинского бассейна. В 2011 г. Gazprom International отказался от продления лицензии на площадь Ренган, поскольку сложные горно-геологические особенности этой площади делают проект её освоения экономически нецелесообразным (расчленённый рельеф местности, крутые склоны, перепады высот более 1000 м). Рассматривается возможность получения лицензий на новые площади Таджикистана, в частности, площадь Нафтмайдон. В 2008 г. канадская компания Tethys Petroleum, имеющая контракты upstream в Казахстане и Узбекистане (в Казахстане ею открыты крупные залежи нефти на блоке Аккулка-Базай в Арало-Устюртском НГБ), приступила к работе в Таджикистане. По соглашению с правительством Таджикистана, подписанном на 13 лет, компания участвует в программе переосвоения 5 нефтяных и газовых месторождений, сейсмогеологического анализа 56 структур по всей территории Верхнеамударьинского бассейна, в разведке, разработке и модернизации 5 северных блоков с прогнозными ресурсами газа около ISSN 2070-5379 Neftegasovaâ geologiâ. Teoriâ i practika (RUS) URL: http://www.ngtp.ru ISSN 2070-5379 Neftegasovaâ geologiâ. Teoriâ i practika (RUS) URL: http://www.ngtp.ru 8 1,5 трлн. м3 (??). В последующем предполагается увеличение инвестиций в южные блоки в приграничном с Афганистаном Пянджском районе. 1,5 трлн. м3 (??). ISSN 2070-5379 Neftegasovaâ geologiâ. Teoriâ i practika (RUS) URL: http://www.ngtp.ru В последующем предполагается увеличение инвестиций в южные блоки в приграничном с Афганистаном Пянджском районе. В 2008-2011 г.г. производилось переосвоение Комсомольского газового месторождения вблизи г. Душанбе путём модернизации старых скважин и бурения единичных наклонных скважин. Остаточные запасы газа на месторождении составляют около 1 млрд. м3 при начальных 2,5 млрд. м3. В 2011 г на юге Таджикистана на разведочной площади Восточный Олимтой из песчаников алайской свиты палеогена получен приток нефти и газа с глубины 3342 м в процессе бурения скв. 1. Проектная глубина скважины 3800 м рассчитана на вскрытие известняков бухарской свиты палеогена. Вероятно, здесь открыто новое нефтяное месторождение в палеогеновых отложениях. В 2011 г. Tethys Petroleum получила от правительства Таджикистана первый контракт upstream о разделе продукции на так называемый участок «Бохтар» площадью 34785 км2, фактически охватывающей всю таджикскую часть Верхнеамударьинского бассейна. Срок действия контракта – до 2033 г. На основе соглашения, заключенного в конце 2012 г, Tethys Petroleum, китайская CNPC и французская Total будут владеть равными долями в проекте «Бохтар». К Таджикистану проявляют интерес и другие иностранные компании (США, Сингапур, Австрия, Швейцария и др.). В декабре 2011 г. специалисты Tethys Petroleum провели проверку «безрисковых перспективных ресурсов глубоких подсолевых зон» участка «Бохтар» т.е. таджикской части Верхнеамударьинского бассейна. Ресурсы УВ составили: нефть и конденсат – 1,14 млрд. барр. н.э. или 155 млн. т н.э.; газ – 7 трлн. кубофутов или 223 млрд. м3 – 178 млн. т н.э.; сумма ресурсов УВ – 333 млн. т н.э.; соотношение жидких и газообразных УВ – 46,5%: 53,5%. Ресурсы УВ подсолевого этажа таджикской части Верхнеамударьинского бассейна (333 млн. т н.э. на площади 35 тыс. км2) оказались сопоставимыми с ресурсами УВ афганской части Верхнеамударьинского бассейна, определёнными ГС США и афганскими специалистами в 644 млн. т н.э. для подсолевого и надсолевого этажа при площади афганской части бассейна 28 тыс. км2. В июле 2012 г. Tethys Petroleum обнародовала новые данные о ресурсах УВ таджикской части Верхнеамударьинского НГБ, включая надсолевой и подсолевой этажи. Новая оценка ресурсов УВ оказалась многократно больше предыдущей и составила: нефть и конденсат – 8,5 млрд. б н.э. или 1156 млн. т н.э.; газ - 114 трлн. кубофутов или 3228 млрд. м3 – 2582 млн. _______________________________________________________________________________________ © Нефтегазовая геология. Теория и практика. – 2013. -Т.8. - №1. - http://www.ngtp.ru/rub/6/6_2013.pdf ISSN 2070-5379 Neftegasovaâ geologiâ. Teoriâ i practika (RUS) URL: http://www.ngtp.ru ISSN 2070-5379 Neftegasovaâ geologiâ. Teoriâ i practika (RUS) URL: http://www.ngtp.ru 9 79 Neftegasovaâ geologiâ. Teoriâ i practika (RUS) URL: http://www.ngtp.ru т н.э.; сумма ресурсов УВ – 3738 млн. ISSN 2070-5379 Neftegasovaâ geologiâ. Teoriâ i practika (RUS) URL: http://www.ngtp.ru т н.э.; соотношение жидких и газообразных УВ – 31%:69%. Эти ресурсы УВ квалифицированы как «валовые безрисковые средние извлекаемые запасы» [Asia Times..., 2012]. Непонятен многократный рост ресурсов УВ с интервалом между оценками всего лишь в полгода, осуществлённый без существенного наращивания обосновывающей геолого- геофизической информации. Огромный рост ресурсов УВ возможен, по нашему мнению, только в случае обнаружения сейсморазведкой крупнейших подсолевых келловей- оксфордских рифов и крупных поднадвиговых палеогеновых объектов. Обнаружение рифов и поднадвиговых объектов возможно, однако маловероятно за столь короткий промежуток времени относительно небольшими объёмами сейсморазведочных работ, проводимых Tethys Petroleum в условиях больших глубин целевых горизонтов (4-7 км) и весьма сложных солевых и надвиговых дислокаций. Подобные рифы – уникальные и крупнейшие газоконденсатные и нефтегазоконденсатные месторождения известны в Амударьинском мегабассейне, соседнем и генетически близком с Верхнеамударьинским – в Мургабской впадине Туркменистана (Галкыныш, Яшлар), на Чарджоуской ступени и в Бешкентском прогибе Узбекистана (Денгизкуль, Уртабулак, Зеварды, Кокдумалак, Шуртан). Проблема выявления подсолевых рифов, как наиболее ёмких ловушек УВ, будет одной из основных и наиболее сложных в Верхнеамударьинском бассейне в целом (т.е. в таджикском, узбекском и афганском секторах) при практической неразбуренности подсолевых карбонатов и, как следствие, неизученности их состава и литофациальной зональности. В пресс-релизе компании «Tethys Petroleum» было отмечено, что «Амударьинский [Верхнеамударьинский?] бассейн является самым богатым …… в мире, который не был расконсервирован». Президент компании Дэвид Робсон заявил, что «эти безрисковые средние перспективные запасы значительно превышают предполагаемые остаточные и безрисковые запасы у британского побережья Северного моря. Проделанная геологическая и геофизическая работа показала, что компания Tethys Petroleum работает в бассейне мирового уровня, имеющем огромный неосвоенный потенциал. Глубокие перспективные участки, на которые направлена работа в Таджикистане, имеют сверхгигантский потенциал и успех их разработки существенно изменит положение компании и этой центральноазиатской страны. Эти дополнительные сейсмические данные помогут определить место для первой в Таджикистане глубокой подсолевой скважины, направленной на чрезвычайно большие перспективные запасы» [Asia Times..., 2012]. _____________________________________________________________________________ азовая геология. Теория и практика. – 2013. -Т.8. - №1. - http://www.ngtp.ru/rub/6/6_2013.pd ISSN 2070-5379 Neftegasovaâ geologiâ. Teoriâ i practika (RUS) URL: http://www.ngtp.ru SN 2070-5379 Neftegasovaâ geologiâ. Teoriâ i practika (RUS) URL: http://www.ngtp.ru 10 Компания Tethys Petroleum обещала в скором времени представить отчёт по новой оценке ресурсов УВ таджикской части Верхнеамударьинского бассейна, из которого, по- видимому, станет понятна реальность обоснования огромного роста ресурсов УВ. Узбекская часть Верхнеамударьинского нефтегазоносного бассейна Узбекская часть Верхнеамударьинского бассейна полностью охватывает Байсунский прогиб (Байсунский инвестиционный блок, осваиваемый малайзийской компанией Petronas) и около половины территории наиболее крупного и перспективного на нефть и газ Сурхандарьинского прогиба (другая его половина расположена в Афганистане). В Сурхандарьинском прогибе выделены три инвестиционных блока с известными инвесторами и операторами: Сурханский блок – Petronas; Восточный Сурханский блок – ExxonMobil; Коштарский блок – ExxonMobil [Абидов и др., 1987; Абидов, 2009; Абдуллаев, Нурматов, 2012]. В Байсунском блоке известно крупное газовое месторождение Гаджак, открытое в 1970 г. На 1991 г здесь были утверждены запасы основной подсолевой залежи (J3ko) и надсолевой залежи (K1h) суммарным объёмом 42,6 млрд. м3. К 2008 г. запасы газа подсолевой залежи увеличились до 61 млрд. м3. Начиная с 2006 г. на Гаджаке и сопредельной территории Байсунского прогиба Petronas осуществил сейсморазведку 2D (1200 пог. км) и 3D, гравимагнитную съёмку, испытание скважин на Гаджаке, интерпретацию накопленных геолого-геофизических данных. На Байсунском блоке намечено оценочное бурение с целью разведки новых месторождений (сообщается об открытии нового газового месторождения Когнисай) и доразведке уже известных. Согласно заключенному СРП, разработка месторождения Гаджак, запасы газа которого предположительно возросли до 100 млрд. м3, начнётся в ближайшее время. Из всех прогибов узбекско-таджикской части Верхнеамударьинского бассейна, потенциал нефтегазоносности мел-палеогенового этажа Сурхандарьинского прогиба выглядит наиболее крупным [Абидов и др., 1987]. Здесь открыто 12 небольших нефтяных месторождений в палеогеновых отложениях и 1 газонефтяное месторождение, в разрезе которого продуктивны также верхнемеловые отложения. Наиболее значительные начальные извлекаемые запасы нефти обнаружены на месторождениях Кокайты (3,447 млн. т) и Ляльмикар (2,698 млн. т). Сурхандарьинская нефть преимущественно тяжёлая, её добыча составила 127 тыс. т в 2007 г. Открыто новое нефтяное месторождение Южный Миршады, которое даст до 40 тыс. т нефти в 2012 г. Объёмы ГРР на надсолевой этаж Сурхандарьинского прогиба в целом невелики, ввиду большой степени освоения данного ISSN 2070-5379 Neftegasovaâ geologiâ. Teoriâ i practika (RUS) URL: http://www.ngtp.ru 11 70-5379 Neftegasovaâ geologiâ. Teoriâ i practika (RUS) URL: http://www.ngtp.ru ISSN 2070-5379 Neftegasovaâ geologiâ. Teoriâ i practika (RUS) URL: http://www.ngtp.ru 11 направления ГРР. В 2012 г. «Узбекнефтегаз» начал программу ГРР по поиску залежей тяжёлой палеогеновой нефти и битумов в пределах Корсаглы-Дагмансянгской зоны. направления ГРР. В 2012 г. «Узбекнефтегаз» начал программу ГРР по поиску залежей тяжёлой палеогеновой нефти и битумов в пределах Корсаглы-Дагмансянгской зоны. SN 2070-5379 Neftegasovaâ geologiâ. Teoriâ i practika (RUS) URL: http://www.ngtp.ru Главные перспективы дальнейшей разведки Сурхандарьинского прогиба связаны с прогнозом крупных газоконденсатных месторождений в глубокозалегающем подсолевом верхнеюрском карбонатном комплексе. Ориентировочная прикидка прогнозных ресурсов подсолевого газа Сурхандарьинского прогиба превышает 1 трлн. м3. В качестве первоочередных объектов подготовки и разведки подсолевых структур называются месторождения и площади Ляльмикар, Кокайты, Учкызыл, Хаудаг, Яилма, Южный Акджар и др. Подсолевое направление ГРР реализуется высокотехнологичной сейсморазведкой 2D- 3D, выполняемой компаниями Petronas и ExxonMobil, и последующим оценочным бурением на глубину 6000-7000 м. ____________________________________________________________________________ © Нефтегазовая геология. Теория и практика. – 2013. -Т.8. - №1. - http://www.ngtp.ru/rub SN 2070-5379 Neftegasovaâ geologiâ. Teoriâ i practika (RUS) URL: http://www.ngtp.ru ISSN 2070-5379 Neftegasovaâ geologiâ. Teoriâ i practika (RUS) URL: http://www.ngtp.ru 12 поисково-разведочном бурении. Пробурено более 150 поисково-разведочных скважин. Геологическая изученность нефтегазоперспективных земель сейсморазведкой (преимущественно малоинформативной модификацией МОВ) составляет 0,1 пог. км/км2, глубоким поисково-разведочным бурением – 5,7 м/км2. поисково-разведочном бурении. Пробурено более 150 поисково-разведочных скважин. Геологическая изученность нефтегазоперспективных земель сейсморазведкой (преимущественно малоинформативной модификацией МОВ) составляет 0,1 пог. км/км2, глубоким поисково-разведочным бурением – 5,7 м/км2. 4. В Амударьинском бассейне, в пределах которого были сосредоточены основные объёмы ГРР, открыты и разведаны 6 нефтяных месторождений (Ангот, Акдарья, Кашкари, Базарками, Замрадсай, Алигул) и 8 газовых месторождений с отдельными подсолевыми газоконденсатными залежами и нефтяными оторочками (Етымдаг, Ходжагугердаг, Ходжабулан, Джаркудук, Шакарак, Башикурд, Джума, Джангаликолон). Ещё на 12 площадях во всех бассейнах Северного и Западного Афганистана получены непромышленные притоки и признаки нефти и газа. Так, например, в Тирпульском бассейне Западного Афганистана притоки нефти получены на Ахмадабадской структуре, в пределах которой, по-видимому, открыта залежь нефти в нижнемеловых отложениях. 4. В Амударьинском бассейне, в пределах которого были сосредоточены основные объёмы ГРР, открыты и разведаны 6 нефтяных месторождений (Ангот, Акдарья, Кашкари, Базарками, Замрадсай, Алигул) и 8 газовых месторождений с отдельными подсолевыми газоконденсатными залежами и нефтяными оторочками (Етымдаг, Ходжагугердаг, Ходжабулан, Джаркудук, Шакарак, Башикурд, Джума, Джангаликолон). Ещё на 12 площадях во всех бассейнах Северного и Западного Афганистана получены непромышленные притоки и признаки нефти и газа. Так, например, в Тирпульском бассейне Западного Афганистана притоки нефти получены на Ахмадабадской структуре, в пределах которой, по-видимому, открыта залежь нефти в нижнемеловых отложениях. 5. Суммарные начальные запасы нефти на разведанных месторождениях оценены советскими и афганскими геологами в 40,8 млн. т (геологические) и в 12,96 млн. т (извлекаемые). Суммарные начальные запасы газа на разведанных месторождениях составляют 174 млрд. м3. 5. Суммарные начальные запасы нефти на разведанных месторождениях оценены советскими и афганскими геологами в 40,8 млн. т (геологические) и в 12,96 млн. т (извлекаемые). Суммарные начальные запасы газа на разведанных месторождениях составляют 174 млрд. м3. Перспективные и прогнозные геологические ресурсы нефти, газа и конденсата Северного Афганистана конкретно не оценивались. По приближённым оценкам они примерно в 2-3 раза превышали разведанные запасы (около 100 млн. т нефти и 400- 500 млрд. м3 газа), причём преобладающая доля ресурсов нефти и газа привязывалась к надсолевому (нефть) и подсолевому (газ) этажам наименее изученного Верхнеамударьинского бассейна. В 2002-2006 гг. ГС США по договору с Правительством Афганистана провела исследование потенциальных ресурсов нефти и газа всей территории Афганистана, базирующееся на анализе советско-афганских геолого-геофизических материалов и на собственных дистанционных работах. В последующие годы были начаты полевые исследования [Аверков, 2006]. Нефтегазоносные бассейны Афганистана Геолого-геофизические исследования нефтегазоносных земель Северного и Западного Афганистана площадью около 100 тыс. км2, разведка и разработка открытых здесь месторождений нефти и газа осуществлялись в 1958-1991 г. г. при полном техническом содействии Советского Союза [по материалам ВНИГНИ, 1978; Браташ и др., 1970; Северный Афганистан..., 1978; Самсонов и др., 1989; Ахмедзянов, 2005]. В крупномасштабный комплекс ГРР входили геологическая и гравимагнитная съёмки, сейсморазведка МОВ, КМПВ, в небольшом объёме – МОГТ, глубокое поисково-разведочное бурение, тематические научно-исследовательские работы. Достигнуты следующие основные результаты реализованного комплекса ГРР: 1. Установлены площади и контуры Амударьинского, Афгано-Таджикского, Калаиморско-Каларинского и Тирпульского нефтегазоносных бассейнов в пределах Северного и Западного Афганистана, перспективных НГБ Гильменд и Кундур (Южный) в Южном Афганистане. 2. Произведено тектоническое и нефтегазогеологическое районирование нефтегазоносных бассейнов Северного и Западного Афганистана, дифференциация территории бассейнов по перспективам нефтегазоносности юрских, меловых и палеогеновых отложений. Получены сведения о продуктивных комплексах и горизонтах юры, мела и палеогена. Выявлены территориальная и глубинная зональности в распределении преимущественно нефтяных и преимущественно газовых месторождений. 3. Обнаружено свыше 450 антиклинальных структур, в разной мере перспективных на газ и нефть. Подготовлено к разведке около 80 структур, около 50 из которых пребывало в SN 2070-5379 Neftegasovaâ geologiâ. Teoriâ i practika (RUS) URL: http://www.ngtp.ru Геологические ресурсы УВ Афганистана в целом оценены ГС США и афганскими геологами следующим образом: нефть – 3,6 млрд. барр. (490 млн. т); газ – 36,5 трлн. кубофутов (1 трлн. м3). Геологические ресурсы УВ Северного Афганистана (включая Западный Гератский регион) составляют: нефть – 1,5 млрд. барр. (204 млн. т); газ – 15,6 трлн. кубофутов ISSN 2070-5379 Neftegasovaâ geologiâ. Teoriâ i practika (RUS) URL: http://www.ngtp.ru 13 13 (442 млрд. м3). (442 млрд. м3). (442 млрд. м3). На территорию Южного Афганистана, включающую регионы Фарах, Гильменд и Катаваз (перспективные бассейны Гильменд и Южный) остаются следующие ресурсы УВ: нефть – 2,1 млрд. барр. (285 млн. т); газ – 20,9 трлн. кубофутов (592 млрд. м3). В оценке потенциальных ресурсов УВ Северного Афганистана, выполненной в 2006 г., происходит значительное повышение ресурсов нефти. Неясно, насколько обоснована повышенная оценка ресурсов УВ Южного Афганистана относительно таковой по Северному Афганистану. Заинтересованность в разведке и освоении афганских нефтегазовых ресурсов проявляли и проявляют в настоящее время ряд международных нефтяных компаний. Так, в 2002 г. «Роснефть» заключила с правительством Афганистана Меморандум о сотрудничестве, предлагала направить в Северный Афганистан специалистов для изучения состояния газовых промыслов, комплексов подготовки газа и газопроводов, обещала содействие в поиске геологоразведочной информации по Северному и Западному Афганистану, вывезенной в СССР. Однако по ряду причин «Роснефть» прекратила свою деятельность в Афганистане. С 1996 г. по настоящее время внимание международного сообщества к Афганистану связано, в основном, с проектом Трансафганского газопровода ТАПИ (Туркменистан- Афганистан-Пакистан-Индия). Пока ещё не решённая практическая реализация проекта ТАПИ ускорит разведку и освоение недр Афганистана на газ и нефть (к газопроводу ТАПИ могут быть подключены новые газовые месторождения Афганистана). В 2009 г. Афганистан объявил тендер на нефтяной блок Кашкари (нефтяные месторождения Кашкари, Ангот и Акдарья) с извлекаемыми ресурсами нефти 64,4 млн. барр. (8,76 млн. т) и прогнозными ресурсами 143,8 млн. барр. (19,48 млн. т) и на газовые блоки Джангаликолон (19 млрд. м3 газа) и Джума-Башикурд (33 млрд. м3). С победителем конкурса предлагалось заключить сервисный контракт на разведку, а если запасы углеводородов окажутся коммерчески выгодными – контракт будет переоформлен на соглашение о разделе продукции. Однако этот тендер, по-видимому, не состоялся. В 2010 г. правительством Афганистана было объявлено об открытии на севере страны крупного нефтяного месторождения с предполагаемыми ресурсами 1,6 млрд. барр. «на границе провинций Балх и Джаузджан, в области Шиберган совместными исследованиями афганских и иностранных [американских?] геологов». По-видимому, здесь речь шла не об открытии конкретного месторождения, а об оценке ресурсов нефти афганской части ISSN 2070-5379 Neftegasovaâ geologiâ. Teoriâ i practika (RUS) URL: http://www.ngtp.ru 14 Верхнеамударьинского бассейна. Исследовательские работы по оценке ресурсов УВ этой территории завершены ГС США и афганскими геологами в 2012 г. [Asia Times..., 2012; ExxonMobil приступит..., 2012]. Геологические ресурсы УВ Верхнеамударьинского бассейна по территории Афганистана составили: нефть – 219 млн. т (1,6 млрд. барр.); газ – 444 млрд. м3 (355 млн. т н.э.); конденсат – 70 млн.т. Суммарные геологические ресурсы УВ – 664 млн. т н.э. В конце 2011 г. (442 млрд. м3). правительство Афганистана подписало контракт с китайской CNPC на разработку 5 нефтяных месторождений (Кашкари, Акдарья, Ангот, Базарками, Замрадсай) в провинциях Сарипуль и Фарьяб с суммарными запасами нефти 87 млн. барр. (11,84 млн. т). На начальной стадии проекта CNPC собирается достичь добычи нефти 1,5 млн. барр. в год (200 тыс. т/год) [Компания CNPC..., 2012]. За пределами нефтяного контракта оказалось месторождение Алигул в провинции Бадгис, расположенное вблизи границы с Туркменистаном. В 2012 г. сообщалось, что канадская компания «Terraseiss» обнаружила новое большое (?) месторождение нефти в провинции Фарьяб, также вблизи границы с Туркменистаном [Канадская компания..., 2012]. Пограничные районы Афганистана и Туркменистана являются высокоперспективными на нефть. В 2012 г. Афганистан провёл тендер на разведку и разработку 6 блоков на севере и западе Афганистана. Западноафганский блок Ахмадабад (1,8 тыс. км2) находится в провинции Герат и принадлежит Тирпульскому ПНГБ, трансграничному с Ираном. Остальные блоки Балх (1,9 тыс. км2), Мазари-Шариф (2,7 тыс. км2), Мухамад Джан Дагар (3,6 тыс. км2), Сандуклы (2,6 тыс. км2), Шамар (2,1 тыс. км2) находятся в пределах Верхнеамударьинского НГБ. Совокупные потенциальные ресурсы жидких УВ этих 6 блоков оцениваются в 200-220 млн. т нефти и 70 млн. т конденсата. Заявки на тендер подали 28 компаний, допущены к тендеру 8 компаний. Тендер проведён в сентябре 2012 г. и предсказуемо выигран американским нефтегазовым гигантом «ExxonMobil». В скором времени Афганистан объявит тендер на разведку и разработку нефтегазовых блоков в Гильмендском ПНГБ и в Южном (Кундурском) ПНГБ, трансграничных с Ираном и Пакистаном. Юго-Восточный Туркменистан. Амударьинский газонефтеносный бассейн. Юго-Восточный Туркменистан. Амударьинский газонефтеносный бассейн. Предкопетдагский газонефтеносный бассейн Предкопетдагский газонефтеносный бассейн Юго-Восточный Туркменистан является главным газоносным регионом Туркменистана, охватывает Мургабскую впадину и прилежащие юго-восточные части Предкопетдагского краевого прогиба (Каахкинская и Меанинская депрессии) и ISSN 2070-5379 Neftegasovaâ geologiâ. Teoriâ i practika (RUS) URL: http://www.ngtp.ru 15 Бахардокского склона (Чанглинская моноклиналь). Определяющими структурами Мургабской впадины являются северная Обручевская и южная Сандыкачинская зоны прогибов, обрамляемый ими внутренний Марыйский пояс поднятий (последний содержит наиболее крупные и высокопродуктивные на газ Мары-Иолотанский, Яшларский, Шатлыкский, Андхойский валы и подсолевые карбонатные рифосодержащие платформы), внешние борта Мургабской впадины – северный Учаджинский и южный Бадхыз- Майманинский [Мелихов, Сибирёв, 2003; Мелихов, 2009]. Главной газоносной областью Юго-Восточного Туркменистана является Мургабская газоносная область (ГО), включающая Марыйский пояс поднятий и Сандыкачинскую зону прогибов. Локализованные разведанные ресурсы газа максимально концентрируются здесь в Мары-Иолотанском газоносном районе (ГР) (запасы газа месторождения Галкыныш оценены в 21 трлн. м3) и в Яшларском ГР (запасы газа Яшларского месторождения оцениваются в 1,5 трлн. м3, возможен их рост до 5 трлн. м3). Высокоперспективен Северо-Карабильский ГР, структуры которого вероятно содержат рифовые постройки и крупные газоконденсатные месторождения. Менее перспективны на газ Учаджинская ГО и Бадхыз-Карабильская ГО. В Учаджинской области повышенная плотность подсолевых локализованных ресурсов газа принадлежит Учаджинскому своду и прилегающей приподнятой части Репетек-Келифской линейной зоны структур. Бадхыз-Карабильская область известна уникальным сложноэкранированным газоконденсатным месторождением Даулетабад-Донмез, начальные запасы газа которого (с учётом новых приращений) достигли 2 тлрн. м3. На Бирлешикском и Дервезекемском выступах области обнаружены крупные структурно-стратиграфические газоконденсатные залежи – газ получен из подразмывной части оксфордских карбонатов, запечатанных глинами валанжина. Слабоизученные Каахкинская и Меанинская депрессии содержат в предгорной полосе две нефтегазоперспективных зоны антиклинальных и дизъюнктивно экранированных объектов – Яшлыкскую (преднадвиг и поднадвиг крупнейшей Гяурсдаг-Зиракевской антиклинали внутреннего борта Предкопетдагского прогиба) и Ходжабуланскую [Жмуд, Мелихов, 2000]. На внешнем борту Каахкинской депрессии выделяется высокопродуктивный на газ Гараджаовлакский погребённый выступ. Запасы его структурно- литологических подсолевых и надсолевых газоконденсатных залежей, «висящих» на южном крыле выступа, оцениваются в 300-400 млрд. м3 и имеют перспективы дальнейшего роста [Мелихов, Сибирёв, 2003; Мелихов, 2009]. _______________________________________________________________________________________ © Нефтегазовая геология. Теория и практика. – 2013. -Т.8. - №1. - http://www.ngtp.ru/rub/6/6_2013.pdf ______________________________________________________________________________ © Нефтегазовая геология. Теория и практика. – 2013. -Т.8. - №1. - http://www.ngtp.ru/rub/6/6 ISSN 2070-5379 Neftegasovaâ geologiâ. Teoriâ i practika (RUS) URL: http://www.ngtp.ru ISSN 2070-5379 Neftegasovaâ geologiâ. Teoriâ i practika (RUS) URL: http://www.ngtp.ru 16 79 Neftegasovaâ geologiâ. Teoriâ i practika (RUS) URL: http://www.ngtp.ru В приосевой части Каахкинской депрессии, на погребённом продолжении Шатлыкского вала расположена высокоамплитудная, вероятно рифовая структура Каахка, содержащая до 100 млрд. м3 перспективных ресурсов газа [Жмуд, Мелихов, 2000]. Предкопетдагский газонефтеносный бассейн Сам крупнейший Шатлыкский вал при отсутствии рифового резервуара оказался малопродуктивным по подсолевым карбонатным отложениям – в его сводовой части, резко «обеднённой» поровыми коллекторами и характеризующейся слабыми притоками газа в десятки тыс. м3/сут с большим количеством воды, приращено всего лишь 90 млрд. м3 газа. По аналогии с Гараджаовлакским выступом, возможны разрастание поровых коллекторов на южном выположенном неразведанном крыле Шатлыкского вала, наличие структурно- литологической ловушки и локализация здесь до 200 млрд. м3 перспективных газовых ресурсов [Мелихов, Сибирёв, 2003]. В пределах пологой Чанглинской моноклинали Бахардокского склона выделяется ряд довольно крупных погребённых подсолевых поднятий и, в том числе, Ишанбейикская атолловидная группа, имеющая сейсмические признаки рифов и в палеотектоническом плане продолжающая на северо-запад Мары-Иолотанский вал. При подтверждении рифовых резервуаров здесь, несомненно, будут открыты крупные газоконденсатные месторождения [Мелихов, Сибирёв, 2003; Мелихов, 2009]. При отсутствии классического количественного прогноза газоносности Юго- Восточного Туркменистана, зональное распределение и общую оценку неразведанных локализованных газовых ресурсов региона по надсолевому и подсолевому комплексам в первом приближении можно выяснить посредством интеграции авторских экспертных оценок ресурсов газа месторождений, подготовленных и выявленных структур, продуктивных и перспективных ареалов и зон. Разведка титон-неокомского надсолевого комплекса Мургабской впадины близка к завершению, поскольку подавляющее число локальных структур региона уже разведано по основному шатлыкскому высокопроницаемому резервуару. Общий потенциал неразведанной газоносности надсолевого этажа оценивается в 300-500 млрд. м3 и принадлежит, в основном, подшатлыкским и надшатлыкским невыдержанным горизонтам средней и слабой проницаемости. Крупные и средние газовые открытия в надсолевом этаже вероятны в слабоизученной юго-восточной части Предкопетдагского прогиба, в Яшлыкской и Ходжабуланской зонах внутреннего борта (комбинации антиклинальных и дизъюнктивно экранированных ловушек), на Каахкинской и др. структурах. Суммарный потенциал неразведанной _______________________________________________________________________________________ © Нефтегазовая геология. Теория и практика. – 2013. -Т.8. - №1. - http://www.ngtp.ru/rub/6/6_2013.pdf 17 ISSN 2070-5379 Neftegasovaâ geologiâ. Teoriâ i practika (RUS) URL: http://www.ngtp.ru газоносности надсолевого этажа Меанинской и Каахинской депрессий составит не менее 500-700 млрд. м3, а в целом по Юго-Восточному Туркменистану прогнозируется на уровне 800-1200 млрд. м3 (в среднем 1 трлн. м3). газоносности надсолевого этажа Меанинской и Каахинской депрессий составит не менее 500-700 млрд. м3, а в целом по Юго-Восточному Туркменистану прогнозируется на уровне 800-1200 млрд. м3 (в среднем 1 трлн. м3). Подавляющая часть перспективных и прогнозных локализованных ресурсов газа Юго- Восточного Туркменистана принадлежит подсолевому келловей-оксфордскому карбонатному комплексу, максимальная концентрация газовых ресурсов карбонатного комплекса происходит в зонах развития рифогенных построек. В рифогенных зонах с максимальной концентрацией газовых ресурсов открыты уникальные газоконденсатные месторождения Галкыныш (второе в Мире по запасам газа и первое в Мире среди месторождений с карбонатным коллектором) и Яшлар. Предкопетдагский газонефтеносный бассейн Предполагаемый геологической службой Туркменистана рост ресурсов газа Яшлара с 1,5 до 5 трлн. м3 (3,5 трлн. м3) может быть соотнесён только с прогнозными локализованными ресурсами. По сейсмическим данным распространение биогермов в карбонатном комплексе предполагается в полосе сочленения Южной Иолотани и Западного Яшлара, на структурах юго-восточного продолжения Яшларского вала, на Елчелекском валу и в Северо- Карабильском прогибе, на Ишанбейикской группе поднятий Чанглинского района и в Каахкинской депрессии (Гараджаовлакский выступ, поднятие Каахка). Признаки биогермов по керну обнаружены на Учаджинском валу. Рифогенные зоны и поднятия являются первоочередными объектами поисково-разведочных работ на газ в Юго-Восточном Туркменистане. Суммарная оценка неразведанных локализованных ресурсов газа подсолевого карбонатного комплекса приближённо составляет 7,5 трлн. м3, включая 3,5 трлн. м3 по Яшларскому месторождению. В подсолевом комплексе можно прогнозировать открытие 5- 10 новых крупнейших газоконденсатных месторождений с запасами более 100 млрд. м3, десятков крупных и средних месторождений. Суммируя неразведанные локализованные ресурсы газа подсолевого этажа (7,5 трлн. м3) и надсолевого этажа (1 трлн. м3), получим общую оценку по Юго-Восточному Туркменистану в объёме 8,5 трлн. м3. Не претендуя на большую точность, эта оценка показывает огромный потенциал разведки региона на газ. Самое значительное газовое открытие для туркменской части Правобережья Амударьи осуществлено в подсолевых карбонатах на давно известном, но не вводившемся в разведку, наиболее крупном поднятии Агайры размерами 35×14 км и высотой 200-300 м. В скв. 21 Оджа, пробуренной в 2011 г китайской CNPC, из нижней, относительно уплотнённой ISSN 2070-5379 Neftegasovaâ geologiâ. Teoriâ i practika (RUS) URL: http://www.ngtp.ru ISSN 2070-5379 Neftegasovaâ geologiâ. Teoriâ i practika (RUS) URL: http://www.ngtp.r SN 2070-5379 Neftegasovaâ geologiâ. Teoriâ i practika (RUS) URL: http://www.ngtp.ru 18 половины карбонатной толщи получен приток газа дебитом 130 тыс. м3/сут (фильтр 3731- 3703 м, 3646-3578 м, штуцер 17 мм). Из верхней органогенной половины карбонатов стабильный среднесуточный приток газа и конденсата составил 1,439 млн. м3 и 36,2 м3. Текущая оценка запасов газа месторождения Оджа (Агайры) достигает 100 млрд. м3 и имеет большие перспективы роста. Предполагается, что крупная структура Агайры объединяет на одном цоколе несколько средних структур – вероятных органогенных банок. После открытия крупнейшего газоконденсатного месторождения Оджа (Агайры) значительно повышаются перспективы газоносности прилегающих крупных поднятий Кувадаг и Керкидаг. Ввиду практической неизученности бурением среднеюрского глубокопогруженного терригенного комплекса, его прогнозные ресурсы газа не определены. Среднеюрские песчаники при достаточной проницаемости будут промышленно газоносными на крупных поднятиях региона – уникальных крупнейших и крупных месторождениях по карбонатному комплексу. Первый промышленный приток газа из среднеюрского терригенного комплекса получен в Мургабской впадине на поднятии Шахмолла, расположенном вблизи границы с Афганистаном [Мелихов, Сибирёв, 2003]. Здесь при опробовании низов 50-метрового песчаного горизонта в подошве бата получен газ дебитом 150 тыс. м3/сут с большим количеством воды (интервал 5088-5100 м, штуцер 14 мм). Опробование низов верхнеюрского карбонатного комплекса дало лишь слабые притоки газа (3-5 тыс. м3/сут), интенсификация которых не проводилась. Ввиду технического несовершенства сводовой скв. 1 крупное поднятие Шахмолла требует повторного ввода в разведку. Высокоперспективным объектом разведки на верхнюю-среднюю юру является также расположенное к северу от Шахмолла крупное поднятие Ширамкуи, замыкающее Андхойский вал. При уникальности газовых ресурсов Юго-Восточного Туркменистана локализованные ресурсы его жидких УВ предполагаются относительно небольшими ввиду невысоких значений конденсатности подсолевых газов и ограниченных перспектив нефтеносности глубокопогруженной подсолевой системы [Халылов и др., 1992; Мелихов, 2009]. Из 20,8 млрд. т официально представляемых геологических НСР нефти Туркменистана 12 млрд. т принадлежит туркменскому шельфу Каспия и около 3 млрд. т – суше Юго-Западного Туркменистана (плиоценовый, миоценовый и мезозойский комплексы отложений), составляющим восточную часть Южно-Каспийского мегабассейна. На Амударьинский мегабассейн остаётся таким образом 6 млрд. т геологических НСР нефти, величина которых вызывает сомнение в виду доказанной преимущественной газоносности мегабассейна. Для ISSN 2070-5379 Neftegasovaâ geologiâ. Teoriâ i practika (RUS) URL: http://www.ngtp.ru ISSN 2070-5379 Neftegasovaâ geologiâ. Teoriâ i practika (RUS) URL: http://www.ngtp.r ISSN 2070-5379 Neftegasovaâ geologiâ. Teoriâ i practika (RUS) URL: http://www.ngtp.ru 19 70-5379 Neftegasovaâ geologiâ. Teoriâ i practika (RUS) URL: http://www.ngtp.ru ISSN 2070-5379 Neftegasovaâ geologiâ. Teoriâ i practika (RUS) URL: http://www.ngtp.ru 19 Амударьинского НГМБ традиционно рассматриваются не ресурсы нефти, а суммарные ресурсы жидких УВ – нефти и конденсата. Амударьинского НГМБ традиционно рассматриваются не ресурсы нефти, а суммарные ресурсы жидких УВ – нефти и конденсата. ______________________________________________________________________________ © Нефтегазовая геология. Теория и практика. – 2013. -Т.8. - №1. - http://www.ngtp.ru/rub/6/6 SN 2070-5379 Neftegasovaâ geologiâ. Teoriâ i practika (RUS) URL: http://www.ngtp.ru Амударьинского НГМБ традиционно рассматриваются не ресурсы нефти, а суммарные ресурсы жидких УВ – нефти и конденсата. Ранее считалось, что большая часть ресурсов жидких УВ (порядка 4 млрд. т) будет сосредоточена в глубокопогруженной подсолевой системе Мургабской впадины по аналогии с эталонным нефтегазоконденсатным месторождением Кокдумалак, расположенном на северо-восточном борту Амударьинского НГМБ и отличающимся аномально высоким, нехарактерным для региона, содержанием жидких УВ (начальные геологические запасы УВ Кокдумалака: газ – 143 млрд. м3; конденсат – 96,4 млн. т; нефть – 98,7 млн. т). Эта аналогия оказалась ошибочной, новые уникальные открытия Мургабской впадины – месторождения Галкыныш и Яшлар – оказались связаны с газом, отличающимся низким содержанием конденсата. Подсолевых скоплений нефти не обнаружено, межсолевые скопления нефти вместе с газом и рапой (Юж. Иолотань и др.) невелики по объёмам. Суммарные ресурсы подсолевого конденсата Мургабской впадины, вероятно, не будут превышать 1 млрд. т. До 2 млрд. т жидких УВ остаются привязанными к малым и умеренным глубинам (1,5-4 км) бортов Мургабской впадины, территорий Предкопетдагского прогиба (преимущественно центральной части), Бахардокского склона (преимущественно западной части), южного фаса Центрально-Каракумского свода, Калаиморско-Каларинского бассейна. Одной из зон бортовой части Мургабской впадины, перспективной на открытие небольших нефтяных и газонефтяных месторождений является полоса сочленения Карабильской моноклинали и Майманинского выступа, трансграничная между Туркменистаном и Афганистаном. В афганской её части, в пределах Джигдалекского приразломного вала открыто нефтяное месторождение Алигул, содержащее 3,6 млн. т извлекаемой нефти. Нефть дебитом 2-25 м3/сут. поступала из известняков и песчаников нижнего апта, залегающих на глубине 847-888 м. Непосредственно к западу от Алигула, в приграничной части Туркменистана К.Н. Кравченко рекомендовал разведку довольно крупного прогнозируемого приразломного поднятия Ходжагугирдоб (27×10 км × 100 м) с привязкой к нему 23 млн. т прогнозных извлекаемых ресурсов нефти и 13 млрд. м3 газа [Кравченко, Иванова, 1997]. «Туркменгеологией», осуществившей здесь бурение двух скважин, название объекта было изменено с Хаджагугирдоба на Тек-Тек. При опробовании в скв. 1 Тек-Тек высокопроницаемых верхнеготеривских песчаников, несогласно перекрывающих сланцы триаса, получен газ дебитом 542 тыс. м3/сут (интервал опробования 1165-1184 м, штуцер 20 мм, пластовое давление 205 кг/см2). Приток газа зафиксирован также из нижнеаптских _______________________________________________________________________________________ © Нефтегазовая геология. Теория и практика. – 2013. -Т.8. - №1. - http://www.ngtp.ru/rub/6/6_2013.pdf ISSN 2070-5379 Neftegasovaâ geologiâ. Teoriâ i practika (RUS) URL: http://www.ngtp.ru ISSN 2070-5379 Neftegasovaâ geologiâ. Teoriâ i practika (RUS) URL: http://www.ngtp.ru 20 известняков, приток нефти дебитом 5 м3/сут – из альбских песчаников. Скв. 2, смещённая на 5 км к западу от скв. ______________________________________________________________________________ © Нефтегазовая геология. Теория и практика. – 2013. -Т.8. - №1. - http://www.ngtp.ru/rub/6/6 SN 2070-5379 Neftegasovaâ geologiâ. Teoriâ i practika (RUS) URL: http://www.ngtp.ru 1, существенно «провалилась» по глубине от прогнозного уровня и оказалась непродуктивной по базальным верхнеготеривским песчаникам – при опробовании в колонне получено 474 м3/сут. воды. Здесь не произведено опробование перспективных по каротажу, но менее проницаемых, барремских и нижнеаптских известняков, альбских песчаников. Возможно скв. 2 попала в опущенный блок поднятия Ходжугугирдоб. Вероятность блокового строения поднятия прямо указана в рекомендации [Кравченко, Иванова, 1997], хотя, ввиду крайней недостаточности обосновывающих данных, была принята его исходная пликативная модель. Скв. 1, 2 не решили задачи оценки нефтегазоносности приразломного поднятия Ходжагугирдоб, его разведку с туркменской, а, возможно, и с афганской стороны целесообразно продолжить заложением новых скважин, исходя из следующих соображений. Скв. 1, 2 не решили задачи оценки нефтегазоносности приразломного поднятия Ходжагугирдоб, его разведку с туркменской, а, возможно, и с афганской стороны целесообразно продолжить заложением новых скважин, исходя из следующих соображений. 1. Поднятие Ходжагугирдоб не подготавливалось к бурению сейсморазведкой ввиду сложного пересечённого рельефа. 2. Задачи подготовки и разведки поднятия рекомендовалось совокупно решить бурением 15 неглубоких (1200-1500 м) структурно-поисковых скважин [Кравченко, Иванова, 1997], что абсолютно реально и малозатратно при использовании лёгкого мобильного бурстанка. Этими пятнадцатью скважинами рекомендовалось разведать как поднятие Ходжагугирдоб, так и расположенное к западу от него предполагаемое поднятие Гельчешме. 3. На структурах Ходжагугирдоб и Гельчешме могут быть приращены значительные запасы газа и, что особенно актуально, нефти. Нефтяные оторочки могут быть обнаружены в продуктивных горизонтах верхнего готерива, баррема и нижнего апта, выяснится ценность альбского нефтяного скопления. К северу от Джигдалекского вала, в направлении от Карабильской моноклинали к Майманинскому выступу намечается ещё несколько воздымающихся субширотных антиклинальных линий (Леккер-Пограничная, Хумлы-Карабаба-Ходжакуль, Дервезекем- Меджитли-Джалаер-Караколь), приподнятые структуры которых высокоперспективны на нефть и подлежат первоочередной разведке как со стороны Туркменистана, так и в пределах Афганистана. В 1965 г. поисковая скв. 1, пробуренная в своде крупной Ходжакульской складки (Афганистан), при глубине 956 м вошла в граниты палеозойского фундамента. Меловой разрез оказался здесь полностью лишённым коллекторов. Таким образом, антиклинальная линия Хумлы-Карабаба-Ходжакуль «закрывается» по восстанию _______________________________________________________________________________________ © Нефтегазовая геология. Теория и практика. – 2013. -Т.8. - №1. - http://www.ngtp.ru/rub/6/6_2013.pdf ISSN 2070-5379 Neftegasovaâ geologiâ. Teoriâ i practika (RUS) URL: http://www.ngtp.ru 21 ISSN 2070-5379 Neftegasovaâ geologiâ. Teoriâ i practika (RUS) URL: http://www.ngtp.ru 21 литологическим экраном, что повышает перспективы нефтегазоносности приграничной, практически неизученной структуры Карабаба. литологическим экраном, что повышает перспективы нефтегазоносности приграничной, практически неизученной структуры Карабаба. Значительное нефтегазовое месторождение предполагается в сводовой части Бадхызского выступа, являющегося платформенным продолжением хребта Восточный Копетдаг. SN 2070-5379 Neftegasovaâ geologiâ. Teoriâ i practika (RUS) URL: http://www.ngtp.ru На основе палеотектонического анализа здесь прогнозируется крупное погребённое поднятие Дузенкыр площадью до 300-400 км2 и высотой 100-150 м [Кравченко и др., 1982]. Прогнозные локализованные ресурсы нижнемеловых отложений Дузенкыра (верхний готерив, баррем, нижний апт) приближённо оцениваются в 100 млрд. м3 газа и в 40 млн. т нефти, не исключается и продуктивность альб-сеноманских отложений. Появление нефтяных скоплений в виде залежей и оторочек ожидается в виду максимального поднятого положения Дузенкырского поднятия на пути восходящей миграции УВ из Мургабской впадины (по аналогии с другими приподнятыми нефтегазовыми объектами Бадхыз- Майманинской области). Пробуренная здесь поисковая скв. 1 оказалась вне сейсмического свода Дузенкырского поднятия и не решила поставленной задачи. Необходимо заложение профиля 3-4 новых скважин глубиной 2000-2100 м, ориентированного вдоль гребня поднятия. (Туркменистан, Афганистан) Калаиморско-Каларинский газонефтеносный бассейна, обычно рассматривается в составе Амударьинского ГНБ, но фактически является самостоятельным. Этот трансграничный между Туркменистаном и Афганистаном бассейн включает Калаиморский, Кайсарский и Каларинский прогибы, Кушкинскую зону поднятий (погруженное продолжение хребта Банди-Туркестан) и ЗП Бандигандао. В пределах Туркменистана наиболее изученными сейсморазведкой, структурным и глубоким бурением является Кушкинская ЗП и Чеменабидский вал, расположенный в осевой зоне Калаиморского прогиба. Афганская часть бассейна исследована незначительно – здесь проведена лишь геологическая съёмка, наметившая ряд структур. В Калаиморском прогибе и в Кушкинской ЗП регионально газоносными являются песчаники батского и байосского ярусов средней юры (газоконденсатные залежи на месторождениях Моргуновское, Ислим, Тореших, Кулишор, притоки газа на Чеменибиде и в Адылбекской зоне выклинивания). В Кушкинской зоне поднятий обнаружены также единичные мелкие газовые залежи в карбонатных коллекторах нижнего апта и верхнего мела. Суммарные запасы газа всех месторождений рассматриваемой территории, не _______________________________________________________________________________________ © Нефтегазовая геология. Теория и практика. – 2013. -Т.8. - №1. - http://www.ngtp.ru/rub/6/6_2013.pdf 70-5379 Neftegasovaâ geologiâ. Teoriâ i practika (RUS) URL: http://www.ngtp.ru 22 ISSN 2070-5379 Neftegasovaâ geologiâ. Teoriâ i practika (RUS) URL: http://www.ngtp.ru 22 превышают, по-видимому, 50 млрд. м3, наиболее крупным месторождением является Торешихское [Одеков, Пашаев, 2001; Мелихов, 2009]. Прогнозные ресурсы газа высокоперспективных средне-нижнеюрских отложений, возможно, достигают 200 млрд. м3. В трёх скважинах месторождения Тореших из байосского горизонта, названного Бурабай («нефтяной»), вместе с газом и конденсатом получены притоки нефти дебитом до 50 м3/сут. [Одеков и др., 2001], что позволяет предположить здесь нефтяную оторочку с запасами в несколько млн. т. На основании этого первого нефтяного открытия прогнозные геологические ресурсы нефти Кушкинской (Тахта-Базарской) зоны поднятий были супероптимистично оценены в 800 млн. т (газета «Нейтральный Туркменистан» от 15.06.2000 г), хотя реально они на порядок меньше. В афганской малоизученной части Калаиморско-Каларинского бассейна первостепенный интерес для разведки на газ и нефть представляют структуры афганского сектора Кушкинской ЗП (Манган-Мургаб и др.), поднятие Бандигандао с поверхностными нефтебитумопроявлениями, Кызылбулакская антиклинальная цепь, разграничивающая Калаиморский и Каларинский прогибы. ______________________________________________________________________________ © Нефтегазовая геология. Теория и практика. – 2013. -Т.8. - №1. - http://www.ngtp.ru/rub/6/6 Тирпульский и Гильмендский бассейны В Предкопетдагском ГНБ Ирану принадлежит Хангиренский блок поднятий и месторождений, а также южная часть Гяурсдаг-Зиракевского блока. На крупнейшем Хангиренском месторождении разведаны две массивные сводовые залежи газа в интервале глубин 3000-3500 м – залежь в песчаниках готерива с запасами 68 млрд. м3 и залежь в известняках оксфорда (свита моздуран) с запасами 294 млрд. м3. Суммарные запасы газа Хангирена первоначально оценивались в 362 млрд. м3 [Справочник по нефтяным..., 1976]. По результатам доразведки и многолетней разработки оценка начальных запасов месторождения значительно возросла. Крупными, вероятно, являются газоконденсатные месторождения Гонбадли (известняки оксфорда) и Серахс (песчаники готерива – иранская часть уникальной «висячей» Донмезской залежи Туркменистана). В 2012 г. в результате сейсморазведки 3D обнаружено новое совместное ирано-туркменское месторождение газа Афшар (Раходж?), где Ираном начато бурение первой разведочной скважины. Площадь иранской части месторождения Афшар составляет примерно 3×3 км2. На отдельных площадях Хангиренского и Гяурсдаг-Зиракевского блоков проводится сейсморазведка современного уровня и возможны новые открытия. _____________________________________________________________________________ азовая геология. Теория и практика. – 2013. -Т.8. - №1. - http://www.ngtp.ru/rub/6/6_2013.pd ISSN 2070-5379 Neftegasovaâ geologiâ. Teoriâ i practika (RUS) URL: http://www.ngtp.ru ISSN 2070-5379 Neftegasovaâ geologiâ. Teoriâ i practika (RUS) URL: http://www.ngtp.ru 23 В 2011 г. в Кучано-Мешхедском межгорном прогибе, вблизи города Мешхед открыто первое газовое месторождение, связанное с известняками оксфорда и имеющее объявленные запасы 63 млрд. м3. Таким образом доказана продуктивность нового Кучано-Мешхедского межгорного газонефтеносного бассейна, имеющего протяжённость 300 км при ширине до 30-40 км. Здесь также проводится сейсморазведка и ожидаются новые открытия. На иранскую территорию приходится около половины площади Тирпульского перспективного НГБ (порядка 7 тыс. км2) и значительная часть Гильмендского ПНГБ. О каких-либо ГРР на нефть и газ в пределах этих элементов не сообщалось. Тирпульский бассейн, как в Афганистане, так и в Иране, вероятно, обладает повышенными перспективами нефтеносности. ______________________________________________________________________________ © Нефтегазовая геология. Теория и практика. – 2013. -Т.8. - №1. - http://www.ngtp.ru/rub/6/6 Литература р ур Абдуллаев Г.С., Нурматов М.Р. Создание сырьевой базы углеводородов на долгосрочную перспективу. Инновации в области геологоразведочных работ // Узбекский журнал нефти и газа. Специальный выпуск к 20-летию НХК "Узбекнефтегаз". – 2012. - С. 16-22. р ур Абдуллаев Г.С., Нурматов М.Р. Создание сырьевой базы углеводородов на долгосрочную перспективу. Инновации в области геологоразведочных работ // Узбекский журнал нефти и газа. Специальный выпуск к 20-летию НХК "Узбекнефтегаз". – 2012. - С. 16-22. Абидов А.А. Нефтегазоносность литосферных плит Мира. – Ташкент: изд-во «Фан». – 2009. – 627 с. Абидов А.А., Убайходжаев Т.И., Абдысадыков А.А, Юлдашев Ж.Ю. Перспективы нефтегазоносности Сурхандарьинского синклинория // Геология нефти и газа. – 1987. - № 7. - С. 20-22. Аверков В. Теперь дорога открыта крупным нефтяным компаниям // Информационный портал "Афганистан.Ру" [Электронный ресурс] – http://www.afghanistan.ru/doc/5421.html/ (дата публикации 28.03.2006). Амурский Г.И., Жабрев И.П., Гончаров В.С., Гончаров Э.С., Иванова В.А., Ледовская Г.И., Кузьмук Л.Г., Соколов В.Я., Соловьёв Н.Н. Геологические предпосылки перспектив газоносности Восточной Туркмении. - М.: Недра. – 1976. - 392 с. Asia Times: В Таджикистане найдены крупные запасы нефти и газа // Информационно- аналитический портал «Нефть России». - [Электронный ресурс] - http://www.oilru.com/news/330866/ (дата публикации 10.08.2012). Афгано-Таджикская и Мургабская впадины. Структурная карта кровли палеоценовых отложений на упрощённой геологической основе / Мелихов В.Н. – М-б 1:500 000. – СПб.: ВСЕГЕИ. - 2007. Ахмедзянов И.Ф. Углеводородные запасы Афганистана // Информационный портал "Афганистан.Ру". - [Электронный ресурс] - http://www.afghanistan.ru/ (дата публикации 23.06.2005). Браташ В.И., Егупов С.В., Печников В.В., Шеломенцев А.И. Геология и нефтегазоносность севера Афганистана. - Недра. – 1970. - 288 с. В Афганистане обнаружено крупное месторождение нефти с запасами в 1,8 млрд. бар. // "RusEnergy.com". - [Электронный ресурс] - http://www.rusenergy.com/ru/ (дата публикации 16.08.2010). ) Гулев В.Л. Геологоразведочные работы на нефтегазоносных площадях Южного Таджикистана // Горный журнал. – 2009. - № 8. - С. 21-22. Давыдов А.Н. Характер сочленения южной окраины Туранской плиты, Северо- Афганской платформы и обрамляющих складчатых сооружений с позиций тектоники литосферных плит // Нефть и газ Туркменистана. – 1997. - №3. - С. 11-16. Емельянов В. Афганистан: краткий справочник для нефтегазового инвестора // Информационно-аналитический портал «Нефть России». - [Электронный ресурс] - http://www.oilru.com/dossier/read/110/ (дата публикации 07.11.2012). Жмуд М.С., Мелихов В.Н. Обновлённая концепция региональной структуры и нефтегазоносности Предкопетдагского прогиба // Нефть и газ Туркменистана. – 2000. - № 2. - С. 10-28. Жмуд М.С., Мелихов В.Н. Обновлённая концепция региональной структуры и нефтегазоносности Предкопетдагского прогиба // Нефть и газ Туркменистана. – 2000. - № 2. - С. 10-28. SN 2070-5379 Neftegasovaâ geologiâ. Teoriâ i practika (RUS) URL: http://www.ngtp.ru 24 На северо-востоке Ирана открыт новый Кучано-Мешхедский НГБ, приуроченный к узкому межгорному прогибу. Открытие здесь крупного газового месторождения повышает перспективы разведки прилежащих Тирпульского (Иран, Афганистан) и Сорхабского (Иран) межгорных бассейнов. Выводы На рассматриваемой территории юга Центральной Азии наиболее крупным потенциалом разведанной (более 27 трлн. м3) и неразведанной (8,5 трлн. м3) газоносности обладает Амударьинский мегабассейн в пределах Юго-Восточного Туркменистана. Относительно менее значимым, но также очень крупным представляется здесь потенциал разведки жидких УВ, приближённо оцениваемый в 3 млрд. т. геологических ресурсов, соотносимый с верхнеюрскими подсолевыми конденсатами и (в меньшей мере) с межсолевыми нефтями погруженной части Мургабской впадины, с юрско-меловыми нефтями и конденсатами Бадхыз-Майманинского борта Мургабской впадины, Предкопетдагского и Калаиморско-Каларинского прогибов, Бахардокского склона. Повышенные перспективы нефтеносности связываются, в частности, с пограничными районами Туркменистана и Афганистана, отдельные объекты которых рекомендуются к разведке. Вторым по нефтегазовому потенциалу бассейном юга Центральной Азии является Верхнеамударьинский, охватывающий сопоставимые по площади сопредельные части Таджикистана, Узбекистана и Афганистана. По приближённым оценкам Верхнеамударьинский НГБ содержит до 3-3,5 трлн. м3 газа в глубокопогруженной подсолевой карбонатной формации верхней юры и до 500-600 млн. т геологических ресурсов жидких УВ – нефти в надсолевом мел-палеогеновом этаже, конденсата в подсолевом этаже. В случае повышенной конденсатности газов, ресурсы жидких УВ бассейна могут возрасти до 700-800 млн. т. Аномально высокая оценка ресурсов УВ, осуществлённая в конце 2012 г. компанией «Tethys Petroleum» по таджикской части Верхнеамударьинского бассейна (газ – 3228 млрд. м3, нефть и конденсат – 1156 млн. т) требует фактического подтверждения. ISSN 2070-5379 Neftegasovaâ geologiâ. Teoriâ i practika (RUS) URL: http://www.ngtp.ru ____________________________________________________________________________ © Нефтегазовая геология. Теория и практика. – 2013. -Т.8. - №1. - http://www.ngtp.ru/rub ISSN 2070-5379 Neftegasovaâ geologiâ. Teoriâ i practika (RUS) URL: http://www.ngtp.ru SN 2070-5379 Neftegasovaâ geologiâ. Teoriâ i practika (RUS) URL: http://www.ngtp.ru Карта нефтегазогеологического, тектонического районирования, локальных поднятий и месторождений Средней Азии и Южного Казахстана / Ред. Клещёв К.А., отв. исполнители Кравченко К.Н., Чернобров Б.С. – М-б 1:1000 000. – М.: ВНИГНИ. - 1989. Карта нефтегазогеологического, тектонического районирования, локальных поднятий и месторождений Средней Азии и Южного Казахстана / Ред. Клещёв К.А., отв. исполнители Кравченко К.Н., Чернобров Б.С. – М-б 1:1000 000. – М.: ВНИГНИ. - 1989. 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Edited by Kleshchev K.A., performed by Kravchenko K.N., Chernobrov B.S. Scale 1:1000000. Moscow: VNIGNI, 1989. Khalylov M., Amurskiy G.I, Solov'ev N.N., Timonin A.N. Obosnovanie perspektiv neftenosnosti i razrabotka rekomendatsiy na provedenie GRR na zhidkie UV na territorii deyatel'nosti GK "Turkmengaz" [Substantiation of oil-bearing prospects and recommendations for liquid hydrocarbons exploration of the "Turkmengaz"]. Ashkhabad: "Turkmengaz", 1992, 62 p. Kompaniya CNPC pristupila k razrabotke neftyanogo mestorozhdeniya v Afganistane [The company CNPC has begun to develop oil field in Afghanistan]. Available at: http://www.afghanistan.ru/doc/53194.html (25th Oct 2012). Kompaniya CNPC pristupila k razrabotke neftyanogo mestorozhdeniya v Afganistane [The company CNPC has begun to develop oil field in Afghanistan]. Available at: http://www.afghanistan.ru/doc/53194.html (25th Oct 2012). 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Neft' i gaz Turkmenistana, 1997, no. 3, p. 11-16. _______________________________________________________________________________________ © Нефтегазовая геология. Теория и практика. – 2013. -Т.8. - №1. - http://www.ngtp.ru/rub/6/6_2013.pdf ____________________________________________________________________________ вая геология. Теория и практика. – 2013. -Т.8. - №1. - http://www.ngtp.ru/rub/6/6_2013.pdf 27 ____________________________________________________________________________ © Нефтегазовая геология. Теория и практика. – 2013. -Т.8. - №1. - http://www.ngtp.ru/rub ISSN 2070-5379 Neftegasovaâ geologiâ. Teoriâ i practika (RUS) URL: http://www.ngtp.ru Prognozno-mineragenicheskaya karta na uglevodorodnoe syr'e Sodruzhestva Nezavisimykh Gosudarstv i sopredel'nykh territoriy [Forecast-minerogenic map ofr hydrocarbons of Commonwealth of Independent States and adjacent areas]. Scale 1:5000 000. VSEGEI, VNIGNI, CIS institutes, 2012. 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[Large oil field with reserves of 1.8 billion barrels discovered in Afghanistan]. Available at: http://www.rusenergy.com/ru/ (16th Aug 2010). V Afganistane obnaruzheno krupnoe mestorozhdenie nefti s zapasami v 1,8 mlrd. bar. [Large oil field with reserves of 1.8 billion barrels discovered in Afghanistan]. Available at: http://www.rusenergy.com/ru/ (16th Aug 2010). V Afganistane obnaruzheno krupnoe mestorozhdenie nefti s zapasami v 1,8 mlrd. bar. [Large oil field with reserves of 1.8 billion barrels discovered in Afghanistan]. Available at: http://www.rusenergy.com/ru/ (16th Aug 2010). Zhmud M.S., Melikhov V.N. Obnovlennaya kontseptsiya regional'noy struktury i neftegazonosnosti Predkopetdagskogo progiba [The updated concept of the regional structure and petroleum potential of Predkopetdagsky trough]. Neft' i gaz Turkmenistana, 2000, no. 2, p. 10-28. Zhmud M.S., Melikhov V.N. 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Extracellular vesicle-derived DNA for performing EGFR genotyping of NSCLC patients
Molecular cancer
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© 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. * Correspondence: kyleemd@kuh.ac.kr 1Lung Cancer Center, Konkuk University Medical Center, Seoul, Republic of Korea 3Department of Pulmonary Medicine, Konkuk University School of Medicine, 120-1 Hwayang-dong, Gwangjin-Gu, Seoul 05030, Republic of Korea Full list of author information is available at the end of the article Abstract Tumor cells shed an abundance of extracellular vesicles (EVs) to body fluids containing bioactive molecules including DNA, RNA, and protein. Investigations in the field of tumor-derived EVs open a new horizon in understanding cancer biology and its potential as cancer biomarkers as well as platforms for personalized medicine. This study demonstrates that successfully isolated EVs from plasma and bronchoalveolar lavage fluid (BALF) of non-small cell lung cancer (NSCLC) patients contain DNA that can be used for EGFR genotyping through liquid biopsy. In both plasma and BALF samples, liquid biopsy results using EV DNA show higher accordance with conventional tissue biopsy compared to the liquid biopsy of cfDNA. Especially, liquid biopsy with BALF EV DNA is tissue-specific and extremely sensitive compared to using cfDNA. Furthermore, use of BALF EV DNA also demonstrates higher efficiency in comparison to tissue rebiopsy for detecting p.T790 M mutation in the patients who developed resistance to EGFR-TKIs. These finding demonstrate possibility of liquid biopsy using EV DNA potentially replacing the current diagnostic methods for more accurate, cheaper, and faster results. Keywords: Liquid biopsy, Bronchoalveolar lavage fluid, Extracellular vesicles, EGFR mutant DNA, Non-small cell lung cancer DNA (cfDNA) are used as supplement tests [3, 4]. The conventional tumor biopsy to assess mutation status can be problematic depending on the location and size of the tumor. Liquid biopsy, a noninvasive way to detect circulat- ing tumor DNA (ctDNA) in the blood, are proposed as an alternative way to detect, evaluate and monitor tumor-drug relation [3, 5]. The integration of liquid biopsy into cancer treatment depends on the precision of detecting ctDNA in blood samples, but plasma cfDNA only contains roughly 1% of ctDNA [6]. Therefore, even with high specificity reported in using ctDNA, varied sensitivity is a problem. For example, some studied reported relatively high sensitiv- ities ranging from 66% to 78%, while other studies resulted low sensitivities ranging from 28.8% to 46%. [7–10]. Extracellular vesicle-derived DNA for performing EGFR genotyping of NSCLC patients Jae Young Hur1,2, Hee Joung Kim1,3, Jong Sik Lee1, Chang-Min Choi4,5, Jae Cheol Lee5, Min Kyo Jung6, Chan Gi Pack6 and Kye Young Lee1,3* Hur et al. Molecular Cancer (2018) 17:15 DOI 10.1186/s12943-018-0772-6 Hur et al. Molecular Cancer (2018) 17:15 DOI 10.1186/s12943-018-0772-6 Open Access Visualization and characterization of EVs isolated from the BALF and plasma NSCLC patients p p Previous studies report that measurements of EV size varies according to the methods of the isolation and meas- urement, but EVs from human body fluids (plasma, urine) are generally found to be in 20~ 300 nm range [15–17]. This study identified the size of BALF EVs to be from 20 to 250 nm (Fig. 1a and Additional file 1: Figure S1) and the size of plasma EV to be smaller ranging from 5 to 15 nm (Fig. 1b and Additional file 1: Figure S2). Visualization of purified EV fraction showed round shape and heterogeneous in size, averaging in 106 nm (SD ± 34 nm) BALF EVs (Fig. 1c and Additional file 1: Figure S3). Image obtained from EM of plasma EVs showed other substances in the background, which are probably pro- teins, low-density lipoproteins (LDL), and high-density li- poproteins (HDL) that exist in plasma (Fig. 1d). Liquid biopsy using BALF EV DNA for EGFR mutation testing is sendant with tissue genotyping g yp g Although, the standard for non-invasive cancer diagnostics is detection of biomarkers circulating in blood, it remains a challenge due to abundant non-cellular contents. Our approach to overcome this problem is to analyze more im- mediate biofluids such as bronchoalveolar lavage fluid (BALF) of NSCLC patients. Proximal biofluids display component specificity and in some cases, they are in direct contact with the site of the disease. Bronchoalveolar wash- ing is not an entirely non-invasive procedure, but often ordered in individuals with and suspected of lung cancer during bronchoscopy. Results of immuno-electron microscopy (immune-EM) identified the presence of dsDNA in the intraluminal por- tion of EVs covered by a multilayered or single-layered membrane (Fig. 1e and Additional file 1: Figure S4). NanoDrop analysis showed that both EV DNA and cfDNA obtained from BALF had similar concentration and purity (Additional file 1: Table S2). Bioanalyzer analysis of dsDNA length showed that the size of both EV DNA and ctDNA are > 3 kb, but abundance of dsDNAs that are longer than 3 kb is greater in EV DNA compared to ctDNA (Fig. 1f, Additional file 1: Table S3 and Figure S5). We compared the sensitivity of EGFR mutation testing between BALF EV DNA and cfDNA by performing PCR. Results showed higher sensitivity of EGFR mutation testing when using EV DNA compared to using cfDNA. For example, a threshold cycle (Ct) value of EV DNA was 32, whereas that of cfDNA could not be determined (Fig. 1g and Additional file 1: Table S4). Higher quantity and quality of EV DNA compared to BALF cfDNA suggested a potential for clinical application of EV DNA for liquid biopsy. Best way to test the specificity and sensitivity of liquid biopsy done with BALF EV DNA is comparing the results with conventional tissue biopsy. Therefore, we tested 23 BALF samples from NSCLC patients with proven EGFR genotyping from tissue biopsy (9 EGFR wild type and 14 EGFR-mutated). EGFR genotyping using BALF EV DNA showed 100% accordance with tissue typing, while detection sensitivity using ctDNA only yielded 71.4% (Table 1). Test results of BALF samples were significantly higher compared to the result of plasma samples in both ctDNA and EV DNA, demonstrating that proximal biofluids better represents tumor status. In addition, as we have seen with the plasma sample, EV DNA resulted in higher specifi- city and sensitivity. Specifically, Kappa coefficient for EGFR Main text have demonstrated that the majority of DNA associated with tumor exosomes is double-stranded in various cancer cell-lines and highlight the translational value of exosomal DNA for its potential usefulness as a circulating biomarker for cancer detection [12]. Main text Lung cancer results in the largest number of cancer- related deaths worldwide and non-small-cell lung cancer (NSCLC) accounts for more than 85% of all lung cancer cases [1]. Most patients are diagnosed at an advanced stage due to lack of efficient diagnostic approaches and asymptomatic characteristic of the disease leading to a poor prognosis [1, 2]. Recent development of target specific drugs such as epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) have slightly improved survival rate, but easy and fast diagnostic assessment of mutation status is important for timely treatment of patients. At present, majority of EGFR genotyping is done through tissue biopsy while liquid biopsies using cell-free The main reason for this high variability of sensitivity ctDNA lies on the unstable nature of cfDNA in the sam- ples [7]. In contrast, DNA inside extracellular vesicle (EV) shed by tumor cells is well protected by dual lipid membranous coating and thus has inherent stability [5, 11, 12]. Along with abundant new discoveries in various Hur et al. Molecular Cancer (2018) 17:15 Page 2 of 6 Page 2 of 6 the outer environment by lipid bilayer structure of EV. We performed EGFR genotyping with 20 plasma samples of NSCLC patients. Comparison of the results with tissue biopsy results showed that plasma cfDNA only had 30% accordance with tissue typing. Even though detection sensitivity with plasma cfDNA was relatively low, we were able to improve detection sensitivity to 55% by using plasma EV DNA (Additional file 1: Table S5). This finding suggests that liquid biopsy using EV DNA is advantageous to conventional use of cfDNA. Recently, Allenson et al. reported that liquid biopsy of exo-DNA was superior to that of cfDNA for detecting mutant KRAS in plasma sam- ples of patients with pancreatic ductal adenocarcinoma [11]. However, isolation and purification of EVs from plasma is associated with some technical difficulties. Char- acteristics of lipoproteins, especially low-density lipopro- teins (LDLs), present in the plasma are very similar to those of EVs; moreover, contamination of isolated EVs samples with LDLs interferes with analysis [18]. Components similar to LDLs were identified in our study after isolating EVs from plasma (Fig. 1d), which could explain the relatively low sensitivity of liquid biopsy when using plasma EV DNA. tumor-derived EVs and EV-derived DNA (EV DNA), they have great potential as cancer biomarkers as well as platforms for personalized medicine [12–14]. For example, Thakur BK, et al. Liquid biopsy of plasma EV DNA resulted in higher accordance rate compared to liquid biopsy of plasma cfDNA BALF was ultracentrifuged to obtain pallets and remove cells and debris, which was resuspended in 200 μl PBS. Sizes of purified EVs were determined using Zetasizer Nano ZS. Average size distribution from six separate experiments is plotted in percentage distribution according to their size. All six distributions are shown in Additional file 1: Figure S1. b. Size distribution of plasma EV. Plasma was ultracentrifuged to obtain pallets and remove cells and debris, which was resuspended in 200 μl PBS. Sizes of purified EVs were determined using Zetasizer Nano ZS. Average size distribution from three separate experiments is plotted in percentage distribution according to their size. All three distributions are shown in Additional file 1: Figure S2. c. EM image of BALF EVs. Samples for EM analysis were negatively stained. The size bar in the EM image indicates 100 nm. d. EM image of plssma EVs. Samples for EM analysis were negatively stained. Plasma EVs indicated by red arrows. The size bar in the EM image indicates 100 nm. e. Detection of dsDNA in BALF EVs by performing immuno-EM. DsDNA was labeled with a mouse monoclonal antibody and colloidal gold-conjugated secondary antibodies. The solid black dots indicate DNA (indicated by red arrows). f. Gel-like images show the size and amount of EV DNA and cfDNA determined using the bioanalyzer. First lane shows the standard size ladder distribution, and numbers on the left indicate corresponding sizes. The second and third lanes show the size and amount of EV DNA and cfDNA, respectively. g. Amplification curve obtained by performing real-time PCR. Exon 19 deletion in EGFR was determined by performing peptide nucleic acid (PNA)-mediated PCR clamping. Both EV DNA and cfDNA were extracted from 1 ml BALF, and 70 ng EV DNA and cfDNA were used for performing PCR turn-around time, which usually takes two or three weeks for EGFR genotyping using tissue biopsy. genotyping by using tumor tissue sample and BALF EV DNA was 1.0 (p < 0.01), which was higher than that for EGFR genotyping by using tumor tissue sam- ple and BALF ctDNA (kappa = 0.705). Our results of liquid biopsy using BALF EV DNA was compatible enough with tissue genotyping that it can replace tis- sue biopsy when obtaining tissue sample is difficult. Additionally, liquid biopsy could significantly reduce Liquid biopsy of plasma EV DNA resulted in higher accordance rate compared to liquid biopsy of plasma cfDNA At present, blood plasma DNA samples are widely used for liquid biopsies and the major concern is the instability of the DNA from the blood sample, which leads to decreased sensitivity. Presumably, using plasma EV DNA instead of cfDNA could increase sensitivity as they are shielded from Hur et al. Molecular Cancer (2018) 17:15 Page 3 of 6 Fig. 1 Characterization of BALF and plasma EV DNA. a. Size distribution of BALF EV. BALF was ultracentrifuged to obtain pallets and remove cells and debris, which was resuspended in 200 μl PBS. Sizes of purified EVs were determined using Zetasizer Nano ZS. Average size distribution from six separate experiments is plotted in percentage distribution according to their size. All six distributions are shown in Additional file 1: Figure S1. b. Size distribution of plasma EV. Plasma was ultracentrifuged to obtain pallets and remove cells and debris, which was resuspended in 200 μl PBS. Sizes of purified EVs were determined using Zetasizer Nano ZS. Average size distribution from three separate experiments is plotted in percentage distribution according to their size. All three distributions are shown in Additional file 1: Figure S2. c. EM image of BALF EVs. Samples for EM analysis were negatively stained. The size bar in the EM image indicates 100 nm. d. EM image of plssma EVs. Samples for EM analysis were negatively stained. Plasma EVs indicated by red arrows. The size bar in the EM image indicates 100 nm. e. Detection of dsDNA in BALF EVs by performing immuno-EM. DsDNA was labeled with a mouse monoclonal antibody and colloidal gold-conjugated secondary antibodies. The solid black dots indicate DNA (indicated by red arrows). f. Gel-like images show the size and amount of EV DNA and cfDNA determined using the bioanalyzer. First lane shows the standard size ladder distribution, and numbers on the left indicate corresponding sizes. The second and third lanes show the size and amount of EV DNA and cfDNA, respectively. g. Amplification curve obtained by performing real-time PCR. Exon 19 deletion in EGFR was determined by performing peptide nucleic acid (PNA)-mediated PCR clamping. Both EV DNA and cfDNA were extracted from 1 ml BALF, and 70 ng EV DNA and cfDNA were used for performing PCR Fig. 1 Characterization of BALF and plasma EV DNA. a. Size distribution of BALF EV. Genotyping using BALF EV DNA is highly promising for p.T790 M detection for acquired resistance patients Hence, precise and swift identification of sec- ondary mutations, p.T790 M, that represent acquired re- sistance is important for planning future treatment. We extended liquid biopsy using BALF to the patients who developed acquired resistance to EGFR-TKIs and need rebiopsy to further test specificity and sensitivity. Nine patients who developed resistance to TKIs were exam- ined for the p.T790 M mutation by performing conven- tional tissue rebiopsy and liquid biopsy with BALF samples. Of these nine patients, suitable cancer cells to perform tissue rebiopsy were obtained from only six pa- tients, while adequate tissues were unobtainable from three patients. Two patients out of six patients were identified to have the p.T790 M mutation from tissue rebiopsy (Fig. 2a and Additional file 1: Table S6). p.L858R acquire resistance to the drug after prolonged treatment. Hence, precise and swift identification of sec- ondary mutations, p.T790 M, that represent acquired re- sistance is important for planning future treatment. We extended liquid biopsy using BALF to the patients who developed acquired resistance to EGFR-TKIs and need rebiopsy to further test specificity and sensitivity. Nine patients who developed resistance to TKIs were exam- ined for the p.T790 M mutation by performing conven- tional tissue rebiopsy and liquid biopsy with BALF samples. Of these nine patients, suitable cancer cells to perform tissue rebiopsy were obtained from only six pa- tients, while adequate tissues were unobtainable from three patients. Two patients out of six patients were identified to have the p.T790 M mutation from tissue rebiopsy (Fig. 2a and Additional file 1: Table S6). Fig. 2 Detection of EGFR mutation by using BALF EV DNA. a. Comparison of EGFR genotyping results performed using tumor tissue, BALF cfDNA, and BALF EV DNA. The top lane indicates EGFR mutation status in the primary tumor tissue of each patient. The second lane shows the result of biopsy of tumor tissues obtained from patients with acquired resistance to EGFR-TKIs. The third and fourth lanes show EGFR mutation status determined by performing liquid biopsy of BALF cfDNA and EV DNA, respectively. Rates of p.T790 M mutation detection by performing tissue rebiopsy and liquid biopsy of BALF cfDNA and BALF EV DNA are shown on the right side of each lane. Abbreviations; E19 del: exon 19 deletion, WT: wild type. b. Diagram showing the contents of an EV. Tumor-derived EVs contain RNA; DNA, including mutant DNA; and proteins Fig. Genotyping using BALF EV DNA is highly promising for p.T790 M detection for acquired resistance patients Unfortunately, most patients prescribed with TKI after identification of mutations such as Exon 19 deletion and Page 4 of 6 Hur et al. Molecular Cancer (2018) 17:15 Page 4 of 6 Table 1 Comparison of the EGFR mutation status between tumor tissue and BALF in EGFR-TKIs naïve patients EGFR genotype Tissue BALF (n = 23) EV DNA cfDNA Mutant type Wild type Mutant type Wild type Mutant type 14 (60.9%) 14 (60.9%) 0 10 (43.5%) 0 Wild type 9 (39.1%) 0 9 (39.1%) 4 (17.4%) 9 (39.1%) Sensitivity(%) (95% CI) 100.0% (85.7–100) 71.4% (51.0–85.7) Specificity (%) (95% CI) 100.0% (85.7–100) 100.0% (85.7–100) Abbreviations: CI Confidence interva Table 1 Comparison of the EGFR mutation status between tumor tissue and BALF in EGFR-TKIs naïve patients When we performed liquid biopsy of BALF cfDNA three patients were identified to have p.T790 M mu- tation including two patients who yielded positive re- sults from the tissue rebiopsy as well as one patient who were not identified by the tissue rebiopsy (Fig. 2a and Additional file 1: Table S6). Sensitivity of liquid biopsy increases even further when tested with BALF EV DNA with total of five patients identified to have p.T790 M mutation including two patients who yielded positive results from the tissue rebiopsy as well as three patients who were not identified by the tissue rebiopsy. Three additionally identified patients consists of two patients who could not provide ad- equate tissue sample for performing tissue rebiopsy and one patient who yielded negative result for p.T790 M mutation from tissue rebiopsy (Fig. 2a and Additional file 1: Table S6). Furthermore, two newly identified patients showed partial response to subse- quent osimertinib treatment (Additional file 1: Table S6). We demonstrated for the first time that EVs iso- lated from BALF of NSCLC patients carry genomic dsDNA and specific mutant EGFR DNA inside the double layered membranous vesicles (Fig. 2b). Clinical usefulness of genotyping using BALF EV DNA was more prominent in the matter of detecting p.T790 M mutation for the prescription of the 3rd generation EGFR-TKIs such as osimertinib [19]. Although the present study did not include a large sample size, preliminary findings of this study suggest that liquid biopsy of BALF EV DNA can overcome limitations associated with tissue rebiopsy, which is widely per- formed for detecting p.T790 M mutation. Abbreviations: CI Confidence interva p.L858R acquire resistance to the drug after prolonged treatment. Conclusions Competing interests p g The authors declare that they have no competing interests. Our results show that EGFR mutation detection in NSCLC patients is possible through EGFR genotyping of EVs present in plasma and BALF. Liquid biopsy of BALF EV DNA is non-invasive, simple and faster testing method that is also high in accuracy and even surpass detection sensitivity compared to tissue biopsy. Sensitivity is shown to be especially high in acquired resistance patients. This study revealed a novel liquid biopsy method of using EV DNA for EGFR genotyping. It has demonstrated potential to serve as a diagnostic and prognostic method in NSCLC patients. Author details 1 1Lung Cancer Center, Konkuk University Medical Center, Seoul, Republic of Korea. 2Department of Pathology, Konkuk University Medical Center, Seoul, Republic of Korea. 3Department of Pulmonary Medicine, Konkuk University School of Medicine, 120-1 Hwayang-dong, Gwangjin-Gu, Seoul 05030, Republic of Korea. 4Department of Pulmonary and Critical Care Medicine, University of Ulsan, College of Medicine, Asan Medical Center, Seoul, Republic of Korea. 5Department of Oncology, University of Ulsan, College of Medicine, Asan Medical Center, Seoul, Republic of Korea. 6Department of Convergence Medicine, University of Ulsan, College of Medicine & Asan Institute for Life Sciences, Asan Medical Center, Seoul, Republic of Korea. Genotyping using BALF EV DNA is highly promising for p.T790 M detection for acquired resistance patients 2 Detection of EGFR mutation by using BALF EV DNA. a. Comparison of EGFR genotyping results performed using tumor tissue, BALF cfDNA, and BALF EV DNA. The top lane indicates EGFR mutation status in the primary tumor tissue of each patient. The second lane shows the result of biopsy of tumor tissues obtained from patients with acquired resistance to EGFR-TKIs. The third and fourth lanes show EGFR mutation status determined by performing liquid biopsy of BALF cfDNA and EV DNA, respectively. Rates of p.T790 M mutation detection by performing tissue rebiopsy and liquid biopsy of BALF cfDNA and BALF EV DNA are shown on the right side of each lane. Abbreviations; E19 del: exon 19 deletion, WT: wild type. b. Diagram showing the contents of an EV. Tumor-derived EVs contain RNA; DNA, including mutant DNA; and proteins Fig. 2 Detection of EGFR mutation by using BALF EV DNA. a. Comparison of EGFR genotyping results performed using tumor tissue, BALF cfDNA, and BALF EV DNA. The top lane indicates EGFR mutation status in the primary tumor tissue of each patient. The second lane shows the result of biopsy of tumor tissues obtained from patients with acquired resistance to EGFR-TKIs. The third and fourth lanes show EGFR mutation status determined by performing liquid biopsy of BALF cfDNA and EV DNA, respectively. Rates of p.T790 M mutation detection by performing tissue rebiopsy and liquid biopsy of BALF cfDNA and BALF EV DNA are shown on the right side of each lane. Abbreviations; E19 del: exon 19 deletion, WT: wild type. b. Diagram showing the contents of an EV. Tumor-derived EVs contain RNA; DNA, including mutant DNA; and proteins Hur et al. Molecular Cancer (2018) 17:15 Page 5 of 6 Page 5 of 6 Abbreviations BALF B h l BALF: Bronchoalveolar lavage fluid; cfDNA: Cell-free DNA; ctDNA: Circulating tumor DNA; DLS: Dynamic light scattering; dsDNA: Double-stranded DNA; EGFR-TKIs: Epidermal growth factor receptor-tyrosine kinase inhibitors; EV DNA: Extracellular vesicle-derived DNA; EV: Extracellular vesicle; HDL: High- density lipoproteins; LDL: Low-density lipoproteins; NSCLC: Non-small cell lung cancer; PNA: Peptide nucleic acid; TEM: Transmission electron microscopy 6. Schwarzenbach H, Hoon DS, Pantel K. Cell-free nucleic acids as biomarkers in cancer patients. Nat Rev Cancer. 2011;11:426–37. 7. Diaz LA Jr, Bardelli A. Liquid biopsies: genotyping circulating tumor DNA. J Clin Oncol. 2014;32:579–86. 7. Diaz LA Jr, Bardelli A. Liquid biopsies: genotyping circulating tumor DNA. J Clin Oncol. 2014;32:579–86. 8. Douillard JY, Ostoros G, Cobo M, Ciuleanu T, Cole R, McWalter G, Walker J, Dearden S, Webster A, Milenkova T, McCormack R. Gefitinib treatment in EGFR mutated caucasian NSCLC: circulating-free tumor DNA as a surrogate for determination of EGFR status. J Thorac Oncol. 2014;9:1345–53. Please contact the corresponding author for all data requests. 11. Allenson K, Castillo J, San Lucas FA, Scelo G, Kim DU, Bernard V, Davis G, Kumar T, Katz M, Overman MJ, et al. High prevalence of mutant KRAS in circulating exosome-derived DNA from early stage pancreatic cancer patients. Ann Oncol. 2017;28:741–7. Funding 10. Takahama T, Sakai K, Takeda M, Azuma K, Hida T, Hirabayashi M, Oguri T, Tanaka H, Ebi N, Sawa T, et al. Detection of the T790M mutation of EGFR in plasma of advanced non-small cell lung cancer patients with acquired resistance to tyrosine kinase inhibitors (West Japan oncology group 8014LTR study). Oncotarget. 2016;7:58492–9. g This research was conducted with the support from AstraZeneca Korea Ltd. (ISSIRES011). This research was conducted with the support from AstraZeneca Korea Ltd. (ISSIRES011). References k 1. Reck M, Rabe KF. Precision diagnosis and treatment for advanced non- small-cell lung cancer. N Engl J Med. 2017;377:849–61. 2. Huang WL, Chen YL, Yang SC, Ho CL, Wei F, Wong DT, Su WC, Lin CC. Liquid biopsy genotyping in lung cancer: ready for clinical utility? Oncotarget. 2017;8:18590–608. 3. Molina-Vila MA. Liquid biopsy in lung cancer: present and future. Transl Lung Cancer Res. 2016;5:452–4. 3. Molina-Vila MA. Liquid biopsy in lung cancer: present and future. Transl Lung Cancer Res. 2016;5:452–4. 4. Kohler C, Radpour R, Barekati Z, Asadollahi R, Bitzer J, Wight E, Burki N, Diesch C, Holzgreve W, Zhong XY. Levels of plasma circulating cell free nuclear and mitochondrial DNA as potential biomarkers for breast tumors. Mol Cancer. 2009;8:10 4. Kohler C, Radpour R, Barekati Z, Asadollahi R, Bitzer J, Wight E, Burki N, Diesch C, Holzgreve W, Zhong XY. Levels of plasma circulating cell free nuclear and mitochondrial DNA as potential biomarkers for breast tumors. Mol Cancer. 2009;8:10 5. Kahlert C, Melo SA, Protopopov A, Tang J, Seth S, Koch M, Zhang J, Weitz J, Chin L, Futreal A, Kalluri R. Identification of double-stranded genomic DNA spanning all chromosomes with mutated KRAS and p53 DNA in the serum exosomes of patients with pancreatic cancer. J Biol Chem. 2014;289:3869–75. 5. Kahlert C, Melo SA, Protopopov A, Tang J, Seth S, Koch M, Zhang J, Weitz J, Chin L, Futreal A, Kalluri R. Identification of double-stranded genomic DNA spanning all chromosomes with mutated KRAS and p53 DNA in the serum exosomes of patients with pancreatic cancer. J Biol Chem. 2014;289:3869–75. 6 Schwarzenbach H Hoon DS Pantel K Cell free nucleic acids as biomarkers Acknowledgments h k h l We thank the Electron Microscopy core facility at the ConveRgence mEDIcine research cenTer (CREDIT), Asan Medical Center for support and instrumentation. 9. Karachaliou N, Mayo-de las Casas C, Queralt C, de Aguirre I, Melloni B, Cardenal F, Garcia-Gomez R, Massuti B, Sanchez JM, Porta R, et al. Association of EGFR L858R mutation in circulating free DNA with survival in the EURTAC trial. JAMA Oncol. 2015;1:149–57. Additional file Additional file 1: Materials and methods. Table S1. Patient demographics and clinical characteristics. Table S2. Concentration and purity (260/280) of BALF EV DNA and BALF cfDNA. Table S3. Concentration of DNA larger than 1kb in EV DNA and cfDNA. Table S4. Ct value of EV DNA and cfDNA samples and their differences. Table S5. Comparison of the EGFR mutation status between tumor tissue and plasma in EGFR-TKIs naïve patients. Table S6. Clinical characteristics of patients who developed acquired resistance to 1st or 2nd generation EGFR-TKIs and underwent rebiopsy. Figure S1. Sizes of purified BALF EVs. Figure S2. Sizes of purified plasma EVs. Figure S3. EM image of BALF EVs. Figure S4. Immuno-EM images show detection of dsDNA in BALF EVs. Red arrows indicate gold particles. Figure S5. Gel-like images show the size and amount of EV DNA and cfDNA determined using the bioanalyzer. (DOCX 900 kb) Received: 8 December 2017 Accepted: 23 January 2018 Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Additional file 1: Materials and methods. Table S1. Patient demographics and clinical characteristics. Table S2. Concentration and purity (260/280) of BALF EV DNA and BALF cfDNA. Table S3. Concentration of DNA larger than 1kb in EV DNA and cfDNA. Table S4. Ct value of EV DNA and cfDNA samples and their differences. Table S5. Comparison of the EGFR mutation status between tumor tissue and plasma in EGFR-TKIs naïve patients. Table S6. Clinical characteristics of patients who developed acquired resistance to 1st or 2nd generation EGFR-TKIs and underwent rebiopsy. Figure S1. Sizes of purified BALF EVs. Figure S2. Sizes of purified plasma EVs. Figure S3. EM image of BALF EVs. Figure S4. Immuno-EM images show detection of dsDNA in BALF EVs. Red arrows indicate gold particles. Figure S5. Gel-like images show the size and amount of EV DNA and cfDNA determined using the bioanalyzer. (DOCX 900 kb) Authors’ contributions Interpreted data, wrote manuscript and acted as corresponding author: KYL; performed experiments and analysis of all samples, interpreted experimental data and wrote manuscript: JYH; interpreted clinical data and wrote manuscript: HJK; produced statistical data and additional file tables: JSL; helped to collect clinical samples and evaluate the manuscript: CMC, JCL; helped to obtain high-resolution electron microscopic images: MKJ, CKP. All authors read and approved the final manuscript. 12. Thakur BK, Zhang H, Becker A, Matei I, Huang Y, Costa-Silva B, Zheng Y, Hoshino A, Brazier H, Xiang J, et al. Double-stranded DNA in exosomes: a novel biomarker in cancer detection. Cell Res. 2014;24:766–9. 13. Li W, Li C, Zhou T, Liu X, Liu X, Li X, Chen D. Role of exosomal proteins in cancer diagnosis. Mol Cancer. 2017;16:145. 14. Balaj L, Lessard R, Dai L, Cho YJ, Pomeroy SL, Breakefield XO, Skog J. Tumour microvesicles contain retrotransposon elements and amplified oncogene sequences. Nat Commun. 2011;2:180. Ethics approval and consent to participate Ethics approval and consent to participate All human samples are collected under Institutional Review Committee (IRB). All human samples are collected under Institutional Review Committee (IRB). 15. Baranyai T, Herczeg K, Onodi Z, Voszka I, Modos K, Marton N, Nagy G, Mager I, Wood MJ, El Andaloussi S, et al. Isolation of Exosomes from blood plasma: qualitative and quantitative comparison of ultracentrifugation and size exclusion chromatography methods. PLoS One. 2015;10:e0145686. 15. Baranyai T, Herczeg K, Onodi Z, Voszka I, Modos K, Marton N, Nagy G, Mager I, Wood MJ, El Andaloussi S, et al. Isolation of Exosomes from blood plasma: qualitative and quantitative comparison of ultracentrifugation and size exclusion chromatography methods. PLoS One. 2015;10:e0145686. 16. Lobb RJ, Becker M, Wen SW, Wong CS, Wiegmans AP, Leimgruber A, Moller A. Optimized exosome isolation protocol for cell culture supernatant and human plasma. J Extracell Vesicles. 2015;4:27031. 16. Lobb RJ, Becker M, Wen SW, Wong CS, Wiegmans AP, Leimgruber A, Moller A. Optimized exosome isolation protocol for cell culture supernatant and human plasma. J Extracell Vesicles. 2015;4:27031. 17. Royo F, Zuniga-Garcia P, Sanchez-Mosquera P, Egia A, Perez A, Loizaga A, Arceo R, Lacasa I, Rabade A, Arrieta E, et al. Different EV enrichment methods suitable for clinical settings yield different subpopulations of urinary extracellular vesicles from human samples. J Extracell Vesicles. 2016;5:29497. 18. Sodar BW, Kittel A, Paloczi K, Vukman KV, Osteikoetxea X, Szabo-Taylor K, Nemeth A, Sperlagh B, Baranyai T, Giricz Z, et al. Low-density lipoprotein mimics blood plasma-derived exosomes and microvesicles during isolation and detection. Sci Rep. 2016;6:24316. 19. Greig SL. Osimertinib: first global approval. Drugs. 2016;76:263–73. Hur et al. Molecular Cancer (2018) 17:15 19. Greig SL. Osimertinib: first global approval. Drugs. 2016;76:263–73. 17. Royo F, Zuniga-Garcia P, Sanchez-Mosquera P, Egia A, Perez A, Loizaga A, Arceo R, Lacasa I, Rabade A, Arrieta E, et al. Different EV enrichment methods suitable for clinical settings yield different subpopulations of urinary extracellular vesicles from human samples. J Extracell Vesicles. 2016;5:29497. 18. Sodar BW, Kittel A, Paloczi K, Vukman KV, Osteikoetxea X, Szabo-Taylor K, Nemeth A, Sperlagh B, Baranyai T, Giricz Z, et al. Low-density lipoprotein mimics blood plasma-derived exosomes and microvesicles during isolation and detection. Sci Rep. 2016;6:24316. Consent for publication Not applicable. Consent for publication Not applicable. Page 6 of 6 Hur et al. 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Genes and Aging: From Bench Side to Bed side
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EDITED BY : Wael M. Y. Mohamed, Chenju Yi, Lilach Soreq and Toshihide Yamashita PUBLISHED IN : Frontiers in Aging Neuroscience GENES AND AGING: FROM BENCH SIDE TO BED SIDE EDITED BY : Wael M. Y. Mohamed, Chenju Yi, Lilach Soreq and Toshihide Yamashita PUBLISHED IN : Frontiers in Aging Neuroscience GENES AND AGING: FROM BENCH SIDE TO BED SIDE EDITED BY : Wael M. Y. Mohamed, Chenju Yi, Lilach Soreq and Toshihide Yamashita PUBLISHED IN : Frontiers in Aging Neuroscience GENES AND AGING: FROM BENCH SIDE TO BED SIDE Frontiers eBook Copyright Statement The copyright in the text of individual articles in this eBook is the property of their respective authors or their respective institutions or funders. The copyright in graphics and images within each article may be subject to copyright of other parties. In both cases this is subject to a license granted to Frontiers. The compilation of articles constituting this eBook is the property of Frontiers. 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By applying the most advanced information technologies, Frontiers is catapulting scholarly publishing into a new generation. Frontiers revolutionizes research publishing by freely delivering the most outstanding research, evaluated with no bias from both the academic and social point of view. By applying the most advanced information technologies, Frontiers is catapulting scholarly publishing into a new generation. Copyright and source acknowledgement notices may not be removed and must be displayed in any copy, derivative work or partial copy which includes the elements in question. About Frontiers Frontiers eBook Copyright Statement What are Frontiers Research Topics? All copyright, and all rights therein, are protected by national and international copyright laws. The above represents a summary only. For further information please read Frontiers’ Conditions for Website Use and Copyright Statement, and the applicable CC-BY licence. Frontiers Research Topics are very popular trademarks of the Frontiers Journals Series: they are collections of at least ten articles, all centered on a particular subject. With their unique mix of varied contributions from Original Research to Review Articles, Frontiers Research Topics unify the most influential researchers, the latest key findings and historical advances in a hot research area! Find out more on how to host your own Frontiers Research Topic or contribute to one as an author by contacting the Frontiers Editorial Office: frontiersin.org/about/contact ISSN 1664-8714 ISSN 1664-8714 ISBN 978-2-88974-974-4 DOI 10.3389/978-2-88974-974-4 ISBN 978-2-88974-974-4 DOI 10.3389/978-2-88974-974-4 April 2022  |  Genes and Aging: From Bench-to-Bedside Frontiers in Aging Neuroscience Citation: Mohamed, W. M. Y., Yi, C., Soreq, L., Yamashita, T., eds. (2022). Genes and Aging: From Bench Side to Bed Side. Lausanne: Frontiers Media SA. doi: 10.3389/978-2-88974-974-4 April 2022  |  Genes and Aging: From Bench-to-Bedside Frontiers in Aging Neuroscience GENES AND AGING: FROM BENCH SIDE TO BED SIDE Topic Editors: Wael M. Y. Mohamed, International Islamic University Malaysia, Malaysia Chenju Yi, Sun Yat-sen University, China Lilach Soreq, University College London, United Kingdom Toshihide Yamashita, Osaka University, Japan Topic Editors: Wael M. Y. Mohamed, International Islamic University Malaysia, Malaysia Chenju Yi, Sun Yat-sen University, China Lilach Soreq, University College London, United Kingdom Toshihide Yamashita, Osaka University, Japan Citation: Mohamed, W. M. Y., Yi, C., Soreq, L., Yamashita, T., eds. (2022). Genes and Aging: From Bench Side to Bed Side. Lausanne: Frontiers Media SA. doi: 10.3389/978-2-88974-974-4 Citation: Mohamed, W. M. Y., Yi, C., Soreq, L., Yamashita, T., eds. (2022). Genes and Aging: From Bench Side to Bed Side. Lausanne: Frontiers Media SA. doi: 10.3389/978-2-88974-974-4 April 2022  |  Genes and Aging: From Bench-to-Bedside Frontiers in Aging Neuroscience April 2022  |  Genes and Aging: From Bench-to-Bedside Keywords: genes, aging, neurodegeneration, dementia, AD Keywords: genes, aging, neurodegeneration, dementia, AD Keywords: genes, aging, neurodegeneration, dementia, AD Genes and Aging: From Bench-to-Bedside Aging is inevitable, and several structural and functional changes occur in the body with advanced aging. Aging affects the brain in a variety of ways, the most obvious of which are changes in motor, sensory, and cognitive function (Jeromin and Bowser, 2017; Ibrahim et al., 2020). Aging has becoming a remarkable public health issue worldwide: by 2050, the proportion of the world’s population over 60 years will reach 22%. This increase in the number of populations aging calls for attention to aging-associated diseases, including Neurodegenerative disorders such as Alzheimer’s Disease (AD) and Parkinson’s Disease (PD) (Gazzaley et al., 2005). In the brain, different cell types, including neurons, astrocytes, oligodendrocytes, as well as microglia, respond to aging differently (von Bartheld et al., 2016; Chapman and Hill, 2020). Studying the transcriptome changes in different cell types during aging, and identification of cell type-specific aging markers would enable us to move the therapeutic window to earlier stages of aging-associated disease progression/premature aging. In addition, understanding the mechanism of how cell-type specific aging genes are involved in the aging process could further shed light on the prevention and therapy of the aging-related diseases (Soreq et al., 2017, 2021). Table of Contents 04 Editorial: Genes and Aging: From Bench-to-Bedside Wael M. Y. Mohamed, Chenju Yi, Lilach Soreq and Toshihide Yamashita 06 Case Report: A Homozygous Mutation (p.Y62X) of Phospholipase D3 May Lead to a New Leukoencephalopathy Syndrome Yi-Hui Liu, Hai-Feng Zhang, Jie-Yuan Jin, Yan-Qiu Wei, Chen-Yu Wang, Liang-Liang Fan and Lv Liu 12 The Frequency of Genetic Mutations Associated With Behavioral Variant Frontotemporal Dementia in Chinese Han Patients Li Liu, Bo Cui, Min Chu, Yue Cui, Donglai Jing, Dan Li, Kexin Xie, Yu Kong, Tianxinyu Xia, Chaodong Wang and Liyong Wu 20 MicroRNAs as Potential Orchestrators of Alzheimer’s Disease-Related Pathologies: Insights on Current Status and Future Possibilities Nermeen Z. Abuelezz, Fayza Eid Nasr, Mohammad Ahmed AbdulKader, Ahmad R. Bassiouny and Amira Zaky 46 Analysis of Genotype-Phenotype Correlations in Patients With Degenerative Dementia Through the Whole Exome Sequencing Lin Sun, Jianye Zhang, Ning Su, Shaowei Zhang, Feng Yan, Xiang Lin, Jie Yu, Wei Li, Xia Li and Shifu Xiao 56 Replenishing the Aged Brains: Targeting Oligodendrocytes and Myelination? Xi Zhang, Nanxin Huang, Lan Xiao, Fei Wang and Tao Li 67 Molecular Insight Into the Therapeutic Potential of Long Non-coding RNA-Associated Competing Endogenous RNA Axes in Alzheimer’s Disease: A Systematic Scoping Review Hani Sabaie, Nazanin Amirinejad, Mohammad Reza Asadi, Abbas Jalaiei, Yousef Daneshmandpour, Omidvar Rezaei, Mohammad Taheri and Maryam Rezazadeh 82 82 Does Chronic Sleep Fragmentation Lead to Alzheimer’s Disease in Young Wild-Type Mice? Li Ba, Lifang Huang, Ziyu He, Saiyue Deng, Yi Xie, Min Zhang, Cornelius Jacob, Emanuele Antonecchia, Yuqing Liu, Wenchang Xiao, Qingguo Xie, Zhili Huang, Chenju Yi, Nicola D’Ascenzo and Fengfei Ding 97 Associations of Alcohol Dehydrogenase and Aldehyde Dehydrogenase Polymorphism With Cognitive Impairment Among the Oldest-Old in China Xurui Jin, Tingxi Long, Huashuai Chen, Yi Zeng, Xian Zhang, Lijing Yan and Chenkai Wu 104 Alzheimer’s Disease: An Update and Insights Into Pathophysiology Murtala Bello Abubakar, Kamaldeen Olalekan Sanusi, Azizah Ugusman, Wael Mohamed, Haziq Kamal, Nurul Husna Ibrahim, Ching Soong Khoo and Jaya Kumar April 2022  |  Genes and Aging: From Bench-to-Bedside Frontiers in Aging Neuroscience EDITORIAL published: 30 March 2022 doi: 10.3389/fnagi.2022.886967 Edited and reviewed by: Allison B. Reiss, Edited and reviewed by: Allison B. Reiss, The present article collection aims at giving a reader the most up-to-date perspective on how the interaction between genes and neuropsychological processes leads to neurological disorders in normal aging and minor and major neurocognitive disorders. It is intended to provide an opportunity for researchers of different perspectives to discuss recent progress in this field. This volume aimed to provide an overview of contributions from basic, and clinical aspects to understand the interplay between genes and aging from Bench-to-Bedside. The authors from different research fields identified numerous existing and emerging genes/aging related topics that provide new and unique perspectives. New York University, United States New York University, United States *Correspondence: Wael M. Y. Mohamed wmy107@gmail.com Specialty section: This article was submitted to Alzheimer’s Disease and Related Dementias, a section of the journal Frontiers in Aging Neuroscience Received: 01 March 2022 Accepted: 14 March 2022 Published: 30 March 2022 Specialty section: This article was submitted to Alzheimer’s Disease and Related Dementias, a section of the journal Frontiers in Aging Neuroscience Received: 01 March 2022 Accepted: 14 March 2022 Published: 30 March 2022 Specialty section: This article was submitted to Alzheimer’s Disease and Related Dementias, a section of the journal Frontiers in Aging Neuroscience Received: 01 March 2022 Accepted: 14 March 2022 Published: 30 March 2022 In this volume, several articles focus on Alzheimer’s Disease. For instance, a review by Abubakar et al. discussed the updates on pathophysiology of Alzheimer’s Disease that introduces a better understanding of novel signaling pathways associated with neural and glial mechanisms involved in AD, elaborates potential links between vascular dysfunction and AD, and recent developments in “omics”-based biomarkers in AD. Also, Sabaie et al. carried out a scoping review according to sic-stage methodology structure and PRISMA guideline to analyse validated loops of competing endogenous (ce)RNA in AD and focus on ceRNA axes associated with lncRNA with the therapeutic potential in AD. In parallel with this, Abuelezz et al. presented an updated analysis of miRNAs role in regulating signaling processes that are involved in AD-related pathologies. They discussed the current challenges against wider use of miRNAs and the future promising capabilities of miRNAs as diagnostic and therapeutic means for better management of AD. Further, this volume discussed AD dementia from different perspectives. Liu L et al. investigated the frequency of Chinese Han Edited and reviewed by: Allison B. Reiss, New York University, United States Edited and reviewed by: Allison B. Reiss, New York University, United States Wael M. Y. Mohamed 1,2*, Chenju Yi 3, Lilach Soreq 4 and Toshihide Yamashita 5 Wael M. Y. Mohamed 1,2*, Chenju Yi 3, Lilach Soreq 4 and Toshihide Yamashita 5 1 Department of Basic Medical Science, International Islamic University Malaysia, Selayang, Malaysia, 2 Department of Clinical Pharmacology, Menoufia University, Shebeen El-Kom, Egypt, 3 Seventh Affiliated Hospital, Sun Yat-sen University, Shenzhen, China, 4 UCL Institute of Neurology, University College London (UCL), London, United Kingdom, 5 Department of Molecular Neuroscience, Osaka University, Suita, Japan FUNDING LS was funded by Rose Trees UK. Citation: analyzed the correlations of genetic features with clinical symptoms in patients with degenerative dementia. They recruited a group of 84 Chinese dementia patients and conducted the whole exome sequencing (WES). The data were analyzed focusing on 153 dementia-related causing and susceptible genes. They concluded that the new variants in dementia-related genes indicated heterogeneity in pathogenesis and phenotype of degenerative dementia. WES could serve as an efficient diagnostic tool for detecting intractable dementia. It is worth mentioning that the current volume contains a basic study conducted by Ba et al. they modeled chronic sleep fragmentation (SF) in young wild-type mice and detected pathological hyperphosphorylated- tau (Ser396/Tau5) and gliosis in the SF hippocampus. In summary, 1.5-month sleep fragmentation could generate AD- like pathological changes including tauopathy and gliosis, mainly linked to stress, as the incremented glucose metabolism observed with PET imaging suggested. Thus SF could eventually lead to chronic neurodegeneration if the stress condition is prolonged in time. This is supported by Zhang et al. as they summarized present knowledge about dynamic changes of oligodendroglial lineage cells during normal aging and discussed their potential roles in age-related functional decline. Especially, they focused on declined myelinogenesis during aging and underlying mechanisms. Their article was clarifying those oligodendroglial (OLG) changes and their effects on neurofunctional decline may provide new insights in understanding aging associated brain. Jin et al. examined the associations of four single nucleotide polymorphisms (SNPs) on aldehyde dehydrogenase (ALDH) and alcohol dehydrogenase (ADH) genes (i.e., ALDH2 rs671, ADH1B rs1229984, ADH1B rs1042026, and ADH1C rs1693482) and cognitive impairment among the oldest-old. They examined data from the Chinese Longitudinal Healthy Longevity Survey genetic sub-study, including 1,949 participants aged over 90 years. They did not observe a significant interaction between those SNPs and alcohol consumption. Finally, Liu Y-H et al. discussed a case report leukoencephalopathy syndrome and they identified a novel homozygous mutation (NM_012268.3: c.186C>G/ p.Y62X) of PLD3 in a consanguineous family with white matter lesions, hearing and vision loss, and kidney disease by whole exome sequencing. This may be the first case report on the homozygous mutation of PLD3 in patients worldwide. Overall, this volume provides a rare opportunity to promote awareness and creativity for neurological disorders using animal models of different species, while stimulating testable theories and establishing a strategic research agenda to facilitate their integration into clinical studies. Citation: Mohamed WMY, Yi C, Soreq L and Yamashita T (2022) Editorial: Genes and Aging: From Bench-to-Bedside. Front. Aging Neurosci. 14:886967. doi: 10.3389/fnagi.2022.886967 March 2022 | Volume 14 | Article 886967 Frontiers in Aging Neuroscience | www.frontiersin.org 14 Editorial: Genes and Aging: From Bench-to-Bedside Mohamed et al. patients harboring genetic Frontotemproal Dementia (FTD) variants. They concluded that there was a high prevalence of genetic variants in Chinese bvFTD patients, highlighting the necessity of genetic testing for bvFTD. Similarly, Sun et al. analyzed the correlations of genetic features with clinical symptoms in patients with degenerative dementia. They recruited a group of 84 Chinese dementia patients and conducted the whole exome sequencing (WES). The data were analyzed focusing on 153 dementia-related causing and susceptible genes. They concluded that the new variants in dementia-related genes indicated heterogeneity in pathogenesis and phenotype of degenerative dementia. WES could serve as an efficient diagnostic tool for detecting intractable dementia. It is worth mentioning that the current volume contains a basic study conducted by Ba et al. they modeled chronic sleep fragmentation (SF) in young wild-type mice and detected pathological hyperphosphorylated- tau (Ser396/Tau5) and gliosis in the SF hippocampus. In summary, 1.5-month sleep fragmentation could generate AD- like pathological changes including tauopathy and gliosis, mainly linked to stress, as the incremented glucose metabolism observed with PET imaging suggested. Thus SF could eventually lead to chronic neurodegeneration if the stress condition is prolonged in time. This is supported by Zhang et al. as they summarized present knowledge about dynamic changes of oligodendroglial lineage cells during normal aging and discussed their potential roles in age-related functional decline. Especially, they focused on declined myelinogenesis during aging and underlying mechanisms. Their article was clarifying those oligodendroglial (OLG) changes and their effects on neurofunctional decline may provide new insights in understanding aging associated brain. Jin et al. examined the associations of four single nucleotide polymorphisms (SNPs) on aldehyde dehydrogenase (ALDH) and alcohol dehydrogenase (ADH) genes (i.e., ALDH2 rs671, ADH1B rs1229984, ADH1B rs1042026, and ADH1C rs1693482) and cognitive impairment among the oldest-old. They examined data from the Chinese Longitudinal Healthy Longevity Survey genetic sub-study, including 1,949 participants aged over 90 patients harboring genetic Frontotemproal Dementia (FTD) variants. They concluded that there was a high prevalence of genetic variants in Chinese bvFTD patients, highlighting the necessity of genetic testing for bvFTD. Similarly, Sun et al. AUTHOR CONTRIBUTIONS WM wrote the original draft and incorporated suggestions from the co-authors. LS edited the paper draft. The other co-authors edited the manuscript. All authors contributed to the article and approved the submitted version. ACKNOWLEDGMENTS We would like to thank our colleagues for dedicating their time, resources and energies to deliver creative and critical perspectives. Their important contributions have provided a comprehensive foundation for this theme of study. It is also a privilege to recognize and thank the editorial team for their expert help and support in creating and producing this volume. Finally, we wish to dedicate this book to all medical practitioners and researchers, with our utmost appreciation and deep respect. We are indebted to their sacrifices and to their unfailing spirit of hope that is our greatest source of inspiration. REFERENCES 150 years of cell counting. J. Comp. Neurol. 524, 3865–3895. doi: 10.1002/cne. 24040 Chapman, T. W., and Hill, R. A. (2020). Myelin plasticity in adulthood and aging. Neurosci. Lett. 715, 134645. doi: 10.1016/j.neulet.2019.134645 Conflict of Interest: 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. Gazzaley, A., Cooney, J. W., Rissman, J., and D’Esposito, M. (2005). Top-down suppression deficit underlies working memory impairment in normal aging. Nat. Neurosci. 8, 1298–1300. doi: 10.1038/nn1543 Ibrahim, N., Yahaya, M. F., Mohamed, W., Teoh, S. L., Hui, C. K., and Kumar, J. (2020). Pharmacotherapy of Alzheimer’s Disease: seeking clarity in a time of uncertainty. Front. Pharmacol. Neuropharmacol. 11, 261. doi: 10.3389/fphar.2020.00261 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. Jeromin, A., and Bowser, R. (2017). Biomarkers in neurodegenerative diseases. Adv. Neurobiol. 15, 491–528. doi: 10.1007/978-3-319-57193-5_20 Soreq, L., Hagai, B., Zvi, I., Hermona, S., and Wael, M. (2021). MicroRNA expression changes in Parkinson’s disease (PD) patients’ leukocytes prior to and following deep brain stimulation (DBS). Am. J Neurodegener. Dis. 10, 28–33. Copyright © 2022 Mohamed, Yi, Soreq and Yamashita. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Soreq, L., Rose, J., Soreq, E., Hardy, J., Trabzuni, D., Cookson, M. R., et al. (2017). Major shifts in glial regional identity are a transcriptional hallmark of human brain aging. Cell Rep. 18, 557–570. doi: 10.1016/j.celrep.2016.12.011 von Bartheld, C. S., Bahney, J., and Herculano-Houzel, S. (2016). TRANSPARENT PEER-REVIEW Editors and reviewers acknowledged by name on published articles REFERENCES The search for true numbers of neurons and glial cells in the human brain: a review of March 2022 | Volume 14 | Article 886967 Frontiers in Aging Neuroscience | www.frontiersin.org 25 Advantages of publishing in Frontiers OPEN ACCESS Articles are free to read for greatest visibility and readership EXTENSIVE PROMOTION Marketing and promotion of impactful research DIGITAL PUBLISHING Articles designed for optimal readership across devices LOOP RESEARCH NETWORK Our network increases your article’s readership Frontiers Avenue du Tribunal-Fédéral 34 1005 Lausanne | Switzerland Visit us: www.frontiersin.org Contact us: frontiersin.org/about/contact FAST PUBLICATION Around 90 days from submission to decision 90 IMPACT METRICS Advanced article metrics track visibility across digital media FOLLOW US @frontiersin TRANSPARENT PEER-REVIEW Editors and reviewers acknowledged by name on published articles HIGH QUALITY PEER-REVIEW Rigorous, collaborative, and constructive peer-review REPRODUCIBILITY OF RESEARCH Support open data and methods to enhance research reproducibility DIGITAL PUBLISHING Articles designed for optimal readership across devices
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Prevalência de internação hospitalar e fatores associados: um estudo de base populacional em um centro urbano no Sul do Brasil
Cadernos de Saúde Pública
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1 Programa de Pós-graduação em Saúde Coletiva, Universidade Luterana do Brasil, Canoas, Brasil. 217 ARTIGO ARTICLE 217 ARTIGO ARTICLE 217 ARTIGO ARTICLE Prevalência de internação hospitalar e fatores associados: um estudo de base populacional em um centro urbano no Sul do Brasil Prevalence and factors associated with hospital admissions in a population-based study in a southern Brazilian city Ligia Regina Garbinato 1 Jorge Umberto Béria 1 Andréia Cristina Leal Figueiredo 1 Beatriz Raymann 1 Luciana Petrucci Gigante 1 Lilian dos Santos Palazzo 1 Denise Rangel Ganzo de Castro Aerts 1 Hospital Services; Hospitalization; Stressful Events Cad. Saúde Pública, Rio de Janeiro, 23(1):217-224, jan, 2007 Correspondência J. U. Béria Programa de Pós-graduação em Saúde Coletiva, Universidade Luterana do Brasil. Av. Farroupilha 801, Canoas, RS 92420-280, Brasil. jberia@terra.com.br Abstract This cross-sectional study investigates the use of health services and associated factors in indi- viduals ≥ 14 years of age in Canoas, Rio Grande do Sul, Brazil. 1,954 persons were interviewed in 40 census tracts. One-year prevalence of hospital admissions was 9.4%. Adjusted data analysis showed that hospitalization was as- sociated with: age ≥ 60 years (RP = 4.14; 95% CI: 2.07-8.25), physician visit in the previous two months (RP = 2.79; 95%CI: 2.03-3.83), and ≥ 2 stressful life events (RP = 1.83; 95%CI: 1.19-2.80). Individual income of ≥ 2.10 times the prevailing minimum wage was associated with decreased likelihood of hospitalization (RP = 0.60; 95% CI: 0.41-0.87). Prevalence of hospital admissions was consistent with other studies. Higher preva- lence of hospitalization in lower-income groups may indicate decreased access to primary health care. Other possible factors are higher morbid- ity and severity of diseases among lower-income groups. Future research should focus on the re- lationship between morbidity and stressful life events. O estudo do padrão de utilização de serviços de saúde pela população é indispensável para o pla- nejamento e desenvolvimento de ações que bus- quem reduzir as desigualdades no adoecer e no acesso aos serviços de saúde 1,2. Correspondência J. U. Béria Programa de Pós-graduação em Saúde Coletiva, Universidade Luterana do Brasil. Av. Farroupilha 801, Canoas, RS 92420-280, Brasil. jberia@terra.com.br O tema utilização de serviços de saúde com- preende diferentes desfechos, incluindo con- sultas médicas curativas, preventivas e interna- ções. Tem-se mostrado crescente atenção para a assistência hospitalar devido ao papel funda- mental nos serviços de saúde e um custo muito elevado. Da mesma forma o entendimento dos fatores associados à utilização de serviços hos- pitalares é essencial para a discussão de políti- cas de saúde. Um estudo de base populacional em um cen- tro urbano no Sul do país, em 1992, encontrou uma prevalência de internações de 9% para a po- pulação adulta 3. Outro estudo que utilizou dados do ano de 1998 da Pesquisa Nacional de Amostra por Domicílios (PNAD) trouxe resultado inferior (6,9%), porém não teve restrições para idade 4. Neste último estudo também foram observados os fatores associados ao desfecho, demonstran- do uma maior utilização dos serviços hospitala- res por indivíduos do sexo feminino (8,7%), gru- po etário de oitenta anos ou mais (18,7%), com maiores necessidades em saúde, menor renda e baixa escolaridade (quando ajusta-se para os fa- tores facilitadores do acesso) 4. Abstract Hospital Services; Hospitalization; Stressful Events Hospital Services; Hospitalization; Stressful Events Cad. Saúde Pública, Rio de Janeiro, 23(1):217-224, jan, 2007 Garbinato LR et al. 218 Ga 218 218 Garbinato LR et al. O presente artigo fundamenta-se em um levantamento de base populacional, realizado em Canoas, Rio Grande do Sul, Brasil, e tem por objetivo estudar a prevalência de internações hospitalares na população de 14 anos ou mais em Canoas, no período de um ano, analisando também as associações deste evento com fatores demográficos, sócio-econômicos, utilização de serviços de saúde, eventos estressantes e percep- ção de saúde. ano; (2) variáveis independentes: demográficas (idade, sexo e situação conjugal), sócio-econô- micas (renda e escolaridade), de necessidade em saúde (auto-avaliação de saúde – utilizou-se três categorias: estado de saúde bom, regular e ruim), referentes aos serviços de saúde (ter ou não aces- so a seguro privado de saúde e realização de consulta médica nos últimos dois meses antes da entrevista) e eventos estressantes (separação conjugal, assalto/roubo, perda do emprego, abu- so sexual e maltrato físico). Os dados foram analisados pelo programa Stata 8.0 (Stata Corporation, College Station, Es- tados Unidos) utilizando-se procedimentos esta- tísticos padronizados. Material e método Este trabalho está integrado a um estudo mais amplo sobre a saúde da população da cidade de Canoas. Foi realizado um estudo transversal, com base populacional, no período de novem- bro de 2002 a junho de 2003. A população-alvo foi composta por residentes da zona urbana da cidade de Canoas, Região Metropolitana de Porto Alegre, Rio Grande do Sul, no Sul do Brasil. A análise estatística foi feita em três etapas: (1) análise descritiva (univariada), incluindo a freqüência de cada variável do estudo; (2) aná- lise bivariada, entre a variável dependente e as variáveis independentes, aplicação do teste qui-quadrado, resultando na determinação da razão de prevalência (RP) e respectivo intervalo de confiança de 95% para cada característica da população estudada; (3) análise multivariada, utilizou o método de regressão de Poisson in- cluindo somente as variáveis associadas ao des- fecho em um nível de significância menor ou igual a 0,2. Essa análise foi feita por níveis de acordo com o modelo de análise hierarquizado (Figura 1), embasado no estudo de Mendoza- Sassi & Béria 6. Quando da inclusão de novas variáveis, a cada nível, utilizou-se o método ba- ckward, permanecendo no modelo as variáveis associadas ao desfecho em um nível de signifi- cância menor que 0,05, no teste de razão de ve- rossimilhança. A análise foi ajustada para efeito de delineamento. A cidade de Canoas possui 391 setores cen- sitários. Destes, foram sorteados aleatoriamente quarenta setores. Foram visitados 26 domicílios por setor censitário. Em cada setor, a partir de um quarteirão previamente sorteado, foi escolhi- da também, de maneira aleatória, a esquina que seria o ponto de partida. A partir dessa esquina, à esquerda do entrevistador em frente da casa de número um, visitou-se a quarta casa, a oitava e a décima segunda casa, sistematicamente. Utilizou-se um questionário padronizado, pré-codificado e testado previamente, aplicado por duplas compostas de um estudante do cur- so de medicina e de uma fonoaudióloga, previa- mente submetidos a programa de treinamento e estudo piloto. Esta pesquisa foi aprovada pelo Comitê de Ética em Pesquisa em Seres Humanos e Animais da Universidade Luterana do Brasil, conforme termo de aprovação n. 2004/078H. Os questionários foram codificados e poste- riormente submetidos à dupla digitação. A en- trada e a edição dos dados foram efetuadas com o auxílio do Programa USCA (Utilização de Ser- viços em Canoas), especialmente desenvolvido para o estudo. Cad. Saúde Pública, Rio de Janeiro, 23(1):217-224, jan, 2007 Cad. Saúde Pública, Rio de Janeiro, 23(1):217-224, jan, 2007 Resultados O banco de dados resultante registrou 2.609 indivíduos de todas as faixas etárias, apresentan- do uma perda de 21%, especialmente entre os homens de 25 a 49 anos. A prevalência geral de internação encontrada na população, no período de um ano, foi de 9,4%. Pouco mais da metade dos indivíduos eram do sexo feminino (57,4%). As faixas etárias relativas aos intervalos de 20 a 39 anos e de 40 a 59 anos apresentaram maior proporção de indivíduos: 38,7% e 34,5%, respectivamente. Das pessoas en- trevistadas, 17,5% possuíam escolaridade de três anos ou menos, e, destas, 3,9% eram analfabe- tas. A renda individual de mais de dois salários mínimos (R$ 400,00 – salário mínimo nacional referente a maio de 2003) foi a mais freqüente (40,6%). O presente estudo selecionou como popu- lação-alvo indivíduos com idade de 14 anos ou mais, incluindo, portanto, um total de 1.954 pes- soas, com perda de 20,3%. Por tratar-se de um estudo satélite, o seu poder estatístico foi calcu- lado posteriormente com o programa Power 5, tendo-se obtido um resultado de 90% para todas as associações. As variáveis utilizadas no estudo foram: (1) variável dependente: hospitalização no último PREVALÊNCIA DE INTERNAÇÃO HOSPITALAR E FATORES ASSOCIADOS 219 Figura 1 Modelo de análise de utilização de serviços hospitalares. 3 Fatores de serviços (convênio/seguros, consulta e local da internação) 3 Necessidade em saúde (autopercepção de saúde) 2 Eventos estressantes (separação, assalto/roubo, perda do emprego, abuso sexual e maltrato físico) Internação hospitalar 1 Fatores sócio-econômicos (renda e escolaridade) 1 Fatores demográficos (idade, sexo e situação conjugal) Modelo de análise de utilização de serviços hospitalares. 1 Fatores sócio-econômicos (renda e escolaridade) 1 Fatores demográficos (idade, sexo e situação conjugal) Internação hospitalar A maioria (67,5%) não referia nenhum dos eventos estressantes estudados e considerava seu estado de saúde como bom (63,2%). venciaram dois ou mais eventos estressantes e os que referiram um estado de saúde ruim, tiveram uma prevalência de internação superior a 15%. A RP bruta encontrada para internação hos- pitalar e para os demais fatores em estudo está disposta na Tabela 1. Foi possível verificar que, entre os fatores demográficos, o sexo feminino apresentou 55% mais chances de internação quando comparado ao sexo masculino. E, como esperado, dentre as faixas etárias estudadas, a de sessenta anos ou mais demonstrou ter uma chance de internação 4,96 vezes mais que a faixa etária utilizada como referência (14-19 anos). Resultados Dentre os indivíduos amostrados, 44,6% infor- maram ter consultado o médico, ao menos uma vez, nos dois meses anteriores à entrevista e, des- tes, 15,1% também internaram no período de um ano. Entre os indivíduos que internaram, menos da metade (47%) o fizeram na cidade de Canoas, a maioria das internações ocorreu em Porto Ale- gre (49,7%) e apenas 3,3% em outras localidades. As prevalências de internação foram maiores entre indivíduos do sexo feminino (11,1%), em comparação com os do sexo masculino (7,1%). A faixa etária de sessenta anos ou mais apresentou a maior prevalência de internação (17,2%) e a de 14-19 anos, a menor (3,5%). Os indivíduos que vi- Dentre os indivíduos amostrados, 44,6% infor- maram ter consultado o médico, ao menos uma vez, nos dois meses anteriores à entrevista e, des- tes, 15,1% também internaram no período de um ano. Entre os indivíduos que internaram, menos da metade (47%) o fizeram na cidade de Canoas, a maioria das internações ocorreu em Porto Ale- gre (49,7%) e apenas 3,3% em outras localidades. As prevalências de internação foram maiores entre indivíduos do sexo feminino (11,1%), em comparação com os do sexo masculino (7,1%). A faixa etária de sessenta anos ou mais apresentou a maior prevalência de internação (17,2%) e a de 14-19 anos, a menor (3,5%). Os indivíduos que vi- Com relação aos fatores sócio-econômicos, verificou-se que um maior número de anos de estudo (oito anos ou mais) foi um fator protetor para internação (RP = 0,56). Indivíduos com ren- Cad. Saúde Pública, Rio de Janeiro, 23(1):217-224, jan, 2007 Garbinato LR et al. 220 Tabela 1 Distribuição de internação hospitalar segundo fatores demográfi cos, sócio-econômicos, eventos estressantes e auto-avaliação de saúde. Canoas, Rio Grande do Sul, Brasil, 2003. Cad. Saúde Pública, Rio de Janeiro, 23(1):217-224, jan, 2007 Resultados Internação Características n % RP bruta IC95% p Sexo 0,003 Masculino 124 11,1 1,00 Feminino 59 7,1 1,55 1,16-2,09 Situação conjugal 0,253 Com companheiro 118 10,0 1,00 Sem companheiro 63 8,5 0,84 0,63-1,12 Faixa etária (anos) 0,0001 14-19 9 3,5 1,00 20-39 71 9,4 2,72 1,37-5,37 40-59 58 8,6 2,48 1,25-4,90 60 ou mais 45 17,2 4,96 2,47-9,93 Analfabeto 0,252 Não 10 13,2 1,00 Sim 173 9,2 1,42 0,78-2,59 Escolaridade (anos completos) 0,013 11 ou mais 46 13,5 0,56 0,38-0,83 8-10 60 9,9 0,56 0,36-0,87 4-7 30 7,6 0,73 0,51-1,04 3 ou menos 47 7,7 1,00 Renda individual (salários mínimos) * 0,0058 2,10 ou mais 63 9,7 0,74 0,52-1,05 0,76-2,00 63 12,5 1,28 0,93-1,77 0,00-0,75 57 7,2 1,00 Convênio 0,359 Sim 98 8,8 1,00 Não 83 10,0 1,13 0,86-1,50 Eventos estressantes ** 0,0163 Nenhum 106 8,4 1,00 Um 49 10,5 1,24 0,89-1,73 Dois ou mais 22 15,5 1,84 1,21-2,80 Estado de saúde auto-avaliado 0,003 Bom 95 7,7 1,00 Regular 73 11,7 1,51 1,13-2,01 Ruim 14 15,4 1,99 1,17-3,39 Consulta *** 0,0001 Não 131 15,1 1,00 Sim 51 4,7 3,19 2,34-4,36 * R$ 200,00; ** Assalto, separação, perda de emprego, maltrato físico, abuso sexual; *** Consulta nos últimos dois meses sofrido nenhum evento estressante àqueles que referiram um evento, os últimos tiveram um au- mento de 24% da chance de internação (apesar do IC95% ter englobado a unidade, o teste para tendência linear foi significativo); enquanto que os indivíduos que sofreram dois ou mais eventos apresentaram 84% mais chances de ocorrência do desfecho. da individual de 2,10 salários mínimos ou mais tiveram menos chances de internação hospitalar (RP = 0,74; IC95%: 0,52-1,05), quando compara- dos aos indivíduos que possuem renda inferior a 0,75 salário mínimo. Salienta-se a relação apresentada entre a va- riável eventos estressantes e o desfecho: com- parando os indivíduos que referiram não ter PREVALÊNCIA DE INTERNAÇÃO HOSPITALAR E FATORES ASSOCIADOS 221 ram uma chance de 83% a mais de internar do que indivíduos sem nenhum evento. Ressalta-se, ainda, que indivíduos com esta- do de saúde auto-avaliado como ruim tiveram 51% mais chances de apresentar o desfecho, quando comparados àqueles que referiram um estado de saúde bom. A chance de internação dos indivíduos que consultaram nos últimos dois meses foi três vezes superior a dos que não consultaram. Resultados A variável consulta também permaneceu após a análise ajustada para as demais variáveis, sugerindo que indivíduos que consultaram no período de dois meses anteriores à entrevis- ta tiveram 2,79 vezes mais chances de internar, quando comparados aos que não consultaram no mesmo período. Os resultados finais da análise multivariada de fatores associados com a ocorrência de hos- pitalizações no último ano estão dispostos na Tabela 2. Das variáveis pertencentes ao primeiro nível do modelo de análise (demográficas e só- cio-econômicas) permaneceram associadas de forma significativa ao desfecho apenas a faixa etária e a renda individual, não demonstrando grandes diferenças na RP ajustada quando com- paradas com a RP bruta. Cad. Saúde Pública, Rio de Janeiro, 23(1):217-224, jan, 2007 Discussão O presente estudo tem delineamento transver- sal, em decorrência disso os resultados obtidos devem ser vistos como fatores associados. É importante salientar, também, que a pesquisa apresentou um maior número de perdas entre os homens de 25 a 49 anos, o que pode ter afetado os resultados, levando a uma superestimação da prevalência geral de internação, pois esta apre- sentou associação com o sexo feminino. Entre os fatores que levaram ao elevado número de per- das salienta-se: (1) necessidade de assinar um A relação encontrada na variável eventos es- tressantes permaneceu após o ajustamento para as demais variáveis (sexo, idade, escolaridade, renda individual, consulta médica e auto-avalia- ção de saúde). Indivíduos que enfrentaram dois ou mais eventos considerados estressantes tive- Internação hospitalar de acordo com variáveis sócio-econômicas, demográfi cas, eventos estressantes e consulta média, análise multivariada. Canoas, Rio Grande do Sul, Brasil, 2003. Internação Características n % RP bruta RP ajustada p (IC95%) (IC95%) Renda individual (salários mínimos) *,** 2,10 ou mais 794 7,2 0,74 (0,52-1,05) 0,60 (0,41-0,87) 0,76-2,00 508 12,5 1,28 (0,93-1,77) 0,92 (0,65-1,29) 0,005 0,00-0,75 652 9,7 1,00 1,00 Faixa etária (anos) ** 14-19 261 3,5 1,00 20-39 756 9,4 2,72 (1,37-5,37) 2,54 (1,27-5,06) 40-59 675 8,6 2,48 (1,25-4,90) 2,46 (1,25-4,81) 0,0001 60 ou mais 262 17,2 4,96 (2,47-9,93) 4,14 (2,07-8,25) Eventos estressantes *** Nenhum 1.267 8,4 1,00 Um 469 10,5 1,24 (0,89-1,73) 1,29 (0,93-1,78) 0,018 Dois ou mais 142 15,5 1,84 (1,21-2,80) 1,83 (1,19-2,79) Consulta #, ## Não 1.078 4,7 1,00 0,0001 Sim 868 15,1 3,19 (2,34- 4,36) 2,79 (2,03-3,83) * R$ 200,00; Internação hospitalar de acordo com variáveis sócio-econômicas, demográfi cas, eventos estressantes e consulta média, análise multivariada. Canoas, Rio Grande do Sul, Brasil, 2003. Garbinato LR et al. 222 Garbinato LR et al. 222 consentimento (estudos populacionais utilizan- do consentimento verbal costumam ter perdas menores) e (2) em alguns setores censitários sor- teados houve dificuldade de acesso dos entrevis- tadores devido à violência. ções que poderiam ser evitadas, caso houvesse uma melhor prática da atenção primária à saúde. Outra questão a ser considerada, refere-se à cha- mada “internação social”, que acontece devido às dificuldades econômicas e sociais do paciente, que é internado para ter acesso ao tratamento adequado (viés de admissão). Outros fatores para a maior hospitalização nos grupos de menor ní- vel sócio-econômico poderiam ser a maior mor- bidade e severidade da doença entre os grupos mais pobres. Discussão A prevalência de internações hospitalares pa- ra o período estudado foi de 9,4% em Canoas, sendo, portanto, muito próxima ao porcentual de 9% encontrado em estudo realizado no Sul do Brasil em 1992 3, porém, superior ao coeficiente de 6,9% encontrado para o Brasil (sem restrições de idade), no estudo de Castro et al. 4, com base em dados do ano de 1998. Salienta-se o fato de que menos da metade das internações hospitalares ocorre na cidade de Canoas, o que demonstra que a capacidade ins- talada de leitos da cidade é insuficiente. Indivíduos com sessenta anos ou mais conti- nuaram fortemente associados ao desfecho, mes- mo na análise da RP ajustada (RP = 4,14; IC95%: 2,07-8,25). Esse fato reforça dados encontrados na literatura referente ao assunto 4,6,7. Conside- rando-se que a população idosa vem crescendo de forma rápida no Brasil nos últimos anos 8, a relevância do resultado anteriormente descrito está justamente em alertar os gestores de saú- de pública para a necessidade de reorganizar e aprimorar os sistemas de atenção à saúde, a fim de que se obtenha um atendimento satisfatório dessa demanda. Estudo realizado em 1992 mostrou existir uma relação positiva entre a internação hospitalar no último ano e a freqüência de consultas médi- cas 3. O presente estudo encontrou uma associa- ção positiva entre essa variável e o desfecho. Sa- be-se, entretanto, que os usuários do SUS ainda encontram dificuldades para conseguir consul- tas com especialistas e, principalmente, para ter acesso a exames complementares, o que também pode contribuir para um aumento nas interna- ções, pois estas representam uma forma de se conseguir realizar exames de alta complexidade e também de ter a assistência do médico espe- cialista durante o período de internação. Assim, acredita-se ser necessário um aprimoramento do nível de atendimento ambulatorial, de modo a alcançar a idealizada integralidade de atenção, que tende a ter, por conseqüência, a redução dos índices de internação hospitalar. A relação existente entre a situação financeira e a internação hospitalar foi analisada a partir da renda individual. Sabe-se que o uso dessa va- riável possibilita a ocorrência de distorções de resultado com relação aos indivíduos que não possuem renda e dependem do chefe da família. Porém, esse foi o dado coletado (com relação à renda) que sofreu o menor número de perdas. A morbidade referida cresce na população na proporção inversa da renda 9,10. Cad. Saúde Pública, Rio de Janeiro, 23(1):217-224, jan, 2007 Discussão Os estudos de Almeida et al. 11 e Travassos et al. 12 afirmam que as desigualdades observadas nesse tipo de ser- viço (internações), no Brasil, são mais sutis que aquelas observadas no consumo geral de servi- ços de saúde. A associação entre percepção de saúde ruim e internação hospitalar não permaneceu após a análise multivariada, o que pode estar ligado à percepção de saúde dos indivíduos com menor renda, porque é possível que essas pessoas decla- rem estado de saúde ruim com menos freqüên- cia. Da mesma forma, a variável possuir seguro de saúde privado não manteve associação, pois alguns dos seguros locais de saúde não cobrem internação hospitalar. ç Porém, deve-se considerar que o fato de uma maior utilização de serviços hospitalares encontrar-se ligada à população com renda in- dividual entre 0,76 e 2 salários mínimos, pode estar relacionado ao modelo de atenção vigente. Isso porque, ainda que se idealize um modelo de atenção à saúde, com base na promoção da saú- de, sabe-se que o Sistema Único de Saúde (SUS) ainda continua, de forma mais intensa, atuando e organizando ações de saúde com vistas a aten- der aqueles que procuram por assistência médi- ca (ambulatórios, centros de saúde) e hospitalar (urgência e emergência), não tendo uma implan- tação satisfatória em termos de atendimento na rede básica. Em decorrência disso, a assistência hospitalar tem sido uma porta de entrada para o SUS, favorecendo um maior número de interna- Os resultados da análise multivariada indi- cam uma relação dose-resposta onde indivídu- os com maior número de eventos estressantes têm uma chance maior de internação hospitalar. Resultado semelhante já foi encontrado em um estudo realizado no Sul do Brasil, porém, com relação à utilização de serviços ambulatoriais 13. Segundo um estudo da Organização Mundial da Saúde (OMS), o evento estressante abuso sexual tem um papel importante na carga global das doenças. Esse mesmo estudo salienta a neces- sidade de estudos em nível micro de fatores de risco como, por exemplo, eventos estressantes e a carga regional de doenças 14. Assim, seria im- Cad. Saúde Pública, Rio de Janeiro, 23(1):217-224, jan, 2007 PREVALÊNCIA DE INTERNAÇÃO HOSPITALAR E FATORES ASSOCIADOS 223 por meio de ações que busquem melhorar as con- dições de vida da população. Resumo Este estudo transversal investiga a utilização de ser- viços hospitalares e fatores associados em indivíduos com 14 anos ou mais em Canoas, Rio Grande do Sul, Brasil. Foram entrevistados 1.954 indivíduos de 40 setores censitários. A prevalência de internação hos- pitalar no período de um ano foi de 9,4%. Na análise ajustada para as demais variáveis, as que permanece- ram associadas a uma maior chance de hospitalização foram: idade de 60 anos ou mais (RP = 4,14; IC95%: 2,07-8,25), realização de consulta médica nos últimos dois meses (RP = 2,79; IC95%: 2,03-3,83), a ocorrência de dois ou mais eventos estressantes (RP = 1,83; IC95%: 1,19-2,80). A renda individual, de 2,10 salários míni- mos ou mais, esteve associada a uma menor chance de hospitalização (RP = 0,60; IC95%: 0,41-0,87). A pre- valência de internações encontrada é compatível com outros estudos. A maior prevalência de hospitalização nos grupos de menor nível sócio-econômico pode in- dicar um menor acesso aos serviços de atenção básica. Outros fatores envolvidos poderiam ser a maior morb- idade e severidade da doença entre os grupos mais po- bres. Salienta-se a importância de investigar a relação entre eventos estressantes e morbidade. L. R. Garbinato participou da análise dos dados e re- dação do artigo. J. U. Béria, B. Raymann e L. P. Gigan- te participaram da coordenação geral do projeto que deu origem ao levantamento de base populacional, que subsidiou este estudo e redação do artigo. A. C. L. Fi- gueiredo coordenou o trabalho de campo e tratamento dos dados do levantamento de base populacional que subsidiou este estudo, e colaborou na análise de dados e redação do artigo. L. P. Gigante participou da coorde- nação do trabalho de campo e tratamento dos dados. L. S. Palazzo foi responsável pelo estudo dos eventos estressantes. D. R. G. C. Aerts contribuiu como respon- sável pela auto-avaliação de saúde. Discussão Assim, salienta-se a importância da formulação e implementação de políticas sociais que apresentem resultados prá- ticos para a população com relação à segurança, à cultura, à educação, ao lazer, à interação social e às oportunidades de trabalho, com significante incremento nos níveis de qualidade de vida e de saúde dos cidadãos. portante desenvolver investigações sobre a re- lação entre eventos estressantes e morbidade, para esclarecer se estes são marcadores de risco ou determinantes. Saúde encontra-se intimamente ligada à qua- lidade de vida e, admitindo-se as dificuldades tí- picas do ambiente urbano em um país com tan- tas desigualdades, enfatiza-se a importância do movimento que incentiva a promoção da saúde Serviços Hospitalares; Hospitalização; Eventos Es- tressantes Cad. Saúde Pública, Rio de Janeiro, 23(1):217-224, jan, 2007 Cad. Saúde Pública, Rio de Janeiro, 23(1):217-224, jan, 2007 Agradecimentos Este estudo faz parte de um estudo mais amplo que contou com apoio de Christoffel Blindenmission, Fun- dação de Amparo à Pesquisa do Estado do Rio Grande do Sul e Conselho Nacional de Desenvolvimento Cien- tífico e Tecnológico. Serviços Hospitalares; Hospitalização; Eventos Es- tressantes Cad. Saúde Pública, Rio de Janeiro, 23(1):217-224, jan, 2007 Garbinato LR et al. 224 Garbinato LR et al. 224 Garbinato LR et al. 224 Referências 1. Fernández de la Hoz K, Leon DA. Self-perceived health status and inequalities in use of health serv- ices in Spain. Int J Epidemiol 1996; 25:593-603. 9. Freeborn DK, Pope CR, Davis MA, Mullooly JP. Health status, socioeconomic status and utiliza- tion of outpatient services for members of a pre- paid group practice. Med Care 1977; 15:115-28. ices in Spain. Int J Epidemiol 1996; 25:593-603. 2. Hulka BS, Wheat JR. Patterns of utilization: the pa- tient perspective. Med Care 1985; 23:438-60. 10. Travassos C, Fernandes C, Perez M. Desigualdades sociais, morbidade e consumo de serviços de saú- de no Brasil. Série Estudos: Política, Planejamento e Gestão em Saúde 1995; 4:5-26. 3. Costa JD, Facchini LA. Utilização de serviços am- bulatoriais em Pelotas: onde a população consulta e com que freqüência. Rev Saúde Pública 1997; 31:360-9. 11. Almeida CM, Travassos C, Porto S, Labra ME. Health sector reform in Brazil: a case study of in- equity. Int J Health Serv 2000; 30:129-62. 4. Castro MSM, Travassos C, Carvalho MS. Fatores associados às internações hospitalares no Brasil. Ciênc Saúde Coletiva 2002; 7:795-811. 12. Travassos C, Viacava F, Fernandes C, Almeida CS. Desigualdades geográficas e sociais na utilização de serviços de saúde no Brasil. Ciênc Saúde Coleti- va 2000; 5:133-49. 5. Dupont WD, Plummer WD. Power and sample size calculation: a review and computer program. Con- trol Clin Trials 1990; 11:116-28. 6. Mendoza-Sassi R, Béria JU. Utilización de los ser- vicios de salud: una revisión sistemática sobre los factores relacionados. Cad Saúde Pública 2001; 17:819-32. 13. Mendoza-Sassi R, Béria JU, Barros AJD. Outpatient health services utilization and associated factors: a population-based study. Rev Saúde Pública 2003; 37:373-8. 14. Ezzati M, Lopez AD, Rodgers A, Vander Hoorn S, Murray CJ; Comparative Risk Assessment Collabo- rating Group. Selected major risk factors and glo- bal and regional burden of disease. Lancet 2002; 360:1347-60. 7. Fernández-Mayoralas G, Rodríguez V, Rojo F. Health services accessibility among Spanish eld- erly. Soc Sci Med 2000; 50:17-26. 14. Recebido em 15/Mai/2005 Versão final reapresentada em 08/Mai/2006 Aprovado em 15/Mai/2006 Agradecimentos Ezzati M, Lopez AD, Rodgers A, Vander Hoorn S, Murray CJ; Comparative Risk Assessment Collabo- rating Group. Selected major risk factors and glo- bal and regional burden of disease. Lancet 2002; 360:1347-60. 8. Lima-Costa MF, Veras R. Saúde pública e enve- lhecimento [Editorial]. Cad Saúde Pública 2003; 19:700-1. 8. Lima-Costa MF, Veras R. Saúde pública e enve- lhecimento [Editorial]. Cad Saúde Pública 2003; 19:700-1. Recebido em 15/Mai/2005 Versão final reapresentada em 08/Mai/2006 Aprovado em 15/Mai/2006
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Multiscale characterisation of chimneys/pipes: Fluid escape structures within sedimentary basins
International journal of greenhouse gas control
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Published In: Publisher Rights Statement: © 2020 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/) Publisher Rights Statement: © 2020 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/) Publisher Rights Statement: © 2020 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/) General rights Copyright for the publications made accessible via the Edinburgh Research Explorer is retained by the author(s) and / or other copyright owners and it is a condition of accessing these publications that users recognise and abide by the legal requirements associated with these rights. Edinburgh Research Explorer Multiscale characterisation of chimneys/pipes: Fluid escape structures within sedimentary basins Edinburgh Research Explorer Citation for published version: Citation for published version: Citation for published version: Robinson, AH, Callow, B, Böttner, C, Yilo, N, Provenzano, G, Falcon-suarez, IH, Marín-moreno, H, Lichtschlag, A, Bayrakci, G, Gehrmann, R, Parkes, L, Roche, B, Saleem, U, Schramm, B, Waage, M, Lavayssière, A, Li, J, Jedari-eyvazi, F, Sahoo, S, Deusner, C, Kossel, E, Minshull, TA, Berndt, C, Bull, JM, Dean, M, James, RH, Chapman, M, Best, AI, Bünz, S, Chen, B, Connelly, DP, Elger, J, Haeckel, M, Henstock, TJ, Karstens, J, Macdonald, C, Matter, JM, North, L & Reinardy, B 2021, 'Multiscale characterisation of chimneys/pipes: Fluid escape structures within sedimentary basins', International Journal of Greenhouse Gas Control, vol. 106, pp. 103245. https://doi.org/10.1016/j.ijggc.2020.103245 g j ( ) 10.1016/j.ijggc.2020.103245 Link: Link to publication record in Edinburgh Research Explorer Link: Link to publication record in Edinburgh Research Explorer Link: Document Version: Publisher's PDF, also known as Version of record Published In: International Journal of Greenhouse Gas Control Multiscale characterisation of chimneys/pipes: Fluid escape structures within sedimentary basins Adam H. Robinson a,*,1, Ben Callow a,*,1, Christoph B¨ottner b, Naima Yilo a, Giuseppe Provenzano a,c, Ismael H. Falcon-Suarez d, H´ector Marín-Moreno d,e, Anna Lichtschlag d, Gaye Bayrakci d, Romina Gehrmann a, Lou Parkes f, Ben Roche a, Umer Saleem g, Bettina Schramm b, Malin Waage h, Aude Lavayssi`ere a, Jianghui Li a, Farid Jedari-Eyvazi a, Sourav Sahoo d, Christian Deusner b, Elke Kossel b, Timothy A. Minshull a, Christian Berndt b, Jonathan M. Bull a, Marcella Dean i, Rachael H. James a, Mark Chapman f, Angus I. Best d, Stefan Bünz h, Baixin Chen g, Douglas P. Connelly d, Judith Elger b, Matthias Haeckel b, Timothy J. Henstock a, Jens Karstens b, Calum Macdonald f, Juerg M. Matter a, Laurence North d, Benedict Reinardy j a University of Southampton, School of Ocean and Earth Science, Southampton, SO14 3ZH, UK b GEOMAR Helmholtz-Centre for Ocean Research Kiel, Kiel, Germany c University of Grenoble Alpes, ISTerre, Grenoble, France d National Oceanography Centre, Waterfront Campus, Southampton, SO14 3ZH, UK e Norwegian Geotechnical Institute, PB 3930 Ullevål Stadion, NO-08906, Oslo, Norway f University of Edinburgh School of Geosciences, Grant Institute, Edinburgh, EH9 3JW, UK g Institute of Mechanical, Process and Energy Engineering, Heriot-Watt University, Edinburgh, EH14 4AS, UK h UiT The Arctic University of Norway, Department of Geosciences, Tromsø, Norway i Shell Global Solutions International BV, Grasweg 31, 1031 HW, Amsterdam, the Netherlands j Stockholm University and Bolin Centre for Climate Research, Stockholm, Sweden a University of Southampton, School of Ocean and Earth Science, Southampton, SO14 3ZH, UK b GEOMAR Helmholtz-Centre for Ocean Research Kiel, Kiel, Germany c University of Grenoble Alpes, ISTerre, Grenoble, France d National Oceanography Centre, Waterfront Campus, Southampton, SO14 3ZH, UK e Norwegian Geotechnical Institute, PB 3930 Ullevål Stadion, NO-08906, Oslo, Norway f University of Edinburgh School of Geosciences, Grant Institute, Edinburgh, EH9 3JW, UK g Institute of Mechanical, Process and Energy Engineering, Heriot-Watt University, Edinburgh, EH14 4AS, UK h UiT The Arctic University of Norway, Department of Geosciences, Tromsø, Norway i Shell Global Solutions International BV, Grasweg 31, 1031 HW, Amsterdam, the Netherlands j Stockholm University and Bolin Centre for Climate Research, Stockholm, Sweden Available online 19 February 2021 1750-5836/© 2020 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). * Corresponding authors. E-mail addresses: A.H.Robinson@soton.ac.uk (A.H. Robinson), Ben.Callow@noc.soton.ac.uk (B. Callow). 1 These authors contributed equally https://doi.org/10.1016/j.ijggc.2020.103245 Received 8 July 2020; Received in revised form 30 November 2020; Accepted 14 December 2020 Contents lists available at ScienceDirect Contents lists available at ScienceDirect Take down policy p y The University of Edinburgh has made every reasonable effort to ensure that Edinburgh Research Explorer content complies with UK legislation. If you believe that the public display of this file breaches copyright please contact openaccess@ed.ac.uk providing details, and we will remove access to the work immediately and investigate your claim. Download date: 24. Oct. 2024 International Journal of Greenhouse Gas Control 106 (2021) 103245 Multiscale characterisation of chimneys/pipes: Fluid escape structures within sedimentary basins Adam H. Robinson a,*,1, Ben Callow a,*,1, Christoph B¨ottner b, Naima Yilo a, Giuseppe Provenzano a,c, Ismael H. Falcon-Suarez d, H´ector Marín-Moreno d,e, Anna Lichtschlag d, Gaye Bayrakci d, Romina Gehrmann a, Lou Parkes f, Ben Roche a, Umer Saleem g, Bettina Schramm b, Malin Waage h, Aude Lavayssi`ere a, Jianghui Li a, Farid Jedari-Eyvazi a, Sourav Sahoo d, Christian Deusner b, Elke Kossel b, Timothy A. Minshull a, Christian Berndt b, Jonathan M. Bull a, Marcella Dean i, Rachael H. James a, Mark Chapman f, Angus I. Best d, Stefan Bünz h, Baixin Chen g, Douglas P. Connelly d, Judith Elger b, Matthias Haeckel b, Timothy J. Henstock a, Jens Karstens b, Calum Macdonald f, Juerg M. Matter a, Laurence North d, Benedict Reinardy j a University of Southampton, School of Ocean and Earth Science, Southampton, SO14 3ZH, UK b GEOMAR Helmholtz-Centre for Ocean Research Kiel, Kiel, Germany c University of Grenoble Alpes, ISTerre, Grenoble, France d National Oceanography Centre, Waterfront Campus, Southampton, SO14 3ZH, UK e Norwegian Geotechnical Institute, PB 3930 Ullevål Stadion, NO-08906, Oslo, Norway f University of Edinburgh School of Geosciences, Grant Institute, Edinburgh, EH9 3JW, UK g Institute of Mechanical, Process and Energy Engineering, Heriot-Watt University, Edinburgh, EH14 4AS, UK h UiT The Arctic University of Norway, Department of Geosciences, Tromsø, Norway i Shell Global Solutions International BV, Grasweg 31, 1031 HW, Amsterdam, the Netherlands j Stockholm University and Bolin Centre for Climate Research, Stockholm, Sweden A R T I C L E I N F O A B S T R A C T Contents lists available at ScienceDirect International Journal of Greenhouse Gas Control journal homepage: www.elsevier.com/locate/ijggc International Journal of Greenhouse Gas Control 106 (2021) 103245 1.1. Overview Carbon dioxide (CO2) capture and subsurface storage (CCS) within sedimentary basins has been identified as an effective solution for reducing anthropogenic CO2 emissions in the atmosphere (IPCC, 2005; The Global CCS Institute, 2019). CCS must form a key component of present and future global climate policy, in order to meet anthropogenic greenhouse gas emission reductions of 80–95 % by 2050, and limit model predictions of likely warming of <2 ◦C relative to pre-industrial levels (IPCC, 2014). Requirements for wide-scale implementation of CCS include: 1) cost-effective CCS technologies; 2) government poli­ cy/incentives for negative emissions technologies; and 3) the need for public acceptance/confidence, all of which are intrinsically linked. The primary technological requirement to widespread implementation of CO2 storage is subsurface site characterisation and containment assur­ ance. Legal regulations governing CCS in Europe exist in the form of the EU CCS Directive on Geological Storage of Carbon Dioxide, 2009/31/EC (2009), which defines requirements for CO2 storage across the lifetime of a storage site, including closure and post-closure obligations. Factors that must be considered for the characterisation and assessment of po­ tential CO2 storage complexes and their surroundings include the role and impacts of potential fluid migration pathways causing loss of containment, and the potential flux rates through these pathways. ll The paper has three main aims: (1) Firstly, we describe the various methods which may be applied to the characterisation of focused fluid conduits, and which allow us to resolve the physical parameters of in­ terest (Section 3). For each individual method, we describe their capa­ bilities, consider their benefits, address uncertainties, and deduce any further developments that may be required. In order to contexualise the various techniques which may be used, we describe their application during our investigation of the Scanner pockmark. (2) Secondly, we discuss the scales of imaging and resolution of the various methods described here, and the co-dependencies which exist between them. This permits integration into a multi-scale approach, and demonstrates how the different techniques may be employed in combination, to ensure appropriate constraint and calibration of the different methods, for complete characterisation of the fluid escape structures (Section 4). 2.1. Focused fluid conduits in seismic data Seismic chimneys (e.g., Hustoft et al., 2010) or pipes (e.g., Moss and Cartwright, 2010a), referred to hereafter only as chimneys, are observed in seismic reflection data as vertical to sub-vertical anomalies with cir­ cular or elliptical planforms, displaying seismic blanking and discon­ tinuous or chaotic reflections (e.g., Løseth et al., 2011). Where free gas is present in the chimney, high amplitude seismic reflections, known as bright spots, with polarity reversals may be observed at discrete in­ tervals, indicating gas accumulation during migration in layers of porous sediments (e.g., Ostrander, 1984). Pull-up of reflectors may also be observed, caused by high seismic velocities, which are commonly attributed to authigenic carbonate accumulations, or where located in the gas hydrate stability zone, to the presence of gas hydrate (e.g., Plaza-Faverola et al., 2010). If CO2 migrates from a sub-seafloor storage reservoir and reaches the base of these chimneys, and if their perme­ ability is sufficiently high, they could act as CO2 pathways towards the seafloor and overlying water column. To provide a reliable prediction of potential seafloor seep sites, the degree to which these pathways are able to transmit fluids (i.e. permeability) needs to be better understood. g p j g , , ; , The location and potential intensity of any possible loss of CO2 containment from a storage reservoir is dependent on the distribution of fluid pathways in the cap rock and overburden, and the ability of these pathways to transmit dissolved, liquid, and/or gaseous CO2, depending on the pressure-temperature conditions and presence of other fluids. Potential pathways may include anthropogenic sources, such as aban­ doned wells (e.g., Watson and Bachu, 2009), formation level inherent structures, including natural migration up-dip along permeable strati­ graphic horizons (e.g., T´oth, 1980; Hindle, 1997), and the formation or reactivation of fluid escape structures (e.g., Nichols et al., 1994; Frey et al., 2009). Such fluid escape, or seal bypass, structures permit pressure-driven, focused fluid flow, which hydraulically connects deeper strata with the seafloor through inter-connected faults, fractures, and porous-permeable sediment layers (Cartwright et al., 2007). Fluid escape can take place as single blow-out events, episodic/pulsed flow, or as continuous seepage flow. The type of flow can vary depending on the subsurface pressure, stress, and lithological conditions. Therefore, the activity of a fluid escape structure may exhibit temporal variability, which may be cyclical over both short timescales such as tidal cycles (e. 2.1. Focused fluid conduits in seismic data g., Boles et al., 2001) or longer-term sea level changes (e.g., Plaza-Fa­ verola et al., 2011; Riboulot et al., 2014). Therefore, the combined un­ derstanding of the presence of fluid pathways and their fluid flow regime is critical for the risk assessment of potential subsurface CO2 escape. l Chimneys have been globally observed by seismic imaging (e.g., Cartwright et al., 2007; Gay et al., 2007; Moss and Cartwright, 2010a, b; Løseth et al., 2011; Plaza-Faverola et al., 2017), including extensively in the North Sea (Hovland and Sommerville, 1985; Cole et al., 2000; Bünz et al., 2003; Karstens and Berndt, 2015; B¨ottner et al., 2019). A comprehensive analysis of 3D seismic reflection volumes in the South Viking Graben, North Sea (an area of 2850 km2; Karstens and Berndt, 2015) identified 46 large-scale (~100−1000 m-wide) chimneys within the shallowest 1000 m of the overburden. 1.1. Overview (3) Thirdly, based on our findings from this study, we describe a framework which can be used to determine the approaches that are needed to un­ derstand potential fluid flow structures in marine environments, in the context of the risk assessment of potential future CO2 geological storage sites (Section 5). l CO2 can be sequestered into porous and permeable subsurface sandstone reservoirs (Bachu, 2000; Benson and Cole, 2008), such as depleted oil and gas reservoirs and saline aquifers. Sandstone reservoirs of this type are commonly overlain by impermeable cap rocks and overburden stratigraphy, which together provide an effective seal that prevents the upward migration of CO2, ensuring safe and permanent storage. Offshore CO2 storage in sandstone reservoirs has been suc­ cessfully demonstrated in Europe and globally (e.g., Sleipner, North Sea; Tomakomi, Japan). Several other commercial-scale offshore CO2 storage projects are also in planning or development stages, such as the North­ ern Lights project (e.g., Furre et al., 2019a; Global CCS Institute, 2019). 1. Introduction Scanner pockmark, which we undertook as part of the European Union Horizon 2020 project Strategies for the Environmental Monitoring of Marine Carbon Capture and Storage (STEMM-CCS; http://www.ste mm-ccs.eu), together with a partner project CHIMNEY (Characterisa­ tion of major overburden leakage pathways above sub-seafloor CO2 storage reservoirs in the North Sea; Bull et al., 2018). STEMM-CCS and CHIMNEY focussed on determining the permeability of subsurface fluid pathways, and developing better techniques to locate fluid escape structures, so that they can be better quantified and constrained, with relevance to potential fluid flow at CO2 storage complexes. A B S T R A C T Keywords: Chimneys Pipes Overburden CO2 sequestration Geological storage North Sea Evaluation of seismic reflection data has identified the presence of fluid escape structures cross-cutting overburden stratigraphy within sedimentary basins globally. Seismically-imaged chimneys/pipes are considered to be possible pathways for fluid flow, which may hydraulically connect deeper strata to the seabed. The properties of fluid migration pathways through the overburden must be constrained to enable secure, long-term subsurface carbon dioxide (CO2) storage. We have investigated a site of natural active fluid escape in the North Sea, the Scanner pockmark complex, to determine the physical characteristics of focused fluid conduits, and how they control fluid flow. Here we show that a multi-scale, multi-disciplinary experimental approach is required for complete charac­ terisation of fluid escape structures. Geophysical techniques are necessary to resolve fracture geometry and sub­ surface structure (e.g., multi-frequency seismics) and physical parameters of sediments (e.g., controlled source electromagnetics) across a wide range of length scales (m to km). At smaller (mm to cm) scales, sediment cores were sampled directly and their physical and chemical properties assessed using laboratory-based methods. Numerical modelling approaches bridge the resolution gap, though their validity is dependent on calibration and constraint from field and laboratory experimental data. Further, time-lapse seismic and acoustic methods capable of resolving temporal changes are key for determining fluid flux. Future optimisation of experiment resource use may be facilitated by the installation of permanent seabed infrastructure, and replacement of manual data processing with automated workflows. This study can be used to inform measurement, monitoring and verification workflows that will assist policymaking, regulation, and best practice for CO2 subsurface storage operations. 1. Introduction A.H. Robinson et al. A.H. Robinson et al. International Journal of Greenhouse Gas Control 106 (2021) 103245 2.3.1. Stratigraphy and seismostratigraphic framework The lithostratigraphy and seismostratigraphic framework of the ~600 m-thick Quaternary sediment succession containing the Scanner pockmark complex was described by B¨ottner et al. (2019) and Stoker et al. (2011) (Fig. 2). Deposited within the Witch Ground Basin (Andrews et al., 1990), this complex is underlain by the Hordland and Nordland Groups, of Palaeogene and Neogene age respectively, and which are composed of claystone with limestone and sandstone in­ terbeds (Judd et al., 1994). The basal Quaternary unit, the Aberdeen Ground Formation (Fm.; unit S1 in Fig. 2), is composed of clay-rich sediments deposited in the Early Pleistocene (up to Marine Isotope Stage, MIS, 13), and displays a laterally continuous, layered seismic character (Stoker et al., 2011; Ottesen et al., 2014). The overlying Ling Bank Fm. (S2) erodes into the top of the Aberdeen Ground Fm., representing a regional glacial un­ conformity, with deposition of glacial tills in the Early to Middle Pleis­ tocene (~1.2−0.5 Ma, MIS 12 to 10; Stewart and Lonergan, 2011; Reinardy et al., 2017; B¨ottner et al., 2019). The glacial tunnel valleys of the Ling Bank Fm. display both a layered and non-layered seismic character. The Coal Pit Fm. (S3) overlies the Ling Bank and Aberdeen Ground Fms., and comprises glacial tills (which include pebbly and muddy sands) deposited in the upper Mid to Late Pleistocene (MIS 6-3; Andrews et al., 1990; Stoker et al., 2011). The Coal Pit Fm. is conformably overlain by Last Glacial Maximum (LGM) deposits, which comprises silty-sandy clays with rare pebbles, deposited during MIS 3−2 (S4). Unit S4 extends upwards to the base of Scanner pockmark. The Coal Pit Fm. and LGM deposits have a dim and chaotic seismic character and are conformably overlain by the Witch Ground Fm. (S5), composed of silty clay deposited during MIS 2 to 1 (Stoker et al., 2011). The Witch Ground Fm. has two main units: the lower (S5.1) and upper (S5.2) Witch Ground Member respectively. The lower Witch Ground Member has an apparent interbedded seismic character. The upper Witch Ground Member conformably overlies the lower Witch Ground Member, thin­ ning or pinching out towards the northeast. The upper Witch Ground Member is composed of sediments of Holocene age (MIS 1) and has a uniform seismic character (Stoker et al., 2011). The Scanner pockmark depression erodes down to the base of the lower Witch Ground Member. 1.2. Aims & objectives Chimney-like features can also be the result of seismic imaging ar­ tefacts. Seismic imaging artefacts may arise both as a result of data acquisition and/or processing (e.g., Tucker and Yorston, 1973). In particular, for locations where gas is present in the subsurface, the effect this has on seismic velocity determination can have a significant impact In this contribution we provide a broad overview of the integrated geophysical, geological, and geochemical methods which can be applied to the characterisation of focused fluid conduits. In order to achieve this, we use as our context an exemplar study of the fluid conduit beneath the 2 A.H. Robinson et al. International Journal of Greenhouse Gas Control 106 (2021) 103245 (Hovland et al., 2010). In this area of the North Sea, large pockmarks (>6 m deep, >250 m long, and >75 m wide; class 1, B¨ottner et al., 2019) are continuously active. Evidence for active methane venting at the Scanner pockmark complex is provided by water column imaging and the presence of methane derived authigenic carbonates (MDACs) at the seabed (Judd et al., 1994; Judd and Hovland, 2009). The West Scanner pockmark (Fig. 1c) releases methane at 1600−2600 kg/day (Li et al., 2020), derived from a combination of biogenic and thermogenic sources (Clayton and Dando, 1996). Smaller pockmarks (class 2) are also distributed across the area (Fig. 1b) with a dominant NNE/SSW orien­ tation (>1500 across 225 km2) and are interpreted as dewatering fea­ tures attributed to localised pressure changes (B¨ottner et al., 2019). The Scanner pockmark complex overlies an area that has been appraised for CO2 storage potential, the East Mey Storage Site (ACT Acorn Con­ sortium, 2018). on both time and depth migration. An example locality where a seismic artefact was interpreted as a chimney is the Goldeneye field, a pro­ spective CO2 storage site in the Central North Sea. Following high res­ olution 3D seismic processing a feature previously interpreted as a fluid escape conduit was later reinterpreted as a seismic imaging artefact caused by a glacial tunnel valley (Dean et al., 2015; Karstens and Berndt, 2015). 2.3.1. Stratigraphy and seismostratigraphic framework Based on the observations from QICS, and other experiments (e.g., Fauria and Rempel, 2011; R¨ass et al., 2018), a conceptual model for the formation mechanism and structure of chimneys in the shallow over­ burden has been developed (Bull et al., 2018). In this model, the first phase of formation is the hydraulic fracturing of low permeability sed­ iments due to high fluid overpressure (Arntsen et al., 2007; Cartwright et al., 2007; Løseth et al., 2009). Seal breaching occurs due to reduced effective stress and leads to either opening of new fractures and/or reactivation of pre-existing fractures, generating a localised connected fracture system. These pathways may permit vertical buoyancy-driven migration of gas-rich pore fluids through the fracture network. Large-scale chimneys (~100−1000 m wide) are therefore hypothesised to represent a series of interconnected sub-vertical or radial fractures, which allow the vertical flow of gas in the shallow subsurface (Bull et al., 2018) due to the elevated permeability relative to the normal ‘back­ ground’ permeability of the host sediment (Cartwright et al., 2007). A transition from fracture to capillary dominant flow behaviour may be observed with increasing depth, due to increased overburden thickness (higher effective stress). In addition to the role of the geometric structures of chimneys in governing fluid flow, coupled physical and chemical processes act within fractures and pores, resulting in complex feedback mechanisms between porosity-permeability and CO2/CH4 reactivity, which may affect the hydro-mechanical response of the system. From a quantitative perspective, little is known regarding the impact that chimneys have on the upwards migration of CO2 and CH4 to the seabed (Liu et al., 2019; Marín-Moreno et al., 2019). 2.2. Formation of chimneys Chimney formation was observed on a small scale during a controlled subsurface CO2 release experiment known as QICS (Taylor et al., 2015; Cevatoglu et al., 2015). In this experiment, CO2 was released into sediments at an increasing rate of 20–210 kg/day, at 12 m depth below the seabed in shallow water (5−30 m) in Ardmucknish Bay, Scotland. Repeated seismic reflection data acquisition prior to, during, and after the gas release showed the temporal development of a chim­ ney, formed by gas propagating upwards by fracture generation and reactivation in fine grained sediment (Cevatoglu et al., 2015). Condi­ tions for hydraulic fracture generation are favourable in shallow (low effective stress) unconsolidated, fine grained sediments, and may be considered a primary mechanism for chimney initiation (Fauria and Rempel, 2011). The upward propagation of fluids may also be facilitated by capillary driven invasion, most prevalent in conditions of high effective stress (Cathles et al., 2010). Further mechanisms for chimney genesis include: erosive fluidisation, localised subsurface volume loss, and syn-sedimentary formation (Lowe, 1975; Sun et al., 2013; Cart­ wright and Santamarina, 2015). 2.3.1. Stratigraphy and seismostratigraphic framework 2.3.1. Stratigraphy and seismostratigraphic framework 3.1. Overview & data acquisition Di­ amonds and triangles indicate locations of RD2 drilling locations and gravity coring sites respectively. Orange outline indicates acquisition during cruise leg MSM78. Black outline indicates acquisition during cruise leg POS518. In support of the STEMM-CCS and CHIMNEY projects, four research expeditions were undertaken for data collection at the Scanner pockmark complex. 2D seismic reflection and refraction data, for use in travel time tomography, were acquired using a GI gun source and 18 ocean bottom seismographs (OBS) by RV Maria S Merian cruise MSM63 (Fig. 3a,d; Berndt et al., 2017), in addition to multi-beam ba­ thymetry data, controlled source electromagnetic (CSEM) data (Fig. 5), and Parasound sub-bottom profiling data (Fig. 2a). RRS James Cook cruise JC152 conducted a wide-ranging seismic experiment over the Scanner and Challenger pockmarks (Figs. 3 & 4). Five different seismic sources were used (Bolt and GI airguns, Squid and Duraspark surface sparkers, and a deep towed sparker; Fig. 3), which were recorded by arrays of 25 and 7 OBSs, at the Scanner and Challenger pockmarks respectively (Fig. 3a; Bull, 2017). The seismic sources were also used to generate multichannel (GI guns, surface sparkers; Fig. 2b) and single channel (deep tow sparker) seismic reflection profiles. During cruises MSM78 (Karstens et al., 2019) and RV Poseidon POS518-2 (Linke and Haeckel, 2018), sediment cores for geological and geochemical analysis were taken from within the Scanner pockmark and a reference site (Fig. 1b,c) using a gravity corer and rock drill (RD2), which acquired core to depths of ~6 and ~33 m below seafloor (mbsf) respectively. Additionally, data from a 3D seismic survey conducted by PGS (Fig. 2c; CNS Mega Survey Plus) were used to further support the study. visco-elastic properties. So long as the geological boundaries correspond to changes in physical properties controlling seismic wave propagation, reflection data imaging provides us with a representation of subsurface sedimentary and tectonic structures with a resolution equal to a fraction of the propagated wavelength (Kallweit and Wood, 1982). Sub-vertical fluid escape structures and sediment deformation (e.g., sediment mobilisation and polygonal faulting) can therefore be imaged, poten­ tially also allowing for a relative dating of geological events by inter­ preting cross-cutting structures and stratal stacking patterns. l Changes in pore fluid type, especially partial gas saturation, have a strong influence on seismic velocities and the absorption of seismic wave energy (White, 1975; Domenico, 1977). 3.1. Overview & data acquisition We have investigated an exemplar natural fluid escape system located near the centre of the Witch Ground Basin, located 190 km off the north-east coast of Scotland (Fig. 1a), within licence block 15/25 of the North Sea. Here, chimneys are observed, which underlie active natural methane venting sites at the seabed, within ~150 m water depth (Fig. 1b) - the Scanner, Challenger and Scotia pockmark complexes (Gafeira and Long, 2015). Pockmarks are seabed depressions, created by release of over-pressured pore-water and gas from the subsurface The methods used to investigate chimneys and their associated fluid flow can be divided into several types (Table 1). These include: seismic reflection imaging, ocean-bottom seismic methods, including seismic tomography and anisotropy analysis, controlled source electromagnetic surveying (CSEM), active acoustics, passive seismic monitoring, sedi­ ment sampling of both the target site and onshore analogues, laboratory rock physics experiments, and process-based numerical modelling and fluid dynamic modelling approaches. 3 International Journal of Greenhouse Gas Control 106 (2021) 103245 A.H. Robinson et al. Fig. 1. STEMM-CCS and CHIMNEY study area. a) Regional map of northern North Sea plotted over GEBCO (2019) bathymetry. Location of the Scanner and Goldeneye experiment sites and active Sleipner CCS storage site are labelled. Yellow box indicates loca­ tion of UK North Sea licence box 15/25. Dashed black line de­ marcates boundary between UK (to W) and Norway (to E) exclusive economic zones (200 nm). b) Ship-acquired swath ba­ thymetry of the study area, around Scanner and Challenger pock­ mark complexes. Dashed black box indicates location of inset. Inset: Detailed bathymetry of Scanner pockmark complex. Di­ amonds and triangles indicate locations of RD2 drilling locations and gravity coring sites respectively. Orange outline indicates acquisition during cruise leg MSM78. Black outline indicates acquisition during cruise leg POS518. Fig. 1. STEMM-CCS and CHIMNEY study area. a) Regional map of northern North Sea plotted over GEBCO (2019) bathymetry. Location of the Scanner and Goldeneye experiment sites and active Sleipner CCS storage site are labelled. Yellow box indicates loca­ tion of UK North Sea licence box 15/25. Dashed black line de­ marcates boundary between UK (to W) and Norway (to E) exclusive economic zones (200 nm). b) Ship-acquired swath ba­ thymetry of the study area, around Scanner and Challenger pock­ mark complexes. Dashed black box indicates location of inset. Inset: Detailed bathymetry of Scanner pockmark complex. 3.1. Overview & data acquisition The bulk effect of gas accu­ mulation within sediment pores is a reduction in the sediment compressibility and, therefore, acoustic impedance (T´oth et al., 2014). Typically this results in strong acoustic impedance contrasts, visible in the seismic data as negative polarity reflections known as bright spots (Cevatoglu et al., 2015). More generally, pore gas manifests itself as local increments in the subsurface reflectivity, as a function of the properties of the encasing medium and the local partial gas saturation (Berndt, 2005; Cartwright, 2007; Løseth et al., 2009; Andresen, 2012; Karstens and Berndt, 2015). Frequency-dependent attenuation and ve­ locity dispersion can also be observed in ultra-high-frequency (in the order of kHz) data, as the seismic frequency approaches the gas bubble characteristic frequency, allowing the detection of gas migration irre­ spective of the presence of reflective anomalies (T´oth et al., 2015). Therefore, not only do reflection data allow us to image potential gas migration pathways in the subsurface, but also to detect gas accumu­ lation pockets and potentially quantify their volume. 3.2.1. Introduction Multichannel seismic reflection data record the wavefield reflected from physical discontinuities in the subsurface at a range of distances from an active source of acoustic energy. By doing so, they carry in­ formation about seismic wave propagation velocity and anomalies in However, as a consequence of the strong energy partitioning at the interface with gas-charged sediments, acoustic blanking may be observed within chimneys (Fig. 2b,c; Fader, 1997), which hampers the 4 International Journal of Greenhouse Gas Control 106 (2021) 103245 A.H. Robinson et al. A.H. Robinson et al. 0.5 km 1 km 1 km SF SF SF 200 220 240 260 200 450 350 400 300 250 100 500 400 300 200 600 a) b) c) TWT (ms) TWT (ms) TWT (ms) 1 3 1 2 5 4 1 2 5 3/4 4 3 5.2 5.1 0 + + + Flank collapse Scanner pockmark Gas accumulation at pockmark base Glacial feature (MSGL) Glacial tunnel valley Sub-vertical fluid migration from top of unit 1/2 Gas accumulation at top of unit 1/2 Acoustic blanking Seabed multiple Seismic chimney/pipe Chaotic, dipping reflectors Seismic chimney/pipe - - Fig. 2. Seismic stratigraphy of the Scanner pockmark region, after B¨ottner et al. (2019). Profiles shown extend from southwest to northeast across the Scanner pockmark complex. a) Sub-bottom profiler seismic reflection data. b) 2D seismic reflection data acquired using Sparker source. c) 3D seismic reflection data. Interpreted seismic units S1 to S5 are shown. S1 – Aberdeen Ground Fm., S2 – Ling Bank Fm., S3-4 – Coal Pit Fm. (S3 – Coal Pit & S4 – Last Glacial Maximum deposits (LGM)), S5 – Witch Ground Fm. (S5.1 – lower Witch Ground Member, S5.2 – upper Witch Ground Member). Light blue line = top S1; green dashed line = top S2; pink dashed line = top S3; blue line = top S4; green solid line = top S5.1 and yellow line = top S5.2 / SF = Seafloor. Outline of a chimney is displayed with white dashed line. as pockmarks and chimneys, this assumption represents a potentially significant source of error. Whilst a chimney can be assumed, in a simple case, to be radially symmetrical around the depth axis, there potentially may be out-of-plane reflections that affect imaging. 3.2.1. Introduction Three-dimensional (3D) seismic data have substantially advanced the knowledge of sub­ surface fluid migration features, which were previously often discarded as poorly imaged zones and seismic artefacts in 2D seismic data (Cart­ wright and Huuse, 2005). The analysis of 3D seismic data is an effective method to map fluid accumulations in the subsurface, identify perme­ ability barriers, and constrain subsurface geometries of entire fluid flow systems (Cartwright et al., 2007; Løseth et al., 2009; Andresen, 2012; Karstens and Berndt, 2015). Where only 2D seismic data are available, application of 3D processing of 2D acquisition geometries (e.g., Lin et al., 2019) has proved extremely useful for improving the quality of the final results, provided that variations of the streamer position during acquisition are monitored within the desired accuracy (Whiteside et al., 2013) or estimated from the data (Clay and Vardy, 2018). TWT (ms) + - However, the resolution of conventional seismic data, laterally (often above 12.5 m) and vertically (~10 m for a dominant frequency of 40 Hz), is often not sufficient to image the seismic expression of fluid flow systems in detail. Recent developments in 3D high-resolution seismic techniques allow imaging of the shallow subsurface in much greater detail than previously (e.g., Planke et al., 2009; Petersen et al., 2010; Plaza-Faverola et al., 2015). An example technology is the P-Cable (e.g., Planke et al., 2009), which has been used to map shallow gas ac­ cumulations, gas hydrate systems, and fluid flow structures, such as chimneys (e.g., Plaza-Faverola et al., 2011; Bünz et al., 2012) down to a resolution of 3 m. In contrast to conventional seismic frequencies of ~5–120 Hz (suitable for monitoring deep reservoirs), high resolution P-Cable uses frequencies of up to 350 Hz, surpassing the resolution of conventional 3D seismic. 1 km SF 500 400 300 200 600 TWT (ms) 1 2 5 3/4 + Gas accumulation at top of unit 1/2 Chaotic, dipping reflectors Seismic chimney/pipe - + - Collectively, the seismic experiments conducted at the Scanner pockmark complex utilise different imaging resolutions and depths, by applying a multiple-frequency 2D surveying approach using a number of seismic sources (Fig. 3b; Bull et al., 2018). Progressive extension of the seismic bandwidth to higher frequencies and shorter shot intervals re­ sults in higher vertical and lateral resolution. 3.2.1. Introduction Surface sparker, deep towed sparker, and sub-bottom profiler data (the latter is single-channel, and may be considered a hydro-acoustic technique, but we include it here with the other seismic approaches as it is used for the same pur­ pose) with frequencies up to several kHz reveal near surface structural features previously not discernable using lower frequency sources, such as sediment slumping/flank collapse within the pockmark, more detailed characterisation of gas accumulation at the top of the Aberdeen Ground Fm., and direct observation of fluid migration towards the base of the pockmark (Fig. 3a,b). Therefore, multi-frequency surveys with different sources (e.g., Bolt Gun, GI Gun, Chirp, Boomer, and Sparker) and high-resolution 2D or P-cable streamers can complement conven­ tional 3D seismic data (e.g., Fig. 3c) and lead to a better understanding of the nature and internal architecture of chimneys, particularly in the shallow overburden stratigraphy. Fig. 2. Seismic stratigraphy of the Scanner pockmark region, after B¨ottner et al. (2019). Profiles shown extend from southwest to northeast across the Scanner pockmark complex. a) Sub-bottom profiler seismic reflection data. b) 2D seismic reflection data acquired using Sparker source. c) 3D seismic reflection data. Interpreted seismic units S1 to S5 are shown. S1 – Aberdeen Ground Fm., S2 – Ling Bank Fm., S3-4 – Coal Pit Fm. (S3 – Coal Pit & S4 – Last Glacial Maximum deposits (LGM)), S5 – Witch Ground Fm. (S5.1 – lower Witch Ground Member, S5.2 – upper Witch Ground Member). Light blue line = top S1; green dashed line = top S2; pink dashed line = top S3; blue line = top S4; green solid line = top S5.1 and yellow line = top S5.2 / SF = Seafloor. Outline of a chimney is displayed with white dashed line. effectiveness of seismic reflection imaging and inversion in such con­ texts. As a result, wide-angle transmission data recorded at the seabed, for example on OBS, may also be required, in order to characterise seismic velocities below the gas layer. Wide-angle transmission also helps resolve the issue of ambiguity between the position of the reflector and the true interval velocity along the wave-path, since the short offset range and the limited bandwidth of seismic reflection data result in a limited sensitivity to interval velocities (Jannane et al., 1989). 3.2.1. Introduction There­ fore, integration with diving wave travel time tomography, in addition to reflection waveform inversion techniques (e.g., Brossier et al., 2015) and seismic-to-well calibration are necessary to compensate for the lack of sensitivity to the kinematic properties of the medium, and provide an accurate depth representation. 3.2.3. Advanced seismic wavefield analysis i In addition to providing reflectivity images of the subsurface, multi- channel seismic data attributes, such as amplitude as a function of offset, can also be exploited to infer kinematic and dynamic properties of the subsurface (Ostrander, 1984). Pre-stack waveform inversion techniques (Virieux and Operto, 2009) can be applied to obtain quantitative char­ acterisation of the effect of partial gas saturation on the elastic proper­ ties (P-wave velocities and Poisson’s ratio). In particular, partial gas saturation has a strong effect on bulk modulus, associated with a rela­ tively low influence on shear modulus, which corresponds to a lowering in P-wave impedance and a reduction of Vp/Vs ratio (Ostrander, 1984; T´oth et al., 2014; Provenzano et al., 2018). These contrasts can in turn be exploited to quantify gas accumulation using appropriately calibrated rock-physics models (T´oth et al., 2014; Provenzano et al., 2018). 3.2.2. 2D vs 3D seismic reflection l Two-dimensional (2D) seismic imaging assumes that subsurface properties are invariant with respect to the direction normal to the survey line. In the case of inherently three-dimensional structures, such 5 International Journal of Greenhouse Gas Control 106 (2021) 103245 A.H. Robinson et al. Table 1 Table 1 Summary of the methods used for the characterisation of a chimney, displaying the assessed parameters and the method specific traits. The parameters used for complete characterisation of a chimney include: 1) fracture geometry, orientation, connectivity, and subsurface structure; 2) physical properties – e.g., porosity, permeability, and resistivity; and 3) fluid presence, distribution, composition, and flux/flow rate. A method can (green) or cannot (red) be used to assess a given parameter. The traits of each method include: a) temporal applicability - whether the method can be applied at different times to observe temporal variability at the site; b) co-dependencies - whether input from another method is required for calibration and/or constraint; c) time and resource intensiveness - whether data can be produced with a given amount of resources on the timescale of weeks (low), months (medium) or greater than six months (high); and d) cost - a relative economic cost scale for completion (that can be qualitatively described as a method requiring desk time (low), laboratory time (medium) and/or ship time (high)). Summary of the methods used for the characterisation of a chimney, displaying the assessed parameters and the method specific traits. The parameters used for complete characterisation of a chimney include: 1) fracture geometry, orientation, connectivity, and subsurface structure; 2) physical properties – e.g., porosity, permeability, and resistivity; and 3) fluid presence, distribution, composition, and flux/flow rate. A method can (green) or cannot (red) be used to assess a given parameter. The traits of each method include: a) temporal applicability - whether the method can be applied at different times to observe temporal variability at the site; b) co-dependencies - whether input from another method is required for calibration and/or constraint; c) time and resource intensiveness - whether data can be produced with a given amount of resources on the timescale of weeks (low), months (medium) or greater than six months (high); and d) cost - a relative economic cost scale for completion (that can be qualitatively described as a method requiring desk time (low), laboratory time (medium) and/or ship time (high)). Fig. 3. Layout of the Scanner pockmark seismic experiments. a) OBS locations for JC152 seismic tomography, anisotropy, and passive seismic experiments (white triangles), and MSM63 seismic tomography experiment (orange triangles). b) Far-field source frequency spectra of the seismic sources used in the JC152 to­ mography and anisotropy experiments. 3.2.4. Time-lapse/4D seismic reflection Vestnesa Ridge, offshore W Svalbard (Bünz et al., 2012; Smith et al., 2014; Panieri et al., 2017), and for experimental fluid injections, including the Sleipner storage site (e.g., Arts et al., 2004; Chadwick et al., 2004, 2019; Boait et al., 2012; Eiken, 2019), the QICS experiment (Cevatoglu et al., 2015), and the STEMM-CCS Goldeneye release experiment (Flohr et al., 2021; Roche et al., in review). Time-lapse seismic data therefore play a key role in the understanding of both naturally occurring fluid flow, and CCS monitoring in pre-, active and post-CO2 injection phases (Lumley, 2010). Time-lapse seismic imaging refers to the acquisition of seismic data at the same location at different points in time, in order to assess tem­ poral changes in the subsurface. Where both seismic datasets are 3D, this may be referred to as 4D seismic imaging. This technique has been applied extensively to subsurface reservoir monitoring, including con­ ventional and unconventional hydrocarbon production (e.g., Watts et al., 1996; Landrø et al., 1999; Barkved et al., 2003). Changes in the subsurface due to fluid flow may change properties such as fluid satu­ ration, temperature, porosity, and pressure, and, hence, the elastic and stress-strain properties and behaviour of the rock, which cause a change in seismic response (Johnston, 2013). Such temporal changes can be used to better understand the formation and development of fluid flow features, as well as providing constraints for multiphase thermo-hydro-mechanical simulations. Recognising temporal changes in subsurface fluid flow systems is integral for monitoring CO2 storage reservoirs and potential leakage in the overburden. Fig. 4. Example OBS data from the JC152 seismic tomography and anisotropy experiments. All records shown are from OBS 1, located within Scanner pockmark, and are approximately E-W oriented (red lines, Fig. 3c). a) Z geophone component, GI source. b) X & Y geophone components, GI source, used for seismic anisotropy analysis. c)-f) Z component (c) Bolt, (d) GI, (e) Duraspark sparker, and (f) Squid sparker, plotted with reduction velocity of 2 km s−1. Note the different horizontal and vertical scales for the airgun vs. sparker sources. Bolt and GI airgun records plotted with a 5-15 Hz low-pass filter. Sparker records plotted with a 100-200 Hz low-pass filter. Arrows show location of emergent (refracted) arrivals. Table 1 c) Seismic acquisition tracks for JC152, with sources labelled, showing the multi- azimuthal geometry of source coverage. Only the airgun (Bolt and GI) and surface sparker (Duraspark and Squid) sources are shown. Blue box shows extent of area shown in a). Red line in­ dicates location of the profiles shown in Fig. 4. d) Acquisition track for the MSM63 acquisition. Fig. 3. L OBS loca passive se tomograp frequency mography tracks fo azimutha and GI) a shown. B dicates lo for the M Fig. 3. Layout of the Scanner pockmark seismic experiments. a) OBS locations for JC152 seismic tomography, anisotropy, and passive seismic experiments (white triangles), and MSM63 seismic tomography experiment (orange triangles). b) Far-field source frequency spectra of the seismic sources used in the JC152 to­ mography and anisotropy experiments. c) Seismic acquisition tracks for JC152, with sources labelled, showing the multi- azimuthal geometry of source coverage. Only the airgun (Bolt and GI) and surface sparker (Duraspark and Squid) sources are shown. Blue box shows extent of area shown in a). Red line in­ dicates location of the profiles shown in Fig. 4. d) Acquisition track for the MSM63 acquisition. International Journal of Greenhouse Gas Control 106 (2021) 103245 A.H. Robinson et al. Fig. 4. Example OBS data from the JC152 seismic tomography and anisotropy experiments. All records shown are from OBS 1, located within Scanner pockmark, and are approximately E-W oriented (red lines, Fig. 3c). a) Z geophone component, GI source. b) X & Y geophone components, GI source, used for seismic anisotropy analysis. c)-f) Z component (c) Bolt, (d) GI, (e) Duraspark sparker, and (f) Squid sparker, plotted with reduction velocity of 2 km s−1. Note the different horizontal and vertical scales for the airgun vs. sparker sources. Bolt and GI airgun records plotted with a 5-15 Hz low-pass filter. Sparker records plotted with a 100-200 Hz low-pass filter. Arrows show location of emergent (refracted) arrivals. 3.3. Seismic tomography and full waveform inversion The P- and S-wave velocity structure of a chimney and its sur­ roundings can be determined by applying travel time tomography and full waveform seismic inversion. These techniques are applied with the principal aims of: locating gas-bearing zones; delineating the shape of the chimney; determining the presence and features of fractures (open or cemented, size and connectivity); and characterising the sediment properties within and outside the chimney. A principal challenge in time-lapse/4D seismic surveying is ac­ counting for the repeatability, such that the produced images represent true temporal changes and not seismic artefacts associated with acqui­ sition and/or processing. Attributes such as the normalised root-mean square noise are used to measure quantitatively the quality of the sur­ vey repeatability. Generally, repeatability is excellent where sediments are well stratified and undisturbed (e.g., Waage et al., 2019). However, since chimneys are often much more chaotic seismic structures, repeatability can be poor, and detection of changes in fluid flow between individual time-lapse surveys requires careful interpretation (Waage et al., 2019). Seismic travel time tomography is an inversion technique in which observed travel times (e.g., Fig. 4b) are compared to those computed through a discretised and parameterised model representation of the subsurface, with defined parameters that control the balance between minimising the data misfit and generating a model with the minimum required structure to fit the data. Travel time tomography approaches may be isotropic (e.g., Zelt and Barton, 1998) or anisotropic (e.g., Dunn et al., 2005), where in the latter the direction and magnitude of velocity anisotropy is fit and calculated directly, whereas in the former the anisotropy manifests as travel time residuals which can be analysed to determine the model anisotropic properties (Dunn and Toomey, 2001). Travel time tomography is a relatively low-resolution (normally Seismic travel time tomography is an inversion technique in which observed travel times (e.g., Fig. 4b) are compared to those computed through a discretised and parameterised model representation of the subsurface, with defined parameters that control the balance between minimising the data misfit and generating a model with the minimum required structure to fit the data. 3.3. Seismic tomography and full waveform inversion Travel time tomography approaches may be isotropic (e.g., Zelt and Barton, 1998) or anisotropic (e.g., Dunn et al., 2005), where in the latter the direction and magnitude of velocity anisotropy is fit and calculated directly, whereas in the former the anisotropy manifests as travel time residuals which can be analysed to determine the model anisotropic properties (Dunn and Toomey, 2001). Travel time tomography is a relatively low-resolution (normally Time-lapse and/or 4D seismic data were not acquired at the Scanner pockmark complex. This technique has, however, been applied to the studies of actively-seeping chimneys, such as at Lomvi pockmark on the 7 International Journal of Greenhouse Gas Control 106 (2021) 103245 A.H. Robinson et al. Fig. 5. Scanner pockmark CSEM experiment. a) Bathymetry map with data acquisition profiles (black) and OBEM receiver locations (circles). Data in c-e) are shown for profiles and OBEM instruments coloured in yellow (c-d) and red (e), respectively. b) Sketch of survey setup, with towed DASI source, towed Vulcan receivers and three component OBEM seafloor receivers. c-d) Example data at 1 Hz from N to S trending profile (yellow line) for the closest towed receiver c) and the OBEM d) (yellow circle on a). The x axis shows the source position (black antenna with white field lines in b)) along profile, with the location of Scanner pockmark set at 0 m. The OBEM in d) is located at ~700 m along profile. The observed data (black dots), the vertical electric field amplitude (Ez) and the total horizontal electric field amplitude (Pmax) respectively, are compared to 2D (c), towed receiver) and 1D (d), OBEM) forward modelled data (coloured lines) for a subsurface with half-space resistivities of 1 to 2 Ωm. The towed receiver data, sensitive to the top ~50-100 m, agree well with predicted data for a 1.2 Ωm subsurface resistivity, while the OBEM data at larger offset to the source agree better with higher resistivities, indicating an increase in resistivity with depth. In c), the location of the grey box corresponds to observed Ez values when the transmitter is above the pockmark. In d), a slight increase of Pmax at ~-200-400 m (highlighted with grey box), however, may indicate a localised increase of resistivities in the subsurface. e) OBEM instrument Pmax data example from SW to NE trending profile (red line). 3.3. Seismic tomography and full waveform inversion i pockmark and 7 at a nearby reference site, located ~1 km southeast of the pockmark, where industry 3D seismic data showed no evidence of the presence of gas (Fig. 3a). These instruments recorded shots from all of the seismic sources used in the experiment (Fig. 3c). Fig. 4c-f shows the shots recorded on an OBS located within Scanner pockmark for an ~E–W oriented profile for each of the airgun and surface sparker sources. The final OBS tomography model can be used as the initial model for full waveform inversion of OBS data (FWI; e.g., Virieux and Operto, 2009). FWI uses the entire recorded seismograms, and, therefore, in­ cludes amplitude and phase information instead of solely using the travel times. As a result, FWI represents a higher resolution technique, which is now widely used in crustal-tectonic (e.g., Morgan et al., 2013; G´orszczyk et al., 2017; Davy et al., 2018) and engineering (e.g., Smi­ thyman et al., 2009) contexts, in addition to being widely adopted across the hydrocarbon sector (e.g., Sirgue et al., 2010; Prieux et al., 2013). The effectiveness of FWI is highly sensitive to the starting model being capable of predicting the travel time within half a dominant period, in order to avoid cycle-skipping (Virieux and Operto, 2009). This approach is, therefore, commonly preceded by travel time tomography. While travel time tomography approaches are not necessarily reliant on a priori information, it is important to consider additional con­ straints, where available, since a poorly constrained inversion may produce a biased result, based on its parameterisation and/or starting model (Zelt et al., 2003). Interpreted multi-channel seismic profiles may be used to inform the initial P-wave velocity model. In turn, the results of travel time tomographic modelling can be employed to improve the processing and interpretation of multi-channel seismic profiles, by providing independent velocity information. The wider-angle trans­ mission regime involved in OBS methods also assists in resolving the issue of acoustic blanking beneath gas accumulations, such as those observed beneath Scanner pockmark (Fig. 2c), and provides a further tool for the verification of whether observed chimneys represent actual subsurface geological features, or reflection imaging artefacts. l Two phases of data acquisition (Fig. 3a) were conducted at the Scanner pockmark complex to acquire data suitable for the application of first arrival seismic travel time tomography and full waveform inversion. 3.3. Seismic tomography and full waveform inversion At about 100-400 m from the Scanner pockmark position, a localised increase of Pmax is observed, potentially related to an increase in resistivity in the subsurface in the vicinity of the seismic chimney. Fig. 5. Scanner pockmark CSEM experiment. a) Bathymetry map with data acquisition profiles (black) and OBEM receiver locations (circles). Data in c-e) are shown for profiles and OBEM instruments coloured in yellow (c-d) and red (e), respectively. b) Sketch of survey setup, with towed DASI source, towed Vulcan receivers and three component OBEM seafloor receivers. c-d) Example data at 1 Hz from N to S trending profile (yellow line) for the closest towed receiver c) and the OBEM d) (yellow circle on a). The x axis shows the source position (black antenna with white field lines in b)) along profile, with the location of Scanner pockmark set at 0 m. The OBEM in d) is located at ~700 m along profile. The observed data (black dots), the vertical electric field amplitude (Ez) and the total horizontal electric field amplitude (Pmax) respectively, are compared to 2D (c), towed receiver) and 1D (d), OBEM) forward modelled data (coloured lines) for a subsurface with half-space resistivities of 1 to 2 Ωm. The towed receiver data, sensitive to the top ~50-100 m, agree well with predicted data for a 1.2 Ωm subsurface resistivity, while the OBEM data at larger offset to the source agree better with higher resistivities, indicating an increase in resistivity with depth. In c), the location of the grey box corresponds to observed Ez values when the transmitter is above the pockmark. In d), a slight increase of Pmax at ~-200-400 m (highlighted with grey box), however, may indicate a localised increase of resistivities in the subsurface. e) OBEM instrument Pmax data example from SW to NE trending profile (red line). At about 100-400 m from the Scanner pockmark position, a localised increase of Pmax is observed, potentially related to an increase in resistivity in the subsurface in the vicinity of the seismic chimney. hundreds of m to km laterally, hundreds of m vertically) imaging tech­ nique, with the resolution ultimately controlled by the shot and receiver spacings and further being dictated by the discretisation and parame­ terisation of the model. 3.4.2. Seismic attenuation While S-wave velocity anisotropy, observed through SWS analysis, is sensitive to both open and closed fracture networks, attenuation anisotropy is primarily sensitive to open fracture networks and the fluids that may be present within these (e.g., Worthington and Hudson, 2000; Chapman, 2009). Attenuation may occur through mechanisms including scattering and wave-induced fluid flow (Baird et al., 2013). Scattering of seismic waves due to aligned heterogeneities has long been recognised to be frequency dependent (e.g., Shapiro and Hubral, 1995; Werner and Shapiro, 1999). Estimating fracture sizes from narrow-band observations of seismic anisotropy may lead to misinterpretations, as there is an ambiguity whereby a medium containing a small number of large fractures will generate the same response as a medium containing a larger number of smaller fractures (e.g., Maultzsch et al., 2003). Therefore, a frequency-dependent approach to studying anisotropy, which is sensi­ tive to the length-scale of the causative mechanism for the anisotropy, is required. Properties such as fracture scale length and fluid saturation can then be inferred from the frequency-dependence of anisotropic at­ tributes, as predicted by theoretical work (Chapman, 2003; Jakobsen and Chapman, 2009). The impact of partial saturation on anisotropy and attenuation in materials of known fracture density and orientation has been studied using models that can link laboratory and field datasets (Amalokwu, 2016), which are required to make robust determinations of permeability. Field observations of attenuation anisotropy have been reported from walkaround vertical seismic profile data (Varela et al., 2006; Maultzsch et al., 2007; Bouchaala et al., 2019), with the observed effects often being consistent with the predictions of poroelastic models ac­ counting for wave-induced fluid flow (Chapman, 2003). If wave-induced fluid flow is the dominant mechanism for the observed attenuation anisotropy, then the phenomenon may provide a link to fracture induced permeability. Chapman (2009) considered the case of multiple sets of fractures having different fluid connectivity. l Application of seismic attenuation and attenuation anisotropy anal­ ysis at Scanner pockmark was directed at investigating both the geom­ etry (cf. SWS) and contents of subsurface fracture networks, where these are present. As with travel time anisotropy, the broad-band frequency nature of the various seismic sources used is required to discriminate between the properties of fractures of different sizes, which may otherwise not be distinguished. The Scanner pockmark anisotropy experiment used several seismic sources with different frequencies, in the range ~10 Hz to 2 kHz, as described above (Fig. 3b). 3.3. Seismic tomography and full waveform inversion The first survey, conducted during cruise MSM63, utilised an array of 18 OBSs, with shots generated using two GI airguns. The OBS and profile locations are shown in Fig. 3a & d respectively. A second, more extensive experiment was conducted during cruise JC152, coin­ cident with the anisotropy experiment (described in section 2.4, below), and utilising an array of 18 OBSs located in and around Scanner l The two seismic approaches, multi-channel seismic reflection imag­ ing and tomography, are highly complementary to one another and may be utilised most effectively when applied synchronously. The results of seismic tomography are also complementary to laboratory scale rock physics experiments, since they place constraints on larger scale in situ 8 A.H. Robinson et al. International Journal of Greenhouse Gas Control 106 (2021) 103245 which may arise in the presence of concentric fractures. physical properties, and, thus, fracture orientations and fracture density (Amalokwu et al., 2017; Jin et al., 2018), which then can be used to complement the geo-mechanical models. The orientation of these symmetry planes and the directions of the fast and slow S-wave arrivals can be used to determine the fracture orientations. The measured delay between the fast and slow S-wave arrivals is used to determine the intensity of anisotropy, which is related to the fracture size and/or density (e.g., Crampin, 1985; Mueller, 1992; Li, 1997). Raw estimates of SWS give only depth-averaged estimates of fracture properties. Therefore, to determine the depth variation of both fracture orientation and density, a layer-stripping approach is required (Haacke et al., 2009), which recursively compensates and removes the anisotropy measured in shallow layers. Shear-wave splitting analysis is applied to the study of the chimney beneath Scanner pockmark in order to map the geometry and extent of the fracture network. This approach allows us to distinguish between different hypotheses for the structure of chimneys. 3.4.3. Ambient noise anisotropy Methods for analysing surface waves from ambient noise utilise lower frequencies, typically <1−2 Hz, than the active seismic sources. This approach further extends the frequency range of frequency- dependent analyses to determine fracture properties. Rayleigh wave velocities can be measured on the vertical and hydrophone components of the OBS. By taking spectrograms of cross-correlations between OBS pairs (processed according to Bensen et al., 2007) located on opposite sides of the chimney, Rayleigh wave phase-velocity dispersion can be measured (Yao et al., 2006) and used to invert for vertical shear-wave velocity structure. Applying Radon transforms to a set of cross-correlations between all possible OBS pairs within an array and summing these over time, a technique known as 2D beamforming (Lacoss et al., 1969), shows the slowness at which Rayleigh waves cross the array at all azimuths. This method facilitates full-azimuth observa­ tions of anisotropy (Alvizuri and Tanimoto, 2011). 3.4. Seismic anisotropy Fractures in sedimentary settings play a crucial role in defining the physical properties of subsurface reservoirs, as they enhance porosity and permeability, or conversely may contribute to reservoir compart­ mentalisation. Thus, fracture orientation, size, volumetric density, and connectivity are of interest to the understanding of subsurface fluid flow. Attribute analysis of stacked seismic images, which includes techniques such as coherence analysis (e.g., Bahorich and Farmer, 1995), can detect larger fractures. However, these techniques are unable to image smaller fractures, below the spatial resolution of the seismic image. Therefore, in order to determine fracture properties at sub-seismic scale, seismic anisotropy analysis can be applied, which utilises the directional dependence of transmitted seismic signals. A range of theories have been developed to describe the elastic response of fractured rocks (e.g., Hudson, 1981; Thomsen, 1995). While these theories generally agree for dry rock, they differ considerably where fluids and fluid flow between cracks and pores are present (Liu et al., 2000). 3.4.2. Seismic attenuation This broadband dataset was specifically designed for the measurement of frequency-dependent anisotropy, to permit enhanced fracture characterisation. In order to achieve maximal azimuthal coverage, which is necessary for determining the direction­ ality of anisotropy, profiles were acquired at multiple orientations through the OBS array (Fig. 3c). Several approaches are available for investigation of frequency-dependent anisotropy associated with chim­ neys. Details of the two active source and one passive source methods that were used at Scanner pockmark are provided below. 3.6. Passive seismic methods Passive seismic monitoring involves the use of seismometers and sound recording devices, such as OBSs, located at the seafloor. These instruments provide continuous recordings, and, therefore, capture events during seismic acquisition interludes. In the Scanner pockmark seismic experiment, passive seismicity was recorded using the same OBS array as for the anisotropy experiment (Fig. 3a). The CSEM method is sensitive to changes in the bulk electric re­ sistivity structure of the Earth, a property that depends on lithology and mineral composition but is particularly sensitive to connected porosity and pore fluid content (Palacky, 1988). Due to this sensitivity to sedi­ ment pore fluids, the marine CSEM method has been widely used for hydrocarbon exploration (e.g., Ellingsrud et al., 2002; Constable, 2010). Electrical resistivity is also an indicator for whether pathways for fluids to the seabed exist, as the presence of aligned permeable and conductive fractures may lead to electrical anisotropy (e.g., Naif et al., 2015). The CSEM method is sensitive to changes in the bulk electric re­ sistivity structure of the Earth, a property that depends on lithology and mineral composition but is particularly sensitive to connected porosity and pore fluid content (Palacky, 1988). Due to this sensitivity to sedi­ ment pore fluids, the marine CSEM method has been widely used for hydrocarbon exploration (e.g., Ellingsrud et al., 2002; Constable, 2010). Various types of events were detected using this approach, including: short (<1 s) duration events, similar to those previously detected in gas seepage areas (Tary et al., 2012; Bayrakci et al., 2014; Batsi et al., 2019), and which may represent collapses and/or the formation of small pockmarks; medium (few seconds) duration events, comparable to a volcano-tectonic tremor (e.g., Latter, 1981; Harrington and Brodsky, 2007), rarely observed in this tectonic context, and which may represent the movements of fluids in conduits in the shallow subsurface; and events related to bubbles escaping at the seafloor. The low frequency content displayed by these types of events (<30 Hz) means it may also be possible to identify these events during periods of, for example, sparker seismic acquisition, due to the much higher frequency content of the source signal (≥200 Hz). Electrical resistivity is also an indicator for whether pathways for fluids to the seabed exist, as the presence of aligned permeable and conductive fractures may lead to electrical anisotropy (e.g., Naif et al., 2015). 3.6. Passive seismic methods y py g The CSEM survey at Scanner pockmark was conducted during cruise MSM63, using the University of Southampton deep-towed active source instrument (DASI; Sinha et al., 1990), towed at 20−40 m above the seafloor. The source signal comprised a ~100 A, 1 Hz square wave. Twelve profiles were acquired, oriented in four different azimuths to assess the electrical anisotropy. Two different types of receiver were utilised to record the induced field: an array of 14 ocean bottom elec­ tromagnetic (OBEM) field receivers (6 three-component receivers and 8 horizontal-component receivers; Fig. 5a), here measuring the electric field only, and two three-axis Vulcan electric field receivers towed behind the source (Fig. 5b; Constable et al., 2016). The CSEM data from both receiver types are processed with a Fourier transform over 1 s-long windows to obtain amplitude and phase data in the frequency domain, and stacked over 60 s-long windows to improve the signal-to-noise ratio (Myer et al., 2011). Estimating the electrical resistivity distribution from the data requires the use of modelling procedures. For the towed re­ ceivers (Fig. 5c), 2D forward modelling for a homogeneous subsurface with one resistivity value (half-space) is performed to compare the modelled data to the observed vertical electric field amplitude (Ez) data and estimate the subsurface resistivity. For the seafloor receivers (Fig. 5d,e), the magnitude of the major axis of the polarisation ellipse traced by the electric field, Pmax, is compared to 1D forward modelling results for half-space resistivities. Pmax is independent of the receiver orientation. Resistivities appear to increase with depth. Inversion algo­ rithms (e.g., Constable et al., 1987) which optimise the fit between the observed and predicted data are required to estimate a heterogeneous resistivity structure (analysis for the towed receivers is presented in Gehrmann et al., in review). For recorded seismicity associated with gas bubble escape at the seafloor, the Minnaert (1933) equation can used to estimate the bubbles’ radii from the frequencies of the events. However, this approach may not be able to distinguish between the acoustic signature of many small bubbles and one big bubble, which highlights the necessity to calibrate this approach using additional methods (e.g., active acoustics). Passive monitoring of seismicity associated with methane venting and/or fluid movement in the subsurface at Scanner pockmark aims to quantify the gas flux through the chimney to the seabed and into the water column. 3.4.1. Shear-wave splitting The measurement of seismic travel time anisotropy using shear-wave splitting (SWS) is an established technique for determining the orien­ tation and density of fracture networks (e.g., Crampin, 1985). Shear-wave splitting occurs when a polarised shear wave enters an anisotropic medium. When this occurs, the shear wave is split into two orthogonal components, oriented perpendicular and parallel to the fracture normal direction. The two split shear waves travel at different speeds through the fractured medium, resulting in differences in their travel time. SWS analysis uses converted P-to-S waves to determine the orientation of a symmetry axis associated with the fracture normal di­ rection. SWS distinguishes between different anisotropic symmetry systems, which produce characteristic patterns that can be observed in transformed radial and transverse components of the horizontal geophone records (Fig. 4b). This approach can be used to differentiate between dominant horizontal transverse isotropy, which may be asso­ ciated with vertically aligned fractures, and vertical transverse isotropy, However, there are limitations on the frequencies that can be observed using passive methods, which arise due to the OBS array ge­ ometry. The largest spacings within the array, termed the aperture, used at Scanner pockmark result in a lowest usable frequency of ~0.5 Hz (Rost and Thomas, 2009). For beamforming there are also challenges arising from the array geometry. While the OBS array located at the Scanner pockmark is relatively symmetrical, the array at the reference 9 A.H. Robinson et al. International Journal of Greenhouse Gas Control 106 (2021) 103245 geological knowledge or constraints from complementary data. For example, seismic reflection data can provide high resolution imaging of geological stratigraphy and structures, while CSEM is able to detect the presence and type of fluids in the pore space, such as hydrocarbons, CO2, and variations in salinity. The difference between changes in pore fluid or lithological variations may be distinguished by interpreting CSEM and seismic data in conjunction (e.g., Hoversten et al., 2006). Geological interpretations benefit from combined analysis with, for example, wide-angle seismic data (e.g., Goswami et al., 2015), seismic reflection data (e.g., Weitemeyer et al., 2011; Attias et al., 2016; Berndt et al., 2019), magnetic data (Gehrmann et al., 2019), and well logs (e.g., Harris et al., 2009). site is not, and so has a highly asymmetric response in the slowness domain, which causes aliasing effects that can cause artificial anisotropy to be observed. 3.5. Controlled source electromagnetic surveying The CSEM method is, overall, a relatively low-resolution method (10−1000 m), due to the diffusive nature of EM fields. The specific resolution and penetration depth depend on the source-receiver geom­ etry, the number of receivers at different offsets to the source, and the frequency spectrum of the source signal (Edwards, 1988). The dual-receiver approach used here includes towed instruments sensitive to the shallow subsurface and OBEM receivers sensitive to the deeper subsurface. For the purposes of understanding potential subsurface fluid escape structures, the CSEM method provides a tool to map variations in resistivity related to connected porosity and fluid/gas content of seafloor sediments. The marine controlled source electromagnetic (CSEM) method is used for mapping variations in the electrical resistivity of the subsurface (e.g., Cheeseman et al., 1987; Edwards, 2005). The CSEM method used in this study involves towing a horizontal electric dipole close to the seabed that transmits an alternating electromagnetic field. The elec­ tromagnetic energy diffuses through the seawater and seafloor and is recorded by electric field receivers that are towed in-line behind the transmitter and others that are stationary on the seafloor (Constable, 2013). The measured field amplitude and phase lag between source and receivers relate to the electrical resistivity of the seafloor. When more resistive Earth materials are encountered (e.g., low porosity sediments), elevated electric fields are observed at the receivers and the phase lag is reduced. 3.4.1. Shear-wave splitting For analysis of azimuthal dependence to be effective, any artificial anisotropy must be removed (Picozzi et al., 2010). i Cross-correlation analysis and 2D beamforming can be used to observe the dispersion and azimuthal dependence of Rayleigh waves and can be inverted for 1D shear wave velocity structure at Scanner pockmark in the 0.5–1.5 Hz range. This technique contributes both to constraining the structure of the chimney and extending the range of frequencies which can be used for considering frequency-dependent anisotropy characteristics. 3.7. Active acoustic methods Hydro-acoustic surveying involves the use of either single- or multi- 3.6. Passive seismic methods Unlike active acoustic methods, this approach represents a continuous monitoring tool. The microseismic events detected through passive monitoring may be associated with the local tidal cycle and, hence, may indicate a possible correlation between tides and the movement of fluids from a subsurface reservoir to the seabed. Further­ more, if the seismicity is a result of fluid movement at depth, it may be applicable to studies of fluid flow beneath the cap rock or overburden, where the fluids do not directly reach the seabed. There are trade-offs between the thickness and resistivity of anom­ alous features (e.g., Edwards, 1997) that can be overcome using prior Hydro-acoustic surveying involves the use of either single- or multi- 10 International Journal of Greenhouse Gas Control 106 (2021) 103245 A.H. Robinson et al. beam echosounders, which transmit one or more beams, respectively, of monochromatic frequency beneath the ship. The delay associated with the returning pulse is typically used to map the underlying bathymetry, while the strength or amplitude of the returning pulse can provide in­ formation on the physical properties of the reflecting medium. Given the strong impedance contrast between water and gas, hydro-acoustic methods are sensitive to the presence of gas bubbles in the water col­ umn (Fig. 6). The gas bubbles rise due to buoyancy, drawing sur­ rounding fluid into and upwards with the plume, with lateral dispersion as a function of bubble rising velocity and water current velocity. The gas plume comprises bubbles of various sizes, with larger bubbles pos­ sessing relatively higher rising velocity and smaller bubbles possessing relatively lower rising velocity. The plume can be observed to rise to the thermocline at ~40 m below sea surface (Fig. 6; B¨ottner et al., 2019; Li et al., 2020). resolution, visual observation of changes in sediment texture and fab­ ric. Changes in sediment colour, e.g., the presence of black layers that form when methane is consumed by anaerobic methane oxidation with sulphate, and the structure of the sediment, e.g., the presence of out­ gassing and fluidisation structures, provide evidence for the presence of gas in the sediment, as well as evidence for palaeo-fluid migration. Further, non-invasive 3D analysis of sediment structure is possible using XCT imaging techniques (Fig. 7b–e), which can highlight the spatial distribution of dense materials such as iron sulphides that can form during anaerobic methane oxidation (Fig. 7c,e). 3.6. Passive seismic methods Geochemical analyses of pore fluids can be used to determine their origin and, hence, the presence of migration pathways and the connectivity of different layers. Geochemical analyses of the solid phase allow understanding of the chemical reactions that are occurring or have occurred in the sediments and can explain physical properties, such as changes in permeability caused by carbonate or iron sulphide precipitation. Methane derived authigenic carbonates in the sediments and on the seafloor are in­ dicators for prolonged presence of fluid flow, and can be dated to derive information about fluid flow history. Multi-beam data can be used to map the three-dimensional extent of gas plumes escaping from pockmarks and, across multiple surveys, observe variations over time. Seabed currents may cause the plume to separate based on differing bubble size distributions, which can be mapped using multi-beam data (Li et al., 2020). Similarly, observing the target strength of gas plumes in single-beam data can be used to estimate the bubble size distribution and gas flux being released from the pock­ mark, provided a range of frequencies covering the bubble resonance frequency are used (Li et al., 2020). Sediment cores from the Scanner pockmark complex and a reference site located ~7 km to the east (Fig. 1b,c) were recovered using the BGS Rockdrill (RD2) and a gravity corer during cruises MSM78 and POS518- 2 (Fig. 7b). RD2 is a remotely operated multi-barrel wireline subsea robotic sampling system that continuously cores 1.72 m-long sections, with a diameter of 6.1 cm. The maximum drilling depth was 33 mbsf at the reference site. Gravity cores were collected from the uppermost 5.75 m of the sediment column using a 12.5 cm-diameter tube driven into the seabed under weight-assisted free-fall (Linke and Haeckel, 2018). Active hydro-acoustic surveying was applied in the study of the active venting at the Scanner pockmark in order to quantify the gas flux from the seabed and to study the gas plume evolution in the water column. Repeat surveys can also be used to determine if there is any temporal variation, for example due to tidal cycles (e.g., Boles et al., 2001; Schneider von Deimling et al., 2010). Temporal patterns may also be compared with observations from passive seismics, which provide a continuous record of gas venting at the seabed, or of the migration of fluids at depth. 3.6. Passive seismic methods Immediately following collection of the sediment cores, pore waters and sediment samples were extracted from both RD2 and gravity cores. Pore waters were collected with 0.2 μm-pore diameter Rhizons (See­ berg-Elverfeldt et al., 2005; Dickens et al., 2007) at approximately 30 cm depth intervals, and preserved for analyses of cations (e.g., dissolved Ca, Fe, Mn, Si, B), anions (e.g., Cl, SO4, total alkalinity), nutrients (NH4, PO4, Si, NOx), and dissolved sulphides. Samples of the sediments were collected with cut-off 5 cm syringes and analysed for their methane concentrations and methane carbon isotopic composition, porosity, grain size, and organic and inorganic carbon content. The cores were then scanned using a GEOTEK multi-sensor core logger (MSCL; Fig. 7a), to measure acoustic wave velocity, density, resistivity, and magnetic susceptibility, before being sectioned horizontally, photographed, and logged. The chemostratigraphy of the sediment was then measured in high resolution (1 mm) with the ITRAX XRF core scanner. 3.8.1. Drilling and coring This frequency band complements the ultrasonic frequencies obtained by the multi-flow CCS rig and helps to constrain improved rock physics models accounting for fluid mobility and frequency-dependent seismic effects (Batzle et al., 2006; Mavko et al., 2009). In addition, the P- and S-wave frequency ranges of the acoustic pulse tube measurements partially overlap with the sparker sources used for the seismic anisotropy assessment of the Scanner pockmark (Fig. 3b), permitting the calibration of the seismic data under controlled conditions in the laboratory. While coring and drilling approaches allow very high (cm-scale) vertical resolution, the horizontal resolution is limited by the number of sediment cores that can be taken within the time frame of an expedition. Therefore, appropriate sampling strategies are required to effectively integrate the information from sediment cores with information gath­ ered using other approaches. For the purpose of our study, previously acquired seismic, real-time fast-track processed seismic lines, and prior information about the shallow geology were used to inform our drilling strategy. However, at the Scanner pockmark drilling locations, RD2 settings were configured for recovery of authigenic carbonates, rather than unconsolidated sediments, and, as a result, sediment recovery was rather low (~50 %), due to washout of loosely consolidated sand intervals. The experimental approach used provides a geophysical and hy­ dromechanical assessment of fluid flow in (i) CO2 reservoir rocks and (ii) the shallowest section of the marine sediment overburden. In the former case, the reservoir rock experiments simulate variable spatial-temporal states during (drainage) and post- (imbibition) CO2 injection, consid­ ering homogeneous (background) and fractured (slip-related features) reservoir samples. In the latter case, the tests focus on the hydrodynamic response of near-seabed sediments subjected to variable CO2 flow rates. The integration of sediment core and geophysical data maximises their inherent value, by providing localised sub-seismic resolution site- specific constraints (Vardy, 2015; Vardy et al., 2017; Provenzano et al., 2018). Multi-sensor core logging of sediment cores permits the measurement of geophysical and geotechnical parameters, providing a way to: calibrate the two-way-time seismic reflection data to depth; constrain seismic inversion and interpretation within a range of plau­ sible values (e.g., Provenzano et al., 2018); interpret and cross-validate the results of seismic tomography and CSEM methods; and interpret physical properties in terms of changes in sediment properties, such as porosity (e.g., Vardy, 2015; Baasch et al., 2017). 3.8.1. Drilling and coring While coring and drilling approaches allow very high (cm-scale) CO2 system: (1) multi-flow Firstly, the multi-flow C signatures (ultrasonic P- and of reservoir rocks subjecte controlled reservoir pressure the hydromechanical chan CO2–brine fluid substitution the acoustic pulse tube rig and attenuation of P-waves quency range of 1−10 kHz a complements the ultrasonic Fig. 7. Gravity cores of sediments recovered from directly beneath East Scanner Pockmark (GC17; Fig. 1c) during the MSM78 expedition. The cores were analysed using a) Multi-Sensor Core Logging (MSCL), which includes density data and b-e) X-ray micro-CT analysis. b) and d) Analysis of a 25 cm length x 12.5 cm diameter core section composed of fine silt. Coarser grained horizons (lighter grey) show evidence of fluidisation structures and layering, picked out by the dotted lines. c) Same image as b) but with lower density material removed, showing the presence of disseminated iron sulphide (bright spots, close-up shown in e) that has precipitated within the coarser grained hori­ zons. The presence of iron sulphide is indicative of transport of methane-rich fluids through the coarser grained sediment horizons. introduced during the drilling procedure. This is particularly important for soft, non-lithified sediments such as those found in our study area, as the coring process may reduce porosity, due to dewatering compaction and shear straining (Bouma and Boerma, 1968; Blomqvist, 1985). Concentrations of some reactive geochemical parameters (e.g., dissolved Fe, dissolved inorganic carbon, H2S and CH4) may further be affected by outgassing or chemical oxidation during the retrieval and subsampling of the cores. These effects can be minimised by subsampling immedi­ ately after core recovery, or by the use of in-situ measurement tech­ niques such as lab-on-chip sensors (e.g., Whitesides, 2006). CO2 system: (1) multi-flow CCS and (2) acoustic pulse tube rigs. Firstly, the multi-flow CCS rig permits the analysis of geophysical signatures (ultrasonic P- and S-wave attributes, and electrical resistivity) of reservoir rocks subjected to brine and CO2 flow-through tests at controlled reservoir pressure and temperature conditions, resulting from the hydromechanical changes occurring in the rock sample due to CO2–brine fluid substitution effects and rock-fluid reactivity. Secondly, the acoustic pulse tube rig allows the measurement of seismic velocity and attenuation of P-waves in sediment and rock samples over a fre­ quency range of 1−10 kHz and S-waves at 500 Hz. 3.8.1. Drilling and coring Appropriate calibration of the instruments is necessary for a meaningful comparison between samples acquired using different coring techniques and core liner ma­ terials, and the need to account for laboratory temperature and humidity conditions. l A study of reservoir rocks was conducted using natural reservoir samples (Utsira sand formation, Sleipner; Falcon-Suarez et al., 2018), and quartzose synthetic sandstones manufactured in the lab following the procedure described in Falcon-Suarez et al. (2019), including ho­ mogeneous (Falcon-Suarez et al., 2016, 2017) and fractured samples (Mu˜noz-Ib´a˜nez et al., 2019). In all tests, an increasing CO2-to-brine partial flow condition was imposed through the originally brine-saturated samples, in order to progressively saturate the sample in CO2. The confining stress, pore pressure, and temperature conditions slightly varied between tests, within the ranges 16−40 MPa, 7−12 MPa, and 20−35 ◦C, respectively. The fluid substitution process and related hydromechanical response of the rock were assessed though the continuous monitoring of ultrasonic P- and S-wave velocity and atten­ uation, electrical resistivity, axial and radial strains, and hydraulic conductivity. 3.8.1. Drilling and coring Direct sampling of seabed sediments via coring allows for a local characterisation of lithology, porosity, and other geophysical and geochemical attributes at centimetre-scale or less resolution, providing a verification of geophysical properties and parameterisation for numer­ ical models. Detailed description of sediments in terms of their lithology, physical, and chemical properties allows determination of the geological origin of the sediments, the extent and characteristics of specific strati­ graphic units, the possible origin of the pore fluids, and a very high- Some caution is required during core logging and interpretation, as artefacts such as small-scale fractures and other disturbances can be Fig. 6. Hydro-acoustic imaging of the Scanner pockmark gas plume, using EM712 echosounder data at a frequency range of 40-100 kHz. Highest backscatter is displayed in red (seabed). The gas plume is clearly visible as a strong (green) zone of backscatter emanating from the pockmark, rising to the thermocline at ~40 m below the sea surface. Figure adapted from B¨ottner et al. (2019). Fig. 6. Hydro-acoustic imaging of the Scanner pockmark gas plume, using EM712 echosounder data at a frequency range of 40-100 kHz. Highest backscatter is displayed in red (seabed). The gas plume is clearly visible as a strong (green) zone of backscatter emanating from the pockmark, rising to the thermocline at ~40 m below the sea surface. Figure adapted from B¨ottner et al. (2019). 11 International Journal of Greenhouse Gas Control 106 (2021) 103245 International Journal of Greenhouse Gas Control 106 (2021) 103245 A.H. Robinson et al. introduced during the drilling procedure. This is particularly important for soft, non-lithified sediments such as those found in our study area, as the coring process may reduce porosity, due to dewatering compaction and shear straining (Bouma and Boerma, 1968; Blomqvist, 1985). Concentrations of some reactive geochemical parameters (e.g., dissolved Fe, dissolved inorganic carbon, H2S and CH4) may further be affected by outgassing or chemical oxidation during the retrieval and subsampling of the cores. These effects can be minimised by subsampling immedi­ ately after core recovery, or by the use of in-situ measurement tech­ niques such as lab-on-chip sensors (e.g., Whitesides, 2006). 3.8.2. Geomechanical testing and sediment core analysis 3.8.2. Geomechanical testing and sediment core analysis Rock physics experiments permit the direct examination of the changes in geophysical, geomechanical, and geochemical sample prop­ erties under controlled conditions in the laboratory. Two rock physics experimental setups were used in combination for the study of coupled thermo-hydro-mechano-chemical phenomena occurring in a rock-brine- Fig. 9 shows a comparison between the results obtained using a synthetic (Falcon-Suarez et al., 2016) and a natural (Falcon-Suarez et al., 2018) sandstone, with similar composition and porosity. The arrival of CO2 in the rock samples lead to increased resistivity, together with a 12 A.H. Robinson et al. International Journal of Greenhouse Gas Control 106 (2021) 103245 Roberts et al., 2017). Following the cessation of active fluid flow, a number of diagenetic and surface processes may alter the physical properties of the rocks, which can include post-dewatering compaction, cementation, groundwater-induced geochemical alteration, weathering, and erosion. XCT techniques can be utilised to acquire 3D digital rock images of the outcrop samples, whereby the cement can be selectively removed using image processing techniques, allowing the study of the physical and hydraulic properties of the rock formation prior to cementation (Fig. 8d; e.g.. B¨ottner et al., in review). ll sudden drop (>5%) in P-wave velocity and increase in the attenuation (more significant in the synthetic sample), while subsequently increasing the CO2 content had only limited further effects. Forced imbibition is used to study the trapping efficiency of the rock through evaluation of the residual CO2 content and the geophysical signatures associated with this value. In this regard, Mu˜noz-Ib´a˜nez et al. (2019) studied the hydrodynamic behaviour of a fractured reservoir analogue sample, and found that non-connected fractures may act as larger cav­ ities than background porosity, and these cavities are energetically favourable for CO2 storage. Therefore, non-connected fractures have the potential to enhance CO2 trapping and the overall sealing efficiency of CO2 storage reservoirs (Mu˜noz-Ib´a˜nez et al., 2019), in contrast to the role played by a connected fracture network (i.e., the hypothesised structure of a chimney). There is often a discrepancy between the scales of fluid flow features observed in onshore outcrops and their seismic manifestations, whereby chimneys observed in seismic data can extend several hundreds of me­ tres in diameter and up to 1000 m in height (Berndt, 2005; Cartwright et al., 2007; Løseth et al., 2009; Andresen, 2012; Karstens and Berndt, 2015). Outcrops of sand-filled fractures in California (Fig. 3.8.2. Geomechanical testing and sediment core analysis 8c), which are sand intrusions that form due to exceedance of lithostatic pressure and subsequent remobilisation and fluidisation of sediments (Huuse et al., 2010; Hurst et al., 2011), have been observed to reach similar di­ mensions to their corresponding seismic discernible features (Huuse et al., 2005; Ross et al., 2014). However, the largest known field out­ crops of hydrocarbon-derived carbonate conduits, located in Varna, Bulgaria, are only up to 10 m high and ~1 m in diameter, and are spread over a comparatively small area (2.4 × 105 m2; De Boever et al., 2006a, 2006b). The lack of outcrop evidence for fracture systems that extend up to 1000 m in vertical height and with large spatial extent is likely a consequence of the preferential preservation of hard parts, as suggested by the observed sand-filled fractures and carbonate pipes in California and Varna respectively (De Boever et al., 2006a; Hurst et al., 2011). However, a benefit of field outcrop analogues is that sub-vertical structures can be mapped at mm-scale resolutions, which is not possible using seismic reflection imaging. Consequently, more accurate quantification of fault/fracture properties, such as measurement of aperture or cement infill type, is achievable using outcrop based studies. The study of outcrops can be complemented with the mapping of a target area, using unmanned aerial vehicles (UAVs) that allow mapping over large distances (km-scale) at very high accuracy (cm-scale). These data can then be used to calculate digital elevation models, image mosaics, and high-resolution point clouds which, once geo-referenced using ground control points, can help close the observational gap between seismic data and geological field sampling. In addition, gravity core samples from cruise POS527 (Achterberg and Esposito, 2018) were used to conduct flow-through experiments, to analyse the coupled hydro-mechanical-chemical response of seafloor sediments subjected to sudden gas release episodes (e.g., pockmarks). These experiments included (i) a set of permeability tests to monitor the sediment reactivity to CO2 (i.e. dissolution/precipitation), and (ii) a simulation of the STEMM-CCS CO2 release experiment (Flohr et al., 2021) to collect linked geophysical-hydromechanical information about the target site under controlled conditions in the laboratory. The data collected during these experiments indicate permeabilities of ~10−16 m2 in the upper part of the sediment column. 3.10. Process-based numerical modelling Process-based numerical modelling, for simplicity called hereafter numerical modelling, can be used to provide quantitative insights into: how chimneys form and propagate through time and space; the distri­ bution and transport of mass (water, CO2, CH4, higher hydrocarbons, salts, grains etc.) and energy (heat) components among different phases (solid, liquid, gas) through pores and fractures; the complex coupled physical and chemical processes between the above components and properties; and the changes in petrophysical (e.g., porosity and perme­ ability) and chemical properties (e.g., dissolved gas distribution in the formation fluid brine). Therefore, numerical modelling approaches are useful in helping to quantitatively understand the governing processes that can explain observations, but they are, in turn, reliant on appro­ priate calibration relating to the target of interest. 3.8.2. Geomechanical testing and sediment core analysis Preliminary analyses of reactive transport models (Marín-Moreno et al., 2019) indicate that the sediment is largely unreactive to the exposure of CO2 saturated brine, while the grain size may be conditioning the CO2 distribution within the sediment; from clay- to sand-rich layers the distribution becomes more homogeneous (Roche et al., in review). For further understanding of hydraulic fracturing and gas migration mechanisms, flow-through experiments were carried out on gravity core samples recovered from locations adjacent to the STEMM-CCS CO2 release site during the RV Poseidon cruise POS534 (Schmidt, 2019). Combining geomechanical testing with XCT imaging (Fig. 10d; Deusner et al., 2016) allows the visualisation of fracture formation in response to pressure accumulation and fluid injection. Fracture formation in the poorly consolidated clay sediments started at a low critical pressure of ~5−15 kPa above the confining stress (Fig. 10a). Fracture propagation was essentially perpendicular to the major principal stress direction, likely resulting from the formation of disk-shaped gas bubbles with high aspect ratios, as expected in clay sediments (Boudreau et al., 2005). Fracture formation was first observed from rapid gas pressure dissipa­ tion (Fig. 10b, event 4) and the appearance of a small gas accumulation in the XCT image projections (Fig. 10c). Mass balancing of the injected gas and changes in confining volumes after fracture opening until the end of the first injection interval indicate a high apparent fracture permeability (Fig. 10b). Flow-through experiments and coupled thermo-hydro-chemo-mechanical studies can provide important insight into fracture formation and propagation in natural sediments, and are an important tool to develop and validate numerical models of chimney evolution. However, mechanical and hydraulic constraints in small-scale laboratory experiments will not provide an exact replication of the in-situ field environment, which must be carefully considered during experimental planning and data interpretation. 3.9. Onshore outcrop analogues As part of the STEMM-CCS and CHIMNEY projects, reactive transport modelling has been conducted (by Marín-Moreno et al., 2019) to examine the migration of injected CO2 through a generic chimney, using Scanner pockmark as a proxy for pressure, temperature, geological, mineral, and chemical conditions. In the model, the chimney extends upwards in the subsurface reservoir through the cap rock to a shallowest depth of 400 mbsf, where it meets the overburden (Fig. 11a). Modelling was conducted in a radially symmetric geometry approximation, with injection of CO2 taking place at 500 mbsf and 0 m radial distance. The time scale of this analysis is 100 years, longer than potential geological In addition to direct sampling of an actively venting system, further characterisation and the long-term development of fluid escape struc­ tures can be determined from analysis of onshore outcrop analogue systems. Field outcrops permit characterisation of physical structure, geometry, and physical properties, and understanding of past physical, chemical, and hydro-mechanical processes, through direct observation and sampling. However, fluid escape outcrop analogues are challenging features for comparative analysis with active subsurface systems (e.g., 13 International Journal of Greenhouse Gas Control 106 (2021) 103245 A.H. Robinson et al. Fig. 8. Field outcrop analogue of a fluid escape system in the Panoche Hills, Central California. a) Map of the Panoche Hills field location. b) Lithostratigraphy of the Panoche Hills which includes the Panoche Fm., comprised of up to 6.5 km of turbiditic sandstone, overlain by greater than 600 m of mudstone and siltstone cap rock/overburden sealing units. c) Map view of sand filled fractures intruding sub-vertically through the sealing units of the Moreno Fm. The map view is analogous to the spatial extent mappable with UAVs. d-f) Direct sampling and observation of a sand-filled fracture at different length scales, which includes d) pore-scale XCT analysis and e) core-scale laboratory analysis. CO2 injection activities. CO2 injection activities. properties, and fluid pathways to a turbulent ocean. In addition, AnsdMF can be applied over a wide range of scales, ranging from micro/ pore- to field-scale (≤10−2 to ≥103 m). The observations from compu­ tational fluid dynamic models can be calibrated against observations derived from active seismics, which provide a measure of fluids expelled from the seabed, and passive seismics, which can additionally detect flow within the subsurface, to compare flux rates of subsurface fluid migration with observed discharge from the seabed. 4. Discussion - A multi-scale, multi-disciplinary approach 4. Discussion - A multi-scale, multi-disciplinary approach The modelling conducted assumes that the injection of CO2 occurs far away from the chimney, in isothermal conditions, and that there are no mechanical changes (Marín-Moreno et al., 2019). However, the non-isothermal upwards migration of CO2 may create stress- and thermal-induced deformations that could affect the aperture of existing fractures, or create new fractures, and so, in turn, affect the migration processes. Thus, assessment of the impact of thermal, hydraulic, and mechanical changes of fractures within chimneys may require further consideration. l 3.9. Onshore outcrop analogues Further, bio- ecosystem models could be coupled with AnsdMF and advanced oceanic hydrodynamic models to assess the impacts of fluid migration on benthic ecosystems and in the water column (Blackford et al., 2020). CO2 injection activities. Modelling indicates that, over these timescales, the porosity (Fig. 11b) and permeability (Fig. 11c) changes due to dissolution and precipitation of minerals are negligible for the representative chimney being modelled (Marín-Moreno et al., 2019). Hence, for this particular location and over the time scale of this analysis, the modelling of chemical reactions (Fig. 11d,e) can be dismissed. For injected CO2 sat­ urations of ≥30 %, and with overburden isotropic matrix permeabilities and chimney effective vertical permeabilities (average permeability of the matrix and the fractures in the chimney) of ≥10−14 m2, the models indicate that CO2 reaches the seabed, at 500 m above the injection point, in less than 100 years (Fig. 11a,f). 4.1. Parameters Electrical resistivity, P-wave velocity, VP, and P-wave attenuation, QP −1, are normalised with respect to the values at the original reservoir conditions (16.4 and 7 MPa of confining and pore pressure, respec­ tively), and plotted together with volumetric strain, εv, versus pore volume (PV, i.e. injected fluid volume normalised to the sample pore volume). The results show the effect of rock consolidation on the elastic and transport properties of reservoir formations with similar petrophysical features but different degrees of cementation (i.e., weakly-cemented Utsira sand formation versus silica- cemented synthetic sandstone), and the evolution with the increasing CO2 content. Figure adapted from Falcon-Suarez et al. (2018). A.H. Robinson et al. International Journal of Greenhouse Gas Control 106 (2021) 103245 A.H. Robinson et al. A.H. Robinson et al. A.H. Robinson et al. frequency-dependent attenuation is a factor in measurements made using a particular method, Table 2 must be considered in this context. An optimal subsurface imaging approach would assess parameters at the highest resolution possible over the areal and depth extent of the chimney. However, where these features are located at depth, beneath overburden and sealing layers, attenuation of higher frequencies results in a depth-resolution trade-off, with increasingly only larger features visible. This represents a key motivation both for the application of a multi-frequency surveying approach in the Scanner pockmark seismic experiment, utilising a range of seismic sources (e.g., airguns, sparkers), and the study location chosen. Together, these allow the development of a cross-scale, sufficiently resolved image of a chimney at shallow depth, where it can be studied in detail, with other techniques (laboratory experiments, numerical modelling) then able to extend the observations to the deeper subsurface, where they may be detected by geophysical techniques at lower resolution. The use of a broad seismic source fre­ quency range for the anisotropy experiment is also critical, due to the frequency dependence of anisotropy detection and interpretation (Chapman, 2003). In addition, some of the methods display differing sensitivities to the magnitudes of the properties being measured (e.g., Constable, 2010). For example, while the elastic properties of sediments are sensitive to both lithological and pore fluid variations, their electrical resistivity is dominated by the pore fluid component. The differing response of these two techniques as gas saturation varies has been shown by Constable (2010) for a 50 % porosity sand. 4.1. Parameters In this paper, a number of methods have been identified which can be utilised and integrated to generate a comprehensive characterisation of subsurface fluid pathways associated with chimneys. The parameters influencing fluid flow within chimneys can be broadly divided into three categories: 1) structural properties including the subsurface strati­ graphic structure, the presence, geometry, distribution and connectivity of fractures, and principal stress magnitude and orientation; 2) physical properties of the sediments, including thermal, hydraulic, mechanical, elastic, and electrical properties; and 3) fluids, which includes their presence, phase, and distribution in the subsurface, chemical composi­ tion, and volume/mass flux calculations (Table 1; Fig. 13). In general, at least one approach from each of the method groupings (Table 1 left-hand column) is required to reach a sufficiently detailed and holistic under­ standing of the structure and processes involved in the venting of gas via a chimney. ii The dynamics of fluids migrating within the shallow overburden sediments can also be assessed using a Navier-Stokes-Darcy multi-fluid flow model (AnsdMF), which simulates the mass and momentum ex­ changes of multi-fluid phases (Behzadi et al., 2004; Bannari et al., 2008) and couples the turbulent flows in the ocean with the flows in sediments (Fig. 12a). The AnsdMF modelling was conducted in 2D, and comprises a sediment layer with a defined thickness, porosity, and fracture proper­ ties (length and aperture; Fig. 12b). These subsurface characteristics may be further constrained using other methods discussed in this study. Above the seabed is a turbulent bottom boundary layer. The location and rate of gas release beneath the seabed are then specified and the gas is tracked as it migrates and disperses through the sediment, and is expelled from the seabed (Fig. 12c). i We have also identified five method traits: resolution, length scale, temporal applicability, co-dependency, and resource use. The method parameters and traits are summarised in Table 1 and are further dis­ cussed below. The AnsdMF model is robust and efficient for simulations of multi- fluid flow in sediments with a complex structure and variable fracture 14 Fig. 9. Laboratory flow-through experimental results. Brine−CO2 flow-through tests were conducted for two samples, a 38 % porosity synthetic sandstone (Syn) and an intact core sample from Utsira Sand formation (Sleipner field). The tests simulate cyclic overpressure scenarios (ΔPP) in a Sleipner-like CO2 storage reservoir during pre-injection, CO2 injection (drainage) and post-injection (imbibition) stages. 4.2. Resolution and scale Table 2 shows (a) the order of magnitude resolutions and (b) length scales (area and/or depth) achievable for each method applied to the study of a chimney. Table 2a demonstrates that there is a resolution gap between the geophysical (seismic, acoustic, CSEM) and geological/ geochemical (rock sampling, laboratory) methods around the m-scale. Numerical modelling approaches allow bridging of the resolution gap, by the integration of data from the geophysical, geological and geochemical methods. Repeat geochemical data collection and seismic surveying can be conducted, commonly on the time scale of every year or few years. However, measurement of true temporal changes using seismic data, rather than seismic artefacts, is limited by the repeatability of the surveying technique. This makes time-lapse seismic studies most suit­ able for application to areas of well stratified and undisturbed sedi­ ments, rather than chaotic features such as chimneys (e.g., Waage et al., 2019). Developments in time-lapse seismic repeatability, such as continued use of true-4D imaging, or the installation of permanent seabed sensing infrastructure such as OBS or fibre optic cables, have the potential to improve this. l There is an inherent trade-off between resolution and length scale, whereby methods capable of achieving higher spatial resolution can typically only be applied at reduced length scales. This is particularly an issue in the vertical dimension. This trade-off arises in seismic and CSEM studies due to an intrinsic relationship between the imaging resolution and depth scale, whereby lower frequency sources have greater depth penetration, due to the stronger attenuation of higher frequency signals over lower frequencies (Resnick, 1990). However, vertical imaging resolution is also related to bandwidth, with lower bandwidth signals resulting in a lower vertical resolution than higher bandwidth signals. Taking the seismic approaches utilised in this study as an example, the lower frequency airgun sources generate the best depth penetration (≥100 s of m), but are limited to high m to 10 s of m or greater vertical resolution, while sparker sources may only penetrate a few 100 s of m, but can generate m-scale resolution, and sub-bottom profiler recording (which we include here with seismic approaches as they are applied for the same purpose) can achieve resolutions on the dm-scale, but with only very limited (10 s of m) depth penetration. 4.3. Temporal applicability The integrated methods described in this paper also influence the temporal scales over which fluid flow through chimneys can be under­ stood (Table 1). Only a few of the methods can be used to determine temporal information related to pore fluid changes in the subsurface (measured at discrete time intervals using time-lapse/4D seismic), fluxes through the subsurface (measured discretely by active acoustics, and measured/modelled continuously using passive seismics and computa­ tional fluid dynamics), and fluid-induced chemical and physical changes (geochemical methods and numerical modelling). 4.1. Parameters The largest effect on P-wave velocity occurs for the first few percent of gas fraction and changes very little above this (White, 1977; Lee, 2004). In contrast, the resistivity response increases exponentially with increases in gas saturation (Archie, 1952). As a result, gas saturations determined using seismic and CSEM methods may differ (e.g., Goswami et al., 2015; Attias et al., 2020; Bialas et al., 2020), with CSEM being more sensitive to the wider saturation-range response measurable. Fig. 9. Laboratory flow-through experimental results. Brine−CO2 flow-through tests were conducted for two samples, a 38 % porosity synthetic sandstone (Syn) and an intact core sample from Utsira Sand formation (Sleipner field). The tests simulate cyclic overpressure scenarios (ΔPP) in a Sleipner-like CO2 storage reservoir during pre-injection, CO2 injection (drainage) and post-injection (imbibition) stages. Electrical resistivity, P-wave velocity, VP, and P-wave attenuation, QP −1, are normalised with respect to the values at the original reservoir conditions (16.4 and 7 MPa of confining and pore pressure, respec­ tively), and plotted together with volumetric strain, εv, versus pore volume (PV, i.e. injected fluid volume normalised to the sample pore volume). The results show the effect of rock consolidation on the elastic and transport properties of reservoir formations with similar petrophysical features but different degrees of cementation (i.e., weakly-cemented Utsira sand formation versus silica- cemented synthetic sandstone), and the evolution with the increasing CO2 content. Figure adapted from Falcon-Suarez et al. (2018). 4.2. Resolution and scale Therefore, where Passive seismic and active acoustics can resolve temporal flow var­ iations on the order of seconds to weeks, providing information on the ascent of individual bubbles, but also how flow is modulated by diurnal tides. Of these, however, only passive seismic provides continuous temporal information over extended periods of time. These two methods have, therefore, different applicabilities to short- and longer-term monitoring activities of fluid escape associated with chimneys. Both methods also play an important role in the calibration of computational fluid dynamic models of fluid migration through the chimney and out of the seabed. These models can then be adapted to provide further 15 International Journal of Greenhouse Gas Control 106 (2021) 103245 10. Fracture formation due to gas migration in poorly consolidated clay sediments. Flow through experiments were carried out on gravity core samples vered during RV Poseidon cruise POS534 (Schmidt, 2019). Injection experiments were carried out with N2 because of its low solubility compared to CO2. a) Gas sure and confining stress. b) Pressure-volume relations during and after fracture formation. c) X-ray projection images at different time points. d) Setup of the y micro-CT imaging system. Gas was injected in intervals (dark blue arrows) followed by pressure dissipation periods. Fracture formation was rapid after gas sure increased above the confining stress (1). After reaching a critical pressure for fracture formation, an increase in confining stress (2) triggered confining fluid oval (3). The gas pressure dissipated rapidly after fracture opening (4), which subsequently resulted in the accumulation of gas at the sample surface. The offset een confining stress and gas pressure during ongoing gas injection (5) likely resulted from measurement inaccuracies because all system components were rated igh-pressure operation. Robinson et al. International Journal of Greenhouse Gas Control 106 (2021) 103245 A.H. Robinson et al. Robinson et al. Fig. 10. Fracture formation due to gas migration in poorly consolidated clay sediments. Flow through experiments were carried out on gravity core samples recovered during RV Poseidon cruise POS534 (Schmidt, 2019). Injection experiments were carried out with N2 because of its low solubility compared to CO2. a) Gas pressure and confining stress. b) Pressure-volume relations during and after fracture formation. c) X-ray projection images at different time points. d) Setup of the X-ray micro-CT imaging system. Gas was injected in intervals (dark blue arrows) followed by pressure dissipation periods. 4.2. Resolution and scale Fracture formation was rapid after gas pressure increased above the confining stress (1). After reaching a critical pressure for fracture formation, an increase in confining stress (2) triggered confining fluid removal (3). The gas pressure dissipated rapidly after fracture opening (4), which subsequently resulted in the accumulation of gas at the sample surface. The offset between confining stress and gas pressure during ongoing gas injection (5) likely resulted from measurement inaccuracies because all system components were rated for high-pressure operation. information about the temporal evolution of fluid flow processes through chimneys in different geological settings. structures. However, the successful application of individual methods relies on a network of co-dependencies between different methods for calibration and constraint (Table 1). In particular, inverse methods, such as travel time tomography and CSEM, are subject to inherent trade-offs between model parameters, such as velocity or resistivity respectively, and layer thickness (Edwards, 1997). Travel time tomography is also problematic when trying to resolve low velocity zones, as the energy is diverted away from them. As a result, use of prior geological knowledge, or constraints from other data, is important in the modelling process (e. g., Harris et al., 2009; Goswami et al., 2015; Attias et al., 2016). i Of the methods that can be used to assess changes in the properties of chimneys over time, only numerical modelling approaches can be extended to much longer timescales (centuries to millennia), compared to experimental, field or laboratory techniques. Field-based outcrop analysis may also be considered to provide additional temporal infor­ mation, allowing understanding of flow over geological timescales, long after active flow has ceased. However, the integration of these tech­ niques enables the development of new multiscale coupled seismic- hydro-mechano-chemical modelling approaches, with the potential of enhancing predictions of chimney behaviour over time. Some co-dependencies are more significant than others. Seismic reflection imaging provides constraints on several of the methods uti­ lised in this study, which explains its widespread use in subsurface exploration and monitoring programmes. Seismic reflection imaging and seismic inversion are co-dependent, although it is possible for these methods to be deployed fully independently. It is advantageous for travel time tomography to have input from reflection imaging of the 4.4. Co-dependencies Table 1 shows that a range of geophysical methods can be used to determine the site parameters of interest to the study of fluid flow 16 International Journal of Greenhouse Gas Control 106 (2021) 103245 A.H. Robinson et al. International Journal of Greenhouse Gas Control 106 (2021) 103245 Fig. 11. Reactive transport modelling of CO2 migration through a generic chimney using the North Sea, Scanner pockmark complex as a proxy for pressure, emperature, geological, mineral, and chemical conditions. The model geometry comprises CO2 injection at a radial distance of 0 m and depth below seabed of 500 m. The chimney extends vertically to the top of the cap rock (400 mbsf). Results are shown after 100 yr, with a constant injected CO2 saturation of 50 %, an overburden sotropic permeability of 10−13 m2 and chimney vertical and horizontal permeabilities of 10−13 m2, and 10-15 m2. Variation of a) CO2 saturation, b) porosity and c) permeability with depth and radial distance. Inset in a) shows the temporal evolution over 100 yr of CO2 saturation with depth at a distance of 1.25 m from the njection point. Red numbers are CO2 saturations. Changes in d) mineralogy and e) concentration of liquid species at a distance of 1.25 m from the injection point. f) Models of CO2 gas reaching the seabed in less than 100 yrs for tested injected CO2 saturations of 10 %, 30 %, and 50 %; overburden isotropic permeabilities of 10−13 m2, and 10-14 m2; and chimney vertical permeabilities ranging from 10-11 m2 to 10-16 m2 (ratios of chimney vertical to horizontal permeability from 1 to 1000 were ested). Figure adapted from Marín-Moreno et al., 2019. A.H. Robinson et al. International Journal of Greenhouse Gas Control 106 (2021) 1 Fig. 11. Reactive transport modelling of CO2 migration through a generic chimney using the North Sea, Scanner pockmark complex as a proxy for pres A.H. Robinson et al. Fig. 11. Reactive transport modelling of CO2 migration through a generic chimney using the North Sea, Scanner pockmark complex as a proxy for pressure, temperature, geological, mineral, and chemical conditions. The model geometry comprises CO2 injection at a radial distance of 0 m and depth below seabed of 500 m. The chimney extends vertically to the top of the cap rock (400 mbsf). 4.4. Co-dependencies Results are shown after 100 yr, with a constant injected CO2 saturation of 50 %, an overburden isotropic permeability of 10−13 m2 and chimney vertical and horizontal permeabilities of 10−13 m2, and 10-15 m2. Variation of a) CO2 saturation, b) porosity and c) permeability with depth and radial distance. Inset in a) shows the temporal evolution over 100 yr of CO2 saturation with depth at a distance of 1.25 m from the injection point. Red numbers are CO2 saturations. Changes in d) mineralogy and e) concentration of liquid species at a distance of 1.25 m from the injection point. f) Models of CO2 gas reaching the seabed in less than 100 yrs for tested injected CO2 saturations of 10 %, 30 %, and 50 %; overburden isotropic permeabilities of 10−13 m2, and 10-14 m2; and chimney vertical permeabilities ranging from 10-11 m2 to 10-16 m2 (ratios of chimney vertical to horizontal permeability from 1 to 1000 were tested). Figure adapted from Marín-Moreno et al., 2019. Fig. 12. Computational fluid dynamic modelling of gas leakage through sub-seabed fracture network. Modelling has been conducted for CO2 fluid escape. (a) Schematic of complex subsurface sediments, gas plume at a potential fluid escape site. (b) Simulation geometry. Lower (black) part of model is sediment (~4 m average thickness) with 1 cm thick, non-connected fractures (white). Upper part is the water column. c) Contoured gas volume fraction at time (snapshot) of gas dispersion through sediments into the ocean. Figure adapted from Saleem (2020). Fig. 12. Computational fluid dynamic modelling of gas leakage through sub-seabed fracture network. Modelling has been conducted for CO2 fluid escape. (a) Schematic of complex subsurface sediments, gas plume at a potential fluid escape site. (b) Simulation geometry. Lower (black) part of model is sediment (~4 m average thickness) with 1 cm thick, non-connected fractures (white). Upper part is the water column. c) Contoured gas volume fraction at time (snapshot) of gas dispersion through sediments into the ocean. Figure adapted from Saleem (2020). 17 A.H. Robinson et al. International Journal of Greenhouse Gas Control 106 (2021) 103245 Fig. 13. Summary of relationships between methods used and parameters investigated in the study of the chimney beneath Scanner pockmark. 4.4. Co-dependencies Triangle plot highlighting the three main parameters (Table 1) used for characterisation of a chimney/fluid escape system, where each method is located within the regions of the triangle corresponding to the parameters it can be used to resolve. subsurface in order to constrain the solution of the non-unique tomo­ graphic problem towards realistic subsurface structures. However, it is also important that modelling is conducted in an objective manner that does not overly bias the result, and suitable modelling approaches can be applied which do not require/depend on this additional starting constraint. The velocity information which is obtained from travel time tomography or FWI can be used to improve the quality of seismic reflection imaging, including assisting with migration and depth con­ version, in which accurate interval velocity information plays a central role. Furthermore, it can provide velocity information in areas of seismic blanking or potential artefacts, such as those associated with the pres­ ence of gas in the chimney. Converted-wave seismic anisotropy may also be partially reliant on seismic reflection data, to identify seismic hori­ zons where mode conversion may occur, and an independent shear- wave velocity constraint is also required to interpret from which layers these signals may be originating, and for layer-stripping (Haacke and Westbrook, 2006). Laboratory experimental approaches can provide insights into the dominant processes governing the response of the system. The experi­ mental strategy and the ability to approximate the real behaviour of subsurface chimneys, or other subsurface fluid migration pathways, depends on suitable calibration and constraints from field geophysical and geochemical experiments. Laboratory experiments are directly dependent on the quality of direct sampling. One way to minimise this dependency is to use synthetic samples to replicate subsurface material. In the study of Scanner pockmark, the main limitation of direct sampling was dewatering of the water saturated, poorly consolidated samples that Fig. 13. Summary of relationships between methods used and parameters investigated in the study of the chimney beneath Scanner pockmark. Triangle plot highlighting the three main parameters (Table 1) used for characterisation of a chimney/fluid escape system, where each method is located within the regions of the triangle corresponding to the parameters it can be used to resolve. Table 2 Summary of the resolution (a) and the length scale (b) over which each geophysical method can be used to assess parameters, for complete characterisation of chimneys. Note that for methods subject to frequency-dependent attenuation with increasing depth penetration (e.g., seismic, CSEM), we show here the full range of resolutions and length scales. Thus, as depth penetration increases, the smallest achievable resolution and length scale will also increase. 18 18 International Journal of Greenhouse Gas Control 106 (2021) 103245 A.H. Robinson et al. likely occurred during the core extraction process, leading to alterations in the physical properties of the core material. Future optimisation of the core extraction process is required, to ensure improved preservation of poorly consolidated cored material. Autoclave pressure coring tools could be used, such as the IODP Pressure Core Sampler (Graber et al., 2002) or MeBo-MDP (Pape et al., 2017), that are able to retrieve sections of a drill core under in-situ pressure, thereby avoiding disturbances of the cored sediment due to degassing that occur during conventional coring. Hence, pressure coring permits the investigation of cored ma­ terials whilst retaining the in-situ physical, structural, and chemical properties (e.g., permeability, gas content, resistivity, salinity). Furthermore, the acquisition of borehole geophysical measurements (e. g., cross borehole surveys using wireline logging tools) is another common method for obtaining in-situ measurements, provided that the drilled borehole is stabilised by a liner (e.g., Crain, 2010). The results from rock physics experiments can be used to derive and validate the results of travel time tomography, FWI, and seismic anisotropy studies. time tomography and seismic anisotropy datasets were collected using the same sets of shots, while the GI, Duraspark and Squid sparker sources were also recorded using a towed seismic streamer. Passive seismicity, for the study of fluid migration through the subsurface and ambient noise, was recorded using the same OBS array as for the JC152 tomog­ raphy and anisotropy experiments (Fig. 3a). However, the parameters of a single seismic experiment may not be able to ideally meet the needs of all the different types of analysis that may be required for full and detailed characterisation of the subsurface feature(s) of interest. For example, for ambient noise studies, larger OBS station spacings would allow lower frequencies to be utilised in the analysis (e.g., Rost and Thomas, 2009). However, this conflicts with the smaller spacing required to achieve the maximum shallow subsurface resolution in tomographic models. Table 2 In a number of cases, the time-based cost of the techniques described in this study is relatively low, due to the relative simplicity of data processing using well-established methods (e.g., seismic reflection data, active seismics, core/field sample analysis). However, while many of the methods discussed in this study are well-established and extensively used in both research and industrial settings, there is potential for future reductions in resource costs. Methods such as travel time tomography, CSEM, passive seismics, and seismic anisotropy involve significant time in manual data picking and/or modelling, resulting in high relative time costs. Future developments in data processing may permit the replace­ ment of manual processing stages with automated workflows, which may be facilitated by machine learning and artificial intelligence (e.g., Kim and Nakata, 2018; Kong et al., 2019), resulting in reduced time costs. Further, from the geophysical experiments at Scanner pockmark, it can be determined that significant processing time could be saved if OBS and OBEM spatial coordinates, degree of tilt, and orientations are more accurately constrained during data acquisition (e.g., Haacke and Westbrook, 2006). Installing permanent OBSs or fibre optic cables would allow easier and more cost-effective implementation of repeat seismic surveying, where multiple cruise legs are required. Compared to offshore exploration costs, laboratory experiments can be run at much lower expense, especially the measurement of basic parameters, while the investigation of the effect of parameter changes (e.g., temperature, pressure) on the system may also be tackled using carefully designed experiments. Multiphase and multicomponent numerical modelling of the ther­ mal, hydrological, mechanical, and chemical response of marine geological systems with chimneys is currently limited by the robustness of available in-situ geophysical and chemical characterisation methods, especially for fracture geometrical, physical, and chemical properties, and the difficulty in accurately recreating these in the models. How well individual processes are represented in the numerical modelling results depends strongly on the in-situ geophysical and geochemical data available to constrain the model input parameters. This further high­ lights that an appropriate direct rock sampling strategy is an essential part of the multi-disciplinary approach for chimney characterisation. ll Active acoustic and passive seismic methods for detecting fluid flow can be applied independently of other approaches (Fig. 13). 4.5. Resource use, challenges and development It is also important to consider resource demand in the study of focused fluid flow conduits, such as the chimneys at Scanner pockmark, and how this may impact future studies relevant to CCS operations. Resources may include both direct monetary costs associated with data acquisition, in addition to less-well defined time-based costs, which cover the data processing and interpretation. Ultimately, there is a trade- off between the field experimental methods which can be performed using a given set of resources (including time), and the total amount of information which can be determined about the subsurface structure. Offshore site characterisations are generally spatially discrete snap­ shots in time. Undertaking studies of this nature requires field expedi­ tions, including ship time, which results in relatively high monetary costs. Furthermore, the spatially discrete nature of offshore site in­ vestigations means that subsurface information must be gathered from all potential future sites of interest to CCS, to understand the geometry of the structures that may be present. i Table 2 A water- column sound velocity profile is required for accurate calibration of the shipboard echo-sounders, and, depending on the subsurface geological structure, a detailed velocity model may be necessary for accurate relocation of micro-seismic events. Cross-calibration of these two approaches is important, in order to extend the temporal observa­ tions of more punctual active acoustic surveys, and to resolve ambigu­ ities in the apparent response of different bubble size distributions. These approaches also provide an important calibration for computa­ tional fluid dynamic modelling, either by indicating the potential flux entering the modelled system or measuring the expulsion from the seabed. Ultimately, the STEMM-CCS and CHIMNEY studies of the chimneys at Scanner pockmark provided significant information on the structure of these features, which may be applied in future studies. The multi-scale and multi-parameter design of these investigations demonstrates that the integration of subsurface geophysical characterisation of a chimney with laboratory experiments, process-based numerical modelling ap­ proaches, and onshore analogues is possible. We also anticipate that our findings will be applicable for future targets, in particular those located at depth, beneath the overburden and sealing cap rocks, where higher resolution subsurface imaging is much more challenging. Therefore, the findings of the STEMM-CCS and CHIMNEY projects have the potential to reduce future resource intensiveness, whereby the pre-existing knowl­ edge of fluid flow systems gained from studying the Scanner pockmark complex can be applied to new sites, reducing the need for additional site specific data acquisition. 5. Relevance to geological CO2 storage If the sediment matrix porosity- permeability or the effects of microfractures were the principal cause of concern, laboratory experiments and/or modelling, using appropriate field analogues to define the input parameters, would be the optimal approach. As a final example, if CO2 emission and flux rate from the seabed over a spatial scale of >1 km were the greatest threat/uncer­ tainty, active acoustics would be the most appropriate method to use. If chimneys are identified as a potential threat from the initial site characterisation (based on existing data), stakeholders would be required to follow a process to determine unknown parameters which may contribute to the containment risk. Fig. 14 demonstrates a potential approach to this, where the findings of our multi-scale approach to determining chimney parameters (Section 4; Tables 1 and 2) are inte­ grated into an existing Measurement, Monitoring and Verification (MMV) risk-based decision framework (Dean and Tucker, 2017). The outstanding risk question is defined as specifically as possible, and the unknown factors which contribute to this are separated from the pa­ rameters which are sufficiently well resolved from pre-existing re­ sources. The remaining unknown factors, which may include fracture connectivity, permeability, and/or fluid volume flux and composition, are then targeted using appropriate methods drawn from Section 3, in order to mitigate the outstanding risks. The selection of which method(s) to use is based on each methods’ suitability for determining the pa­ rameters of interest (Table 1; Fig. 13), at the appropriate resolution and scale for the problem (Table 2), and any potential dependencies it may have (Table 1). Tailored deployment of new resources is then conducted, in a monitoring step. This may, for example, involve further data acquisition and/or modelling studies. Alternatively, this could also include the re-processing or re-appraisal of existing data or models. In order to optimise the risk assessment process, this step should factor both the additional cost and time of further acquisition against the benefits of obtaining the new information using these approaches. The staging of resource deployment is also important at this stage, such that the maximum amount of new information is resolved using the mini­ mum necessary additional resource cost/time. It would not, therefore, necessarily be either feasible or desirable to use all of the methods dis­ cussed in this study, highlighting that stakeholders need to efficiently determine the parameter-specific traits they require for appropriate de-risking of the chimney. 5. Relevance to geological CO2 storage The study of chimneys as potential fluid flow systems is highly relevant to the risk assessment of future CCS operations. For the assessment of a prospective CO2 storage site, a site characterisation is first conducted to understand the potential containment risks, based on pre-existing seismic, well, and environmental baseline data (e.g., Furre et al., 2019b). As the project progresses in maturity, additional data may be acquired based on identified risks or uncertainties. Containment risks are commonly identified using a bowtie risk assessment, where moni­ toring and modelling activities are implemented as barriers to reduce Depending on the availability of prior information about the specific imaging target, such as its spatial and depth scale, and the practicalities of implementing different imaging techniques, it may be possible to conduct experiments where multiple data sets are acquired concur­ rently, reducing the time-cost of the operation. For example, during the cruise JC152 seismic experiments at Scanner pockmark both the travel 19 A.H. Robinson et al. International Journal of Greenhouse Gas Control 106 (2021) 103245 sufficiently, the process should be repeated until they have. In this respect, the risk assessment and mitigation process can be considered to be a cycle (Fig. 14). the likelihood of a top event (Dean and Tucker, 2017). A top event may be described as the unintended migration of CO2 from a primary or a secondary sealing layer. In this context, chimneys could be considered as one such risk element. Fluid escape through abandoned/legacy wells and faults are other common risk elements that require consideration (IPCC, 2005). i Each prospective CO2 storage site will have unique characteristics and, therefore, will be different. However, potential risks need to be assessed holistically, which will result in different resource deployment choices for different sites. Based on the outline workflow we have described, and using Tables 1 and 2 as a guide, we can consider a number of simple examples which could be resolved using this approach. If a fracture network at a spatial scale of 10 s of m was deemed to be the greatest threat/uncertainty for prospective CCS operations, then seismic reflection or seismic tomography would be the most suit­ able method to use, while CSEM data may also assist in determining fracture connectivity and fluid content. References In this paper we have described an approach for the multi-scale, multi-method geophysical characterisation of chimneys within sedi­ mentary basins, using the Scanner pockmark as a case study. In the development of this multi-method approach, we note the following: Achterberg, E., Esposito, M., 2018. RV POSEIDON Fahrtbericht / Cruise Report POS527: Baseline Study for the Environmental Monitoring of Subseafloor CO2 Storage Operations, Kiel-Kiel (Germany), 15.8.-3.9.2018. https://doi.org/10.3289/geomar_ rep_ns_45_2018. • While individual experimental approaches, conducted in the field or in the lab, are able to determine one or more of the physical prop­ erties of chimneys and their role in fluid flow, integration between different methods is required to fully characterise the system. ACT Acorn Consortium, 2018. D08 East Mey CO2 Storage Site Development Plan 10196ACTC-Rep-26-01. https://actacorn.eu/sites/default/files/ACT%20Acorn% 20East%20Mey%20CO2%20Storage%20Site%20Storage%20Development%20Plan %201.0.pdf. p Alvizuri, C., Tanimoto, T., 2011. Azimuthal anisotropy from array analysis of Rayleigh waves in Southern California. Geophys. J. Int. 186 (3), 1135–1151. https://doi.org/ 10.1111/j.1365-246X.2011.05093.x. • Many of the described approaches rely on having suitable constraints and calibration from other methods. It is necessary, therefore, to ground-truth the methods, both in the field and in the laboratory, wherever possible, in order to ensure that conclusions determined are robust. j Amalokwu, K., 2016. Saturation Effects on Frequency-dependent Seismic Anisotropy in Fractured Porous Rocks. Doctoral thesis. University of Southampton, p. 168. http ://eprints.soton.ac.uk/id/eprint/391107. Amalokwu, K., Papageorgiou, G., Chapman, M., Best, A.I., 2017. Modelling ultrasonic laboratory measurements of the saturation dependence of elastic modulus: new insights and implications for wave propagation mechanisms. Int. J. Greenh. Gas Control. 59, 148–159. https://doi.org/10.1016/j.ijggc.2017.02.009. l • The method approaches available are applicable at different physical length scales and resolution, with a common trade-off between these two factors. Hence, it is also important that the techniques used to study fluid flow structures are applied across scale and resolution ranges, as well as different temporal scales, in order to build a ho­ listic and sufficiently detailed understanding. Andresen, K.J., 2012. Fluid flow features in hydrocarbon plumbing systems: What do they tell us about the basin evolution? Mar. Geol. 332, 89–108. https://doi.org/ 10.1016/j.margeo.2012.07.006. j g Andrews, I.J., Long, D., Richards, P.C., Thomson, A.R., Brown, S., Chesher, J.A., McCormac, M., 1990. The Geology of the Moray Firth, Vol. 3. HMSO, London. htt p://pubs.bgs.ac.uk/publications.html?pubID=B01844. Acknowledgements This work has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No.654462 (STEMM-CCS) and the Natural Environment Research Council (CHIMNEY; NERC Highlight Topic; NE/N016130/1). We would like to thank all those involved in the planning and acquisition of data during research cruises MSM63, JC152, POS518 and MSM78, including the officers, engineers and crews, the scientific parties, and all seagoing technicians and engineers. The NERC Ocean-Bottom Instrumentation Facility (Minshull et al., 2005) provided the OBSs and OBEs and their technical support at sea during JC152. OBSs and technical support at sea during MSM63 were provided by GEOMAR. RD2 is operated by the British Geological Survey. We are grateful for the support of Applied Acoustics Ltd during Sparker data acquisition. We thank Steven Constable from Scripps Institution of Oceanography for lending eight CSEM loggers, David Myer for CSEM data analysis routines, Kerry Key for the CSEM inversion and modelling routines and giving input to the data analysis, and Karen Weitemeyer for input to the discussion of CSEM data, and guidance on the analysis and modelling of the Ocean Bottom instrument data. We thank Jeroen Snippe for input to the discussion of reactive transport modelling. We thank the editors, and Alan Orpin and an anonymous reviewer for their positive and constructive comments. Overall, our integrated multi-scale experimental approach can be incorporated into pre-existing measurement, monitoring and verifica­ tion approaches (e.g., Dean and Tucker, 2017). Future monitoring ap­ proaches must also be supported by robust baseline surveys and detailed understanding of the risks of CO2 emissions that extend into the marine environment (Blackford et al., 2014, 2015; Flohr et al., 2021). Our un­ derstanding of fluid flow through chimneys must be improved to decrease containment risks at prospective storage sites and thereby realise the potential of CCS for mitigating global warming. 5. Relevance to geological CO2 storage The outcomes of the monitoring step are then used to re-appraise and verify the containment risk-level attributed to the chimney, before a decision is made. If this verification stage in­ dicates that all of the outstanding risks have not yet been constrained Alternatively, further data acquisition may not be required. In the case of the Goldeneye prospective storage site, located in the Outer Moray Firth Basin, Central North Sea, a chimney-like feature was observed during the risk assessment phase. However, rather than further data acquisition, the first monitoring step applied was to conduct high resolution re-processing of existing data, alongside pre-stack interpre­ tation. Together, these approaches resolved the issue sufficiently, finding that the observed feature instead represented a seismic imaging artefact caused by a glacial tunnel valley, without the need to acquire additional data (Dean et al., 2015). Active natural methane escape is observed directly within the shallow subsurface of the exemplar chimney at the Scanner pockmark complex in the North Sea. However, prospective CO2 storage reservoirs are more commonly located at greater depth and are capped by several sealing layers, for example the storage units at Sleipner are located at a depth of ~800−1000 mbsf. Therefore, understanding the role of chim­ neys in applied CCS environments may also represent a deeper subsur­ face problem. Compared to shallow chimneys, deeper chimneys may exhibit a number of differences, such as 1) increased stress - fluid pressure - temperature conditions; 2) the fluid of interest may be in a different phase (liquid compared to gas); and 3) changes in the sediment Fig. 14. Outline of a decision-making cycle illustrating the steps in the risk-based assessment of a site where chimneys may pose a risk to potential CCS operations. The multi-parameter, multi-scale approach discussed in Sections 3 and 4 of this paper should be used in steps 3 and 4, the mitigating and monitoring steps, in order to optimise the decision making process. Fig. 14. Outline of a decision-making cycle illustrating the steps in the risk-based assessment of a site where chimneys may pose a risk to potential CCS operations. The multi-parameter, multi-scale approach discussed in Sections 3 and 4 of this paper should be used in steps 3 and 4, the mitigating and monitoring steps, in order to optimise the decision making process. 20 A.H. Robinson et al. International Journal of Greenhouse Gas Control 106 (2021) 103245 6. Summary Chimneys (also referred to as seismic chimneys and/or pipes) may represent an important class of subsurface fluid escape pathway. The study and understanding of chimneys are highly important to risk as­ sessments for potential CCS applications. To this end, the STEMM-CCS and CHIMNEY research programmes have investigated chimneys in the North Sea. The results of these investigations will contribute to the further development of models for the understanding of fluid flow through such structures. CRediT authorship contribution statement and pore fluid composition. At greater depths (higher effective stresses) there is likely to be more consolidated material, which may result in reduced porosity-permeability of the sediment matrix. Moreover, frac­ ture apertures tend to decrease with increasing effective stress (increasing depth). Therefore, if a chimney existed at greater depths, it is more likely to have lower effective permeability, although this is dependent on the rock material properties and microscopic fracture roughness (e.g., McDermott and Kolditz, 2006; Rutqvist, 2015). At greater depths the chimney would need to contain considerable stress-persistent fracture apertures to be deemed a risk. Further research is required to more accurately constrain the differences expected be­ tween deeper and shallower chimneys. The study of deeper chimneys may also alter the experimental approach. For example, a number of the applied geophysical methods lose resolution with depth, due to the increasing attenuation of higher frequency signals that are required to image structures at shorter length scales (Resnick, 1990). However, by applying the principles discussed here and in Fig. 14, a multi-component, appropriate target-scale risk appraisal approach can be developed for a chimney structure located at greater depth. Investigation, Methodology, Formal analysis, Validation, Writing - original draft, and Writing - review & editing were performed by: AHR, BCa, CB¨o, NY, GP, IHFS, HMM, ALi, GB, RG, LP, BRo, US, BS, MW, ALa, JL, FJE, SS, CD, EK, TAM, CBe, JMB, MD, RHJ, MC, AIB, SB, BCh, DPC, JE, MH, TJH, JK, CM, JMM, LN, and BRe. Investigation, Methodology, Formal analysis, Validation, Writing - original draft, and Writing - review & editing were performed by: AHR, BCa, CB¨o, NY, GP, IHFS, HMM, ALi, GB, RG, LP, BRo, US, BS, MW, ALa, JL, FJE, SS, CD, EK, TAM, CBe, JMB, MD, RHJ, MC, AIB, SB, BCh, DPC, JE, MH, TJH, JK, CM, JMM, LN, and BRe. Investigation, Methodology, Formal analysis, Validation, Writing - original draft, and Writing - review & editing were performed by: AHR, BCa, CB¨o, NY, GP, IHFS, HMM, ALi, GB, RG, LP, BRo, US, BS, MW, ALa, JL, FJE, SS, CD, EK, TAM, CBe, JMB, MD, RHJ, MC, AIB, SB, BCh, DPC, JE, MH, TJH, JK, CM, JMM, LN, and BRe. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. References Tectonic controls on gas hydrate distribution off SW Taiwan. J. Geophys. Res. Solid Earth 146 (1). https://doi.org/10.1029/2018JB016213, 54–21. 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Thematic set: full waveform inversion: the next leap forward in imaging at Valhall First Break. 28 (4), 65–70. https://doi.org/10.3997/1365-2397.2010012. 25 International Journal of Greenhouse Gas Control 106 (2021) 103245 Geophys. J. Int. 166 (2), 732–744. https://doi.org/10.1111/j.1365- 246X.2006.03028.x. Zelt, C.A., Barton, P.J., 1998. Three-dimensional seismic refraction tomography: a comparison of two methods applied to data from the Faeroe Basin. J. Geophys. Res. Solid Earth 103 (B4), 7187–7210. https://doi.org/10.1029/97JB03536. Zelt, C.A., Sain, K., Naumenko, J.V., Sawyer, D.S., 2003. Assessment of crustal velocity models using seismic refraction and reflection tomography. Geophys. J. Int. 153 (3), 609–626. https://doi.org/10.1046/j.1365-246X.2003.01919.x. A.H. Robinson et al. A.H. Robinson et al. 26
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Risk of Microangiopathy in Patients with Epilepsy under Long-term Antiepileptic Drug Therapy
Frontiers in neurology
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Nai-Ching Chen1†, Chih-Hsin Chen2†, Tsu-Kung Lin1, Shang-Der Chen1,3, Meng-Han Tsai1, Chiung-Chih Chang1, Wan-Chen Tsai1 and Yao-Chung Chuang1,3,4,5* Nai-Ching Chen1†, Chih-Hsin Chen2†, Tsu-Kung Lin1, Shang-Der Chen1,3, Meng-Han Tsai1, Chiung-Chih Chang1, Wan-Chen Tsai1 and Yao-Chung Chuang1,3,4,5* 1 Department of Neurology, Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Kaohsiung, Taiwan, 2 Department of Ophthalmology, Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Kaohsiung, Taiwan, 3 Institute for Translational Research in Biomedicine, Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Kaohsiung, Taiwan, 4 Department of Neurology, Faculty of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan, 5 Department of Biological Science, National Sun Yat-sen University, Kaohsiung, Taiwan Background: Long-term antiepileptic drug (AED) therapy is considered a risk factor of ath- erosclerosis. Furthermore, the duration of therapy contributes to acceleration of large-vessel atherosclerosis. Therefore, in this study, we tested the hypothesis that long-term AED therapy plays a crucial role in the pathogenesis of microangiopathy in patients with epilepsy. Background: Long-term antiepileptic drug (AED) therapy is considered a risk factor of ath- erosclerosis. Furthermore, the duration of therapy contributes to acceleration of large-vessel atherosclerosis. Therefore, in this study, we tested the hypothesis that long-term AED therapy plays a crucial role in the pathogenesis of microangiopathy in patients with epilepsy. Reviewed by: Reviewed by: Scott Mintzer, Thomas Jefferson University, United States Stephan Schuele, Northwestern University, United States Carli Lorraine Roulston, University of Melbourne, Australia results: The ratio of urine albumin to creatine and OCT findings showed that patients with epilepsy had higher abnormal microalbuminuria and narrowing retinal vein diame- ters, respectively. Multiple linear regression analysis showed that increased triglyceride and hs-CRP levels might contribute to microalbuminuria. In addition, serum creatinine, duration of AED therapy, enzyme-inducing AED therapy, and duration of enzyme-induc- ing AED therapy were candidate risk factors for retinal vein narrowing. *Correspondence: Yao-Chung Chuang ycchuang@cgmh.org.tw †These authors have equal contribution as co-first authors. Specialty section: This article was submitted to Epilepsy, a section of the journal Frontiers in Neurology conclusion: Patients with epilepsy are at a higher risk for microangiopathy presented as retinopathy and nephropathy. Long-term AED therapy, particularly with enzyme-inducing AEDs; high triglyceride levels, and inflammatory processes play an important role in the development of microangiopathy in patients with epilepsy. Received: 01 April 2017 Accepted: 14 February 2018 Published: 12 March 2018 Keywords: antiepileptic drug therapy, atherosclerosis, microangiopathy, retinopathy, microalbuminuria Keywords: antiepileptic drug therapy, atherosclerosis, microangiopathy, retinopathy, microalbuminuria Original Research published: 12 March 2018 doi: 10.3389/fneur.2018.00113 Edited by: Mark J. Cook, University of Melbourne, Australia Methods: We recruited 120 patients with epilepsy (age, 18–60 years) and 40 healthy controls. Optical coherence tomography (OCT) was used to measure the central mac- ular thickness and diameters of the retinal artery and vein to evaluate atherosclerotic retinopathy; microalbumin and creatinine levels in urine were assessed to evaluate ath- erosclerotic nephropathy. In addition, high-sensitivity C-reactive protein (hs-CRP), lipid profiles, homocysteine, folate, uric acid, and body mass index were determined. Citation: Chen N-C, Chen C-H, Lin T-K, Chen S-D, Tsai M-H, Chang C-C, Tsai W-C and Chuang Y-C (2018) Risk of Microangiopathy in Patients with Epilepsy under Long-term Antiepileptic Drug Therapy. Front. Neurol. 9:113. doi: 10.3389/fneur.2018.00113 More than 30% of patients with epilepsy do not experience seizure remission despite appropriate and adequate antiepileptic drug (AED) therapy (1). Therefore, long-term or lifelong AED treatment is often required for those patients with intractable epilepsy. Prolonged AED therapy, particularly with enzyme-inducing AEDs (EIAEDs), may promote the pathogenesis of atherosclerosis and increase atherosclerotic risk arising from cerebrovascular events or ischemic heart disease resulting in high morbidity and mortality in patients with epilepsy (2–5). March 2018  |  Volume 9  |  Article 113 1 Frontiers in Neurology  |  www.frontiersin.org Microangiopathy and AED Therapy Chen et al. healthy volunteers who received an annual physical checkup were recruited as normal controls. Patients who had discontinued their medication within 2 years were excluded from this study. The other exclusion criteria for patients and controls included cerebrovascular diseases; ischemic heart diseases; hypertension; diabetes mellitus; clinical history of retinopathy; hematologic, endocrine, and autoimmune diseases; and nephritis, nephrotic syndrome, and chronic kidney disease (3, 5). Patients with a documented diagnosis of gouty arthritis, manifestation of tophi, or nephrolithiasis associated with hyperuricemia were also excluded. We also excluded patients who consumed vitamins, tobacco, antihypertensive drugs, oral hypoglycemic agents, or those who received medication that affected the metabolism of uric acid or lipids. Meanwhile, patients with retinal vascular abnormalities, such as diabetic retinopathy, hypertensive retin- opathy, retinal vein occlusion, age-related macular degeneration, and other retinal vascular disorders, were excluded. Atherosclerosis is a chronic systemic condition involving the wall of the arteries and multiple factors account for the patho- genesis and progress of atherosclerosis (6). We have previously shown (3, 5) that the duration of AED therapy is an important independent factor affecting the acceleration of atherosclerosis by increasing the common carotid artery intima media thickness (CCAIMT) and metabolic disturbances. These results suggest that the cumulative effects of long-term treatment with AEDs play a crucial role in the pathogenesis of atherosclerosis-related macroangiopathy in large blood vessels. Long-term treatment with AEDs may also cause microangiopathy in target organs, such as the retina and kidneys, which may lead to atherosclerosis- related retinopathy and nephropathy. Citation: Microangiopathy has been found to be associated with diabetes mellitus, hypertension, and other conditions that increase the risk of cerebrovascular diseases (7–10); however, microvascular complications present in patients with epilepsy under prolonged AED therapy remains unclear. Microangiopathies, including retinal proliferation and micro- albuminuria, have been reported as indicators of atherosclerotic retinopathy and nephropathy (8–11). However, the reported inci- dence of retinopathy and nephropathy in patients with epilepsy is limited. To investigate atherosclerosis-related microangiopathy in retinal vessels, optical coherence tomography (OCT) has been used for assessing atherosclerotic plaques in the walls of the retinal blood vessels (12, 13). In addition, biomarkers of kidney function, including serum creatinine, estimated glomerular filtration rate (eGFR), and microalbuminuria, can predict atherosclerosis-related nephropathy (8, 9, 14). Recent studies have shown that microalbuminuria may represent endothelial dysfunction in renal vessels and may be associated with increased risk of atherosclerosis-related renal microangiopathy (8–10, 14).h All patients were subjected to a general physical and neuro- logical examination as well as clinical interviews. Clinical demo- graphic data, including the age at onset of seizures, seizure types, etiology of epilepsy, seizure frequency (per month), duration of AED therapy, and any current AEDs therapy were recorded. To identify any confounding high-risk factors for atherosclerosis and to exclude epilepsy that arises from asymptomatic cerebro- vascular diseases, magnetic resonance imaging of the brain was conducted for all the patients.h The AEDs being used by the enrolled patients included lamotrigine, carbamazepine, phenytoin, valproate acid, topira- mate, phenobarbital, levetiracetam, vigabatrin, and gabapentin. Carbamazepine, phenytoin, and phenobarbital have been defined as enzyme inducers of the liver cytochrome P450 (CYP450) system (3, 11). Lamotrigine, levetiracetam, valproate, topira- mate, vigabatrin, and gabapentin are considered non-enzyme inducers (3, 11). We defined the duration of EIAED therapy as the time period during which patients received continuous treatment with enzyme inducers. The duration of non-EIAED therapy was defined as the time period during which patients received sustained treatment with or switched to non-enzyme inducers. This study evaluates the hypothesis that prolonged treatment with AEDs may play a crucial role in the pathogenesis of micro- vascular endothelial cell damage in the end organs, particularly in the kidney and retina, which result in atherosclerosis-related retinopathy and nephropathy in patients with epilepsy. Blood Collection and Analysis of Circulating Biochemical Markerst This is a case-control study conducted at a single center. The study hospital, Kaohsiung Chang Gung Memorial Hospital, is a tertiary medical center in southern Taiwan. Ethical approval for this study (Ethical Committee NO100-2011B) was provided by the Chang Gung Medical Foundation Institutional Review Board, and writ- ten informed consent was obtained from both the patients as well as from healthy controls. All procedures performed in this study involving human participants were in accordance with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. After overnight fasting, blood samples were obtained from the patients between 8 and 10 a.m. and analyzed at the Central Laboratory of Kaohsiung Chang Gung Memorial Hospital for serum levels of total cholesterol, high-density lipoprotein cho- lesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), triglycerides, folate, homocysteine, blood glucose, glycated hemo- globin (HbA1c), high-sensitive C-reactive protein (hs-CRP), uric acid, and creatinine levels. All circulating biochemical markers were analyzed according to our previous reports (3, 5, 15).i y g p p Estimated glomerular filtration rate was calculated as: Statistical Analysish at the time of study ranged from 2 to 57 years; the duration of non-EIAED therapy in the other 47 patients ranged from 3 to 28 years. Based on statistical analysis, the duration of therapy did not show significance between the EIAED and non-EIAED groups (p = 0.069). According to etiology, 75 patients (62.5%) had idiopathic or cryptogenic epilepsy and 45 patients (37.5%) had symptomatic epilepsy. In terms of basic demographic data, age and gender between patients and controls did not show a significant difference (Table 2). Three separate statistical analyses were performed. First, gender and microalbumiuria (defined as urine albumin/creatinine ratio ≥30) were compared between patients with epilepsy and controls by using a Chi-square test. Comparisons of continuous variables with normal distribution between the two study groups were made using the Student’s t-test. Comparison of continuous vari- ables without normal distribution between the two study groups were made using the Mann–Whitney Test. Second, correlation analysis was used to evaluate the relationship between urine albu- min/creatinine ratio; vein diameter; and variables that included age, body mass index (BMI), duration of AED therapy, duration of EIAED and non-EIAED, number and type of AED therapy, lipid profile, hs-CRP, folate, homocysteine, HbA1c, and serum creatinine levels. Third, factors with p value <0.3 from the cor- relation analysis were used to evaluate the relationships between baseline clinical factors associated with the urine albumin/cre- atinine ratio and vein diameter by using the stepwise method of multiple linear regression. All statistical analysis was conducted using the Statistical Package for Social Sciences software package (version 13 for Windows®, SPSS Inc., Chicago, IL, USA). Characteristics and Demographic Data of Patients with Epilepsyh The characteristics and demographic data of 120 patients with epilepsy are listed in Table 1. The age at onset of epilepsy ranged from 3 months to 49 years, and the duration of epilepsy ranged from 2 to 57 years. Their average seizure frequency ranged from 0 to 80 per month. The duration of AED therapy at the time of study ranged from 2 to 56 years, and it was significantly corre- lated to the duration of epilepsy (p < 0.001). 57 patients (47.5%) received monotherapy and 63 (52.5%) were on multiple AED therapy. Seventy-three patients with epilepsy received EIAEDs for seizure control. The duration of EIAED therapy in 73 patients RESULTS Table 2 also shows that BMI, total cholesterol, LDL-C, triglyc- eride, homocysteine, and hs-CRP were significantly increased, and folate was decreased in patients with epilepsy. Other results included fasting blood sugar, uric acid, HDL-C, and serum cre- atinine did not show significant differences between the patient and control groups. Assessment of Retinal Microangiopathy g p y Retinal microangiopathy was measured by a third-generation OCT (Model 3000, Carl-Zeiss Instruments, Dublin, CA, USA). OCT using a manually assisted technique was conducted for all the patients by using a previously reported methodology (17, 18) by experienced examiners in order to measure the central macu- lar thickness, retinal artery diameter, and retinal vein diameter (17, 18). We routinely measured the diameter of the retinal artery and vein over the superior-temporal arcade one-disk distance away from the upper disk margin. Microangiopathy and Vascular Risk Factors Are Increased in Patients with Epilepsyh The retinal artery diameter and central macular thickness on either side of the eyes did not show a significant difference between the patients and controls. Univariate analysis (Table 2) revealed that the retinal vein diameters on either side were significantly narrower in patients with epilepsy. In addition, the urine albu- min/creatinine ratio was significantly increased in patients with epilepsy. A urine albumin/creatinine ratio over 30 was not found in the control group; however, 11.7% of patients with epilepsy had a urine albumin/creatinine ratio over 30. Measurement of Microalbuminuria Urine samples obtained from the patients were analyzed for creatinine and microalbuminuria levels. The ratio of albumin to creatinine levels was also calculated. Microalbuminuria was defined as a ratio of urine albumin to urine creatinine of 30–300 g/mg (16). Routine urinary analysis was also conducted to exclude urinary tract infections and other etiologies of abnor- mal microalbuminuria. Subjects From October 2011 to September 2013, 120 patients (aged between 18 and 65 years) who received AED treatment for more than 2  years were enrolled through the Epilepsy Outpatient Clinic of Kaohsiung Chang Gung Memorial Hospital. Forty Male 14 age body weight serum creatinine 72 Female : [( ) ]/( ) : 0 − × × 14 age body weight serum creatinine 72 85. [( ) ]/( ) . 0 0 − × × × Male 14 age body weight serum creatinine 72 Female : [( ) ]/( ) : 0 − × × 14 age body weight serum creatinine 72 85. [( ) ]/( ) . 0 0 − × × × March 2018  |  Volume 9  |  Article 113 Frontiers in Neurology  |  www.frontiersin.org 2 Chen et al. Microangiopathy and AED Therapy TABLE 1 | Baseline characteristics of 120 patients with epilepsy enrolled in this study. Characteristics Age at onset (years) 21.8 ± 11.7 Duration of epilepsy (years) 18.6 ± 8.1 Type of seizures, n (%) Generalized 12 (10%) Partial 108 (90%) Seizure free,* n (%) 51 (42.5%) Etiology, n (%) Idiopathic/cryptogenic 75 (62.5%) Symptomatic 45 (37.5%) Duration of AED therapy (years) 17.8 ± 10.8 Duration of EIAED therapy (years) 14.5 ± 12.4 Duration of non-EIAED therapy (years) 13.7 ± 6.4 Mode of AED therapy, n (%) Single 57 (47.5%) Multiple 63 (52.5%) EIAEDs 73 (60.8%) Values are expressed in mean ± SD. *Seizure free for more than 12 months under current AED therapy. AED, antiepileptic drug, EIAEDs, enzyme-inducing antiepileptic drugs. Risk Factors Associated with Microangiopathyh The patients with epilepsy were further divided into two groups based on whether or not they received long-term EIAED therapy. Based on the statistical analysis, uric acid (p = 0.007), total cholesterol (p  =  0.004), HDL-C (p  =  0.003), and urine March 2018  |  Volume 9  |  Article 113 Frontiers in Neurology  |  www.frontiersin.org 3 Microangiopathy and AED Therapy Chen et al. TABLE 2 | Demographic data and vascular risk factors in patients with epilepsy vs. controls. Controls (n = 40) Patients (n = 120) p Value Age (years) 38.7 ± 10.6 38.6 ± 9.6 0.936 Gender (female/male) 20/20 60/60 1 Body mass index (kg/m2) 23.3 ± 3.4 25.0 ± 4.8* 0.027 Fasting blood sugar (mg/dL) 92.2 ± 7.3 94.9 ± 18.8 0.679 Glycated hemoglobin (HbA1c) (%) 5.5 ± 0.3 5.5 ± 0.7 0.336 Uric acid (mg/dL) 5.6 ± 1.5 5.0 ± 1.7 0.051 Cholesterol (mg/dL) Total 169.8 ± 21.8 192.2 ± 32.5* <0.001 HDL-C 64.3 ± 15.3 67.1 ± 17.9 0.416 LDL-C 89.9 ± 19.9 103.7 ± 27.1* 0.001 Triglyceride (mg/dL) 77.4 ± 39.8 115.1 ± 83.1* 0.003 Homocysteine (umol/L) 12.8 ± 8.5 14.9 ± 10.4* 0.049 Folate (ng/mL) 8.2 ± 2.9 7.1 ± 3.5* 0.012 hs-CRP (mg/L) 1.5 ± 3.2 4.2 ± 9.5* 0.009 Creatinine (mg/dL) 0.8 ± 0.2 0.8 ± 0.2 0.273 Urine albumin/creatinine ratio (mg/g) 7.0 ± 4.5 20.2 ± 50.9* 0.006 Urine albumin/creatinine ratio ≥30, n (%) 0 (0%) 14 (11.7%)* 0.023 Central macular thickness, right eye (μm) 197.7 ± 8.2 197.4 ± 13.0 0.995 Central macular thickness, left eye (μm) 198.4 ± 7.8 198.3 ± 12.7 0.934 Retinal artery diameter, right eye (μm) 65.5 ± 1.6 65.9 ± 2.4 0.458 Retinal artery diameter, left eye (μm) 66.6 ± 2.3 66.5 ± 2.9 0.596 Retinal vein diameter, right eye (μm) 88.3 ± 2.7 86.2 ± 3.6* 0.001 Retinal vein diameter, left eye (μm) 88.6 ± 2.7 86.6 ± 3.8* 0.004 Values are expressed in mean ± SD. AED, antiepileptic drug, HDL-C, high-density lipoprotein cholesterol, LDL-C, low- density lipoprotein cholesterol, hs-CRP, high-sensitivity C-reactive protein; BMI, body mass index. *p < 0.05 vs. normal control. TABLE 3 | Correlation analysis of the effects of candidate risk factors on microangiopathy in patients with epilepsy and controls. Risk Factors Associated with Microangiopathyh Urine albumin/ creatinine ratio Retinal vein diameter Variables Correlation coefficient p Value Correlation coefficient p Value Age −0.080 0.195 −0.221* 0.008 Body mass index 0.058 0.266 0.055 0.277 Duration of AED therapy 0.030 0.374 −0.250* 0.003 EIAEDs −0.162* 0.040 0.155* 0.047 Duration of EIAED therapy 0.080 0.193 −0.327* 0.001 Duration of non-EIAED therapy −0.085 0.179 −0.034 0.359 AED number 0.040 0.333 −0.020 0.416 Homocysteine 0.004 0.482 0.109 0.119 hs-CRP 0.206* 0.013 0.037 0.345 Total cholesterol 0.217* 0.009 −0.027 0.386 LDL-C 0.167* 0.036 0.045 0.315 Triglyceride 0.313* 0.001 0.083 0.187 Glycated hemoglobin (HbA1c) 0.084 0.182 −0.021 0.410 Creatinine −0.044 0.318 0.272* 0.001 AED, antiepileptic drug, EIAEDs, enzyme-inducing antiepileptic drugs, hs-CRP, high- sensitivity C-reactive protein; LDL-C, low-density lipoprotein cholesterol; BMI, body mass index. The duration of AED therapy was considered 0 in the control group. *p < 0.05. TABLE 2 | Demographic data and vascular risk factors in patients with epilepsy vs. controls. TABLE 3 | Correlation analysis of the effects of candidate risk factors on microangiopathy in patients with epilepsy and controls. ographic data and vascular risk factors in patients with epilepsy TABLE 4 | Multiple linear regression analysis of association of microangiopathy with duration of AED therapy and other candidate risk factors. | p g y g p y with duration of AED therapy and other candidate risk factors. Urine albumin/creatinine ratio Retinal vein diameter Variables B SE p Value B SE p Value Age −0.119 −1.366 0.175 −0.117 −1.373 0.173 Body mass index −0.074 −0.791 0.431 0.069 0.818 0.415 Duration of AED therapy −0.020 −0.227 0.821 0.515 3.340 0.001* EIAEDs −0.122 −1.399 0.165 0.203 2.143 0.034* Duration of EIAED therapy 0.047 0.539 0.591 −0.526 −5.764 <0.001* Duration of non- EIAED therapy −0.062 −0.702 0.484 0.093 1.113 0.268 AED number 0.081 0.920 0.360 0.086 1.036 0.303 hs-CRP 0.199 2.297 0.023* 0.075 0.933 0.353 Total cholesterol 0.137 1.521 0.133 0.007 0.092 0.927 LDL-C 0.086 0.964 0.337 0.003 0.036 0.972 Triglyceride 0.308 3.559 0.001* 0.070 0.818 0.415 Glycated hemoglobin (HbA1c) 0.028 0.322 0.748 0.079 0.982 0.328 Creatinine −0.151 −1.672 0.097 0.230 2.833 0.005* AED, antiepileptic drug, EIAEDs, enzyme-inducing antiepileptic drugs, hs-CRP, high- sensitivity C-reactive protein; LDL-C, low-density lipoprotein cholesterol; BMI, body mass index. The duration of AED therapy was considered 0 in the control group. *p < 0.0.5. albumin/creatinine ratio (p = 0.009) showed significant differ- ences between the two patient subgroups. Risk Factors Associated with Microangiopathyh Other factors, such as age, gender, fasting blood sugar, glycated hemoglobin, and bilateral retinal artery and vein diameter did not show significant differences between the patient subgroups. Correlation analysis (Table 3) revealed that the urine albu- min/creatinine ratio was significantly correlated with blood levels of EIAEDs, hs-CRP, total cholesterol, LDL-C, and triglycerides. Moreover, bilateral retinal vein diameter showed a significant correlation with age, serum creatinine, EIAED therapy, duration of AED therapy, and duration of EIAED therapy. Variables with p < 0.03 were further analyzed by a multiple linear regression test to identify the factors that promote retinal and renal microangiopathy in patients with epilepsy. Multiple lin- ear regression analysis showed that hs-CRP and triglyceride levels were significantly correlated to the urine albumin/creatinine ratio (Table 4). In addition, creatinine, EIAED therapy, duration of AED therapy, and duration of EIAED therapy were significantly correlated with retinal vein diameter. DISCUSSION microangiopathy, including higher risk of retinopathy and nephropathy as revealed by decreased retinal vein diameters and increased urine albumin/creatinine ratio when compared with healthy controls. Our results further show that serum creatinine levels, EIAED therapy, duration of AED therapy, and duration In this study, we used two non-invasive parameters, OCT and urine albumin/creatinine ratio, to examine atherosclerotic microangiopathy in patients with epilepsy. Our findings confirm that patients with chronic epilepsy who are under prolonged AED therapy, particularly with EIAEDs frequently exhibit In this study, we used two non-invasive parameters, OCT and urine albumin/creatinine ratio, to examine atherosclerotic microangiopathy in patients with epilepsy. Our findings confirm that patients with chronic epilepsy who are under prolonged AED therapy, particularly with EIAEDs frequently exhibit March 2018  |  Volume 9  |  Article 113 Frontiers in Neurology  |  www.frontiersin.org 4 Microangiopathy and AED Therapy Chen et al. of EIAED therapy might contribute to the narrowing of retinal veins. In addition, a high lipid profile and elevated hs-CRP levels were associated with a high urine albumin/creatinine ratio in patients with epilepsy.f also revealed that atherosclerosis-related retinopathy, indicated by narrowing of the retinal vein diameter, may occur in patients with epilepsy under long-term AED therapy. Furthermore, in addition to contributing to retinal vein narrowing, duration of AED therapy, administration of EIAEDs, and duration of EIAED therapy were found to be significantly associated with atheroscle- rosis-related retinopathy in our study group. Thus, OCT might be a useful tool for early detection of retinal microangiopathy in patients with epilepsy. Microangiopathy is an angiopathy that affects small blood vessels; the damage is common in the small arteries, arterioles, venules, and capillaries of end organs. The reduced blood supply leads to atherosclerosis-related retinopathy and nephropathy in the retina and kidney (10) or cerebral and coronary small ves- sel diseases in the brain and heart (19). In addition to diabetes mellitus, which is the most common cause of microangiopathy (9), conditions, including low HDL-C plus elevated triglycer- ide levels, hyperhomocysteinemia, hypertension, lupus, and hemolytic uremic syndrome have been found to be correlated with microangiopathy (8, 14, 20). We (3, 5) and others (2, 4, 21–23) have identified a number of biomarkers in circulation that are related to increased risk of large-vessel atherosclerosis. These biomarkers include lipid profiles, lipoproteins, hs-CRP, homocysteine, and uric acid levels in patients with epilepsy who received prolonged AED therapy. DISCUSSION Based on the measurement of CCA IMT, we showed that the duration of AED therapy is an important independent factor affecting CCA IMT (3, 5). Since long-term exposure to AEDs alters various vascular risk factors and contributes to atherosclerosis-related macroangiopathy, we reason that long-term exposure to AEDs may also contribute to microangiopathy. Our results showed that cholesterol, LDL-C, triglyceride, and hs-CRP levels were major risk factors for microalbumi- nuria. Previous studies have shown that triglyceride (20, 30) and hs-CRP (7, 16) levels are correlated with the structure and function of blood vessels and increased microalbuminuria. Atherosclerosis is a process that involves intravascular develop- ment of a chronic inflammatory condition that results from local interactions between modified lipoproteins, inflammatory cells, and cytokines within the walls of veins and arteries (7, 31). Our results, therefore, suggest that chronic inflammatory reactions and lipid disturbances in circulation may contribute to microan- giopathy in patients with epilepsy. Hypertension, hyperlipidemia, and diabetes are traditional risk factors attributable to disturbance of retinal microcirculation and change in retinal vessel diameters (10, 27, 32, 33). Recent evidence showed that the signs of retinopathy, including changes in retinal microvasculature, may be a risk marker for stroke and coronary microvascular disease (34, 35). However, whether retinal micro- vasculature may predict risk of atherosclerosis in patients with epilepsy under prolonged AED therapy remains unclear. In this study, age, serum creatinine, EIAEDs, duration of AED therapy, and duration of EIAED therapy were found to be correlated with narrow retinal vein diameter. We are aware that age may also influence the diameter of retinal vessels (27). However, after adjustment for age, the duration of AED therapy, administration of EIAEDs, and duration of EIAED therapy remain significantly correlated with the change of retinal vein thickness and vascular factors. On the other hand, non-EIAED therapy did not affect the retinal vein thickness and vascular risk profiles. We noted that the duration of therapy was 14.5 ± 12.4 years in patients who received EIAEDs with or without combination of other AEDs, the duration of non-EIAED therapy in patients without combination of EIAEDs was 13.7 ± 6.4 years. Since the duration of therapy between the EIAED and non-EIAED groups was similar, we rea- son that EIAEDs may play an important role in the pathogenesis of change in retinal microvasculature. Frontiers in Neurology  |  www.frontiersin.org REFERENCES 11. Klein R, Myers CE, Lee KE, Gangnon R, Klein BE. Changes in retinal vessel diameter and incidence and progression of diabetic retinopathy. Arch Ophthalmol (2012) 130(6):749–55. doi:10.1001/archophthalmol.2011.2560 11. Klein R, Myers CE, Lee KE, Gangnon R, Klein BE. Changes in retinal vessel diameter and incidence and progression of diabetic retinopathy. Arch Ophthalmol (2012) 130(6):749–55. doi:10.1001/archophthalmol.2011.2560 1. Kwan P, Brodie MJ. Early identification of refractory epilepsy. N Engl J Med (2000) 342(5):314–9. doi:10.1056/NEJM200002033420503 1. Kwan P, Brodie MJ. Early identification of refractory epilepsy. N Engl J Med (2000) 342(5):314–9. doi:10.1056/NEJM200002033420503 12. Douma K, Megens RT, van Zandvoort MA. Optical molecular imaging of athero­ sclerosis using nanoparticles: shedding new light on the darkness. Wiley Interdiscip Rev Nanomed Nanobiotechnol (2011) 3(4):376–88. doi:10.1002/ wnan.139 2. Mintzer S. Metabolic consequences of antiepileptic drugs. Curr Opin Neurol (2010) 23(2):164–9. doi:10.1097/WCO.0b013e32833735e7 2. Mintzer S. Metabolic consequences of antiepileptic drugs. Curr Opin Neurol (2010) 23(2):164–9. doi:10.1097/WCO.0b013e32833735e7 f 3. Chuang YC, Chuang HY, Lin TK, Chang CC, Lu CH, Chang WN, et al. Effects of long-term antiepileptic drug monotherapy on vascular risk factors and atherosclerosis. Epilepsia (2012) 53(1):120–8. doi:10.1111/j.1528-1167.2011. 03316.x 13. Matcher SJ. Practical aspects of OCT imaging in tissue engineering. Methods Mol Biol (2011) 695:261–80. doi:10.1007/978-1-60761-984-0_17 14. Maharjan BR, Bhandary S, Sunuwar L, Shrestha A, Ranjitkar N. Association of hypertension with microalbuminuria and lipid profile in the local population of Patan. Nepal Med Coll J (2013) 14(2):157–62. 4. Mintzer S, Skidmore CT, Abidin CJ, Morales MC, Chervoneva I, Capuzzi DM, et al. Effects of antiepileptic drugs on lipids, homocysteine, and C-reactive protein. Ann Neurol (2009) 65(4):448–56. doi:10.1002/ana.21615 p 15. Tseng YL, Huang CR, Lin CH, Lu YT, Lu CH, Chen NC, et al. Risk factors of hyperammonemia in patients with epilepsy under valproic acid therapy. Medicine (Baltimore) (2014) 93(11):e66. doi:10.1097/MD.0000000000000066 5. Tan TY, Lu CH, Chuang HY, Lin TK, Liou CW, Chang WN, et al. Long-term antiepileptic drug therapy contributes to the acceleration of atherosclerosis. Epilepsia (2009) 50(6):1579–86. doi:10.1111/j.1528-1167.2009.02024.x i 16. Donnelly R, Yeung JM, Manning G. Microalbuminuria: a common, inde- pendent cardiovascular risk factor, especially but not exclusively in type 2 diabetes. J Hypertens Suppl (2003) 21(1):S7–12. 6. Rafieian-Kopaei M, Setorki M, Doudi M, Baradaran A, Nasri H. Atherosclerosis: process, indicators, risk factors and new hopes. Int J Prev Med (2014) 5(8):927–46. 17. Chen CH, Liu YC, Wu PC. The combination of intravitreal bevacizumab and phacoemulsification surgery in patients with cataract and coexisting diabetic macular edema. ETHICS STATEMENT Ethical approval for this study (Ethical Committee NO100- 2011B) was provided by the Chang Gung Medical Foundation Institutional Review Board. The pathogenesis of microangiopathy and macroangiopathy are different (36). Microangiopathy begins earlier than mac- roangiopathy, has no symptoms, and progresses more rapidly than macroangiopathy (36). It thus provides novel insights into early and easy diagnosis of atherosclerosis in patients with epi- lepsy under long-term AED therapy, particularly with EIAEDs. Furthermore, strategies to decelerate the course of endothelium- damaging atherosclerosis due to the cumulative effects of pro- longed AED therapy should be promptly initiated in patients with high-vascular risks. FUNDING This work was supported in part by research grant CMRPG8A0371 to Y-CC from Kaohsiung Chang Gung Memorial Hospital, Taiwan. This work was supported in part by research grant CMRPG8A0371 to Y-CC from Kaohsiung Chang Gung Memorial Hospital, Taiwan. AUTHOR CONTRIBUTIONS YCC and NCC participated in the design of the study, drafted the manuscript, and performed the statistical analysis. YCC, NCC, CHC, MHT, SDC, WCT, CCC and TKL participated in the clinical evaluation of patients, and drafting of the manuscript. YCC helped draft the work and revised it critically for important intellectual content. All the authors have read and approved the final manuscript. In conclusion, this study shows that two microangiopathies, retinopathy and nephropathy, which are associated with high levels of triglyceride and hs-CRP, duration of AED therapy, EIAEDs and duration of EIAED in patients with epilepsy who received long-term AED therapy. This information is important to our understanding of the pathogenesis of microangiopathy in DISCUSSION March 2018  |  Volume 9  |  Article 113 5 Microangiopathy and AED Therapy Chen et al. In general, retinopathy and microalbuminuria are well-known microvascular complications, but they have different pathophysi- ologies and risk factors. Taking together with the risk factors with microalbuminuria, it is reasonable to suggest that atherosclerosis- related microangiopathy in patients with epilepsy is effected by long-term AED therapy, particularly with EIAEDs, in association with disturbances in cholesterol, triglyceride, homocysteine or folate metabolism, and elevation of the inflammation marker hs-CRP. Thus, CYP450 EIAEDs should be used cautiously in patients with epilepsy who also have atherosclerosis-related vascular disorders.h patients with epilepsy, and offers a guide for the choice of drug for patients with epilepsy under long-term AED therapy. A regular work-up of lipid profiles, metabolic status, microalbuminuria, and retinal examination should be considered, particularly in aged and high-vascular risk individuals. DISCUSSION Thus, our results suggest that the duration of AED treatment and the enzyme inducers may contribute to the atherosclerosis-related retinopathy in patients with epilepsy. We further propose that retinal microvascular abnormalities and microalbuminuria may be biomarkers for the screening of the risk of atherosclerosis-related diseases in patients receiving prolonged EIAED therapy. Several studies show that microalbuminuria leads to endothelial dysfunction in renal vessels and increases the risk for atherosclerosis-related renal microangiopathy (7, 16, 24, 25). In type 1 diabetes, which is associated with high cardiovascular complications, microalbuminuria is found to increase the risk of cardiovascular disease (26). In this study, we found a significantly increased urine albumin/creatinine ratio, indicating the presence of nephropathy in patients with epilepsy who received long-term AED therapy. In addition, our analysis revealed that disturbance of lipid profiles and elevated hs-CRP levels were associated with nephropathy in patients with epilepsy. Microangiography in the retina was previously assessed by visualizing dilated pupils on retinal photographs and classified as mild nonproliferative, moderate nonproliferative, severe nonpro- liferative, or proliferative retinopathy (11, 12). Retinopathy can now be assessed using OCT, a new tool that can directly measure the diameter of the retinal artery and vein for further analysis of retinal microangiography (27, 28). Of the few studies (27–29) that employed OCT to assess atherosclerotic retinopathy, the results are inconsistent. The change in retinal venular diameters might be an independent factor in predicting cardiovascular disorders (29). Using OCT, we showed that the diameter of the retinal vein was narrower than that of the retinal artery in patients with epilepsy. This result is in agreement with previous studies (27, 28) which showed that narrowing of the retinal venous diam- eter, while the arterial diameter remained unchanged provides early evidence of incidence and progression of atherosclerosis- related microangiopathy. It follows that our results suggest that early risk of retinal angiopathy may be presented as decreased retinal vein diameter before changes in the diameter of the retinal artery are observed in patients with epilepsy. Our OCT findings In our previous studies (3, 5), we found that patients under long-term AED therapy may show disturbances in lipid profiles, total homocysteine levels, and folate metabolism, and increased inflammatory reaction and oxidative stress. Whereas, microan- giopathy may lead to both atherosclerosis-related retinopathy and nephropathy, these events may not occur at the same time. 11. Klein R, Myers CE, Lee KE, Gangnon R, Klein BE. Changes in retinal vessel diameter and incidence and progression of diabetic retinopathy. Arch Ophthalmol (2012) 130(6):749–55. doi:10.1001/archophthalmol.2011.2560 REFERENCES doi:10.3109/00207454.2014.949704 J Kidney Dis (2005) 46(2):214–24. doi:10.1053/j.ajkd.2005.05.005 33. Lee JY, Yoon YH, Kim HK, Yoon HS, Kang SW, Kim JG, et  al. Baseline characteristics and risk factors of retinal vein occlusion: a study by the Korean RVO Study Group. J Korean Med Sci (2013) 28(1):136–44. doi:10.3346/ jkms.2013.28.1.136 24. Tseng CH. Differential dyslipidemia associated with albuminuria in type 2 diabetic patients in Taiwan. Clin Biochem (2009) 42(10–11):1019–24. doi:10.1016/j.clinbiochem.2009.03.013 j 25. Weir MR. Microalbuminuria and cardiovascular disease. Clin J Am Soc Nephrol (2007) 2(3):581–90. doi:10.2215/CJN.03190906 34. Ikram MK, de Jong FJ, Bos MJ, Vingerling JR, Hofman A, Koudstaal PJ, et al. Retinal vessel diameters and risk of stroke: the Rotterdam Study. Neurology (2006) 66(9):1339–43. doi:10.1212/01.wnl.0000210533.24338.ea h 26. de Boer IH, Gao X, Cleary PA, Bebu I, Lachin JM, Molitch ME, et  al. Albuminuria changes and cardiovascular and renal outcomes in type 1 dia- betes: the DCCT/EDIC study. Clin J Am Soc Nephrol (2016) 11(11):1969–77. doi:10.2215/CJN.02870316 35. McClintic BR, McClintic JI, Bisognano JD, Block RC. The relationship between retinal microvascular abnormalities and coronary heart disease: a review. Am J Med (2010) 123(4):.e1–7. doi:10.1016/j.amjmed.2009.05.030 27. Myers CE, Klein R, Knudtson MD, Lee KE, Gangnon R, Wong TY, et  al. Determinants of retinal venular diameter: the Beaver Dam Eye Study. Ophthalmology (2012) 119(12):2563–71. doi:10.1016/j.ophtha.2012.06.038 36. Gartner V, Eigentler TK. Pathogenesis of diabetic macro- and microangiopa- thy. Clin Nephrol (2008) 70(1):1–9. doi:10.5414/CNP70001 28. Wong TY, Cheung N, Tay WT, Wang JJ, Aung T, Saw SM, et al. Preval­ ence and risk factors for diabetic retinopathy: the Singapore Malay Eye Study. Ophthalmology (2008) 115(11):1869–75. doi:10.1016/j.ophtha.2008. 05.014 Conflict of Interest Statement: The authors declare that the research was con- ducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer CR and handling editor declared their shared affiliation. 29. Ikram MK, de Jong FJ, Vingerling JR, Witteman JC, Hofman A, Breteler MM, et al. Are retinal arteriolar or venular diameters associated with markers for cardiovascular disorders? The Rotterdam Study. Invest Ophthalmol Vis Sci (2004) 45(7):2129–34. doi:10.1167/iovs.03-1390 Copyright © 2018 Chen, Chen, Lin, Chen, Tsai, Chang, Tsai and Chuang. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). Frontiers in Neurology  |  www.frontiersin.org REFERENCES J Ocul Pharmacol Ther (2009) 25(1):83–9. doi:10.1089/ jop.2008.0068 7. Goud BK, Nayal B, Devi OS, Devaki RN, Avinash SS, Satisha TG, et  al. Comparison of microalbuminuria with hs-CRP and low density lipoprotein levels in nondiabetic, nonhypertensive myocardial infarction patients. J Cardiovasc Dis Res (2012) 3(4):287–9. doi:10.4103/0975-3583.102702 18. Chen CH, Wu PC, Liu YC. Intravitreal bevacizumab injection therapy for per- sistent macular edema after idiopathic macular epiretinal membrane surgery. J Ocul Pharmacol Ther (2011) 27(3):287–92. doi:10.1089/jop.2010.0166 h 8. Hermans MP, Ahn SA, Rousseau MF. The atherogenic dyslipidemia ratio [log(TG)/HDL-C] is associated with residual vascular risk, beta-cell function loss and microangiopathy in type 2 diabetes females. Lipids Health Dis (2012) 11:132. doi:10.1186/1476-511X-11-132 i h 19. Gray SP, Jandeleit-Dahm K. The pathobiology of diabetic vascular com- plications – cardiovascular and kidney disease. J Mol Med (Berl) (2014) 92(5):441–52. doi:10.1007/s00109-014-1146-1 9. Kumar HK, Kota S, Basile A, Modi K. Profile of microvascular disease in type 2 diabetes in a tertiary health care hospital in India. Ann Med Health Sci Res (2013) 2(2):103–8. doi:10.4103/2141-9248.105654 20. Vaya A, Sanchez F, Todoli J, Calvo J, Alis R, Collado S, et al. Homocysteine levels in patients with primary and secondary Raynaud’s phenomenon. Its association with microangiopathy severity. Clin Hemorheol Microcirc (2013) 56(2):153–9. doi:10.3233/CH-131681 10. Romero P, Salvat M, Fernandez J, Baget M, Martinez I. Renal and retinal microangiopathy after 15 years of follow-up study in a sample of type 1 diabetes mellitus patients. J Diabetes Complications (2007) 21(2):93–100. doi:10.1016/j.jdiacomp.2006.04.001 21. Hamed SA. Atherosclerosis in epilepsy: its causes and implications. Epilepsy Behav (2014) 41:290–6. doi:10.1016/j.yebeh.2014.07.003 March 2018  |  Volume 9  |  Article 113 Frontiers in Neurology  |  www.frontiersin.org 6 Chen et al. Microangiopathy and AED Therapy subjects: a cross-sectional study. Kidney Blood Press Res (2009) 32(6):434–9. doi:10.1159/000266477 22. Hamed SA, Hamed EA, Hamdy R, Nabeshima T. Vascular risk factors and oxidative stress as independent predictors of asymptomatic atherosclerosis in adult patients with epilepsy. Epilepsy Res (2007) 74(2–3):183–92. doi:10.1016/j. eplepsyres.2007.03.010 subjects: a cross-sectional study. Kidney Blood Press Res (2009) 32(6):434–9. doi:10.1159/000266477 32. Edwards MS, Wilson DB, Craven TE, Stafford J, Fried LF, Wong TY, et al. Associations between retinal microvascular abnormalities and declining renal function in the elderly population: the cardiovascular health study. Am J Kidney Dis (2005) 46(2):214–24. doi:10.1053/j.ajkd.2005.05.005 23. Talaat FM, Kamel T, Rabah AM, Ahmed SM, El-Jaafary SI, Abdelaziz GH. Epilepsy and antiepileptic drugs: risk factors for atherosclerosis. Int J Neurosci (2015) 125(7):507–11. REFERENCES The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner 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. 30. Reddy SC, Kihn YM, Nurjahan MI, Ramil A. Retinopathy in type 2 diabetic patients with microalbuminuria. Nepal J Ophthalmol (2013) 5(9):69–74. doi:10.3126/nepjoph.v5i1.7830 31. Shantha GP, Kumar AA, Bharadhi MK, Arthur P. Role of gender in the associations of microalbuminuria with inflammatory markers in hypertensive March 2018  |  Volume 9  |  Article 113 Frontiers in Neurology  |  www.frontiersin.org
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Correction to: RIP1 protects melanoma cells from apoptosis induced by BRAF/MEK inhibitors
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C O R R E C T I O N O p e n A c c e s s Lei et al. Cell Death and Disease (2019) 10:225 https://doi.org/10.1038/s41419-019-1301-2 Lei et al. Cell Death and Disease (2019) 10:225 https://doi.org/10.1038/s41419-019-1301-2 Cell Death & Disease Cell Death & Disease Correction to: RIP1 protects melanoma cells from apoptosis induced by BRAF/MEK inhibitors Correction to: Cell Death and Disease (2018) 9: 679 https://doi.org/10.1038/s41419-018-0714-7 published online 07 June 2018 patient#3(post)), and Fig. 5F (the RIP1 of Mel-CV.S and Mel-RMu.S, the GAPDH of Mel-CV and Mel-RMu). As a result of the misfiling of the data during preparation of figures, incorrect images were inadvertently inserted in these figures. The correct figures are given below. The corrections do not alter the conclusions of the paper. Since publication of this paper, the authors have noticed that there were errors in Fig. 2A (the GAPDH of Mel-CV, Mel-CV.S, Mel-RMu and Mel-RMu.S), Fig. 2C (the GAPDH of Mel-CV.S and Mel-RMu.S), Fig. 3F (the GAPDH of Mel-CV.S and Mel-RMu.S), Fig. 3J (the GAPDH of Mel-RMu.S), Fig. 5C (the ERK1/2 of The authors would like to apologize for any incon- venience this may have caused. Correspondence: Xin Han Zhao (zhaoxinhan@mail.xjtu.edu.cn) or Xu Dong Zhang (Xu.Zhang@newcastle.edu.au) 1Department of Medical Oncology, The First Affiliated Hospital of Xi’an Jiaotong University, Xi’an, Shaanxi 710061, China 2School of Medicine and Public Health, The University of Newcastle, Newcastle, NSW 2308, Australia 3School of Life Science, Anhui Medical University, Hefei, Anhui 230032, China 4Department of Molecular Biology, Shanxi Cancer Hospital and Institute, Taiyuan, Shanxi 030013, China These authors contributed equally: Fu Xi Lei, Lei Jin Edited by: G Dewson Correspondence: Xin Han Zhao (zhaoxinhan@mail.xjtu.edu.cn) or Xu © The Author(s) 2019 2 75kDa- -RIPK1 -GAPDH 37kDa- pre post pre post pre post pre post pre post #1 patient #2 #3 #4 #5 110kDa- -CYLD 44/42kDa- -p-ERK B A B 75kDa- -R -G 37kDa- C Mel CV.S Mel RMu.S 0 10 20 30 40 50 60 70 80 90 100 Apoptotic cells(%) + - - + + - RIP1 siRNA2 necrostatin1 (10μM) PLX4720 (3μM) + - + + + + Mel-CV.S Mel-RMu.S + - + + + + + - - + + - ** ** * * C C D 0 10 20 30 40 50 60 70 80 90 100 * * * * Apoptotic cells(%) Ctrl siRNA RIP1 siRNA1 RIP1 siRNA2 PLX4720 (3μM) + - - - - + - - - - + - - - - + + - - + - + - + - - + + - - - - Patient #1(post) 0 10 20 30 40 50 60 70 80 90 100 * * * * Apoptotic cells(%) Ctrl siRNA RIP1 siRNA1 RIP1 siRNA2 PLX4720 (3μM) + - - - - + - - - - + - - - - + + - - + - + - + - - + + - - - - Patient #3(post) Ctrl siRNA RIP1 siRNA1 RIP1 siRNA2 PLX4720 (3μM) + - - - - + - - - - + - - - - + + - - + Apoptotic cells(%) Mel-CV.S - + - + - - + + - - - - ** ** 0 10 20 30 40 50 60 70 80 * * Mel-RMu.S Apoptotic cells(%) ** Ctrl siRNA RIP1 siRNA1 RIP1 siRNA2 PLX4720 (3μM) + - - - - + - - - - + - - - - + + - - + - + - + - - + + - - - - *** 0 10 20 30 40 50 60 70 80 90 100 * ** Fig. © The Author(s) 2019 © The Author(s) 2019 OpenAccessThisarticleislicensedunderaCreativeCommonsAttribution4.0InternationalLicense,whichpermitsuse,sharing,adaptation,distributionandreproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. 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Official journal of the Cell Death Differentiation Association Page 2 of 4 Lei et al. © The Author(s) 2019 Cell Death and Disease (2019) 10:225 D F G 0 10 20 30 40 50 60 70 80 90 100 * * * * Apoptotic cells(%) Ctrl siRNA RIP1 siRNA1 RIP1 siRNA2 PLX4720 (3μM) + - - - - + - - - - + - - - - + + - - + - + - + - - + + - - - - Patient #1(post) 0 10 20 30 40 50 60 70 80 90 100 * * * * Apoptotic cells(%) Ctrl siRNA RIP1 siRNA1 RIP1 siRNA2 PLX4720 (3μM) + - - - - + - - - - + - - - - + + - - + - + - + - - + + - - - - Patient #3(post) UT Ctrl siRNA RIP1 siRNA2 Mel-CV.S PLX4720 (3μM) UT Ctrl siRNA RIP1 siRNA2 Mel-RMu.S PLX4720 (3μM) 75kDa- -RIPK1 -GAPDH 37kDa- pre post pre post pre post pre post pre post #1 patient #2 #3 #4 #5 110kDa- -CYLD 44/42kDa- -p-ERK E Relative Cell Viability(%) SM-406(1μM) PLX4720(3μM) - - + - - + + + - - + - - + + + Mel-CV.S Mel-RMu.S * * 0 20 40 60 80 100 120 + - - + + - RIP1 siRNA2 z-VAD-fmk (50μM) PLX4720 (3μM) Apoptotic cells(%) + - + + + + Mel-CV.S Mel-RMu.S * 0 10 20 30 40 50 60 70 80 90 100 + - + + + + + - - + + - * ** ** Ctrl siRNA RIP1 siRNA1 RIP1 siRNA2 PLX4720 (3μM) + - - - - + - - - - + - - - - + + - - + Apoptotic cells(%) Mel-CV.S - + - + - - + + - - - - ** ** 0 10 20 30 40 50 60 70 80 * * Mel-RMu.S Apoptotic cells(%) ** Ctrl siRNA RIP1 siRNA1 RIP1 siRNA2 PLX4720 (3μM) + - - - - + - - - - + - - - - + + - - + - + - + - - + + - - - - *** 0 10 20 30 40 50 60 70 80 90 100 * ** 0 10 20 30 40 50 60 70 80 90 100 Apoptotic cells(%) + - - + + - RIP1 siRNA2 necrostatin1 (10μM) PLX4720 (3μM) + - + + + + Mel-CV.S Mel-RMu.S + - + + + + + - - + + - ** ** * * B A C Fig. © The Author(s) 2019 3 A Relative activity of NF- κB Mel-CV Mel-CV.S Mel-RMu Mel-RMu.S 0 1 2 3 4 5 6 7 8 9 * * B I -p- IκBα -IκBα -GAPDH 37kDa- 37kDa- 37kDa- Mel-CV Mel-CV.S Mel-RMu Mel-RMu.S 3*Flag-empty 3*Flag-IκBα S32AS36A Ctrl siRNA RIP1 siRNA2 Mel CV S Relative cell viability(%) Mel RMu S - - - - + - - - - + - - - + + - - + - + 0 20 40 60 80 100 120 - - - - + - - - - + - - - + + - - + - + * * I 3*Flag-empty 3*Flag-IκBα S32AS36A Ctrl siRNA RIP1 siRNA2 Mel-CV.S Relative cell viability(%) Mel-RMu.S - - - - + - - - - + - - - + + - - + - + 0 20 40 60 80 100 120 - - - - + - - - - + - - - + + - - + - + * * C 75kDa- 37kDa- 37kDa- Dox(1μg/ml) -- + -- + -- + -- + Mel-CV.S RIP1 IKD -GAPDH -p-IκBα -RIP1 Mel-RMu.S RIP1 IKD Mel-CV RIP1 IKD Mel-RMu RIP1 IKD -IκBα -p-P65 37kDa- 75kDa- C J D 0 2 4 6 8 10 12 14 16 18 * * Mel-CV RIP1 IKD Mel-CV.S RIP1 IKD Mel-RMu RIP1 IKD Mel-RMu.S RIP1 IKD Dox(1μg/ml) - + - + - + - + * ** Relative abundance ofcFLIP mRNA D A J F K F Relative cell viability(%) Dox(1μg/ml ) Bay-11-7082 (3μM) PS1145 (10μM) PLX4720 (3μM) - - - - + - - - + + - - + - + - - - - + + - - + + + - + + - + + ** 0 20 40 60 80 100 120 * K Relative cell viability(%) Dox(1μg/ml ) Bay-11-7082 (3μM) PS1145 (10μM) PLX4720 (3μM) - - - - + - - - + + - - + - + - - - - + + - - + + + - + + - + + ** 0 20 40 60 80 100 120 * F K E Relative activity of NF- κB 0 1 2 3 4 5 6 7 8 9 ** * * Mel-CV RIP1 IKD Mel-CV.S RIP1 IKD Mel-RMu RIP1 IKD Mel-RMu.S RIP1 IKD Dox(1μg/ml) - + - + - + - + *** E 9 Mel CV.S RIP1 IKD Relative cell viability(%) Dox(1μg/ml ) Bay-11-7082 (3μM) PS1145 (10μM) PLX4720 (3μM) - - - - + - - - + + - - + - + - - - - + + - - + + + - + + - + + Mel-RMu.S RIP1 IKD *** 0 20 40 60 80 100 120 * -GAPDH Mel-RMu.S - - - - + - - - - + - - - + + - - + - + -RIP1 -3*Flag-IκBα-S32AS36A 37kDa- 3*Flag-vector 3*Flag-IκBα-S32AS36A Ctrl siRNA RIP1 siRNA2 Mel-CV.S - - - - + - - - - + - - - + + - - + - + 75kDa- 37kDa- G Relative cell viability(%) * ** Dox(1μg/ml) Ctrl siRNA IκBα siRNA PLX4720 (3μM) - - - + + - - + + + - + + - + + - - - - + - - - + + - - + - + - 0 20 40 60 80 100 120 Mel-CV.S RIP1 IKD * ** ** * Relative cell viability(%) 0 20 40 60 80 100 120 Dox(1μg/ml) Ctrl siRNA IκBα siRNA PLX4720 (3μM) - - - + + - - + + + - + + - + + - - - - + - - - + + - - + - + - Mel-RMu.S RIP1 IKD * *** Relative cell viability(%) Dox(1μg/ml ) Bay-11-7082 (3μM) PS1145 (10μM) PLX4720 (3μM) - - - - + - - - + + - - + - + - - - - + + - - + + + - + + - + + Mel-RMu.S RIP1 IKD *** 0 20 40 60 80 100 120 * H Fig. © The Author(s) 2019 2 D Ctrl siRNA RIP1 siRNA1 RIP1 siRNA2 PLX4720 (3μM) + - - - - + - - - - + - - - - + + - - + Apoptotic cells(%) Mel-CV.S - + - + - - + + - - - - ** ** 0 10 20 30 40 50 60 70 80 * * F Relative Cell Viability(%) SM-406(1μM) PLX4720(3μM) - - + - - + + + - - + - - + + + Mel-CV.S Mel-RMu.S * * 0 20 40 60 80 100 120 D F Mel-RMu.S Apoptotic cells(%) ** Ctrl siRNA RIP1 siRNA1 RIP1 siRNA2 PLX4720 (3μM) + - - - - + - - - - + - - - - + + - - + - + - + - - + + - - - - *** 0 10 20 30 40 50 60 70 80 90 100 * ** G UT Ctrl siRNA RIP1 siRNA2 Mel-CV.S PLX4720 (3μM) UT Ctrl siRNA RIP1 siRNA2 Mel-RMu.S PLX4720 (3μM) G 0 10 20 30 40 50 60 70 80 90 100 * * * * Apoptotic cells(%) Ctrl siRNA RIP1 siRNA1 RIP1 siRNA2 PLX4720 (3μM) + - - - - + - - - - + - - - - + + - - + - + - + - - + + - - - - Patient #1(post) P Official journal of the Cell Death Differentiation Association Official journal of the Cell Death Differentiation Association Page 3 of 4 Lei et al. © The Author(s) 2019 Cell Death and Disease (2019) 10:225 A B C D I J Relative activity of NF- κB Mel-CV Mel-CV.S Mel-RMu Mel-RMu.S 0 1 2 3 4 5 6 7 8 9 * * -p- IκBα -IκBα -GAPDH 37kDa- 37kDa- 37kDa- Mel-CV Mel-CV.S Mel-RMu Mel-RMu.S K E -GAPDH Mel-RMu.S - - - - + - - - - + - - - + + - - + - + -RIP1 -3*Flag-IκBα-S32AS36A 37kDa- 3*Flag-vector 3*Flag-IκBα-S32AS36A Ctrl siRNA RIP1 siRNA2 Mel-CV.S - - - - + - - - - + - - - + + - - + - + 75kDa- 37kDa- F 3*Flag-empty 3*Flag-IκBα S32AS36A Ctrl siRNA RIP1 siRNA2 Mel-CV.S Relative cell viability(%) Mel-RMu.S - - - - + - - - - + - - - + + - - + - + 0 20 40 60 80 100 120 - - - - + - - - - + - - - + + - - + - + * * Relative activity of NF- κB 0 1 2 3 4 5 6 7 8 9 ** * * Mel-CV RIP1 IKD Mel-CV.S RIP1 IKD Mel-RMu RIP1 IKD Mel-RMu.S RIP1 IKD Dox(1μg/ml) - + - + - + - + *** G Relative cell viability(%) * ** Dox(1μg/ml) Ctrl siRNA IκBα siRNA PLX4720 (3μM) - - - + + - - + + + - + + - + + - - - - + - - - + + - - + - + - 0 20 40 60 80 100 120 Mel-CV.S RIP1 IKD * ** ** * Relative cell viability(%) 0 20 40 60 80 100 120 Dox(1μg/ml) Ctrl siRNA IκBα siRNA PLX4720 (3μM) - - - + + - - + + + - + + - + + - - - - + - - - + + - - + - + - Mel-RMu.S RIP1 IKD * *** Relative cell viability(%) Dox(1μg/ml ) Bay-11-7082 (3μM) PS1145 (10μM) PLX4720 (3μM) - - - - + - - - + + - - + - + - - - - + + - - + + + - + + - + + Mel-CV.S RIP1 IKD ** 0 20 40 60 80 100 120 * Relative cell viability(%) Dox(1μg/ml ) Bay-11-7082 (3μM) PS1145 (10μM) PLX4720 (3μM) - - - - + - - - + + - - + - + - - - - + + - - + + + - + + - + + Mel-RMu.S RIP1 IKD *** 0 20 40 60 80 100 120 * H 75kDa- 37kDa- 37kDa- Dox(1μg/ml) -- + -- + -- + -- + Mel-CV.S RIP1 IKD -GAPDH -p-IκBα -RIP1 Mel-RMu.S RIP1 IKD Mel-CV RIP1 IKD Mel-RMu RIP1 IKD -IκBα -p-P65 37kDa- 75kDa- 0 2 4 6 8 10 12 14 16 18 * * Mel-CV RIP1 IKD Mel-CV.S RIP1 IKD Mel-RMu RIP1 IKD Mel-RMu.S RIP1 IKD Dox(1μg/ml) - + - + - + - + * ** Relative abundance ofcFLIP mRNA Fig. © The Author(s) 2019 Cell Death and Disease (2019) 10:225 A B C D F E I Ctrl siRNA RIP1 siRNA2 - - + - - + Mel-CV.S - - + - - + Mel-RMu.S 37kDa- -ERK12 37kDa- -p-ERK1/2 37kDa- -GAPDH 75kDa- -RIP1 37kDa- -SNAIL1 Mel-CV Mel-CV.S Mel-RMu Mel-RMu.S 37kDa- -p-ERK1/2 37kDa- -GAPDH 37kDa- -ERK1/2 37kDa- Ctrl siRNA ERK1/2 siRNA - - + - - + Mel-CV.S - - + - - + Mel-RMu.S 37kDa- 75kDa- - - + - - + Mel-CV - - + - - + Mel-RMu -ERK12 -GAPDH -RIP1 100kDa- 37kDa- Ctrl siRNA ERK1/2 siRNA2 - - + - - + Mel-CV.S - - + - - + Mel-RMu.S 37kDa- 37kDa- -ERK1/2 -CYLD -SNAIL1 -GAPDH - - + - - + Mel-CV - - + - - + Mel-RMu 37kDa- Ctrl siRNA ERK1/2 siRNA M98CTVector pEZ-M98-SNAIL1 Flag-HA-GFP Flag-HA-CYLD -ERK12 37kDa- 37kDa- -GAPDH 75kDa- -RIP1 -SNAIL1 100kDa- -CYLD - - - - - - + - - - - - - + - - - - - + + - - - - + - + - - - + - + + - - + - + - + Mel-CV.S - - - - - - + - - - - - - + - - - - - + + - - - - + - + - - - + - + + - - + - + - + Mel-CV H G J - - + - Mel-CV AZD6244(1μM) SCH772984(1μM) - + 37kDa- -p-ERK1/2 75kDa- -RIP1 100kDa- -CYLD 37kDa- -SNAIL1 37kDa- -GAPDH 37kDa- -ERK1/2 Mel-CV.S - - + - - + Relative Cell Viability(%) PLX4720(3μM) AZD6244(1μM) SCH772984(1μM) - - - + - - + + - + - + Mel-CV.S Mel-RMu.S * * - - - + - - + + - + - + 0 20 40 60 80 100 120 * * Mel-CV.S Ctrl siRNA ERK1/2 siRNA Snail1 siRNA1 PLX4720(3μM) - - - - + - - - - + - - - - + - - - - + + - - + - + - + - - + + Mel-RMu.S Relative Cell Viability(%) * * ** ** * * ** * - - - - + - - - - + - - - - + - - - - + + - - + - + - + - - + + 0 20 40 60 80 100 120 Fig. © The Author(s) 2019 3 G Official journal of the Cell Death Differentiation Association Page 4 of 4 Lei et al. © The Author(s) 2019 5 A C D 37kDa- -SNAIL1 Mel-CV Mel-CV.S Mel-RMu Mel-RMu.S 37kDa- -p-ERK1/2 37kDa- -GAPDH 37kDa- -ERK1/2 37kDa- Ctrl siRNA ERK1/2 siRNA - - + - - + Mel-CV.S - - + - - + Mel-RMu.S 37kDa- 75kDa- - - + - - + Mel-CV - - + - - + Mel-RMu -ERK12 -GAPDH -RIP1 B Ctrl siRNA RIP1 siRNA2 - - + - - + Mel-CV.S - - + - - + Mel-RMu.S 37kDa- -ERK12 37kDa- -p-ERK1/2 37kDa- -GAPDH 75kDa- -RIP1 B E 100kDa- 37kDa- Ctrl siRNA ERK1/2 siRNA2 - - + - - + Mel-CV.S - - + - - + Mel-RMu.S 37kDa- 37kDa- -ERK1/2 -CYLD -SNAIL1 -GAPDH - - + - - + Mel-CV - - + - - + Mel-RMu C E C D G Mel-CV.S Ctrl siRNA ERK1/2 siRNA Snail1 siRNA1 PLX4720(3μM) - - - - + - - - - + - - - - + - - - - + + - - + - + - + - - + + Mel-RMu.S Relative Cell Viability(%) * * ** ** * * ** * - - - - + - - - - + - - - - + - - - - + + - - + - + - + - - + + 0 20 40 60 80 100 120 G F F I 37kDa- Ctrl siRNA ERK1/2 siRNA M98CTVector pEZ-M98-SNAIL1 Flag-HA-GFP Flag-HA-CYLD -ERK12 37kDa- 37kDa- -GAPDH 75kDa- -RIP1 -SNAIL1 100kDa- -CYLD - - - - - - + - - - - - - + - - - - - + + - - - - + - + - - - + - + + - - + - + - + Mel-CV.S - - - - - - + - - - - - - + - - - - - + + - - - - + - + - - - + - + + - - + - + - + Mel-CV H J - - + - Mel-CV AZD6244(1μM) SCH772984(1μM) - + 37kDa- -p-ERK1/2 75kDa- -RIP1 100kDa- -CYLD 37kDa- -SNAIL1 37kDa- -GAPDH 37kDa- -ERK1/2 Mel-CV.S - - + - - + Relative Cell Viability(%) PLX4720(3μM) AZD6244(1μM) SCH772984(1μM) - - - + - - + + - + - + Mel-CV.S Mel-RMu.S * * - - - + - - + + - + - + 0 20 40 60 80 100 120 * * Mel-CV.S ERK1/2 siRNA Snail1 siRNA1 PLX4720(3μM) - - - - - - + - - - + - - - + - - + + - + - + + Mel-RMu.S - - - - - - + - - - + - - - + - - + + - + - + + Fig. © The Author(s) 2019 5 H Relative Cell Viability(%) PLX4720(3μM) AZD6244(1μM) SCH772984(1μM) - - - + - - + + - + - + Mel-CV.S Mel-RMu.S * * - - - + - - + + - + - + 0 20 40 60 80 100 120 * * H I - - + - Mel-CV AZD6244(1μM) SCH772984(1μM) - + 37kDa- -p-ERK1/2 75kDa- -RIP1 100kDa- -CYLD 37kDa- -SNAIL1 37kDa- -GAPDH 37kDa- -ERK1/2 Mel-CV.S - - + - - + I J Official journal of the Cell Death Differentiation Association
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https://www.frontiersin.org/articles/10.3389/fenvs.2018.00132/pdf
English
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Changes in the Microbial Community Structure and Soil Chemical Properties of Vertisols Under Different Cropping Systems in Northern China
Frontiers in environmental science
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Changes in the Microbial Community Structure and Soil Chemical Properties of Vertisols Under Different Cropping Systems in Northern China Xiuli Song 1,2†, Bo Tao 1*, Jing Guo 1†, Jingjing Li 1† and Guofeng Chen 2 1 Agricultural College, Northeast Agricultural University, Harbin, China, 2 Qiqihar Branch of Heilongjiang Academy of Agricultural Sciences, Qiqihar, China The predominant cropping system and management practices play an important role in soil physico-chemical properties and microbiome composition and diversity. This study analyzed the changes in soil fertility and the microbial community in four soybean-based cropping systems over 12 years. Amplification subsequencing techniques were used to compare soil community structures among the systems and identify significant positive and negative fertility factors. Soybean cropping favored the accumulation of OM and the available N, K DTPA Fe, Mn, Zn and Cu contents in soil but not fixed available P. The WS and CS cropping systems were conducive to the fixed available P, but they consumed OM, DTPA K, and Zn. The SC exhibited the lowest soil bacterial and archaeal abundance and diversity but high fungal abundance and diversity. The dominant Proteobacteria in the SC were significantly positively correlated with soil DTPA Fe and Mn. The dominant Actinobacteria were positively correlated with available P, DTPA Cu and Mn. The FS cropping system contained 764 unique bacterial species, 724 unique fungal species and the highest relative abundance of Protista. The FS had high microbial diversity, with high relative abundances of Bacteroidetes and Zygomycota and a significantly lower relative abundance of Actinobacteria. The Bacteroidetes were significantly correlated with available N, OM and DTPA Fe and negatively correlated with available P and DTPA Cu. Zygomycota was negatively correlated with available P and DTPA Cu. In the CS and WS, the soil bacterial abundance and diversity were moderate. The dominant Acidobacteria was significantly negatively correlated with soil DTPA, Fe and Mn. The CS exhibited the lowest fungal abundance and diversity. Furthermore, the relative abundance of Ascomycota was significantly improved in the WS and significantly positively correlated with available P and DTPA Cu. Decreases in the available P, K, DTPA Cu, and Mn of Vertisols greatly affect microbial community structure, and these nutrients regulate bacterial and fungal abundance and diversity. Compared to the SC, the FS, WS, and CS had more balanced soil fertility and microbial stability, but diverse cropping systems are most conducive to soil productivity. These findings are of great relevance for protecting the ecological environment. Keywords: microbial community, microbial diversity, soybean, high-throughput sequencing, 16S rRNA gene, soil Edited by: Sudhakar Srivastava, Banaras Hindu University, India Edited by: Sudhakar Srivastava, Banaras Hindu University, India Sudhakar Srivastava, Banaras Hindu University, India Reviewed by: Saurabh Yadav, Hemwati Nandan Bahuguna Garhwal University, India Arnab Majumdar, Indian Institute of Science Education and Research Kolkata, India *Correspondence: Bo Tao botaol@163.com *Correspondence: Bo Tao botaol@163.com *Correspondence: Bo Tao botaol@163.com †These authors have contributed equally to this work Specialty section: This article was submitted to Agroecology and Ecosystem Services, a section of the journal Frontiers in Environmental Science Received: 02 May 2018 Accepted: 24 October 2018 Published: 13 November 2018 ORIGINAL RESEARCH published: 13 November 2018 doi: 10.3389/fenvs.2018.00132 Edited by: Sudhakar Srivastava, Banaras Hindu University, India Citation: Song X, Tao B, Guo J, Li J and Chen G (2018) Changes in the Microbial Community Structure and Soil Chemical Properties of Vertisols Under Different Cropping Systems in Northern China. Front. Environ. Sci. 6:132. doi: 10.3389/fenvs.2018.00132 November 2018 | Volume 6 | Article 132 Frontiers in Environmental Science | www.frontiersin.org 1 Cropping Systems Affect Microbial Communities Song et al. INTRODUCTION The rotation of corn and soybeans was found to have a great effect on the microbial community structure of rhizosphere soil microbial groups, with greater impacts being observed on fungi than on bacteria (Benitez et al., 2017). In a plant system containing soy, combining the appropriate crop management techniques is necessary for sustainable production (Fierer et al., 2009). Proteobacteria, Acidobacteria, and Actinobacteria were the main phyla in soybean continuous cropping (SC), soybean-corn, and corn- soybean rotation systems in all soil samples across succession. With the accumulation of soil organic matter (OM) and nutrients, the relative abundance of Actinobacteria decreased, while the relative abundance of Proteobacteria increased (Zeng et al., 2017). High-throughput sequencing provides a useful means to understand the soil microbial community structure and microbial diversity. The microbial community structure is influenced by the physical and chemical properties of the soil (Xun et al., 2015; Cai et al., 2017) as well as by the species and even the genotype of the host plant (Ofek et al., 2014). Heilongjiang Province is the main soybean production area in northern China. From 1999 to 2016, the average soybean production accounted for 36.21% of the national average, while Nenjiang is the main production area in Heilongjiang Province. Therefore, in this study, high-throughput sequencing (i.e., Illumina MiSeq) was conducted to analyze the microorganism community structure and diversity as well as the interactions of the community with soil nutritional profiling under different cropping systems, i.e., the fallow-soybean cropping system (FS), corn-soybean rotation cropping system (CS), wheat-soybean rotation cropping system (WS), and soybean continuous cropping system (SC), across multiple years. The objectives were to (1) identify the appropriate cropping system for maintaining soil fertility, (2) determine the appropriate agricultural cropping system for protecting the soil microbial community, and (3) identify how to enhance the microbial community structure according to the nutritional preferences under different cropping systems. The results of this work will provide a foundation for the regulation of soil nutritional profiling and microorganism community structure, guide cropping system decisions, and protect soil ecology. INTRODUCTION among different cropping systems, soil fertility and microbial communities, especially on the effects of mineral elements on microbial communities in soil, is rare. Thus, the potential biological control of crop plants is of great relevance for reducing the amount of fertilizer used (Cai et al., 2015). The microbial community in soil drives important nutrient cycling processes that can influence the soil nutritional profile, crop productivity, and environmental sustainability. Agricultural management affects the chemical, physical, and biological processes in the soil and triggers many changes in the microbial community structure (Fierer et al., 2012; Zhang et al., 2016). The abnormal accumulation or excessive consumption of soil nutrients can result in the rapid multiplication of pathogenic microorganisms, imbalances in the soil microbial population structure, and decreased crop yield and quality (Xing et al., 2011). crop productivity, and environmental sustainability. Agricultural management affects the chemical, physical, and biological processes in the soil and triggers many changes in the microbial community structure (Fierer et al., 2012; Zhang et al., 2016). The abnormal accumulation or excessive consumption of soil nutrients can result in the rapid multiplication of pathogenic microorganisms, imbalances in the soil microbial population structure, and decreased crop yield and quality (Xing et al., 2011). Continuous monocultures reduce soil microbial biodiversity and alter the microbial community structure, which decreases its buffering capability to tolerate biotic and abiotic stress, thus increasing the incidence of disease and subsequently affecting plant health (Wang et al., 2011; Mo et al., 2016; Xiong et al., 2016). Continuous cropping could reduce the quantity of bacteria in the soil and increase the relative proportion of fungi. Soil fungal diversity was found to be a good and sensitive indicator of soil fertility (Hobbs et al., 2008; Fierer et al., 2012). Reduced tillage is a sustainable plant management system that can lead to increased surface microbial activity and biomass and protect soil, water, and air quality as well as biodiversity (Tian et al., 2015; Wang et al., 2015; Benitez et al., 2017). Anaerobic bacteria, such as Clostridia and other dominant anaerobic methane-producing archaea, were found in only no-till systems (Luo et al., 2016). However, crop rotation can also modify soil mass and fertility factors and thus influence microbial community structure (Benitez et al., 2017). Microbial diversity is reportedly higher in plots in which oat and maize are grown (Fierer et al., 2012). Frontiers in Environmental Science | www.frontiersin.org Description of the Experimental Field Description of the Experimental Field This study was based on soil samples collected during a 12-year experiment conducted in the experimental ground of the 5th squadron of the fourth field of the Nenjiang River in northern China, known as China’s soybean country. The soil type belongs to the Vertisol class according to the American soil classification system (Soil Survey Staff, 2014). This site has a temperate monsoon climate and the following geographical coordinates: 125◦41′ E longitude and 49◦N latitude. The local annual mean temperature is 0.8 to 1.4◦C, and the average rainfall is 480 to 512 mm. The winter is cold, long, and dry, with a minimum temperature of −47.3 to −43.7◦C. The summer is hot and rainy, with a maximum temperature of 33.9 to 37.4◦C. The soil is classified as black, and the region is the main production area of wheat and soybean in China. Lu et al. (2011) found that increasing the crop rotation of legumes could release nutrients more quickly, leading to higher nitrogen (N) and potassium (K) contents and lower available phosphorus (P) content. Fierer et al. (2009) found that the P, total N, and mineral N contents were significantly improved on the soil surface by no-tillage management systems relative to traditional farming. Rotation could also increase the amount of OM and N in soil, and increasing N enriches the bacterial microorganism composition. In previous studies, microbial communities were significantly correlated with the OM and available N, and external carbon (C), and N additions to a system significantly influenced the microbial community structure (Fierer et al., 2012). However, research on the interactions MATERIALS AND METHODS Description of the Experimental Field Chemical Analyses Soil samples were dried at room temperature and ground into pieces (that passed through a 0.2-mm sieve). The soil micronutrients Fe, Mn, Zn, and Cu were extracted with diethylenetriaminepentaacetic acid (DTPA) followed by determination by atomic absorption spectrophotometry (Lindsay and Norvell, 1978). Available boron (B) was determined by K imine colorimetry (Malekani and Cresser, 1998), and the pH was measured at a soil-to-water ratio of 2.5:1. Soil organic C was determined following wet digestion (Walkley and Black, 1934), and the values were multiplied by a factor of 1.724 to obtain OM values. The basic N solution was determined using the diffusion method (Bremner and Keeney, 1966), and available P was determined using the 0.5 mol. L−1 NaHCO3 leaching-molybdenum antimony colorimetric method (Föhse et al., 1991). Available K was measured with the 1 mol. L−1 NH4OAc extraction-flame photometric method (Petersen and Corey, 1966). Statistical and Bioinformatics Analyses Statistical and Bioinformatics Analyses Fastx was used to remove bases with end masses lower than Q15; the paired sequences obtained by double-end sequencing were merged into a longer sequence by FLASH in an overlapping relationship; and the obtained splicing sequence (merge) was obtained. Cutadapt software was used to delete the miscellaneous sequence at the end of the short sequence, and the resulting sequence was filtered with USEARCH software to obtain the valid sequence (Primer Filter). The singleton sequence was removed by USEARCH software, and the reference standard was compared to remove the chimeric sequence. The UPARSE clustering method was performed to merge the quality-controlled sequences (97-degree similarity between sequences) into an operational taxonomic unit (OTU) cell for subsequent OTU classification annotations (Edgar, 2013). Mothur software was used to compare the RDP, and the UNITE database was used for taxonomic analysis (Caporaso et al., 2010). All sequences were deposited in the NCBI Sequence Read Archive (SRA) database (accession numbers SRP155104), and the quality of the raw data sequencing is shown in Supplementary Table 1. Microbial community composition and species diversity analyses were performed using Lefse in R (R Development Core Team, 2008) and with Mothur software, and regression analyses of the relationships between microorganisms and soil physico- chemical properties were performed using R (Tian et al., 2015). Analyses of variance (ANOVAs) and Duncan’s multiple range tests were performed with SPSS 17.0 to test the significance level (p < 0.05) of the differences among the treatments. Experimental Design and Treatments Experimental Design and Treatments Soybean was planted in experimental plots before March 2005, and rotation trials began in April 2005. Four cropping systems were arranged in a randomized block design: fallow- soybean cropping system (FS), corn-soybean rotation cropping system (CS), wheat-soybean rotation cropping system (WS), November 2018 | Volume 6 | Article 132 Frontiers in Environmental Science | www.frontiersin.org 2 Cropping Systems Affect Microbial Communities Song et al. XP Kit (Beckman Coulter, CA, USA). The purified products were indexed in the 16S V4-V5 or ITS1 library. The library quality was assessed on the Qubit@2.0 Fluorometer (Thermo Scientific) and Agilent Bioanalyzer 2100 systems. Finally, the pooled library was sequenced on an Illumina MiSeq 250 Sequencer, generating 2 × 250 bp paired-end reads (Zhu et al., 2016). and soybean continuous cropping system (SC). Each year, the dosage of ammonium phosphate was 349.5 kg ha−1, the dosage of urea was 199.5 kg ha−1, and the dosage of K phosphate was 199.5 kg ha−1. The cropping lasted for 12 years. Soil samples were collected after the cropping of soybeans in 2016. DNA Extraction, Library Construction, and Sequencing Sequencing Total DNA was extracted using the MoBio PowerSoil DNA Isolation Kit (MoBio, Carlsbad, CA, USA) according to the recommended protocol. The concentration and purity of the DNA were determined using a NanoDrop spectrophotometer, and the DNA integrity was determined using 0.8% agarose gel electrophoresis. The primers 515F (5′-GTGCCAGCMGCC GCGG-3′) and 907R (5′-CCGTCAATTCMTTTRAGTTT-3′) were used to amplify the V4-V5 hypervariable regions of the 16S rRNA gene. The primers ITS1F (5′-CTTGGTCAT TTAGAGGAAGTAA-3′) and ITS1R (5′- GCTGCGTTCTTC ATCGATGC-3′) were used to amplify the fungal internal transcribed spacer (ITS1) region. A parallel amplification of E. coli/yeast genomic DNA served as the positive control, and sterile water was used as the negative control. Each sample was independently amplified three times. Finally, the PCR products were evaluated by agarose gel electrophoresis, and PCR products from the same sample were pooled. The pooled PCR product was used as a template, and index PCR was performed using index primers to add the Illumina index to the library. The amplification products were evaluated using gel electrophoresis and purified using the Agencourt AMPure Soil Sampling Soil samples were collected at seven different sampling points per plot. The soil cores were obtained using a 5-cm-diameter soil auger that collected soil between depths of 0 and 20 cm. The cores were removed by hand and separated into two parts. One part was air dried and sieved through a 2-mm sieve for soil fertility analysis, while the second part was stored at −80◦C for DNA extraction. Frontiers in Environmental Science | www.frontiersin.org Soil Properties p Analysis of the collected soil samples showed that the four cropping systems (i.e., FS, CS, WS, and SC) had significantly varied fertility characteristics (p < 0.05; Table 1). The soils of the tillage modes in SC and FS exhibited higher contents of OM, available N, and K and were thus not conducive to the accumulation of available soil P. These results indicated that soybean cultivation resulted in the accumulation of available OM, N, K, Zn, Fe, and Mn in the soil, which resulted in the overconsumption of P. FS tillage reduced the available soil Cu content and the accumulation of Mn, resulting in the loss of available P. The WS cropping system improved the soil available P and the Cu content, but it consumed greater quantities of OM, available N and Fe. The contents of available K and Zn were the lowest in CS. November 2018 | Volume 6 | Article 132 Frontiers in Environmental Science | www.frontiersin.org 3 Cropping Systems Affect Microbial Communities Song et al. TABLE 1 | Soil physico-chemical properties and microbial diversity by cropping system. Soil Properties Soil properties FS CS WS SC Organic Matter (g/kg) 6.00 ± 0.84ab 5.41 ± 0.14bc 4.60 ± 0.19c 7.40 ± 0.36a Available N (ppm) 364 ± 2.08ab 311 ± 7.54b 248 ± 8.39c 387 ± 37.54a Available P (ppm) 12.33 ± 0.88d 38.33 ± 1.20b 47.33 ± 0.88a 25.00 ± 0.58c Available K (ppm) 228 ± 0.58b 152 ± 11.32c 231 ± 2.52b 323 ± 1.76a pH 6.13 ± 0.05b 6.25 ± 0.01a 6.19 ± 0.04ab 6.24 ± 0.01ab DTPA Cu (ppm) 0.65 ± 0.02c 1.33 ± 0.04b 1.84 ± 0.04a 1.29 ± 0.01b DTPA Zn (ppm) 0.46 ± 0.09b 0.14 ± 0.04c 0.50 ± 0.03b 0.69 ± 0.02a DTPA Fe (ppm) 244 ± 4.02b 208 ± 9.10c 196 ± 3.47c 304 ± 0.69a DTPA Mn (ppm) 10.91 ± 0.23d 13.95 ± 0.72c 16.48 ± 0.54b 32.54 ± 0.19a DTPA B (ppm) 0.30 ± 0.00a 1.02 ± 0.64a 0.96 ± 0.11a 0.70 ± 0.27a Bacteria Chao1 5161 ± 204a 4415 ± 148b 4371 ± 46b 4315 ± 159b ACE 5112 ± 132a 4358 ± 143b 4362 ± 38b 4307 ± 135b Shannon 6.71 ± 0.08a 6.49 ± 0.08ab 6.53 ± 0.03ab 6.42 ± 0.06b Fungi Chao1 1222 ± 69a 884 ± 51b 906 ± 69b 1256 ± 21a ACE 1202 ± 64a 873 ± 59b 901 ± 57b 1244 ± 9ab Shannon 4.67 ± 0.123a 3.57 ± 0.138c 4.11 ± 0.067b 4.37 ± 0.070ab SC, soybean continuous cropping system; FS, fallow-soybean cropping system; CS, corn-soybean rotation cropping system; and WS, wheat-soybean rotation cropping system. The mean value ± standard deviation (n = 3). For each line, different lowercase letters (a–d) indicate significant differences (p < 0.05). Values with the same letter are not significantly different (p < 0.05). Chao1 and ACE: abundance of the Chao1 and ACE estimator and operational taxonomic units. Shannon: nonparametric Shannon diversity index. TABLE 1 | Soil physico-chemical properties and microbial diversity by cropping system. SC, soybean continuous cropping system; FS, fallow-soybean cropping system; CS, corn-soybean rotation cropping system; and WS, wheat-soybean rotation cropping system. The mean value ± standard deviation (n = 3). For each line, different lowercase letters (a–d) indicate significant differences (p < 0.05). Values with the same letter are not significantly different (p < 0.05). Chao1 and ACE: abundance of the Chao1 and ACE estimator and operational taxonomic units. Shannon: nonparametric Shannon diversity index. Microbial Colony Structure and Diversity Analysis The dominant bacteria (i.e., the relative abundance) in the various soil samples were mainly distributed in Proteobacteria, Acidobacteria, Bacteroidetes, Actinobacteria, Planctomycetes, Chloroflexi, Gemmatimonadetes, candidate division WPS-1, Verrucomicrobia, Armatimonadetes, and Nitrospirae. The differences among the microbial communities in the different cropping systems were significant. Proteobacterium was the most abundant in all samples, with a relative abundance of more than 20% of the total number of OTUs (Figure 2A). y Bacterial and fungal principal coordinate analysis (PCoA) showed significant differences among the four different cropping systems, but the WS and SC colony structures were similar (Figures 1A,D). The abundance (Chao1/ACE) and diversity (Shannon) of bacteria and fungi in FS were significantly higher (p < 0.05). The diversity and abundance of soil bacteria in SC were the lowest among all the cropping systems analyzed, but the diversity of fungi was higher in SC than in the CS and WS (Table 1). The diversity of fungi in CS was the lowest among all the cropping systems analyzed (p < 0.05). The archaea in WS exhibited enhanced formation, and the lowest abundance of archaea was found in SC (p < 0.05; Figure 1B). The different cropping systems affected the formation of the soil microbial colony structure and diversity. In total, 764 unique bacterial species and 724 unique fungal species were identified in FS, and these values were much higher than those observed in the other treatments. Additionally, 305 bacterial species and 379 fungal species were processed in SC (Figures 1C,F). The FS cropping system was the most conducive to the formation of soil microbial colony diversity among all the cropping systems analyzed, especially in terms of protozoan formation (Figure 1E). ( g ) The dominant bacteria in the SC soil had significantly more members of the phyla Proteobacteria, Bacteroidetes and Actinobacteria than the other cropping systems, and significantly fewer bacteria from the phyla Acidobacteria, Planctomycetes, Gemmatimonadetes, Chloroflexi, and Nitrospirae were observed. The dominant bacteria in WS soil included more members from the phyla Planctomycetes and Acidobacteria than in the other cropping systems, and WS had a significantly lower relative abundance of Bacteroidetes than that found in the other cropping systems. Additionally, candidate division WPS-1 was significantly higher and Verrucomicrobia was significantly lower in the WS cropping system than in the other cropping systems analyzed. Microbial Colony Structure and Diversity Analysis The dominant bacteria in the SC soil had significantly more members of the phyla Proteobacteria, Bacteroidetes and Actinobacteria than the other cropping systems, and significantly fewer bacteria from the phyla Acidobacteria, Planctomycetes, Gemmatimonadetes, Chloroflexi, and Nitrospirae were observed. The dominant bacteria in WS soil included more members from the phyla Planctomycetes and Acidobacteria than in the other cropping systems, and WS had a significantly lower relative abundance of Bacteroidetes than that found in the other cropping systems. Additionally, candidate division WPS-1 was significantly higher and Verrucomicrobia was significantly lower in the WS cropping system than in the other cropping systems analyzed. Microbial diversity was greater in the FS cropping system, which presented higher relative abundances of Chloroflexi, Verrucomicrobia, Nitrospirae, and Bacteroidetes and a significantly lower relative abundance of Actinobacteria. There was a significant percentage (7.2%) of bacteria in the “No_Rank” category in FS. Microbial Community Abundance Analysis The bacteria and archaea identified in this study belong to 33 phyla (classified reads = 94%), 61 classes (84%), 73 orders (56%), 164 families (50%), 385 genera (61%), and 626 species (41%). The fungi and protists identified in this study belong to 7 phyla (classified reads = 95%), 31 classes (93%), 88 orders (85%), 191 families 81%), 371 genera (74%), and 453 species (46%; Supplementary Table 2). The fungi in all samples were mainly distributed in Ascomycota, Basidiomycota and Zygomycota, encompassing 90% of the total samples. Fungi from Ascomycota were significantly more common in WS than in the other systems. The quantity of Basidiomycota was significantly higher in the Frontiers in Environmental Science | www.frontiersin.org November 2018 | Volume 6 | Article 132 4 Cropping Systems Affect Microbial Communities Song et al. FIGURE 1 | Microbial community structure. (A–C): bacteria. (D–F): fungi. (A,D): PCoA. (B,E): histogram. (C,F): Venn diagrams. PCoA: in the PCoA figure, a shorter distance (close) indicates that the difference between the samples in terms of the microbial community composition is small, whereas a larger difference between samples in terms of microbial community diversity is indicated by a longer distance between the samples. The more similar samples (the same sample group) will eventually converge. Histogram: relative abundance (%) of super kingdoms in the soil samples. Venn diagram: the number of species in the overlapping areas of different colors indicate the total number of species. Microbial Colony Structure and Diversity Analysis SC, soybean continuous cropping system; FS, fallow-soybean cropping system; CS, corn-soybean rotation cropping system; and WS, wheat-soybean rotation cropping system. FIGURE 1 | Microbial community structure. (A–C): bacteria. (D–F): fungi. (A,D): PCoA. (B,E): histogram. (C,F): Venn diagrams. PCoA: in the PCoA figure, a shorter distance (close) indicates that the difference between the samples in terms of the microbial community composition is small, whereas a larger difference between samples in terms of microbial community diversity is indicated by a longer distance between the samples. The more similar samples (the same sample group) will eventually converge. Histogram: relative abundance (%) of super kingdoms in the soil samples. Venn diagram: the number of species in the overlapping areas of different colors indicate the total number of species. SC, soybean continuous cropping system; FS, fallow-soybean cropping system; CS, corn-soybean rotation cropping system; and WS, wheat-soybean rotation cropping system. Flavobacterium, Gp3, Rhizomicrobium, Rhodanobacter, Opitutus, and Pedobacter genera, but most bacteria were not identified at the species level (i.e., No_Rank; Figure 2D). The dominant bacteria in the Gp6 genus in FS was negatively correlated with the DTPA Fe and Mn contents. The dominant bacterial Gp4 found in the CS was negatively correlated with the available K and DTPA Zn, Fe, and Mn contents. The dominant bacteria in WS were Gemmatimonas, Gaiella, and incertae sedis WPS-1, which were significantly related to the available P and DTPA Cu contents. Gp1 was negatively related to the available N content. CS cropping system than in the other cropping systems. The relative abundance of Zygomycota in FS was significantly higher (relative abundance of 19.7%) than those in the SC (9.7%) and crop rotation (6.1%) cropping systems (Figure 2B). Proteobacteria was significantly positively correlated with the DTPA Fe and Mn contents in the soil, whereas Acidobacteria was significantly negatively correlated with these factors. Bacteroidetes was significantly correlated with the available N, OM, and Fe contents and significantly negatively correlated with the available P and DTPA Cu contents. Actinobacteria was positively correlated with the available P and DTPA Cu and Mn contents (Figure 2C). The dominant bacteria Rhizomicrobium, Rhodanobacter, and Pedobacter in SC were significantly positively correlated with the available K, DTPA Mn and DTPA Fe contents (Table 2). Fungal Ascomycota was significantly correlated with the available P and DTPA Cu contents. Zygomycota and Cercozoa (Protista) were significantly negatively correlated with the available P and DTPA Cu contents (Figure 2E). Frontiers in Environmental Science | www.frontiersin.org Microbial Colony Structure and Diversity Analysis The relative abundance values of fungal genera varied significantly among the samples (Figure 2F). The dominant fungus Mortierella in the FS was negatively correlated with the DTPA Cu and available P contents, and the dominant fungus Peziza was significantly negatively correlated with the available Bacteria in the Vertisol samples were mainly distributed in the Gp4, Gp6, Gemmatimonas, Gp1, Gaiella, incertae sedis WPS-1, November 2018 | Volume 6 | Article 132 Frontiers in Environmental Science | www.frontiersin.org 5 Cropping Systems Affect Microbial Communities Song et al. FIGURE 2 | Analysis of microbial community abundance. (A,C,D): bacteria. (B,E,F): fungi. (A,B): histograms of the phyla. (D,F): histograms of the genera. (C,E): heat map. The histograms show the relative abundances (%) of the phyla or genera. Heat map: redundancy analysis identified ten selected soil physico-chemical variables (Continued) FIGURE 2 | Analysis of microbial community abundance. (A,C,D): bacteria. (B,E,F): fungi. (A,B): histograms of the phyla. (D,F): histograms of the genera. (C,E): heat map. The histograms show the relative abundances (%) of the phyla or genera. Heat map: redundancy analysis identified ten selected soil physico-chemical variables (Continued) November 2018 | Volume 6 | Article 132 6 Cropping Systems Affect Microbial Communities Song et al. FIGURE 2 | and the microbial biomasses among the microbial communities. Each column represents a different soil physico-chemical property, each row represents a different species, and the different colors indicate the sizes of two correlation coefficients. A darker blue indicates a stronger negative correlation, whereas a more intense orange represents a greater positive correlation. *indicates significance at p < 0.05, **indicates significance at p < 0.01. SC, soybean continuous cropping system; FS, fallow-soybean cropping system; CS, corn-soybean rotation cropping system; and WS, wheat-soybean rotation cropping system. FIGURE 2 | and the microbial biomasses among the microbial communities. Each column represents a different soil physico-chemical property, each row represents a different species, and the different colors indicate the sizes of two correlation coefficients. A darker blue indicates a stronger negative correlation, whereas a more intense orange represents a greater positive correlation. *indicates significance at p < 0.05, **indicates significance at p < 0.01. SC, soybean continuous cropping system; FS, fallow-soybean cropping system; CS, corn-soybean rotation cropping system; and WS, wheat-soybean rotation cropping system. TABLE 2 | Relationships between dominant bacterial species and soil physico-chemical properties. Microbial Colony Structure and Diversity Analysis Genus Phylum Higher Lower N P K OM pH Cu Zn Fe Mn B Gp4 Acidobacteria CS SC −0.66 0.49 −0.94 −0.63 0.08 0.21 −0.86 −0.88 −0.78 0.18 Gp6 Acidobacteria FS SC −0.44 −0.01 −0.62 −0.66 −0.61 −0.21 −0.52 −0.72 −0.92 −0.14 Gp1 Acidobacteria WS −0.67 0.37 0.04 −0.31 −0.20 0.46 0.17 −0.42 −0.20 −0.07 Gemmatimonas Gemmatimonadetes WS −0.65 0.73 −0.33 −0.76 0.02 0.65 −0.14 −0.73 −0.35 0.38 WPS-1 candidate_division_WPS-1 WS −0.81 0.84 −0.38 −0.66 0.20 0.77 −0.16 −0.76 −0.33 0.28 Gaiella Actinobacteria WS −0.68 0.94 −0.36 −0.68 0.29 0.85 −0.29 −0.67 −0.12 0.55 Gp3 Acidobacteria −0.18 −0.21 0.03 0.16 −0.36 −0.17 −0.04 −0.03 −0.16 −0.22 Rhizomicrobium Proteobacteria SC 0.24 0.22 0.71 0.28 0.38 0.47 0.63 0.52 0.87 0.02 Rhodanobacter Proteobacteria SC FS −0.02 0.19 0.38 0.46 0.46 0.34 0.21 0.45 0.72 0.16 Pedobacter Bacteroidetes SC FS 0.62 −0.17 0.72 0.62 0.33 0.04 0.55 0.80 0.90 −0.10 Opitutus Verrucomicrobia FS 0.86 −0.92 0.39 0.67 −0.20 −0.84 0.35 0.75 0.22 −0.30 Flavobacterium Bacteroidetes FS WS 0.59 −0.84 0.25 0.34 −0.35 −0.81 0.35 0.43 −0.14 −0.43 Notes: the most abundant OTUs in each taxonomic level were correlated with the tillage system and soil physico-chemical properties (i.e., pH, OM, available N, P, K, DTPA Fe, Mn, Zn, and Cu) by correlation with a significance level. The red boxes indicate significant positive correlations, and the blue boxes represent significant negative correlations (p < 0.01). The red digits represent positive correlations, and the blue digits represent negative correlations (p < 0.05). WPS-1: WPS-1_genera_incertae_sedis. TABLE 2 | Relationships between dominant bacterial species and soil physico-chemical properties. B Notes: the most abundant OTUs in each taxonomic level were correlated with the tillage system and soil physico-chemical properties (i.e., pH, OM, available N, P, K, DTPA Fe, Mn, Zn, and Cu) by correlation with a significance level. The red boxes indicate significant positive correlations, and the blue boxes represent significant negative correlations (p < 0.01). The red digits represent positive correlations, and the blue digits represent negative correlations (p < 0.05). WPS-1: WPS-1_genera_incertae_sedis. Different cropping systems cause significant differences in soil fertility. Thus, the microbial community structure is further influenced. The microbial community structure in FS was significantly affected by the low contents of available P, DTPA Cu and Mn. The soil microbial community structure in the SC cropping system was mainly affected by high available K, DTPA Mn and Fe contents. Microbial Colony Structure and Diversity Analysis The microbial community structure in the CS cropping system was mainly affected by low available N and OM contents and high available P and DTPA Cu contents. The soil microbial community structure in the WS cropping system was mainly affected by low available N, OM, and DTPA Fe contents and by high available P and DTPA Cu contents. The crop species and soil characteristics coopted and promoted the growth of microbial communities. The microbial community structure could be altered by regulating the variety of crops and increases and decreases in soil characteristics. P and DTPA Cu contents. Chaetomium was significantly less prominent in FS, and Chaetomium was positively correlated with the available P content. The dominant bacterium Tetracladium in SC soil was positively correlated with the DTPA Mn content. The dominant bacterium Gibberella was positively correlated with the available N content, and the dominant bacterium Lectera was positively correlated with the available K and DTPA Zn and Mn contents. In the CS and WS rotation soils, the dominant fungi Alternaria and Hydropisphaera were positively correlated with the available P and DTPA Cu contents but negatively correlated with the available N and DTPA Fe contents (Table 3). P and DTPA Cu contents. Chaetomium was significantly less prominent in FS, and Chaetomium was positively correlated with the available P content. The dominant bacterium Tetracladium in SC soil was positively correlated with the DTPA Mn content. The dominant bacterium Gibberella was positively correlated with the available N content, and the dominant bacterium Lectera was positively correlated with the available K and DTPA Zn and Mn contents. In the CS and WS rotation soils, the dominant fungi Alternaria and Hydropisphaera were positively correlated with the available P and DTPA Cu contents but negatively correlated with the available N and DTPA Fe contents (Table 3). Significant Differences in Species Under the different cropping systems, the genera (Figures 3A,B) that formed were distinct for the different colony structures. The Geobacter, Geothrix, and Azonexus bacteria compositions were significantly different. In FS, significant differences in fungi existed and included Eocronartium, Pluteus, Slopeiomyces, and Acaulospora. These microorganisms were significantly correlated with low available P and low DTPA Cu content in the soil. The soil microbial community is well correlated with soil physico-chemical properties (Figures 4A,B; Table 5). The bacterial microbial community was significantly correlated with the available P and DTPA Mn contents in the soil, with a correlation coefficient of 0.93; additionally, the community was correlated (0.91) with the available P, DTPA Cu and Mn contents. The effects of fungi and soil on the available P, K, and DTPA Mn contents were significant, with a correlation coefficient of 0.85 (Table 5). Microorganisms that produced significant differences in SC were mainly distributed in the bacteria Sphingobacterium, Brevundimonas, and Mucilaginibacter and in Auxarthron, the fungi Entorrhiza, unclassified Bionectriaceae, and unclassified Corticiaceae. These microorganisms were significantly associated with a high OM content, high available K content, and high DTPA Fe and Mn contents in SC soil. Microbes and physico- chemical properties have a corresponding relationship (Table 4), and soil physico-chemical property extremes (i.e., extremely high/extremely low) determine the microbial community structure. Frontiers in Environmental Science | www.frontiersin.org Bacterial KEGG Functional Differences The red digits represent positive correlations, and the blue digits represent negative correlations (p < 0.05). Notes: the most abundant OTUs in each taxonomic level were correlated with the tillage system and soil physico-chemical properties (i.e., pH, OM, available N, P, K, DTPA Fe, Mn, Zn, and Cu) by correlation with a significance level. Higher: indicates that the species is significantly more abundant in that cropping system. Lower: indicates that the species is significantly less abundant in that cropping system (p < 0.05). The red boxes represent significant positive correlations, and the blue boxes represent significant negative correlations (p < 0.01). The red digits represent positive correlations, and the blue digits represent negative correlations (p < 0.05). significantly, which is consistent with the results of Li et al. (2010). Single cropping may increase the relative abundance of fungi in the soil and increase the occurrence probability of soil-borne diseases. In this study, the FS cropping system was characterized by a more diverse microbial ecosystem, confirming that reducing agricultural cropping can increase soil microbial diversity (Wang et al., 2011). The diversity of soil bacteria in the WC and CS cropping systems was greater than that in the SC cropping system, which is consistent with previous studies (Ai et al., 2015). Proteobacteria and Bacteroidetes can inhibit the occurrence of diseases (Ai et al., 2015), and Acidobacteria has the ability to break down complex soil OM and plant-derived polysaccharides (Ward et al., 2009). The dominant bacteria (i.e., the relative abundance) distributed in various samples of cold-ground Vertisols were previously found to be from Proteobacteria, Acidobacteria, Bacteroidetes, and Actinobacteria (Knelman et al., 2015), and the results of the present study confirmed these conclusions. Proteobacteria was the most abundant in all treatments, accounting for ∼20% of the total number of OTUs (Figure 2A), and the Proteobacteria associated with symbiotic rhizobia was significantly more abundant in the SC cropping system. With the accumulation of soil OM and nutrients, the relative abundance of Proteobacteria increased (Freedman and Zak, 2015; Zeng et al., 2017). Acidobacteria was significantly less abundant in SC but significantly more abundant in the WS cropping system. Bacteroidetes was significantly higher in the FS cropping system. Actinobacteria was significantly higher in the SC cropping system but significantly lower in the FS cropping system. Bacterial KEGG Functional Differences Janssen (2006) found that the content of Gemmatimonadetes in soil bacterial communities was 2% (Janssen, 2006), and in the present study, Gemmatimonadetes was significantly more abundant in the WS cropping system and was detected in ∼0.91% of reads. We also found that Verrucomicrobia was significantly more abundant in the FS cropping system. Bergmann et al. (2011) studied undisturbed soil and found that 23% of bacterial sequences belonged to Verrucomicrobia microbes (Bergmann et al., 2011). In the present study, the bacteria phylum Chloroflexi was abundant in FS and WS and was nearly twice as abundant as that in more abundant in the SC cropping system than in the other cropping systems (Figure 4E). Additionally, a range of archaeal methanogens, such as Methanomassiliicoccus and Methanosarcina (both anaerobes of Euryarchaeota), were significantly higher in FS than in the other cropping systems. However, these groups were not detected in samples from SC and WS (Figure 4E). Azotobacter N-fixing bacteria existed in only FS, and Nitrospira and Anaerolinea were significantly more abundant in FS than in the other cropping systems. Azotobacter bacteria existed in only FS, and Nitrospira and Anaerolinea were significantly more abundant in FS than in the other cropping systems (Figure 4E). Frontiers in Environmental Science | www.frontiersin.org Bacterial KEGG Functional Differences Bacterial KEGG Functional Differences The bacterial microbial communities of the different cropping systems had significant functional differences, while the functional differences in the fungal communities were not obvious (Figures 4C,D). Cyanobacteria were significantly Frontiers in Environmental Science | www.frontiersin.org November 2018 | Volume 6 | Article 132 7 Cropping Systems Affect Microbial Communities Song et al. TABLE 3 | Relationships between dominant fungal species and soil physico-chemical properties. Genus Phylum Higher Lower N P K OM pH Cu Zn Fe Mn B Mortierella Zygomycota FS 0.44 −0.69 0.12 0.33 −0.11 −0.67 0.33 0.28 −0.23 −0.38 Alternaria Ascomycota WS CS −0.73 0.89 −0.51 −0.62 0.20 0.71 −0.53 −0.77 −0.29 0.52 Chaetomium Ascomycota FS −0.34 0.59 0.01 0.11 0.50 0.57 −0.19 −0.05 0.41 0.28 Sistotrema Basidiomycota CS −0.18 0.08 −0.44 0.09 0.29 0.02 −0.40 0.01 −0.01 0.33 Tetracladium Ascomycota SC 0.27 0.00 0.52 0.63 0.43 0.16 0.33 0.66 0.85 0.25 Gibberella Ascomycota SC CS 0.63 −0.07 0.12 0.19 0.46 −0.09 −0.13 0.32 0.39 −0.02 Peziza Ascomycota FS 0.67 −0.93 0.55 0.63 −0.43 −0.79 0.52 0.68 0.16 −0.44 Lectera Ascomycota SC 0.33 0.17 0.63 0.16 0.07 0.35 0.61 0.39 0.66 −0.14 Hydropisphaera Ascomycota WS CS −0.78 0.92 −0.37 −0.55 0.25 0.79 −0.39 −0.74 −0.18 0.45 Notes: the most abundant OTUs in each taxonomic level were correlated with the tillage system and soil physico-chemical properties (i.e., pH, OM, available N, P, K, DTPA Fe, Mn, Zn, and Cu) by correlation with a significance level. Higher: indicates that the species is significantly more abundant in that cropping system. Lower: indicates that the species is significantly less abundant in that cropping system (p < 0.05). The red boxes represent significant positive correlations, and the blue boxes represent significant negative correlations (p < 0.01). The red digits represent positive correlations, and the blue digits represent negative correlations (p < 0.05). TABLE 3 | Relationships between dominant fungal species and soil physico-chemical properties. Notes: the most abundant OTUs in each taxonomic level were correlated with the tillage system and soil physico-chemical properties (i.e., pH, OM, available N, P, K, DTPA Fe, Mn, Zn, and Cu) by correlation with a significance level. Higher: indicates that the species is significantly more abundant in that cropping system. Lower: indicates that the species is significantly less abundant in that cropping system (p < 0.05). The red boxes represent significant positive correlations, and the blue boxes represent significant negative correlations (p < 0.01). DISCUSSION In this study, the OM and available N contents in soil were highest in SC, which is consistent with previous results for soybean cropping (Lu et al., 2011). In addition, the study showed that SC increased the contents of available K, DTPA Zn, Fe and Mn in soil, which is consistent with previous single-cropping results (Cai et al., 2017). In the soil under the FS cropping system, the available P, DTPA Cu and Mn contents decreased significantly. In contrast, the contents of available P and DTPA Cu increased significantly in the WS soil, but the contents of available N and DTPA Fe decreased significantly. In the soil under the CS cropping system, the contents of available K and DTPA Zn were significantly lower than under other cropping systems. Thus, different cropping systems generated different soil physico-chemical properties, which is consistent with the results of previous studies (Fierer et al., 2009; Xun et al., 2015), demonstrating that the cropping system can regulate soil physico-chemical properties. The abundance and diversity of soil microbes determine the stability of the soil microbial community and its ability to resist pathogens, which is crucial to the function and sustainable development of the soil ecosystem (Smith et al., 2008; Pang et al., 2017). In this study, the abundance of fungal microorganisms in the SC cropping system increased November 2018 | Volume 6 | Article 132 8 Cropping Systems Affect Microbial Communities Song et al. FIGURE 3 | Analysis of significant species. (A): bacteria. (B): fungi. (A,B): LefSe. LefSe analysis diagram: the nodes indicate that the microbiota (with significant differences from the other groups) played an important role in the group, and the yellow nodes indicate the absence of significant microbial groups in the group. The (Continued) FIGURE 3 | Analysis of significant species. (A): bacteria. (B): fungi. (A,B): LefSe. LefSe analysis diagram: the nodes FIGURE 3 | A l i f i ifi t i (A) b t i (B) f i (A B) L fS L fS l i di th d i di t th t th i bi t ( ith i ifi t FIGURE 3 | Analysis of significant species. (A): bacteria. (B): fungi. (A,B): LefSe. LefSe analysis diagram: the nodes indicate that the microbiota (with significant differences from the other groups) played an important role in the group, and the yellow nodes indicate the absence of significant microbial groups in the group. DISCUSSION The (Continued) Frontiers in Environmental Science | www.frontiersin.org November 2018 | Volume 6 | Article 132 9 Cropping Systems Affect Microbial Communities Song et al. FIGURE 3 | English letters in the image on the right are represented in each group and play an important role in the outline of the species name level. The names on the outer ring of the letters between groups play an important role in the door at the species name level; thus, each layer of nodes from the inside runs through the phylum, class, order, family, genus, and species. Each species layer represents the process from inside extroversion to the said phylum, class, order, family, genus, and species. The diameter of the node is proportional to the relative abundance. SC, soybean continuous cropping system; FS, fallow-soybean cropping system; CS, corn-soybean rotation cropping system; and WS, wheat-soybean rotation cropping system. TABLE 4 | Relationships between species and soil physico-chemical properties. Community Taxon Physico-chemical properties OM N P K Zn Fe Cu Mn pH FS Bacteria Geobacter −0.70 −0.83 Geothrix −0.80 −0.83 −0.75 Azonexus −0.70 −0.80 −0.61 Fungi Eocronartium −0.63 −0.65 −0.63 Pluteus Slopeiomyces −0.73 −0.77 Acaulospora −0.66 −0.71 CS Bacteria Segetibacter 0.85 0.80 0.65 Niastella 0.80 0.71 Paralcaligenes 0.65 −0.59 −0.65 0.60 Fungi Plenodomus −0.77 −0.83 Unclassified_Xylariaceae WC Bacteria Bradyrhizobium −0.62 0.71 0.83 Microlunatus −0.74 −0.81 0.74 −0.61 −0.86 0.61 Bacillus −0.63 −0.69 0.91 −0.61 0.87 Angustibacter −0.67 0.86 0.89 Terrisporobacter −0.59 Fungi Unclassified_Pezizaceae −0.59 −0.77 0.69 0.74 Septoglomus −0.74 0.64 0.72 SC Bacteria Sphingobacterium 0.75 0.64 0.82 0.63 0.90 0.92 Brevundimonas 0.66 0.71 0.80 Mucilaginibacter 0.59 0.82 Fungi Auxarthron 0.68 0.62 0.83 0.67 0.87 0.95 Entorrhiza 0.69 0.64 0.69 0.74 Unclassified_Bionectriaceae 0.75 0.77 0.61 0.82 0.89 Unclassified_Corticiaceae 0.58 0.70 0.78 0.64 0.82 0.89 Notes: the red background represents the highest content of soil physico-chemical properties, and the orange color represents a high content physico-chemical properties. Yellow represents a medium content of physico-chemical properties, and blue represents the lowest content of physico-chemical properties. The value represents the correlation. “+” indicates a positive correlation, and “–” indicates a negative correlation. The higher the value is, the stronger the correlation. TABLE 4 | Relationships between species and soil physico-chemical properties. TABLE 4 | Relationships between species and soil physico-chemical properties. Notes: the red background represents the highest content of soil physico-chemical properties, and the orange color represents a high content physico-chemical properties. Frontiers in Environmental Science | www.frontiersin.org DISCUSSION Yellow represents a medium content of physico-chemical properties, and blue represents the lowest content of physico-chemical properties. The value represents the correlation. “+” indicates a positive correlation, and “–” indicates a negative correlation. The higher the value is, the stronger the correlation. the SC cropping system. These microbes can perform anaerobic photosynthesis and withdraw electrons from hydrogen sulfide (Eisen et al., 2002). Nitrospirae was significantly more abundant in the FS cropping system and significantly lower in SC than in the other cropping systems. Cyanobacteria were more abundant in the SC cropping system; members of this phylum can fix N gas into ammonia (NH+ 4 ), nitrite (NO− 2 ) or nitrate (NO− 3 ) as well as significantly reduce carbon aerobically (Flores, 2008). microbes and anaerobic methanogens as well as many other species (Lei et al., 2017). The genera Methanomassiliicoccus and Methanosarcina of Euryarchaeota were detected in only FS, but other Euryarchaeota were almost five times more abundant in the SC cropping system. Collectively, these data support the notion that the abundance of anaerobic microorganisms increased in the FS because of less aeration due to low soil disturbance. In addition, the OM in the FS soil maintains a certain water storage capacity, providing anaerobic habitats for anaerobic organisms (Minamisawa et al., 2004). Thaumarchaeota and a Candidatus Nitrososphaera comprise the soil NH+ 4 -oxidizing archaea genera; NH+ 4 is oxidized by producing ammoxase (AMO; Zhalnina et al., 2012). Thaumarchaeota was significantly more abundant in the WS cropping system. Euryarchaeota was significantly abundant in FS, and this group includes extreme halophiles, sulfate-reducing The fungi in all samples were mainly distributed in Ascomycota, Basidiomycota and Zygomycota, which accounted for 90% of the total sampled fungi. This result confirms previous findings (Xu et al., 2012). Fungi from Ascomycota were Frontiers in Environmental Science | www.frontiersin.org November 2018 | Volume 6 | Article 132 10 Cropping Systems Affect Microbial Communities Song et al. FIGURE 4 | Microbial community differences. (A,D): bacteria, (B,C): fungi. (A,B): RDA. (C,D): body figure. (E): relative abundance of genera. RDA: The image indicates multiple physico-chemical properties and a sample/microflora RDA analysis diagram. The arrows represent different physico-chemical properties. A longer ray indicates a greater influence of the soil physico-chemical properties. DISCUSSION For each sample, the angle between the center and the arrow represents the relationship between the sample and soil physico-chemical properties (acute angles show positively correlated relationships, obtuse angles indicate negatively correlated relationships, and rectangles indicate irrelevance); the points in different colors represent different samples. The different samples are perpendicular to the arrows for different physico-chemical properties. Closer projection points indicate that the soil physico-chemical properties are more similar in the sample, and the value of the physico-chemical properties increases along the direction of the arrow. The horizontal coordinates in the above C and D images represent different cropping system, and the vertical coordinates indicate the distance between the sample groups. Blue boxes: SC; green boxes: FS; orange boxes: and CS; black boxes: WS. The P-values indicate the differences between the groups. The horizontal coordinates in the unit E graph represent different classifications. The ordinate values represent the relative abundances; the relative abundance of Cyanobacteria and Nitrospira is expressed in %, whereas the quantitative value of the relative abundance times 100 is shown for the other genera. SC, soybean continuous cropping system; FS, fallow-soybean cropping system; CS, corn-soybean rotation cropping system; and WS, wheat-soybean rotation cropping system. FIGURE 4 | Microbial community differences. (A,D): bacteria, (B,C): fungi. (A,B): RDA. (C,D): body figure. (E): relative abundance of genera. RDA: The image indicates multiple physico-chemical properties and a sample/microflora RDA analysis diagram. The arrows represent different physico-chemical properties. A longer ray indicates a greater influence of the soil physico-chemical properties. For each sample, the angle between the center and the arrow represents the relationship between the sample and soil physico-chemical properties (acute angles show positively correlated relationships, obtuse angles indicate negatively correlated relationships, and rectangles indicate irrelevance); the points in different colors represent different samples. The different samples are perpendicular to the arrows for different physico-chemical properties. Closer projection points indicate that the soil physico-chemical properties are more similar in the sample, and the value of the physico-chemical properties increases along the direction of the arrow. The horizontal coordinates in the above C and D images represent different cropping system, and the vertical coordinates indicate the distance between the sample groups. Blue boxes: SC; green boxes: FS; orange boxes: and CS; black boxes: WS. The P-values indicate the differences between the groups. The horizontal coordinates in the unit E graph represent different classifications. DISCUSSION The ordinate values represent FIGURE 4 | Microbial community differences. (A,D): bacteria, (B,C): fungi. (A,B): RDA. (C,D): body figure. (E): relative abundance of genera. RDA: The image indicates multiple physico-chemical properties and a sample/microflora RDA analysis diagram. The arrows represent different physico-chemical properties. A longer ray indicates a greater influence of the soil physico-chemical properties. For each sample, the angle between the center and the arrow represents the relationship between the sample and soil physico-chemical properties (acute angles show positively correlated relationships, obtuse angles indicate negatively correlated relationships, and rectangles indicate irrelevance); the points in different colors represent different samples. The different samples are perpendicular to the arrows for different physico-chemical properties. Closer projection points indicate that the soil physico-chemical properties are more similar in the sample, and the value of the physico-chemical properties increases along the direction of the arrow. The horizontal coordinates in the above C and D images represent different cropping system, and the vertical coordinates indicate the distance between the sample groups. Blue boxes: SC; green boxes: FS; orange boxes: and CS; black boxes: WS. The P-values indicate the differences between the groups. The horizontal coordinates in the unit E graph represent different classifications. The ordinate values represent the relative abundances; the relative abundance of Cyanobacteria and Nitrospira is expressed in %, whereas the quantitative value of the relative abundance times 100 is shown for the other genera. SC, soybean continuous cropping system; FS, fallow-soybean cropping system; CS, corn-soybean rotation cropping system; and WS, wheat-soybean rotation cropping system. FIGURE 4 | Microbial community differences. (A,D): bacteria, (B,C): fungi. (A,B): RDA. (C,D): body figure. (E): relative abundance of genera. RDA: The image indicates multiple physico-chemical properties and a sample/microflora RDA analysis diagram. The arrows represent different physico-chemical properties. A longer ray indicates a greater influence of the soil physico-chemical properties. For each sample, the angle between the center and the arrow represents the relationship between the sample and soil physico-chemical properties (acute angles show positively correlated relationships, obtuse angles indicate negatively correlated relationships, and rectangles indicate irrelevance); the points in different colors represent different samples. The different samples are perpendicular to the arrows for different physico-chemical properties. Closer projection points indicate that the soil physico-chemical properties are more similar in the sample, and the value of the physico-chemical properties increases along the direction of the arrow. DISCUSSION The digits represent r values (p < 0.05) on between each physico-chemical property (i.e., the combination of soil physico-chemical properties) and the community. The greater the he correlation and the greater the influence on the community. The digits represent r values (p < 0.05). The red character stands for outstanding Each line represents the level of correlation between each physico-chemical property (i.e., the combination of soil physico-chemical properties) and the community. The greater the correlation absolute value is, the stronger the correlation and the greater the influence on the community. The digits represent r values (p < 0.05). The red character stands for outstanding numeral. and amounts of soil samples studied. However, there was a significant correlation between the microbial community and the soil physico-chemical properties. significantly more common in the WS cropping system. The relative abundance of Basidiomycota did not significantly differ among the samples. The distribution of Zygomycota fungi was significantly greater in the FS soil, with a relative abundance of 19.7%, which was significantly higher than that under the SC (9.7%) and crop rotation (6.1%) cropping systems. In this study, microbial community structure was influenced by high or low soil fertility in the soils of different cropping systems. In fact, 33 of the 40 microbial phyla were significantly positively or negatively correlated with one or several soil fertility parameters (p < 0.05). The bacterial community structure was significantly correlated with the physico-chemical properties of soil available P, DTPA Cu, and Mn. The fungal community was significantly correlated with available P, K, and DTPA Cu. Soil fertility affects soil microbial community structure (Liu et al., 2014; Zhang et al., 2016). The increased OM and N in soil could promote bacterial diversity (Shen et al., 2014; Li et al., 2016; Zeng et al., 2017). An interesting phenomenon was observed in this experiment: in the SC and FS cropping systems, the contents of OM and N were very high, but the abundance and diversity of bacteria differed significantly, indicating that the abundance and diversity of soil bacteria may be related to other factors in addition to OM and N. The diversity of fungi under SC was significantly higher than that under rotation, and Hooper et al. proposed that this might be related to the long-term accumulation of soil nutrients (Hooper et al., 2005). DISCUSSION These changes in the microbial communities are related to the nutrients and plant biomass available in the soil (Bokhorst et al., 2017). Many studies have concluded that soil and physico- chemical properties shape community structure (Xun et al., 2015; Pang et al., 2017), and as confirmed experimentally, the cropping system, soil fertility, and soil microbial community structure are interrelated and can be adjusted concomitantly (Ofek et al., 2014). In the present study, the cropping system altered the soil properties and the community structure, and we found that there was no perfect cropping system. The FS, WS, and CS systems were capable of balancing soil fertility and microbial stability. Therefore, institutional diversity is most conducive to soil productivity (Kuramae et al., 2012; Yuan et al., 2015). We found that the available P, K, DTPA Cu, and Mn contents were closely related to the microorganism communities. In the FS cropping system, Bacteroidetes and Verrucomicrobia were significantly positively correlated with the OM content, and Bacteroidetes, Verrucomicrobia, and Latescibacteria were significantly negatively correlated with the available P and DTPA Cu contents. Nitrospirae was significantly negatively correlated with the available P, K, and DTPA Mn contents, and Actinobacteria was significantly positively correlated with the available P, DTPA Cu, and Mn contents. In the SC cropping system, Proteobacteria was significantly positively correlated with the available Fe and DTPA Mn contents. In the WS cropping system, Planctomycetes was negatively correlated with the DTPA Zn content. Zygomycota and Cercozoa of Protista were significantly more abundant in the FS cropping system and were significantly negatively correlated with the P and Cu contents. Ascomycota was significantly more abundant in the WS cropping system and was positively correlated with the DTPA Fe and Cu contents. Frontiers in Environmental Science | www.frontiersin.org DISCUSSION The horizontal coordinates in the above C and D images represent different cropping system, and the vertical coordinates indicate the distance between the sample groups. Blue boxes: SC; green boxes: FS; orange boxes: and CS; black boxes: WS. The P-values indicate the differences between the groups. The horizontal coordinates in the unit E graph represent different classifications. The ordinate values represent the relative abundances; the relative abundance of Cyanobacteria and Nitrospira is expressed in %, whereas the quantitative value of the relative abundance times 100 is shown for the other genera. SC, soybean continuous cropping system; FS, fallow-soybean cropping system; CS, corn-soybean rotation cropping system; and WS, wheat-soybean rotation cropping system. Frontiers in Environmental Science | www.frontiersin.org November 2018 | Volume 6 | Article 132 Frontiers in Environmental Science | www.frontiersin.org Frontiers in Environmental Science | www.frontiersin.org 11 Cropping Systems Affect Microbial Communities Song et al. TABLE 5 | Relationships between microorganisms and soil physico-chemical properties. Physico-chemical properties combination Size correlation Physico-chemical properties combination Size correlation Bacteria Fungi P 0.71 P 0.78 P Mn 0.93 P Mn 0.83 P Cu Mn 0.91 P K Cu 0.86 P Cu Fe Mn 0.89 P K Cu Mn 0.85 P K Cu Fe Mn 0.85 P K pH Cu Mn 0.82 P K pH Cu Fe Mn 0.84 N P K pH Cu Mn 0.80 N P K pH Cu Fe Mn 0.82 N P K pH Cu Zn Mn 0.80 N P K OM pH Cu Fe Mn 0.81 N P K pH Cu Zn Fe Mn 0.77 N P K OM pH Cu Zn Fe Mn 0.78 N P K OM pH Cu Zn Fe Mn 0.74 N P K OM pH Cu Zn Fe Mn B 0.74 N P K OM pH Cu Zn Fe Mn B 0.70 Each line represents the level of correlation between each physico-chemical property (i.e., the combination of soil physico-chemical properties) and the community. The greater the correlation absolute value is, the stronger the correlation and the greater the influence on the community. The digits represent r values (p < 0.05). The red character stands for outstanding numeral. TABLE 5 | Relationships between microorganisms and soil physico-chemical properties. Each line represents the level of correlation between each physico-chemical property (i.e., the combination of soil physico-chemical proper correlation absolute value is, the stronger the correlation and the greater the influence on the community. REFERENCES photosynthetic, anaerobic, green-sulfur bacterium. Proc. Natl. Acad. Sci. U.S.A. 99, 9509–9514. doi: 10.1073/pnas.132181499 photosynthetic, anaerobic, green-sulfur bacterium. Proc. Natl. Acad. Sci. U.S.A. 99, 9509–9514. doi: 10.1073/pnas.132181499 Ai, C., Liang, G., Sun, J., Wang, X., He, P., Zhou, W., et al. (2015). Reduced dependence of rhizosphere microbiome on plant-derived carbon in 32-year long-term inorganic and organic fertilized soils. Soil Biol. Biochem. 80, 70–78. doi: 10.1016/j.soilbio.2014.09.028 Fierer, N., Lauber, C. L., Ramirez, K. S., Zaneveld, J., Bradford, M. A., and Knight, R. (2012). Comparative metagenomic, phylogenetic and physiological analyses of soil microbial communities across nitrogen gradients. 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This study was financially supported by the National Key Research and Development Plan of China (2016YFD0200500; 2016YFD070030103). CONCLUSION The cropping system affects the physical and chemical properties of soil and the microbiome structure. (1) Soybean cropping favored the accumulation of OM and the available N, K DTPA Fe, Mn, Zn, and Cu contents in soil but not fixed available P. The WS and CS cropping systems were conducive to the fixed available P, but they consumed OM, DTPA K, and Zn. (2) The FS cropping system could increase the abundance and These results are inconsistent with those of Cai et al. (2017) and Pang et al. (2017), which may be due to the different types November 2018 | Volume 6 | Article 132 Frontiers in Environmental Science | www.frontiersin.org 12 Cropping Systems Affect Microbial Communities Song et al. diversity of bacteria. The SC cropping system could decrease the abundance and diversity of bacteria but increase the abundance and diversity of fungi. The WS and CS cropping systems could reduce fungal abundance and diversity. (3) There was a significant correlation between microbial community structure and the physico-chemical properties of soil, and the most significant correlation was between the bacterial community and available P, DTPA, and Mn (0.93). The fungal community was most correlated with available P, DTPA K, and Cu, with a correlation coefficient of 0.8. In summary, increasing the diversity of the cropping system was more favorable to maintaining soil fertility. The cropping system changes soil properties and microbial diversity, and the microbial community in turn manipulates nutrient cycling processes and alters soil fertility, plant productivity and environmental sustainability. 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Identification of the mulberry genes involved in ethylene biosynthesis and signaling pathways and the expression of MaERF-B2-1 and MaERF-B2-2 in the response to flooding stress
Functional & integrative genomics
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Identification of the mulberry genes involved in ethylene biosynthesis and signaling pathways and the expression of MaERF-B2-1 and MaERF-B2-2 in the response to flooding stress Jingzhe Shang & Penghua Song & Bi Ma & Xiwu Qi & Qiwei Zeng & Zhonghuai Xiang & Ningjia He Received: 13 March 2014 /Revised: 2 September 2014 /Accepted: 7 September 2014 /Published online: 18 September 2014 # The Author(s) 2014. This article is published with open access at Springerlink.com Received: 13 March 2014 /Revised: 2 September 2014 /Accepted: 7 September 2014 /Published online: 18 September 2014 # The Author(s) 2014. This article is published with open access at Springerlink.com Abstract The phytohormone ethylene is essential to plant growth and development. It plays crucial roles in responses to biotic and abiotic stress. The mulberry tree is an important crop plant in countries in which people rear silkworms for silk production. The availability of the mulberry genome has made it possible to identify mulberry genes involved in ethylene biosynthesis and signal pathways. A total of 145 mulberry genes were identified by both homology-based and hidden Markov model (HMM) search, including 29 genes associated with ethylene biosynthesis and 116 genes in the AP2/ERF family. Studies on gene structure have provided a genetic basis for understanding the functions of these genes. The differ- ences in gene expression were also observed in different tissues. The expression of two mulberry genes in the AP2/ ERF family, MaERF-B2-1 and MaERF-B2-2, was found to be associated with the response to flooding stress. nodulation, and responses to biotic and abiotic stress (Johnson and Ecker 1998; Lin et al. 2009). As early as 1910, ethylene was used to accelerate the ripening of bananas (Abeles et al. 1992). In Arabidopsis, ethylene signaling has been found to be important to flooding and salt response (Zhang et al. 2011; Voesenek and Blom 1989). Recent work has indicated that ethylene promotes hypocotyl elongation in light and suppresses it in darkness (Zhong et al. 2012). Ethylene biosynthesis and signaling networks are well understood in plants. Biosynthesis of ethylene occurs in three steps. First, the amino acid methionine is converted to S- adenosylmethionine (AdoMet) by S-adenosylmethionine syn- thetase (Yang and Hoffman 1984). AdoMet is then converted to 1-aminocyclopropane-1-carboxylic acid (ACC) and 5′-de- oxy-5′methylthioadenosine (MTA) by 1-aminocyclopropane- 1-carboxylase synthase (ACS) (Adams and Yang 1979). The formation of the ACC is the rate-limiting step in ethylene biosynthesis, and it requires pyrodoxal-5′-phosphate (PLP) as a cofactor. Funct Integr Genomics (2014) 14:767–777 DOI 10.1007/s10142-014-0403-2 Funct Integr Genomics (2014) 14:767–777 DOI 10.1007/s10142-014-0403-2 ORIGINAL PAPER Identification of the mulberry genes involved in ethylene biosynthesis and signaling pathways and the expression of MaERF-B2-1 and MaERF-B2-2 in the response to flooding stress When a large amount of ethylene is produced, MTA is recycled to methionine through the Yang cycle to supplement the methionine pool (Miyazaki and Yang 1987). In the third step, ACC is transformed into ethylene, CO2, and cyanide by 1-aminocyclopropane-1-carboxylase oxidase (ACO). The cyanide is detoxified into β-cyanoalanine by β- cyanoalanine synthase (Blumenthal et al. 1968). Ethylene can be detected by a family of membrane-localized receptors. A copper cofactor is necessary for the transduction of this signal (Rodrıguez et al. 1999). When ethylene is present, the confor- mation of the compound of ethylene receptor and constitutive triple response 1 (CTR1) changes and the suppression ends (Gao et al. 2003). Ethylene-insensitive 2 (EIN2) is then acti- vated, resulting in the activation of EIN3 transcription factors (Alonso et al. 1999). Proteins EIN3 and EIN3-like 1 (EIL1) are specifically bound to the promoter of ethylene response Keywords Ethylene . Plant hormone . Mulberry . Gene expression J. Shang: P. Song: B. Ma: X. Qi: Q. Zeng: Z. Xiang: N. He (*) State Key Laboratory of Silkworm Genome Biology, Southwest University, Beibei, Chongqing 400715, China e-mail: hejia@swu.edu.cn Introduction Ethylene plays important roles in various aspects of plant growth and development including fruit ripening, germina- tion, flowering, sex determination, leaf senescence, root Electronic supplementary material The online version of this article (doi:10.1007/s10142-014-0403-2) contains supplementary material, which is available to authorized users. J. Shang: P. Song: B. Ma: X. Qi: Q. Zeng: Z. Xiang: N. He (*) State Key Laboratory of Silkworm Genome Biology, Southwest University, Beibei, Chongqing 400715, China e-mail: hejia@swu.edu.cn 768 Funct Integr Genomics (2014) 14:767–777 sequences identified in this way was determined by (http://smart.embl-heidelberg.de/) (SMART) analyses (Letunic et al. 2012). sequences identified in this way was determined by (http://smart.embl-heidelberg.de/) (SMART) analyses (Letunic et al. 2012). factor 1 (ERF1) in AP2/ERF family, and ERF1 plays important roles in the transcriptional regulation of a variety of biological processes associated with growth and development and with biotic and abiotic stress responses. ERF1 binds to the promoters that contain the GCC box. These promoters are present in many inducible ethylene genes (Fujimoto et al. 2000). The related apetala 2.2 gene from Arabidopsis (RAP2.2, At3g14230) has an ERF-bearing AP2 domain (Hinz et al. 2010). Overexpression of RAP2.2 has been shown to improve plant survival under hypoxia stress conditions. The ERF-associated amphiphilic repression (EAR) motif is a repression domain in the C- terminal region of repressor-type ERF proteins. These genes are implicated in developmental, hormonal and stress signaling pathways and can be negatively regulated through the EAR motif (Ohta et al. 2001; Kagale et al. 2010). Sequence alignments and construction of the phylogenetic tree Multiple alignment analysis was performed with ClustalX and subsequently adjusted manually by Genedoc (Larkin et al. 2007). A phylogenetic tree of AP2/ERF was constructed using maximum-likelihood method in PhyML 3.0 using a Perl script. Web tools at the Gene Structure Display Server (http://gsds.cbi.pku.edu.cn/help.php) were used to draw the structures of the genes (Guo et al. 2007). Mulberry trees are deciduous and woody. They are widely used to produce crops for the sericulture industry. The domes- ticated silkworm, Bombyx mori, feeds only on mulberry leaves. In this way, the silk industry depends on the quality and quantity of available mulberry leaves. Mulberry fruits are also used to make jam, juice, and wine. Ethylene has been shown to affect seasonal leaf abscission and fruit ripening (Adams- Phillips et al. 2004). The genes involved in ethylene signaling and biosynthesis are important targets for the improvement of leaf and fruit yield. RNA extractions and quantitative RT-PCR Total RNA was isolated from mulberry roots, barks, leaves, male flowers, female flowers, and fruits using RNAiso Plus (Takara, Japan) according to the manufacturer’s instructions. The quality and concentration of RNA samples were mea- sured using a ND-1000 UV spectrophometer (Nanodrop Technologies, USA). Reverse transcription was performed following the manufacturer’s instructions (Takara, Japan). Primers were designed using Primer Premier 5 software (http://www.primer-e.com/). Polymerase chain reactions were performed in a 96-well plate with a StepOne Plus System apparatus, amplified with SYBR® Green II (Takara, Japan) according to the manufacturer’s instructions. Cycling conditions were as follows: 90 °C for 30 s; 40 cycles of 95 °C for 5 s and 60 °C for 30 s. Amplification specificity was verified by a dissociation curve. To compare data from differ- ent PCR runs, cycle threshold (CT) values for samples were normalized to that of the mulberry ribosomal protein gene (Morus024083). The cDNA quantity was calculated using 2ΔCT ; here, ΔCT is the difference in the number of cycles between tests and controls. The change in gene expression after flooding treatment was measured using 2ΔΔCT , where ΔΔCT=(ΔCT sample−ΔCT control). Gene-specific primers Introduction In China’s Three Gorges Reservoir Region, the most serious type of environmental stress limiting mulberry crop productivity is water-logging. ERFs have been reported to play important roles in the regulation of biotic and abiotic stress (Dietz et al. 2010). One ERF subfamily, the VIIERFs, responds to hypoxia stress and promotes the expression of hypoxia- related genes (Hinz et al. 2010). In the present study, we found the expression patterns of two mulberry genes in the AP2/ERF family, MaERF-B2-1 and MaERF-B2-2, changed in association with flooding treatment. Analysis of the expression of the genes involved in ethylene biosynthesis and signal transduction The reads per kilobase of exon model per million mapped reads (RPKM) was used to analyze the expression levels of all predicted mulberry genes using RNA sequencing data (Mortazavi et al. 2008). The clustering analyses were carried out using Gene Cluster 3.0. The data were adjusted by log transformation and the mean center method. Hierarchical clustering (HCL) with average linkage was set to calculate K-medians with five clusters. Heat maps illustrating the gene expression data were generated using Java TreeView. Materials and methods Database searches and sequence analysis Prediction of cis-acting elements Mulberry species, Husang-32 (Morus alba L. var. multicaulis), maintained at the Mulberry Germplasm Nursery of Southwest University (China), was used in the present study. Husang-32 seedlings were grown in plastic pots filled with soil, vermiculite, and perlite mixture (6:1:1) in incubators (25 °C; 16 h day, 8 h night; 75 % relative humid- ity). After being incubated for 2 months, the seedlings with 15-cm length were subjected to flooding treatment. Plants in To identify cis-regulatory elements, upstream regions within about 2 kb of the transcription start sites of three genes, MnERF-B1, MnERF-B2, and MnERF-B3, were analyzed. All promoter sequences were placed in the PlantCare database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html), which has 435 different plant transcription binding sites. The search results are divided into four categories including stress Table 1 Genes involved in ethylene biosynthesis and signaling pathways in the M. notabilis genome Genes Accession no. Protein size CDS length Gene length Exons Scaffold. no. Database searches and sequence analysis The mulberry genome database was downloaded from MorusDB (http://morus.swu.edu.cn/morusdb/). The Arabidopsis genome database was obtained from TAIR (http://www.arabidopsis.org/). The majority of amino acid sequences of the Arabidopsis thaliana genes involved in the ethylene biosynthesis and signaling pathways were retrieved from Uniprot (http://www.uniprot.org/). The A. thaliana AP2/ ERF genes were downloaded from the DATF website (http:// datf.cbi.pku.edu.cn/index.php). All of the A. thaliana sequences were used as templates in a search of the mulberry genome using BLASTP. Hidden Markov model (HMM) was used to identify motifs of mulberry proteins. The presence of AP2 domains in the mulberry 769 Funct Integr Genomics (2014) 14:767–777 response, hormone response, light response, and others (Lescot et al. 2002). response, hormone response, light response, and others (Lescot et al. 2002). used for the real-time RT-PCR are listed in Supplementary Table 2. All results are representative of three independent experiments. used for the real-time RT-PCR are listed in Supplementary Table 2. All results are representative of three independent experiments. SAM S-adenosylmethionine synthase, ACS 1-aminocyclopropane-1-carboxylate synthase, ACO 1-aminocyclopropane-1-carboxylate oxidase, ETR ethylene receptor, CTR1 constitutive triple response-1, EIN2 ethylene-insensitive 2, EIN3 ethylene-insensitive 3, EBF EIN3-binding F-box protein, ERF ethylene-responsive element binding factor. y p g a The transaminases in ACS family b b The genes located at the same scaffold Prediction of cis-acting elements (strand) Start codon Stop codon MnSAM1 Morus003267 393 1182 1182 1 1843(+) 66,189 67,370 MnSAM2 Morus013867 393 1182 1182 1 498(+) 76,865 78,046 MnSAM3 Morus025140 393 1182 1182 1 172(+) 571,546 572,727 MnSAM4 Morus022555 390 1173 1173 1 124(+) 13,770 14,942 MnACS1 Morus012919 496 1491 2732 4 298(+) 185,423 188,154 MnACS2 Morus024218 486 1461 2196 4 297(+) 4647 6842 MnACS3 Morus007775 471 1416 1782 4 300(−) 229,933 231,714 MnACS4 Morus007092 467 1404 2044 4 991(+) 117,590 119,633 MnACS5 Morus027243 446 1341 1660 3 329(−) 186,543 188,202 MnACS10a Morus023174 557 1674 3428 5 87(+) 408,302 411,729 MnACS12a Morus012967 504 1515 1791 4 399(+) 330,934 332,724 MnACO1 Morus027261 306 921 1127 3 329(+) 408,750 409,876 MnACO2 Morus014137 322 969 1526 4 1292(−) 16,575 18,100 MnACO3 Morus004820 319 960 1682 4 528(−) 23,279 24,960 MnACO4 Morus013401 310 933 1272 3 1379(+) 391,862 393,133 MnETR1 Morus018344 738 2217 5082 6 521(−) 266,232 271,313 MnETR2 Morus008145 793 2382 2949 2 606(−) 247,113 250,061 MnERS1 Morus007485 617 1854 2872 5 1526(+) 209,537 212,408 MnEIN4 Morus024538 764 2295 2558 2 205(−) 594,177 596,734 MnCTR1 Morus003569 861 2586 6563 17 548(+) 106,465 113,027 MnCTR2 Morus016797 666 2001 6117 12 112(−) 213,548 219,664 MnEIN2 Morus024376 1306 3921 5614 7 342(−) 402,131 407,744 MnEBF1 Morus000805 697 2094 2590 2 1037(+) 4387 6976 MnEBF2 Morus002248 642 1929 2397 2 1513(−) 59,843 62,239 MnEIN3 Morus002490 617 1854 1854 1 3287(−)b 31,544 33,397 MnEIL1 Morus002491 607 1824 1824 1 3287(−)b 50,362 52,185 MnEIL3 Morus007978 611 1836 1836 1 18(−) 151,180 153,015 MnEIL4 Morus016592 478 1437 1910 2 841(+)b 82,684 84,593 MnEIL5 Morus016593 543 1632 1632 1 841(+)b 86,287 87,918 SAM S-adenosylmethionine synthase, ACS 1-aminocyclopropane-1-carboxylate synthase, ACO 1-aminocyclopropane-1-carboxylate oxidase, ETR ethylene receptor, CTR1 constitutive triple response-1, EIN2 ethylene-insensitive 2, EIN3 ethylene-insensitive 3, EBF EIN3-binding F-box protein, ERF ethylene-responsive element binding factor. a The transaminases in ACS family Table 1 Genes involved in ethylene biosynthesis and signaling pathways in the M. notabilis genome Funct Integr Genomics (2014) 14:767–777 770 the flooding-treated group were submerged 2–3 cm under the surface of the water. The roots and leaves from both treated and control groups of Husang-32 were collected after the treatments lasting 1 h, 3 h, 5 h, 7 h, 1 day, 3 days, 5 days, and 7 days. Plant materials were frozen in liquid N2, and stored at −80 °C until further use for total RNA isolation. has four exons (Fig. 1). Prediction of cis-acting elements The motifs for binding the cofactor (H-X-D-X-H) and the cosubstrate (R-X-S) are also conserved in MnACOs. In the C-terminus, E-R-E is essential to enzyme activity, particularly R, which may be involved in the mech- anism of CO2 activation (Supplementary Figure 2). the flooding-treated group were submerged 2–3 cm under the surface of the water. The roots and leaves from both treated and control groups of Husang-32 were collected after the treatments lasting 1 h, 3 h, 5 h, 7 h, 1 day, 3 days, 5 days, and 7 days. Plant materials were frozen in liquid N2, and stored at −80 °C until further use for total RNA isolation. Ethylene signal transduction is important to ethylene syn- thesis. Many genes take part in this process. Ethylene recep- tors (ETRs) form a family of membrane-localized receptors. There are four MnETRs in the mulberry genome. The N- terminal of MnETRs includes three highly conserved trans- membrane regions. This region has been demonstrated to be essential to the ethylene binding activity of MnETRs. The C- terminal region of all MnETRs except MnERS1, which has a truncated C-terminal, contains a receiver domain. In addition, the N-terminal regions of MnETR2 and MnEIN4 are much longer than those of other receptors. SMART prediction re- vealed that they contain four transmembrane regions (Supplementary Figure 3). EIN3 and EIN3-like proteins are essential to the ethylene signal pathway. They are positive regulators, and they bind to the promoter regions of the downstream genes to regulate their expression. Five EIN3s were found here in the mulberry genome. They all have a conserved amino-terminal acidic domain (AD), pro-rich re- gion (PR), and five small basic domains (BDI-V). The Gln- rich and Asn-rich regions, found in mung bean plants, are only conserved in MnEIN3 and MnEIL1 (Supplementary Figure 4). MnEIN3, MnEIL1, MnEIL4, and MnEIL5 were all located on the same scaffold in the mulberry genome (Table 1). Results Identification of genes involved in the mulberry ethylene biosynthesis and signaling pathways In total, 145 genes were predicted to belong to the ethylene biosynthesis and signaling pathways. Of these, 29 genes were found to be involved in ethylene biosynthesis and signal transduction (Table 1). The AP2/ERF family contains 116 genes (Supplemental Table 1). The MnACS family contains 7 genes, of which MnACS10 and 12 are transaminases. Four of the remaining five MnACS genes each had four exons. MnACS5 only had three exons (Fig. 1). Sequence alignment indicated that each MnACS protein contained seven con- served boxes also found in ACS proteins from other plant species. The conserved glutamate (E) residue in box 1, which determines substrate specificity, is present in all the members of the ACS gene family. Except for MnACS5, the MnACS proteins all shared a Ser residue in the C-terminal, which produced a shorter C-terminus than other ACS isozymes (Supplemental Figure 1). ACO is a member of the Fe II- dependent family of oxidases. They require ascorbate as a substrate and Fe (II) as a cofactor to exert enzymatic activity. Four MnACOs were identified in the mulberry genome. MnACO1 and MnACO4 have three exons, and MnACO2/3 Expression of mulberry ethylene biosynthesis and signal pathway genes Expression of mulberry ethylene biosynthesis and signal pathway genes Genes whose expression was detected by qPCR are listed in Fig. 2. Results showed that the genes in these four families Fig. 1 Exon/intron structures of ACS, ACO, ETR, and EIN3 genes in mulberry plants. Gene structures were plotted using the GSDS server. The intron phases indicate the position of the intron within a codon. The scale bar indicates 1 kb Fig. 1 Exon/intron structures of ACS, ACO, ETR, and EIN3 genes in mulberry plants. Gene structures were plotted using the GSDS server. The intron phases indicate the position of the intron within a codon. The scale bar indicates 1 kb Funct Integr Genomics (2014) 14:767–777 771 Fig. 2 Expression patterns of 18 genes involved in the ethylene biosyn- thesis and signal transduction pathways. Six tissues were used. Relative levels of gene expression by qRT-PCR were normalized against the mulberry ribosomal protein gene Morus024083. Data are represented as mean±standard error of three replicates Funct Integr Genomics (2014) 14:767–777 771 Fig 2 Expression patterns of 18 genes involved in the ethylene biosyn mulberry ribosomal protein gene Morus024083 Data are represented as Fig. 2 Expression patterns of 18 genes involved in the ethylene biosyn- thesis and signal transduction pathways. Six tissues were used. Relative levels of gene expression by qRT-PCR were normalized against the mulberry ribosomal protein gene Morus024083. Data are represented as mean±standard error of three replicates have diverse expression profiles across different tissues. MnACS2, MnACO2, MnACO3, MnETR2, MnEIN3, and MnEIL1 have higher levels of expression. On the contrary, the expression of MnACS3, MnACS4, MnACO4, MnERS1, MnEIN4, MnEIL3, and MnEIL4 was relatively repressed. In addition, MnACS5 exhibited tissue-specific expression in fe- male flowers. Among ACO genes, MnACO1 and MnACO2 exhibited tissue-biased expression in fruit, although the ex- pression levels of MnACO1 were only about one tenth of those of MnACO2. MnEIN3 and MnEIL1 showed more ex- pression than the rest of the EIN3 family in roots and fruits. of AP2 domains and their structural features, these 116 pro- teins can be divided into five subfamilies. There are 58 genes in the ERF subfamily, 33 genes in the DREB subfamily, 21 genes in the AP2 subfamily, 3 genes in the RAV subfamily, and 1 gene in the Soloist subfamily. A phylogenetic tree was constructed using the amino acid sequences of AP2/ERF transcription factors of Morus and Arabidopsis. Expression of mulberry ethylene biosynthesis and signal pathway genes This tree was separated into 15 groups. Groups I–IV represent the DREB subfamily, and V–X represent the ERF subfamily (Supplementary Figure 5). All groups except for the V group have lower numbers of genes in mulberry tree than those in Arabidopsis. There are 11 genes in the V group of mulberry and 5 in the V group of Arabidopsis. Expression of MnERF genes To investigate the levels of expression of AP2/ERF genes in different tissues, RPKM data of these genes were analyzed in roots, barks, leaves, flowers, and buds. Heat maps were con- structed based on the RPKM data. Sixteen MnERF genes, including five genes in MnERF-B1, four in MnERF-B3, three in MnERF-B2, two in MnERF-B4, and one each in MnERF- B5 and MnERF-B6, had relatively high levels of expression in five tissues (Fig. 4a). This was also true of 8 of the 33 genes in the DREB subfamily, 6 of the 21 genes in the AP2 subfamily, and 1 of the 3 genes in the RAV subfamily. In addition, several genes were also found to be expressed in a tissue-biased manner. For example, MnERF-B3-21 was expressed solely in male flowers (Fig. 4a). MnDREB-A4-7 was more abundant- ly expressed in leaves than in other tissues (Fig. 4b). MnAP2-5 had high levels of expression in male flower (Fig. 4c). However, the expression of nine genes (MnERF-B6-1/8, MnDREB-A3-1, MnDREB-A4-4, MnDREB-A6-1/2, MnAP2- 13/18/21) was not detectable in any tissue. Phylogenetic and structural analysis of the AP2/ERF transcription factor family The pattern of exon/intron splicing usually provides infor- mation useful to understanding of the emergence and evolu- tion of a gene family. Structural analyses of genes indicated that all genes in the AP2 subfamily had different numbers of In the present study, 116 AP2/ERF transcription factors were identified in the mulberry genome. According to the number Funct Integr Genomics (2014) 14:767–777 772 Fig. 3 EAR motif-like sequences of AP2/ERF in mulberry plants. a The genes contain DLNXXP motif. b The genes contain LXLXL motif. c The genes contain LDLNLXPP motif. Multiple sequence alignments were performed using ClustalX. The letters C, M, and N prior to protein names indicate location in the C-terminal, middle of the sequence, and N-terminal Sequence analyses revealed the 19 genes that bear LXLXL motif found in mulberry proteins. Sequence analyses revealed the 19 genes that bear LXLXL motif found in mulberry proteins. introns ranging from zero to twelve. However, 28 of the 33 genes in the DREB subfamily, 44 of the 58 genes in the ERF subfamily, and 2 of the 3 genes in the RAV subfamily had no introns. Results showed that most genes in the AP2 family shared similar patterns of exon/intron splicing (Supplementary Figure 6). Genes in the AP2/ERF family containing the EAR motif The ERF-associated amphiphilic repression (EAR) motif (DLNxxP or LXLXL) has been reported to be a repres- sion domain in repressor-type ERF proteins. These pro- teins negatively regulate genes involved in developmen- tal, hormonal, and stress signaling pathways. There are three types of EAR motifs in mulberry plants, DLNXXP, LXLXL, and LDLNLXPP. These three motifs have been found in ERF, DREB, AP2, and RAV sub- family (Fig. 3). All of MnERF proteins bearing EAR motif belong to MnERF-B1 group except MnERF-B4-3 and MnERF-B6-8. Only four genes in MnDREB sub- family contain EAR motif, and the DLNXXP motif has been identified in MnDREB-A5-4 and MnDREB-A5-5. 773 Funct Integr Genomics (2014) 14:767–777 ig. 4 Heat maps of hierarchical clustering of mulberry genes in the P2/ERF family. a ERF subfamily; b DREB subfamily; c AP2 subfam- y; and d RAV and Soloist subfamilies. The clustering analyses were arried out using Gene Cluster 3.0. The data were adjusted by log transformation and the mean center method. Hierarchical clusteri (HCL) with average linkage was set to calculate K-medians with fi clusters. Heat maps illustrating the gene expression data were generat using Java TreeView transformation and the mean center method. Hierarchical clustering (HCL) with average linkage was set to calculate K-medians with five clusters. Heat maps illustrating the gene expression data were generated using Java TreeView transformation and the mean center method. Hierarchical clustering (HCL) with average linkage was set to calculate K-medians with five clusters. Heat maps illustrating the gene expression data were generated using Java TreeView Fig. 4 Heat maps of hierarchical clustering of mulberry genes in the AP2/ERF family. a ERF subfamily; b DREB subfamily; c AP2 subfam- ily; and d RAV and Soloist subfamilies. The clustering analyses were carried out using Gene Cluster 3.0. The data were adjusted by log Funct Integr Genomics (2014) 14:767–777 774 Response of MaERF-B2-1 and MaERF-B2-2 to flooding stress Genes in MnERF-B subfamily were expressed at relatively higher levels in different tissues. All members of this subfam- ily have a conserved N-terminal motif MCGGAV/II, which is considered to be involved in the regulation of low-oxygen response in plants (Fig. 5). Analyses of the promoter regions of MnERF-B2-1, MnERF-B2-2, and MnERF-B2-3 showed that they contain consensus sequences of transcription factor binding sites. As shown in Table 2, four catalogues of putative regulatory elements (stress response, hormone response, light Fig. Genes in the AP2/ERF family containing the EAR motif 5 Alignment of amino acid sequences of ERF VII subfamily genes from mulberry, Arabidopsis and rice using the ClustalX program. HRE1/2, RAP2.2, RAP2.3, and RAP2.12 belong to VII group of Arabidopsis. Sub1A-1, Sub1B-1, and Sub1C-1 belong to Oryza sativa 774 Funct Integr Genomics (2014) 14:767–777 Fig. 5 Alignment of amino acid sequences of ERF VII subfamily genes from mulberry, Arabidopsis and rice using the ClustalX program. HRE1/2, RAP2.2, RAP2.3, and RAP2.12 belong to VII group of Arabidopsis. Sub1A-1, Sub1B-1, and Sub1C-1 belong to Oryza sativa Fig. 5 Alignment of amino acid sequences of ERF VII subfamily genes from mulberry, Arabidopsis and rice using the ClustalX program. HRE1/2, RAP2.2, RAP2.3, and RAP2.12 belong to VII group of Arabidopsis. Sub1A-1, Sub1B-1, and Sub1C-1 belong to Oryza sativa Response of MaERF-B2-1 and MaERF-B2-2 to flooding stress Response of MaERF-B2-1 and MaERF-B2-2 to flooding stress considered to be involved in the regulation of low-oxygen response in plants (Fig. 5). Analyses of the promoter regions of MnERF-B2-1, MnERF-B2-2, and MnERF-B2-3 showed that they contain consensus sequences of transcription factor binding sites. As shown in Table 2, four catalogues of putative regulatory elements (stress response, hormone response, light Genes in MnERF-B subfamily were expressed at relatively higher levels in different tissues. All members of this subfam- ily have a conserved N-terminal motif MCGGAV/II, which is 775 Funct Integr Genomics (2014) 14:767–777 Table 2 Cis-elements in MnERF-B2-1, MnERF-B2-2, and MnERF-B2-3 promoters Stress responses Hormone response Light response Others M BS TC-r ich repe ats HS E GC-m otif A RE LT R GA RE- moti f C E3 TGA - elem ent TCA-ele ment TGACG- motif CGTCA- motif AB RE AC E ATCT- motif B ox 4 AE- box G-b ox GAG- motif GT1- motif I-b ox B ox I MN F1 CATT- motif AT-r ich elem ent CAAT- box TATA- box 5UTRPy -rich stretch MB SI B ox II I MnERF- B2-1 Morus00 1004 + - + + + + - - + - + + + - + + + + + - - + - + - + + + - + MnERF- B2-2 Morus00 2477 + + + - - - - - + + + + + - + + - + + + + - + - - + + + + - MnERF- B2-3 Morus00 5243 - + + + + - + + - - - + + + + + - + + + - - - - + + + + - + Table 2 Cis-elements in MnERF-B2-1, MnERF-B2-2, and MnERF-B2-3 promoters 32 is widely planted in the Three Gorges Reservoir area. As shown in Fig. 6a, MaERF-B2-2 has higher expression than MaERF-B2-1 or MaERF-B2-3. To understand the changes in gene expression that takes place after flooding, the expression of the MaERF-B2-1 and MaERF-B2-2 was detected after treatment lasting 1 h, 3 h, 5 h, 7 h, 1 day, 3 days, 5 days, and 7 days (Fig. 6c). Results showed that MaERF-B2-1 was up-regulated in roots and leaves after 1 day of flooding. The change took place faster in leaves than that in roots. MaERFB2-2 showed fast up-regulation in roots after 1 h of flooding. Response of MaERF-B2-1 and MaERF-B2-2 to flooding stress MaERF-B2-1 showed a greater fold change than 32 is widely planted in the Three Gorges Reservoir area. As shown in Fig. 6a, MaERF-B2-2 has higher expression than MaERF-B2-1 or MaERF-B2-3. To understand the changes in gene expression that takes place after flooding, the expression of the MaERF-B2-1 and MaERF-B2-2 was detected after treatment lasting 1 h, 3 h, 5 h, 7 h, 1 day, 3 days, 5 days, and 7 days (Fig. 6c). Results showed that MaERF-B2-1 was up-regulated in roots and leaves after 1 day of flooding. The change took place faster in leaves than that in roots. MaERFB2-2 showed fast up-regulation in roots after 1 h of flooding. MaERF-B2-1 showed a greater fold change than response, and others) were detected in the flanking regions of three genes. GARE, CE3, ABRE, TCA, and TGACG-motifs are involved in the signaling of mulberry hormones. Typical heat shock element (HSE) and low temperature stress re- sponse element (LTR) were also observed. It is noteworthy that ARE and GC motifs, which are known to be responsive to the hypoxemia and anaerobic conditions, were detected in the promoters of MnERF-B2-1 and MnERF-B2-3. In this context, two of these three genes from Husang-32, MaERF-B2-1 and MaERF-B2-2, were cloned, sequenced, and their expression patterns were further investigated. Mulberry cultivar Husang- Fig. 6 Expression analyses of MaERF-B2-1/2/3 genes. Expression of MaERF-B2-1, MaERF-B2-2, and MaERF-B2-3 in roots and leaves (a, b). The qPCR data of MaERF-B2-1 and MaERF-B2-2 in roots and leaves after flooding treatment (c). Fold inductions are calculated by dividing the value of treatment with that of control in corresponding time point. Data are represented as mean±standard error of three replicates after flooding treatment (c). Fold inductions are calculated by dividing the value of treatment with that of control in corresponding time point. Data are represented as mean±standard error of three replicates Fig. 6 Expression analyses of MaERF-B2-1/2/3 genes. Expression of MaERF-B2-1, MaERF-B2-2, and MaERF-B2-3 in roots and leaves (a, b). The qPCR data of MaERF-B2-1 and MaERF-B2-2 in roots and leaves Funct Integr Genomics (2014) 14:767–777 776 transcriptional factors (Sub1A, Sub1B, and Sub1C) (Jung et al. 2010). In Arabidopsis, the RAP2.12 and RAP2.2 genes became active in response to water-logging. In flooding, the expression of RAP2.12 is up-regulated and the product accumulates in the nucleus to activate the expression of hypoxia genes (Licausi et al. 2011). Discussion Genes such as ACS, ACO, and EIN3 are essential targets in the biosynthesis of ethylene and the transduction of this signal. In Arabidopsis, ACS is considered a rate-limiting synthase in ethylene biosynthesis (Jakubowicz and Sadowski 2002). The phosphorylation sites of ACS are important to rapid turnover during the response to various kinds of stimulation (Joo et al. 2008). Previous research indicated CmACS-7 affects the de- velopment of the stamina in female flowers (Boualem et al. 2008). The expression of MnACS5 was restricted to female flowers in mulberry plants, suggesting that MnACS5, rather than an ACC synthase, may play a different role. ACO genes are responsible for ethylene production. In the present study, the expression profiles of four putative MnACOs were ana- lyzed and variation was observed in their levels of expression. Mulberry ACO genes, such as MnACO1 and MnACO2, whose expression takes place mainly in fruit, might be involved in the regulation of ethylene during fruit ripening. In ethylene signal transduction, EIL and EIN3 proteins are crucial tran- scriptional factors. They are bound to the promoters of down- stream genes. In this way, they regulate a wide range of stress responses (Chao et al. 1997). MnEIN3, MnEIL1, MnEIL3, and MnEIL5 have no introns. MnEIN3, MnEIL1, MnEIL4, and MnEIL5 form tandem arrays in the mulberry genome, and the genes in these two pairs are not only similar in sequence but in pattern of expression, suggesting the existence of complicated regulation affecting those processes. Acknowledgments This project was funded by the research grants from the National Hi-Tech Research and Development Program of China (No. 2013AA100605-3), the Fundamental Research Funds for the Cen- tral Universities (No. 2362014xk05), the “111” Project (B12006), and the Science Fund for Distinguished Young Scholars of Chongqing (Grant No. cstc2011jjjq0010). 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. Response of MaERF-B2-1 and MaERF-B2-2 to flooding stress The overexpression of RAR2.2 improves plant survival under hypoxia stress conditions (Hinz et al. 2010). Three mul- berry genes, MaERF-B2-1, MaERF-B2-2, and MaERF-B2-3, appear to be the counterparts of RAP2.12, RAP2.2, and RAP2.3 based on the sequences. All of them contain conserved N- terminal sequences, which are important in response to hypoxia. Moreover, the presences of ARE and GC cis-elements in the promoters of MnERF-B2-1 and MnERF-B2-3 could lead to a proposal that these two genes might be involved in dealing with hypoxia. Our data further indicated that the patterns of expression of MaERF-B2-1 and MaERF-B2-2 changed in response to flooding treatment. MaERF-B2-2. These results indicated that MaERF-B2-1 and MaERFB2-2 may play important roles in Husang-32’s re- sponse to flooding. 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Evaluation of A Novel Split-Feeding Anaerobic/Oxic Baffled Reactor (A/OBR) For Foodwaste Anaerobic Digestate: Performance, Modeling and Bacterial Community
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Shaojie Wang1,*, Liyu Peng1,*, Yixin Jiang1, Petros Gikas2, Baoning Zhu3 & Haijia Su1 Shaojie Wang1,*, Liyu Peng1,*, Yixin Jiang1, Petros Gikas2, Baoning Zhu3 & Haijia Su1 To enhance the treatment efficiency from an anaerobic digester, a novel six-compartment anaerobic/ oxic baffled reactor (A/OBR) was employed. Two kinds of split-feeding A/OBRs R2 and R3, with influent fed in the 1st, 3rd and 5th compartment of the reactor simultaneously at the respective ratios of 6:3:1 and 6:2:2, were compared with the regular-feeding reactor R1 when all influent was fed in the 1st compartment (control). Three aspects, the COD removal, the hydraulic characteristics and the bacterial community, were systematically investigated, compared and evaluated. The results indicated that R2 and R3 had similar tolerance to loading shock, but the R2 had the highest COD removal of 91.6% with a final effluent of 345 mg/L. The mixing patterns in both split-feeding reactors were intermediate between plug-flow and completely-mixed, with dead spaces between 8.17% and 8.35% compared with a 31.9% dead space in R1. Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR- DGGE) analysis revealed that the split-feeding strategy provided a higher bacterial diversity and more stable bacterial community than that in the regular-feeding strategy. Further analysis indicated that Firmicutes, Bacteroidetes, and Proteobacteria were the dominant bacteria, among which Firmicutes and Bacteroidetes might be responsible for organic matter degradation and Proteobacteria for nitrification and denitrification. Food waste, which is generated by the feedstock sorting, peeling, cooking and dining processes, usually accounts for 30% (w/w) of organics1. As a crowded capital of more than 21 million residents, Beijing has a daily food waste generation of more than 2000 t/d2. Traditional solid waste disposal technologies, such as landfill, composting and incineration, are mostly incapable of treating food waste because the waste has high contents of water and bio- degradable organics3. Anaerobic digestion has been claimed as an alternative technology in China to dispose of food waste and produce renewable biogas energy using a continuous stirred tank reactor (CSTR)1. However, only around 40% of the total solids in food waste can be degraded. In addition, a large amount of extra water has to be added into this system to maintain a constant water content, resulting in a huge amount of digestate. Therefore, the digestate is necessary to be treated before it is discharged into the environment1,4,5. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports received: 27 May 2016 accepted: 16 September 2016 Published: 06 October 2016 Scientific Reports | 6:34640 | DOI: 10.1038/srep34640 Evaluation of A Novel Split-Feeding Anaerobic/Oxic Baffled Reactor (A/OBR) For Foodwaste Anaerobic Digestate: Performance, Modeling and Bacterial Community received: 27 May 2016 accepted: 16 September 2016 Published: 06 October 2016 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Parameter Concentration* COD 4250 ±​ 520 mg/L Total nitrogen (TN) 110.8 ±​ 6.3 mg/L Ammonia nitrogen (NH4-N) 30.2 ±​ 8.4mg/L Total phosphor (TP) 8 ±​ 3 mg/L NaCl 13.8 ±​ 0.6 g/L pH 4.1 ±​ 0.5 Table 1. Characteristics of the food waste digestate used as influent to the A/OBRs. *Average ±​ standard deviation for three independent samples. Parameter Concentration* COD 4250 ±​ 520 mg/L Total nitrogen (TN) 110.8 ±​ 6.3 mg/L Ammonia nitrogen (NH4-N) 30.2 ±​ 8.4mg/L Total phosphor (TP) 8 ±​ 3 mg/L NaCl 13.8 ±​ 0.6 g/L pH 4.1 ±​ 0.5 Table 1. Characteristics of the food waste digestate used as influent to the A/OBRs. *Average ±​ standard deviation for three independent samples. Table 1. Characteristics of the food waste digestate used as influent to the A/OBRs. *Average ±​ standard deviation for three independent samples. Table 1. Characteristics of the food waste digestate used as influent to the A/OBRs. *Average ±​ standard deviation for three independent samples. ability of the reactor. Their results suggested that the integrated process improved anaerobic digestion in terms of volatile solids reduction, biogas production and reactor stability6–8.f ability of the reactor. Their results suggested that the integrated process improved anaerobic digestion in terms of volatile solids reduction, biogas production and reactor stability6–8.f Since the integration of multiple biological technologies shows positive effects on the degradation of digestate, a specific reactor layout should be designed to carry out such novel process. The anaerobic baffled reactor (ABR) shows the potential because it has already been successfully applied to many wastewater treatment plants due to its effective chemical oxygen demand (COD) removal, high tolerance towards loading shock, and capability to contain various biological metabolism phases9–11.h g p The ABR was initially developed by McCarty and coworkers12. A traditional ABR consists of a series of ver- tical baffles which force the wastewater flow under and over them as it passes from the inlet to the outlet13. Most importantly, the ABR is able to separate acidogenesis and methanogenesis horizontally along the reactor, thereby allowing the reactor to behave as a two-phase system14. This multi-compartmental structure encourages different bacterial groups to develop under their most favorable conditions, and enables the ABR to maintain a high bio- mass concentration. However, the accumulation of toxic residual substrates in upstream compartments will result in the inhibition of bacterial growth and metabolism15–17. www.nature.com/scientificreports/ A deficient amount of substrates in the downstream compartments could also lead to reduction of the reaction rate18,19. The degradation efficiency of organics is usu- ally lower than that of aerobic processes since the traditional ABR process is operated under anaerobic conditions. y p p p Microorganisms play an important role in wastewater treatment, and understanding the microbial commu- nity structure is of great importance for improving reactor performance20. Recently, the PCR-denaturing gradient gel electrophoresis (PCR-DGGE) of 16S rRNA genes has been used to rapidly monitor shifts in microbial com- munity compositions21,22. The ABR, designed to achieve the separation of acid and methanogenic microbes with the best activity, was characterized by microbial alternation in different compartments along the flow direction23. Nachaiyasit and Stukey found that most microbes in the first compartment were butyric acid-producing bacteria, and Methanogens were dominant in downstream compartments24. Peng et al. investigated the spatial succession of functional microbial communities in a five-compartment ABR. The results showed that the acidogenesis stage and acetogenesis stage were located in the first two compartments, where H2-producing acetogens (19.7%) and H2-utilizing acetogens (8.3%) were the dominant bacteria. However, cloning and 16S rRNA gene analyses of the integrated system where the anaerobic and aerobic populations co-existed are still limited25.fl g y p p In this study, a novel anaerobic/oxic baffled reactor (A/OBR) process was developed by adding a series of oxic compartments to a traditional ABR to enhance biodegradation. Operational parameters and various feeding strategies were investigated to reduce excessive loading shock, to optimize the distribution of substrate, and to improve COD removal. The objectives of this work were to evaluate the performance of the A/OBR, identify the optimal feeding strategy, and analyze its hydrodynamic characteristics. In addition, the development and compo- sition of the bacterial community structure based on PCR-DGGE analysis were also determined to evaluate the process performance. Scientific Reports | 6:34640 | DOI: 10.1038/srep34640 Shaojie Wang1,*, Liyu Peng1,*, Yixin Jiang1, Petros Gikas2, Baoning Zhu3 & Haijia Su1 g y g Various studies have been conducted on the biological treatment of sewage sludge to reduce organic contents, but few focused on the treatment of anaerobic digestate. This is mainly because that digestate consists of high contents of salts with less content of nutrients (N, P and K), which are hard to be removed. The selection of suita- ble technologies has become a critical issue for the practical application in the digestate treatment. An increasing number of studies has focused on the integration of the aerobic and anaerobic process to enhance the degradation 1Beijing Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical Technology, Beijing, 100029, PR China. 2School of Environmental Engineering, Technical University of Crete, 73100, Chania, Greece. 3Beijing Higher Institution Engineering Research Center of Environmental Pollution Control and Resource Utilization, Beijing University of Chemical Technology, No. 15 Beisanhuan East Road, Chaoyang District, Beijing 100029, PR China. *These authors contributed equally to this work. Correspondence and requests for materials should be addressed to B.Z. (email: bnzhu@mail.buct.edu.cn) or H.S. (email: Suhj@mail.buct.edu.cn) Scientific Reports | 6:34640 | DOI: 10.1038/srep34640 Results Effi i The results showed that both two split-feeding reactors R2 and R3 could significantly improve the overall COD removal of stage I, II, and III (all P <​ 0.05) compared with regular-feeding reactor R1 (Table S1 in Supplementary Information). Among the three A/OBRs, R2 showed the best performance with the average effluent of 345 ±​ 27 mg/L and COD removal of 91.7 ±​ 0.7% at stage III (P <​ 0.05). with R1. The average COD removals of R2 and R3 were 84.1 ±​ 6.9% and 78.7 ±​ 3.8% at stage II respectively and slightly increased at stage III (91.7 ±​ 0.7% and 82.1 ±​ 3.5%, respectively). Considering the fact that the high var- iations of feedstock might cause fluctuation of COD removal, the one-way analyses of variance (ANOVA) were used to determine the significance of differences between reactors at different stages. The results showed that both two split-feeding reactors R2 and R3 could significantly improve the overall COD removal of stage I, II, and III (all P <​ 0.05) compared with regular-feeding reactor R1 (Table S1 in Supplementary Information). Among the three A/OBRs, R2 showed the best performance with the average effluent of 345 ±​ 27 mg/L and COD removal of 91.7 ±​ 0.7% at stage III (P <​ 0.05). Hydraulic characteristics of A/OBRs. Residence time distribution (RTD) analysis was carried out to investigate the hydraulic characteristics of A/OBRs. Figure 2 showed the normalized concentration of K+ in the effluent against the normalized time (C-curve). The data from the C-curve were analyzed with a two-phase dis- persion model and tanks-in-series (TIS) model26. Results are shown in Table 3. Mixing patterns. Mixing patterns were analyzed by observing the variance of the C-curve. In the dispersion model, the findings were incorporated to calculate the dispersion number (D/μL)27. For D/μL above 0.2, the sys- tem is considered as completely-mixed, while D/μL below 0.02 it is considered as a plug-flow system28. l As shown in Table 3, dispersion numbers (D/μL) of two split-feeding strategies (6:3:1 and 6:2:2) were 0.14 and 0.15, respectively. These values were significantly lower than the D/μL of 0.33, which was calculated from the regular-feeding strategy. The results indicated that the mixing pattern in the regular-feeding reactor R1 was close to completely-mixed, while both two split-feeding reactors R2 and R3 were intermediate between plug-flow and completely-mixed, tending towards the latter. Results Effi i Efficient COD removal by the novel A/OBR. A novel A/OBR was specially designed based on traditional ABR for the treatment of high salt and low pH foodwaste anaerobic digestate (Table 1). Three stages were divided artificially during the operation process according to the reactor and sludge characteristics. Stage I (day 1~14), the start-up stage, where microbes began to adapt to the environment and the characteristics of the reactors were quite unstable. The biomass increased with the increase of time and small granular particles or flocs formed. Stage II (day 15~28), the steady stage, where the reactor operation was relatively stable and granular sludge formed. The biomass concentration reached its maximum and a variety of bacteria were dominant in different compartments. Stage III (day 29~35), the final stage, where the operation system might be fluctuated or remain stable. Some of the dominant bacteria might fade and new dominant bacteria might appear.hlflf g g pp The influent and effluent COD concentrations of the A/OBRs at different stages are illustrated in Fig. 1. Successful start-up of the A/OBR using regular-feeding strategy (R1) had been achieved during a 14-day oper- ation at stage I, while the start-up time of both two split-feeding strategies (R2 and R3) was decreased to about 7 days due to the relief of the organic loading rate (OLR) in the upstream compartments. After the start-up, the operation of the three reactors was relatively stable at stage II. However, the COD removal in R1 at stage III became unstable due to the high OLR and some sludge bulking appeared, while that of R2 and R3 remained stable and efficient.hflf fi The average effluent and COD removal at different stages in the three A/OBRs are summarized in Table 2. In our study, COD removals at stage II and stage III were only 68.7 ±​ 9.5% and 66.4 ±​ 6.5% respectively in R1 (Table 2). However, two split-feeding reactors (R2 and R3) showed better efficiency and less variation compared Scientific Reports | 6:34640 | DOI: 10.1038/srep34640 2 www.nature.com/scientificreports/ Figure 1. Influent and effluent COD concentrations of all A/OBRs at different stages. ( )R1: regular- feeding; ( ) R2: split-feeding at ratio of 6:3:1, and ( ) R3: split-feeding at ratio of 6:2:2. All three A/OBRs were domesticated for one month before the experiments and were operated in the same conditions. Figure 1. Influent and effluent COD concentrations of all A/OBRs at different stages. Results Effi i ( )R1: regular- feeding; ( ) R2: split-feeding at ratio of 6:3:1, and ( ) R3: split-feeding at ratio of 6:2:2. All three A/OBRs were domesticated for one month before the experiments and were operated in the same conditions. ReactorNo. Stage I Stage II Stage III Effluent (mg/L) Removal (%) Effluent (mg/L) Removal (%) Effluent (mg/L) Removal (%) R1 3174 ±​ 898 32.8 ±​ 12.6 1281 ±​ 390 68.7 ±​ 9.5 1390 ±​ 267 66.4 ±​ 6.5 R2 1208 ±​ 1038 81.2 ±​ 10.4 653 ±​ 281 84.1 ±​ 6.9 345 ±​ 27 91.7 ±​ 0.7 R3 1178 ±​ 619 77.2 ±​ 8.9 872 ±​ 154 78.7 ±​ 3.8 740 ±​ 145 82.1 ±​ 3.5 Table 2. Average effluent and total COD removals at different stages in three A/OBRs. R1: regular-feeding; R2: split-feeding at ratio of 6:3:1; R3: split-feeding at ratio of 6:2:2. ReactorNo. Stage I Stage II Stage III Effluent (mg/L) Removal (%) Effluent (mg/L) Removal (%) Effluent (mg/L) Removal (%) R1 3174 ±​ 898 32.8 ±​ 12.6 1281 ±​ 390 68.7 ±​ 9.5 1390 ±​ 267 66.4 ±​ 6.5 R2 1208 ±​ 1038 81.2 ±​ 10.4 653 ±​ 281 84.1 ±​ 6.9 345 ±​ 27 91.7 ±​ 0.7 R3 1178 ±​ 619 77.2 ±​ 8.9 872 ±​ 154 78.7 ±​ 3.8 740 ±​ 145 82.1 ±​ 3.5 Table 2. Average effluent and total COD removals at different stages in three A/OBRs. R1: regular-feeding; R2: split-feeding at ratio of 6:3:1; R3: split-feeding at ratio of 6:2:2. Table 2. Average effluent and total COD removals at different stages in three A/OBRs. R1: regular-feedin R2: split-feeding at ratio of 6:3:1; R3: split-feeding at ratio of 6:2:2. Table 2. Average effluent and total COD removals at different stages in three A/OBRs. R1: regular-feeding; R2: split-feeding at ratio of 6:3:1; R3: split-feeding at ratio of 6:2:2. with R1. The average COD removals of R2 and R3 were 84.1 ±​ 6.9% and 78.7 ±​ 3.8% at stage II respectively and slightly increased at stage III (91.7 ±​ 0.7% and 82.1 ±​ 3.5%, respectively). Considering the fact that the high var- iations of feedstock might cause fluctuation of COD removal, the one-way analyses of variance (ANOVA) were used to determine the significance of differences between reactors at different stages. Scientific Reports | 6:34640 | DOI: 10.1038/srep34640 Results Effi i aDispersion number (D/uL): a higher value represents the flow pattern is closer to completely-mixed. bTanks-in-series number (N): the number of theoretical stirred tanks, a higher value represents the flow pattern is closer to plug-flow. Reactor No. HRT (Hour) Variance of C-curve D/uLa (Dispersion model) Nb (Tanks-in-series model) Dead space (%) R1 44.6 0.47 0.33 1.35 31.9 R2 57.2 0.28 0.14 3.58 8.2 R3 53.4 0.30 0.15 3.34 8.6 Table 3. Results from RTD analyses for three A/OBRs. aDispersion number (D/uL): a higher value represents the flow pattern is closer to completely-mixed. bTanks-in-series number (N): the number of theoretical stirred tanks, a higher value represents the flow pattern is closer to plug-flow. Table 3. Results from RTD analyses for three A/OBRs. aDispersion number (D/uL): a higher value represents the flow pattern is closer to completely-mixed. bTanks-in-series number (N): the number of theoretical stirred tanks, a higher value represents the flow pattern is closer to plug-flow. Hydraulic dead space. Dead space is divided into the categories of hydraulic dead space and biological dead space. The hydraulic dead space is a function of the flow rate and the number of compartments in the reactor while the biological dead space is a function of the biomass concentration and activity26. In order to better under- stand the effects of different feeding strategies on the hydraulic characteristics in A/OBRs, all three reactors were carried out without inoculation to eliminate the influence of biological dead space in RTD studies.h l The evaluation of the hydraulic dead space mainly depends on the mean of the C-curve and the area under the curve between θ​ =​ 0 and θ​ =​ 226. A lower dead space represents a better mixing in reactor. As shown in Table 3, the dead space in R1 was calculated as 31.9%. However, the dead spaces in R2 and R3 were 8.35% and 8.17%, respectively, which were significantly lower than that of R1. Bacterial community analyses by PCR-DGGE in A/OBRs. Samples from all compartments (C1–C6) in each reactor at different stages were taken, and the functional bacteria were analyzed by PCR-DGGE (Fig. 3). Twenty-nine predominant bands from DGGE gels of the anaerobic and aerobic compartments were sequenced for identification by BLAST analysis (Table 4) and the phylogenetic tree showing the phylogenetic identities of the 16S rRNA gene fragments was constructed (Fig. 4). Spatio-temporal distribution of bacterial community. Results Effi i In the TIS model, the reactor was characterized by a series of N equally sized CSTRs. When the value of N tends to ∞​, the flow pattern of the reactor approaches plug-flow, and when N tends to 1, the reactor behaves as completely mixed28. Mixing patterns fitted by the TIS model showed the same trend as fitted by the dispersion model. However, the TIS model was the more useful since it could predict the degree of back-mixing in the reactor26. The larger the number of N in the reactor was, the smaller the amount of back-mixing that will occur28. Table 3 showed that the number of N in R1 was 1.35, indicating a large amount of back-mixing occurred in a regular-feeding strategy. However, both two split-feeding strategies showed significantly larger numbers of N (3.58 in R2 and 3.34 in R3 respectively). The larger number of baffles (N) inside the reactor inhibited back-mixing between compartments, although each individual compartment would be well-mixed. Therefore, mixing pat- terns of two split-feeding strategies were intermediate between plug-flow and completely-mixed, but closer to the completely-mixed pattern. Scientific Reports | 6:34640 | DOI: 10.1038/srep34640 3 www.nature.com/scientificreports/ Figure 2. C-curves for the A/OBRs using regular-feeding and split-feeding strategies without biomass. Reactors were washed, filled with deionized water and then 0.56 mg K+/L was fed impulsively. Water samples of three reactors were collected at every 6 h intervals. Figure 2. C-curves for the A/OBRs using regular-feeding and split-feeding strategies without biomass. Reactors were washed, filled with deionized water and then 0.56 mg K+/L was fed impulsively. Water samples of three reactors were collected at every 6 h intervals. Reactor No. HRT (Hour) Variance of C-curve D/uLa (Dispersion model) Nb (Tanks-in-series model) Dead space (%) R1 44.6 0.47 0.33 1.35 31.9 R2 57.2 0.28 0.14 3.58 8.2 R3 53.4 0.30 0.15 3.34 8.6 Table 3. Results from RTD analyses for three A/OBRs. aDispersion number (D/uL): a higher value represents the flow pattern is closer to completely-mixed. bTanks-in-series number (N): the number of theoretical stirred tanks, a higher value represents the flow pattern is closer to plug-flow. Reactor No. HRT (Hour) Variance of C-curve D/uLa (Dispersion model) Nb (Tanks-in-series model) Dead space (%) R1 44.6 0.47 0.33 1.35 31.9 R2 57.2 0.28 0.14 3.58 8.2 R3 53.4 0.30 0.15 3.34 8.6 Table 3. Results from RTD analyses for three A/OBRs. Results Effi i Generally, the distribution of bacterial composition in the A/OBR showed that most identified bacterial populations belonged to Actinobacteria, Bacteroidetes, Chloroflexi, Firmicutes and Proteobacteria (Table 4). This result was similar to previous studies of treating nitrobenzene23 and livestock wastewater20. Bacterial community changes in the regular-feeding reactor were observed as shown in Fig. 3A. No dom- inant bands appeared at the start-up stage (stage I) while bright dominant bands were clearly observed in the fingerprints at stage II, which indicated that the microorganisms were gradually adapted to the environment and dominant bacteria formed. It was reported that the shift of community structure required some time to be visible because it would need time for microbes to fade out than to shut down their functional capability20. Bands a1, a2 and a3 were dominant in the first three compartments. Bands a6 and a7 were dominant in C4 and C5 at stage II, but decreased at stage III.t A community shift was also found in the split-feeding reactors (Fig. 3B,C). Band b5 was dominant in C4–C6 at stage II, especially in the aerobic compartment C5 and C6. Band b6 became dominant in C1 and C3 at stage III. Moreover, a new band b7 appeared in C6 at stage III (Fig. 3B). However, unlike for the regular-feeding strategy in A/OBR, most of the dominant bacteria in split-feeding at ratio of 6:3:1 were relatively stable with only a slight change at stage II and stage III, which indicated that the split-feeding strategies could promote the formation of a Scientific Reports | 6:34640 | DOI: 10.1038/srep34640 4 www.nature.com/scientificreports/ www.nature.com/scientificreports/ steady bacterial community Similar trend in split feeding at ratio of 6 2 2 could also be seen in Fig 3C Bands c1 Figure 3. The DGGE profile for the bacterial communities analyses of (A) R1, (B) R2 and (C) R3 at different stages. Bands at the same horizontal position were the same species by further sequencing. Figure 3. The DGGE profile for the bacterial communities analyses of (A) R1, (B) R2 and (C) R3 at different stages. Bands at the same horizontal position were the same species by further sequencing. steady bacterial community. Similar trend in split-feeding at ratio of 6:2:2 could also be seen in Fig. 3C. Bands c1, c5 and c10 were quite stable and only band c4 in C2 disappeared at stage III. steady bacterial community. Results Effi i Similar trend in split-feeding at ratio of 6:2:2 could also be seen in Fig. 3C. Bands c1, c5 and c10 were quite stable and only band c4 in C2 disappeared at stage III. Phylogenetic analysis. Though all reactors possessed almost the same dominant phylum, the detailed analysis showed significant differences in the terms of certain bacterial groups between the reactors (Fig. 4). Among a total of 29 bands of bacterial DGGE (Table 4), bands a2, a13, b5 and b9 belonged to Clostridium spp. Band b3, which was dominant in the downstream compartments (C4–C6) in split-feeding at ratio of 6:3:1, was closely related to Anaerofilum sp. These species were reported as acidogenic bacteria and could produce various organic acids as Scientific Reports | 6:34640 | DOI: 10.1038/srep34640 5 www.nature.com/scientificreports/ Band No.* Closest sequences Identity (%) a1 Uncultured bacterium 100 a2 Clostridium sp. 99 a3 Uncultured bacterium 93 a4 Flavobacterium sp. 98 a6 Arcobacter sp. 100 a7 Lactobacillus sp. 100 a8 Uncultured Prevotella sp. 98 a10 Pseudomonas sp. 100 a12 Lactobacillus sp. 99 a13 Clostridium sp. 99 a14 Uncultured bacterium 100 a15 Uncultured Bacteroidetes bacterium 100 b1 Lysinibacillus sp. 99 b2 Uncultured Acidovorax sp. 99 b3 Anaerofilum sp. 94 b4 Uncultured bacterium 100 b5 Uncultured Clostridiales bacterium 99 b6 Uncultured Acidovorax sp. 99 b7 Pectinatus sp. 100 b8 Dechlorosoma sp. 99 b9 Clostridium sp. 98 c1 Uncultured bacterium 100 c3 Comamonadaceae sp. 96 c4 Pseudomonas sp. 100 c5 Uncultured bacterium 100 c7 Uncultured Chloroflexi bacterium 99 c8 Uncultured Bacteroidetes bacterium 98 c9 Uncultured Erysipelothrix sp. 100 Table 4. 16S rRNA gene sequence of DGGE bands in A/OBR. *Bands a5, a9, a11, b10, c2, c6 and c10 were not detected. Band No.* Closest sequences Identity (%) a1 Uncultured bacterium 100 a2 Clostridium sp. 99 a3 Uncultured bacterium 93 a4 Flavobacterium sp. 98 a6 Arcobacter sp. 100 a7 Lactobacillus sp. 100 a8 Uncultured Prevotella sp. 98 a10 Pseudomonas sp. 100 a12 Lactobacillus sp. 99 a13 Clostridium sp. 99 a14 Uncultured bacterium 100 a15 Uncultured Bacteroidetes bacterium 100 b1 Lysinibacillus sp. 99 b2 Uncultured Acidovorax sp. 99 b3 Anaerofilum sp. 94 b4 Uncultured bacterium 100 b5 Uncultured Clostridiales bacterium 99 b6 Uncultured Acidovorax sp. 99 b7 Pectinatus sp. 100 b8 Dechlorosoma sp. 99 b9 Clostridium sp. 98 c1 Uncultured bacterium 100 c3 Comamonadaceae sp. 96 c4 Pseudomonas sp. Results Effi i (band a10 and c4), Dechlorosoma sp. (band b8) and Acidovorax sp. (band b2 and b6) were also found. Though bands a3, a14, a15, b4, c5, c7, and c8 could be classified as shown in Fig. 4, their functions are still unknown. Results Effi i 100 c5 Uncultured bacterium 100 c7 Uncultured Chloroflexi bacterium 99 c8 Uncultured Bacteroidetes bacterium 98 c9 Uncultured Erysipelothrix sp. 100 Table 4 16S rRNA gene sequence of DGGE bands in A Table 4. 16S rRNA gene sequence of DGGE bands in A/OBR. *Bands a5, a9, a11, b10, c2, c6 and c10 were not detected. Table 4. 16S rRNA gene sequence of DGGE bands in A/OBR. *Bands a5, a9, a11, b10, c2, c6 and c10 were detected. well as hydrogen29,30. Band b1 was identified as Lysinibacillus sp., which was a protein-fermentation-related genus due to its ability to secret α​-chymotrypsin and oxidize various amino acids31,32. Some species of Lysinibacillus have been used in bioaugmentation to enhance anaerobic digestion of food wastewater33. Band a1 belonged to phylum Actinobacteria, most of which are heterotrophs, indicating that it might play an important role in reduc- ing organic carbon34. Bands a8 and c1 were close to Prevotella sp., which was considered as a hydrogen-producing microorganism from organics and could also consume by-products such as acetate, succinate, or lactate35. Flavobacterium (band a4) as well as Pseudomonas were significant genera of flocforming bacteria due to its pro- duction of glue-like extracellular polymers and ability to bind cells together36. Besides, some denitrifying bacteria such as Arcobacter sp. (band a6), Comamonadaceae (band c3) Pseudomonas sp. (band a10 and c4), Dechlorosoma sp. (band b8) and Acidovorax sp. (band b2 and b6) were also found. Though bands a3, a14, a15, b4, c5, c7, and c8 could be classified as shown in Fig. 4, their functions are still unknown. well as hydrogen29,30. Band b1 was identified as Lysinibacillus sp., which was a protein-fermentation-related genus due to its ability to secret α​-chymotrypsin and oxidize various amino acids31,32. Some species of Lysinibacillus have been used in bioaugmentation to enhance anaerobic digestion of food wastewater33. Band a1 belonged to phylum Actinobacteria, most of which are heterotrophs, indicating that it might play an important role in reduc- ing organic carbon34. Bands a8 and c1 were close to Prevotella sp., which was considered as a hydrogen-producing microorganism from organics and could also consume by-products such as acetate, succinate, or lactate35. Flavobacterium (band a4) as well as Pseudomonas were significant genera of flocforming bacteria due to its pro- duction of glue-like extracellular polymers and ability to bind cells together36. Besides, some denitrifying bacteria such as Arcobacter sp. (band a6), Comamonadaceae (band c3) Pseudomonas sp. Scientific Reports | 6:34640 | DOI: 10.1038/srep34640 Discussion Many literatures have demonstrated that the first two compartments (C1 and C2) were crucial for the degradation of macromolecular organics and accounted for more than 50% COD removal in a ABR37,38. Nevertheless, exces- sive and/or toxic influent might greatly inhibit the degradation ability of C1 and C2. As a consequence, C1 and C2 only accounted for 19.3% (18.6% plus 0.7%) of the total COD removal in the regular-feeding reactor R1 in this study (Fig. 5). For this reason, C1 and C5 were changed into aerobic compartments to enhance the degradation ability, and a split-feeding strategy was employed to further reduce the inhibitions in the front compartments of the reactor. Consequently, the novel split-feeding A/OBR was established.if q y p g A pre-experiment was firstly conducted to determine the optimal feed ratio of C1, and three different feed ratios (70%, 60% and 50% of the initial influent that was fed into C1) were examined respectively. The results showed that the average COD removals in the last 7 days of the first two compartments were 38.3%, 51.4% and 43.2% respectively after a four-week operation (Table S2). This further indicated that C1 and C2 were still inhib- ited when 70% of influent was fed. On the other hand, less than 50% of the initial influent into C1 was meaning- less though it might reduce the inhibitions, as the maximum removals of the first two compartments in theory were only 50%. Therefore, two split-feeding ratios (6:3:1 and 6:2:2) were chosen for this study. h Microbial growth and granule development were relatively lower in the anaerobic process than that in the aerobic process, and it usually took about 45~160 days to start-up in a traditional ABR39,40. However, the start-up period of the reactor would be greatly accelerated when a part of the anaerobic compartments were replaced by Scientific Reports | 6:34640 | DOI: 10.1038/srep34640 6 www.nature.com/scientificreports/ Figure 4. Neighbor-joining phylogenetic tree of 16S rRNA gene sequences from DGGE profile. Bands of ( ) regular-feeding, ( ) split-feeding at ratio of 6:3:1 and ( ) split-feeding at ratio of 6:2:2 at different stages were collected. Sequences were aligned using Clustal X 1.8 and MEGA 5.0 was used to construct phylogenetic tree. The bar represents 2% sequence divergence. The numbers in parentheses indicate GenBank accession number. Figure 4. Neighbor-joining phylogenetic tree of 16S rRNA gene sequences from DGGE profile. Discussion In our study, the high salt concen- tration (1.35~1.45%) might lead to a low COD removal in R1. Besides, there might be some other undetectable toxic compounds that could inhibit the microbes in the reactor due to the complex and fluctuant composition of foodwaste digestate. Therefore, a further increase in degradation efficiency could be expected after sharing the OLR and toxic substrate into the downstream of the reactor. As a result, COD removals of R2 and R3 increased to 91.7 ±​ 0.7% and 82.1 ±​ 5.5% respectively at stage III.h p y g The ammonia-nitrite-nitrate conversion in three reactors at stage II was further analyzed and shown in Figures S1 and S2. Ammonium would be produced from the proteolysis of the proteinaceous substrates in the upstream compartments (C1–C3) and be assimilated by bacteria for amino acid synthesis. Excess ammonium then passed through downstream compartments and was utilized by denitrifying bacteria, resulting in a decrease in ammonium concentration (Figure S1)43. Figure S2 showed that denitrification occurred almost in the first compartment of all three reactors. Notably, ammonia increased in C5 with a slight decrease in nitrite/nitrate, indicating nitrification and denitrification process might occur simultaneously. Nevertheless, similar variations of ammonia, nitrite/nitrate were observed among three A/OBRs (Figures S1 and S2), resulting in a similar trend of pH (Fig. 5). Therefore, these factors might not be the main reasons for three reactors showing different COD removal efficiencies.h fi The average volatile fatty acid (VFAs) concentrations for each compartment of all reactors at stage II were shown in Figure S3. In a traditional ABR, most of the substrates were degraded into VFAs in upstream compart- ments, which were then utilized by methanogens to produce CH4 and CO2 in downstream compartments44,45. Similar trends could also be found in all three A/OBRs at stage II (Figure S3). Besides, Figure S3 showed that the VFAs of each compartment in R2 and R3 were relatively lower than that in R1 (all P <​ 0.05). This further demon- strated that the split-feeding strategy could relieve the OLR and toxic substrates for microbes in the upstream compartments and split it into downstream compartments, thus appearing as an increase in COD removal and decrease in start-up time. p As mentioned above, most of COD was degraded in the front compartments of the A/OBR, especially at rela- tively low organic loading rate (OLR). Polprasert et al. Discussion Bands of ( ) regular-feeding, ( ) split-feeding at ratio of 6:3:1 and ( ) split-feeding at ratio of 6:2:2 at different stages were collected. Sequences were aligned using Clustal X 1.8 and MEGA 5.0 was used to construct phylogenetic tree. The bar represents 2% sequence divergence. The numbers in parentheses indicate GenBank accession number. Scientific Reports | 6:34640 | DOI: 10.1038/srep34640 7 www.nature.com/scientificreports/ Figure 5. Average pH and COD removals for each compartment of all reactors at stage II. Average values and error bars were calculated from all daily data of stage II. The removal data for each compartment were calculated as: COD removal =​ (influent of this compartment – effluent of this compartment)/initial influent ×​ 100%. Error bars represent SEM; P <​ 0.05 according to one-way ANOVA was considered significant and is indicated by */#; **/##P <​ 0.01; ***/###P <​ 0.001 (*Significantly different from R1, #Significantly different between R2 and R3). Figure 5. Average pH and COD removals for each compartment of all reactors at stage II. Average values and error bars were calculated from all daily data of stage II. The removal data for each compartment were calculated as: COD removal =​ (influent of this compartment – effluent of this compartment)/initial influent ×​ 100%. Error bars represent SEM; P <​ 0.05 according to one-way ANOVA was considered significant and is indicated by */#; **/##P <​ 0.01; ***/###P <​ 0.001 (*Significantly different from R1, #Significantly different between R2 and R3). aerobic compartments. As shown in Fig. 1, the integration of aerobic and anaerobic processes in R1 could sig- nificantly shorten the start-up time to approximately 14 days. Moreover, the start-up time of two split-feeding reactors could be markedly reduced by 50%, which in turn led to the improvement in COD removal and stability of the A/OBR system. As summarized in Table 2, the COD removal in R1 was only 66.4 ±​ 6.5% at the end of the operation, which might be caused by the inhibition of excessive organic loading and high level toxic substances in upstream compartments. Yang et al. reported that it would be toxic to uncultivated microorganisms if the salt concentration in wastewater exceeded 1.0%41. Ji et al. used an ABR to treat heavy oil polluted water with high con- centration of salt (1.15~1.46%), but the average COD removal was only 65%42. Scientific Reports | 6:34640 | DOI: 10.1038/srep34640 Discussion However, with the supplement of substrate from the C5 into the C6, more abundant microorganisms appeared in C6, thus showing a better removal. removals in the last two compartments were totally different between R2 and R3 (P <​ 0.001). In R2, COD remov- als of C5 and C6 were 13.0% and 1.8%, while that in R3 were 0.3% and 8.5%, respectively. This difference might be caused by the high feeding rate to C5 in R3 (twice as much as that in R2), which would result in shorter retention time compared with R2. Therefore, there was no sufficient time for efficient COD removal. However, with the supplement of substrate from the C5 into the C6, more abundant microorganisms appeared in C6, thus showing a better removal. Mixing patterns of the three A/OBRs were predicted by observing the variance in C-curve (Fig. 2). Date were then fitted into the two-phase dispersion model and the tank-in-series (TIS) model and results were summarized in Table 3. The results fitted by two models showed the same trend that the mixing pattern in the regular-feeding R1 was close to completely-mixed, while both two split-feeding reactors R2 and R3 were intermediate between plug-flow and completely-mixed, tending towards the latter. Moreover, split-feeding reactors R2 and R3 showed significantly advantage over regular-feeding reactor R1 in term of hydraulic dead space (8.35% and 8.17% versus 31.9%). Furthermore, it was reported that an increase in the (hydraulic) dead space was expected with a decrease in HRT because of more channeling in the reactor bed at high HRT (>​20 h)26. Consistent with this conclusion, the calculated HRT in R1 was 44.6 h, which was much lower than that in R2 (57.2 h) and R3 (53.4 h), indicating that a significant amount of by-pass channeling took place in R1. Correspondingly, the fraction of dead spaces in R2 and R3 were much lower. Considering the fact that all three reactors were identical and operated in the same conditions except for the feeding ratios, it was reasonable to conclude that the split-feeding strategy could prevent channeling from taking place by creating a greater degree of fluidization in the reactor, and thus decreased the fraction of dead space.f p In order to further study the function changes of different compartments, the diversity of bacterial commu- nities determined by the Shannon-Wiener index (H’) is shown in Table 5. Discussion The Shannon-Wiener diversity index reflects the variety of bacterial communities and lower H’ values represents a lower α​-diversity48. Table 5 showed that the H’ in R1 at stage II decreased constantly from C1 to C4, and then increased gradually in the last two com- partments. As previously stated, the high OLR as well as high salinity inhibited the growth of microorganisms in upstream compartments, as reflected in a decrease in H’ value. On the contrary, H’ in R2 and R3 increased from C1 to C4, but decreased in the last two compartments. Compared to the R1, H’ values in R2 and R3 were relatively higher with less variation, which again provided the evidence that the split-feeding strategy could relieve the OLR and enhanced the degradation efficiency. Noticeably, the lowest H’ value of R3 was observed in C5, which agreed well with the above result that almost zero COD removal was obtained in C5. Further phylogenetic analysis indicated that the most important organic matter degradation bacteria belonged to Firmicutes, Bacteroidetes as well as some of Actinobacteria, whilst the Proteobacteria played a crucial role in nitrification and denitrification. It has been proved that many Clostridium spp. such as Clostridium aceticum, Clostridium formicoaceticum, Clostridium thermoaceticum and Clostridium cylindrosporum harbored H2-utilizing ability and dominated in the acidogenesis/acetogenesis stage25. Aydina et al.49 also found that Clostridium spp. represented 93% of Firmicutes members in the seed sludge. Ren et al.20 indicated that Firmicutes was the most predominant and was more abundant in samples performing high COD removal efficiency. These dominant bacteria were relatively stable and played an important part in COD removal. Notably, some strictly anaerobic bacteria such as Clostridium sp. (band a2, a13, b5 and b9) and Anaerofilum sp. (band b3) were observed and even represented a major component in aerobic compartments C1 and C5, which may contribute to the formation of granular sludge. Similar results were also obtained in other aerobic reactors such as SBR and UASB50,51. This granulation allowed the gradient distribution of oxygen, thus making it possible for various aerobic, anoxic and anaerobic bacteria to be distributed throughout the granule layers. Moreover, this spatial distribution promoted the mass transfer to a certain extent and allowed nitrification and denitrification process occur simultaneously52. Figure 4 showed that Arcobacter sp. (band a6), Comamonadaceae (band c3) Pseudomonas sp. (band a10 and c4), Dechlorosoma sp. (band b8) and Acidovorax sp. Discussion reported that COD was removed in the first compartment at an OLR of 0.87 kg/m3d. However, when the OLR increased initially to 1.82 kg/m3d and then to 4.73 kg/m3d, the larger fraction of COD was degraded both in the first and second compartments46. Manariotis and Grigoropoulos used an ABR to treat low-strength wastewater and found that most organic matter was degraded in the first two compartments, as the COD removals in the first three compartments were measured as 56.1%, 22.4% and 5.3%, respectively47. However, COD removal in each compartment was obviously different, though no apparent differ- ence between R2 and R3 was observed in terms of total COD removal at stage II. In regular-feeding reactor R1, the first two compartments C1 and C2 only accounted for 19.3% (18.6% plus 0.7%) of the total COD removal, while it significantly increased to 51.2% (40.0% plus 11.2%, P <​ 0.05) and 48.5% (30.7% plus 17.8%, P <​ 0.05) in two split-feeding reactors R2 and R3 (Fig. 5). It should be highlighted that the ratio to the downstream com- partments was important and an excess feed might reduce the COD removal ability. As shown in Fig. 5, COD 8 Scientific Reports | 6:34640 | DOI: 10.1038/srep34640 www.nature.com/scientificreports/ Compartment No. Shannon-Wiener index (H’)* R1 R2 R3 C1 1.06 1.10 1.10 C2 0.92 1.18 1.12 C3 0.79 1.30 1.14 C4 0.73 1.30 1.20 C5 1.19 1.21 0.97 C6 1.17 1.27 1.12 Table 5. Shannon-Wiener index of bacterial communities in different reactors at stage II. *Shannon index (H’): a higher value represents more diversity. Compartment No. Shannon-Wiener index (H’)* R1 R2 R3 C1 1.06 1.10 1.10 C2 0.92 1.18 1.12 C3 0.79 1.30 1.14 C4 0.73 1.30 1.20 C5 1.19 1.21 0.97 C6 1.17 1.27 1.12 Table 5. Shannon-Wiener index of bacterial communities in different reactors at stage II. *Shannon index (H’): a higher value represents more diversity. removals in the last two compartments were totally different between R2 and R3 (P <​ 0.001). In R2, COD remov- als of C5 and C6 were 13.0% and 1.8%, while that in R3 were 0.3% and 8.5%, respectively. This difference might be caused by the high feeding rate to C5 in R3 (twice as much as that in R2), which would result in shorter retention time compared with R2. Therefore, there was no sufficient time for efficient COD removal. Discussion (band b2 and b6) were denitrifying bacteria, which could use organic acids as electron donors and nitrate as an electron acceptor for denitrification53–55. Some of these deni- trifying bacteria were also proven to have the anammox ability56, which might support the above inference that nitrification and denitrification might occur simultaneously in A/OBR. ii g y It should be noticed that although PCR-DGGE was an excellent, highly reproducible, comparative community analysis tool57–60, the V3 region of the 16S rRNA might limit the amount of sequence information for precise identification. Therefore, only the genus level of bacteria was discussed in this study. Other nucleic sequencing method for cultivation-independent community analysis (e.g., 454 pyrosequencing, Illumina sequencing and Scientific Reports | 6:34640 | DOI: 10.1038/srep34640 9 www.nature.com/scientificreports/ Figure 6. Schematic diagram of anaerobic/oxic baffled reactor (A/OBR) used in the experiments. Three peristaltic pumps were used to supply feedstock to C1, C2 and C5 at a total flow rate of 0.37 L/h. The flow rates provided by each pump were calculated according to the feeding ratios in different feeding strategies. All three reactors were carried out as calculated HRT of 48 hours under constant temperature of 35 °C. Figure 6. Schematic diagram of anaerobic/oxic baffled reactor (A/OBR) used in the experiments. Three peristaltic pumps were used to supply feedstock to C1, C2 and C5 at a total flow rate of 0.37 L/h. The flow rates provided by each pump were calculated according to the feeding ratios in different feeding strategies. All three reactors were carried out as calculated HRT of 48 hours under constant temperature of 35 °C. metagenomic approaches) can be used in future research to develop a mechanistic understanding of the relation- hips between reactor operational strategies, microbial community structure, and reactor performance.fifl In summary, a novel, quick start-up and efficient anaerobic/oxic baffled reactor (A/OBR) was developed by incorporating an aerobic and anaerobic process for treating foodwaste anaerobic digestate. An effective split-feeding strategy was moreover presented to enhance the performance of A/OBR. Three aspects, the COD removal, the hydraulic characteristics and the bacterial community, were systematically investigated, compared and evaluated between the regular-feeding and split-feeding strategy. Compared with the regular-feeding reactor R1, the two split-feeding reactors R2 and R3 could greatly shorten the start-up time (~7 days), enhance the COD removal ability (more than 25%), reduce the hydraulic dead space (more than 20%) and maintain a more stable bacterial community. Discussion These characters implied that this new split-feeding A/OBR system might provide an effec- tive and sustainable solution for treating foodwaste anaerobic digestate and other high COD and toxic wastewater. However, the feeding ratio should be tested to achieve the maximal removal efficiency before the application of this new system. Besides, some other wastewater treatment systems such as membrane bio-reactor (MBR), could also be combined with this A/OBR system to further enhance the treatment capability. Materials and Methods R d i d i Reactor design and operation. The anaerobic/oxic baffled reactor (A/OBR), with dimensions of 600 mm long, 200 mm wide, 200 mm high, and an effective volume of 17.88 L, were used in the present study (Fig. 6). Each reactor was comprised of six compartments marked as C1, C2, C3, C4, C5 and C6, while each compartment was sub-divided by a vertical baffle into down-flow and up-flow sections with a volume ratio of 1:4. In order to increase the degradation efficiency of the reactor, an air diffuser was sunk into C1 to provide an oxic-anoxic-an- aerobic-anaerobic condition for C1–C4. The main function of C1 and C2 was to decompose the macromolecular organic matter (such as starch, fat and protein) into small molecular substances (such as VFAs and amino acids). These small molecular substances were then converted to CH4, CO2 and H2 by anaerobic bacterium in C3 and C4. C5 was designed to further remove the residual organics by adding double air diffusers and C6 was designed as a settling compartment. Each air diffuser can provide an aeration rate of 1.25 L/min.hff www.nature.com/scientificreports/ www.nature.com/scientificreports/ The A/OBRs were inoculated with dewatered anaerobic sludge collected from a wastewater treatment plant in the Shunyi District, Beijing, China. The inoculums had a VS/TS ratio of 0.32 and were initially fed into the empty reactors at a volume ratio of 30% of the reactor’s effective volume. All three reactors were operated under the same conditions after one month domestication before the experiments. Statistical analysis. Data analysis was performed with SigmaStat 3.5 and Excle. The one-way ANOVA were used to determine the significance of differences between groups, and P <​ 0.05 was considered as significant. Investigation of hydraulic characteristics. The hydraulic characteristics of the reactor were determined based on the residence time distribution (RTD) study by tracer stimulus-response technology24. The reactor was firstly filled with deionized water, and then KCl solution with concentration of 0.56 mg/L was fed intermittently. Water samples of three reactors were collected at every 6 h intervals. K+ concentrations were determined by atomic absorption spectrophotometer (SpectrAA55b, Varian, USA).f p p p ( p ) To compare the mixing patterns at different HRTs, the unit of time is normalized (dimensionless): θ = t HRT (1) θ = t HRT (1) where, θ is the normalised time, t is the time. Effluent samples are collected at regular intervals from the time o mpulse feeding to 3 times of nominal HRT (θ =​ 3), where the tracer concentration is too low to measure. = θ C C t C ( ) (2) 0 (2) where, Cθ is the normalized tracer concentration at dimensionless time θ, C(t) is the tracer concentration at time C0 is the initial tracer concentration.h where, Cθ is the normalized tracer concentration at dimensionless time θ, C(t) is the tracer concentration at time t, C0 is the initial tracer concentration. The C curves (C vs θ) determined as a function of the normalized tracer concentration (Eq (2)) against the t, C0 is the initial tracer concentration. The C-curves (C vs θ​), determined as a function of the normalized tracer concentration (Eq. (2)) against the 0 The C-curves (C vs θ​), determined as a function of the normalized tracer concentration (Eq. (2)) against the ormalized time (Eq. (1)), are shown in Fig. 2.h These curves were then further analyzed, as the calculated the mean (θm) (Eq. (3)) and variance (σθ 2) (Eq. www.nature.com/scientificreports/ (4)) of the curve, the fraction of dead space in the reactor (Vd/VT), the overall dispersion number (D/uL), and the equivalence number of perfectly-mixed tanks in series (N)26. ∫ θ θ θ = ⋅ θ C d (3) m ∫ θ θ θ = ⋅ θ C d m (3) ∫ σ θ θ θ = − θ C d (4) m m 2 2 2 (4) The fraction of dead space in the reactor was calculated using Eq. (5) as follows: The fraction of dead space in the reactor was calculated using Eq. (5) as follows: θ = − V V 1 (5) T m d (5) where, Vd is dead space and VT is total volume. In a dispersion model, where, Vd is dead space and VT is total volume. In a dispersion model, where, Vd is dead space and VT is total volume. In a dispersion model, θ µ µ =      −      ×    −    µ − D L D L e 2 2 1 (6) m L D 2 2fil (6) where, D is the axial dispersion coefficient, μ is the average fluid velocity and L is the axial distance of the reactor. D/μL is the reciprocal of the Peclet number Pe. where, D is the axial dispersion coefficient, μ is the average fluid velocity and L is the axial distance of the reactor. D/μL is the reciprocal of the Peclet number Pe. k d l In a tank-in-series model, θ = N 1 (7) m 2 θ = N 1 m 2 (7) Reactor design and operation. g pf p Three identical A/OBRs were employed to evaluate the effects of different feeding strategies in the present study. The regular-feeding strategy, in which the feedstock was loaded only into compartment C1 by one pump at the flow rate of 0.37 L/h, was tested in the first reactor (control group, labeled as R1). For the second and third reactors, two split-feeding strategies were carried out by splitting the total flow of 0.37 L/h into three streams and pumping into C1, C3 and C5 at volume ratio of 6:3:1 and 6:2:2, reactively (test group, labeled as R2 and R3). The purpose of split-feeding strategies was to relieve the loading shock in the first compartment. The hydraulic reten- tion time (HRT) of the three reactors was 48 h. All three reactors were operated in the same conditions at 35 °C, except for the specifically applied feeding ratios. Characteristics of feedstock and inoculums. As feedstock for the present study, the discharged efflu- ent was collected from a 100 m3 food waste anaerobic digester in the Changping district, Beijing. Samples were centrifuged at 5000 rpm for 5 min in a micro centrifuge (TGL-16G Centrifuge, Anting Scientific Apparatus Co., China). The pH was monitored by an ion meter (MP 523 pH/ISE meter, SANXIN Co., China). The COD, total nitrogen (TN), ammonia nitrogen (NH4 +-N), nitrite (NO2 −-N), nitrate (NO3 −-N) and total phosphorus (TP) were analyzed according to the standard method61. The concentration of sodium was measured according to the standard method50 by atomic absorption spectrophotometer (SpectrAA55b, Varian, USA), as Chinese food usually contained high content of kitchen salt. The characteristics of the feedstock (Table 1) showed relatively high variations on element concentrations, which might be attributed to the feedstock contents and operation patterns of the food waste digester. Scientific Reports | 6:34640 | DOI: 10.1038/srep34640 10 where, N is the number of on continuous stirred tanks in series. where, N is the number of on continuous stirred tanks in series. where, N is the number of on continuous stirred tanks in series. 16S rRNA gene amplification and denaturing gradient gel electrophoresis (DGGE). The V3 region of the 16S rRNA gene was amplified by PCR using the primers 341F-GC (5′​-CGC CCG CCG CGC GCG GCG GGC GGG GCG GGG GCA CGG GGG GCC TAC GGG AGG CAG CAG-3′​) and 534R (5′​-ATT ACC GCG GCT GCT GG-3′​) for the bacteria62. The PCR protocol included 5 min pre-degeneration at 94 °C, followed by 30s denaturation for 30 cycles at the same temperature. Then the sample was annealed for 30s at 55 °C and then was extended for 30s at 72 °C, followed by 5 min additional extension at the same temperature. DGGE was performed on a DCode universal mutation detection system (Bio-Rad, USA). Samples of PCR products were loaded onto 8% polyacrylamide (Amresco, Ohio, United States) gels (37.5:1, acrylamide/bisacrylamide), and urea (Amresco, Ohio, United States) and deionized formamide (Amresco, Ohio, United States) (containing 7 mol/L urea and 40% formamide, defined as the denaturant concentration 100%) were added in one of the two solutions. The gel was prepared by using the Bio-Rad gradient mixing device (Bio-Rad, California, United States), to make the bacterial denaturant concentration range of approximately 40 to 65%, wherein the concentration of denaturant was decreasing from bottom to top. The sheet was put into a completely solidified electrophoresis Scientific Reports | 6:34640 | DOI: 10.1038/srep34640 11 www.nature.com/scientificreports/ tank containing 1×​ Tris-Acetate-EDTA buffer (Biotopped, Beijing, China), and the temperature was maintained at 60 °C during the whole electrophoresis. Pre-run was performed for 20 minutes under conditions of 180 V, in order to remove impurities in the gel. The 30 μ​L samples were loaded to the inlet. After electrophoresis at 180 V for 5.5 hours, the gel was stained for 30 minutes using a 3×​ GelRed (Biotium, California, United States) for further analysis. For identification of DGGE bands, each band was eluted into 40 μ​L of deionized and distilled water and then the mixture was incubated overnight at 4 °C to extract the DNA from DGGE bands. References J Environ Manage. 90, 2488–2493 (2009). 10. Thanwised, P., Wirojanagud, W. & Reungsang, A. Effect of hydraulic retention time on hydrogen production and chemical oxygen demand removal from tapioca wastewater using anaerobic mixed cultures in anaerobic baffled reactor (ABR). Int J Hydrogen Energy 37 15503–15510 (2012) 0. Thanwised, P., Wirojanagud, W. & Reungsang, A. Effect of hydraulic retention time on hydrogen production and chemical oxygen demand removal from tapioca wastewater using anaerobic mixed cultures in anaerobic baffled reactor (ABR). Int J Hydrogen Energy 37, 15503–15510 (2012).h 11. Baloch, M., Akunna, J. C. & Collier, P. J. The performance of a phase separated granular bed bioreactor treating brewery wastewater. Bioresour Technol. 98, 1849–1855 (2007).fl 12. 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Solution was then used as the template in the reamplification reaction using the same primer without GC-lamp, the specific primers, 341F (5′​-TAC GGG AGG CAG CAG-3′​) and 534R (5′​-ATT ACC GCG GCT GCT GG-3′​)63.hh ( ) ( ) The PCR products were sent to Hanyu biotech Co. Ltd. (Shanghai, China) for sequencing. The sequences were blasted with the nucleotide sequence databases in the GenBank and the BLAST program (http://www.ncbi. nlm.nih.gov/BLAST/). The migration and intensity of each band was analyzed using Quantity One 4.6.2 soft- ware (Bio-Rad, USA). The phylogenetic tree was constructed by MEGA version 5.1, using the neighbor-joining method. 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Anaerofilum pentosovorans gen. nov., sp. nov., and Anaerofilum agile sp. nov., two new, strictly anaerobic, mesop acidogenic bacteria from anaerobic bioreactors. Int J Syst Bacteriol. 46, 871–875 (1996). g y 30. Mohan, S. V. et al. Firmicutes with iron dependent hydrogenase drive hydrogen production in anaerobic bioreactor using dist wastewater. Int J Hydrogen Energy 36, 8234–8242 (2011). p y g wastewater. Int J Hydrogen Energy 36, 8234–8242 (2011). J y g gy , ( ) 31. Ahmed, I., Yokota, A., Yamazoe, A. & Fujiwara, T. Proposal of Lysinibacillus boronitolerans gen. nov. sp. nov., and transfer of Bacillus fusiformis to Lysinibacillus fusiformis comb. nov. and Bacillus sphaericus to Lysinibacillus sphaericus comb. nov. Int J Syst Evol Microbiol. 57, 1117–1125 (2007). 32. Guo, Z. et al. Enhanced short chain fatty acids production from waste activated sludge conditioning with typical agricul residues: carbon source composition regulates community functions. Biotechnol Biofuels 8, 1 (2015). p g y f 3. Goud, R. K., Sarkar, O., Chiranjeevi, P. & Mohan, S. V. Bioaugmentation of potent acidogenic isolates: A strategy for enhancing biohydrogen production at elevated organic load. Bioresour Technol. 165, 223–232 (2014). y g p g 4. Servin, J. A., Herbold, C. W., Skophammer, R. G. & Lake, J. A. Evidence excluding the root of the tree of life from the actinobacteria Mol Biol Evol. 25, 1–4 (2008). 5. Tapia-Venegas, E. et al. Adaptation of acidogenic sludge to increasing glycerol concentrations for biohydrogen production. App Microbiol Biotechnol. 99, 8295–8308 (2015).f 6. Li, A. J., Yang, S. F., Li, X. Y. & Gu, J. D. Microbial population dynamics during aerobic sludge granulation at different organic loading rates. Water Res. 42, 3552–3560 (2008).fl 37. Sarathai, Y., Koottatep, T. & Morel, A. Hydraulic characteristics of an anaerobic baffled reactor as onsite wastewater treatment system. J Environ Sci-China 22, 1319–1326 (2010).fl y 38. Hahn, M. J. & Figueroa, L. A. Pilot scale application of anaerobic baffled reactor for biologically enhanced primary treatment of raw municipal wastewater. Water Res. 87, 494–502 (2015).fl p 9. Boopathy, R. & Tilche, A. Anaerobic digestion of high strength molasses wastewater using hybrid anaerobic baffled reactor. Wate Res. 25, 785–790 (1991).ifl 40. Bayrakdar, A., Sahinkaya, E., Gungor, M., Uyanik, S. & Atasoy, A. D. Performance of sulfidogenic anaerobic baffled reactor (ABR) treating acidic and zinc-containing wastewater. Bioresour Technol. 100, 4354–4360 (2009). g g 41. Yang, L. & Lai, C. www.nature.com/scientificreports/ Biological treatment of mineral oil in a salty environment. Water Sci Technol. 42, 369–375 (2000).fl 42. Ji, G., Sun, T., Ni, J. & Tong, J. Anaerobic baffled reactor (ABR) f 42. Ji, G., Sun, T., Ni, J. & Tong, J. Anaerobic baffled reactor (ABR) for treating heavy oil produced water with high concentrations and poor nutrient. Bioresour Technol. 100, 1108–1114 (2009).ifli 43. Barber, W. P. & Stuckey, D. C. Nitrogen removal in a modified anaerobic baffled reactor (ABR): 2, nitrification. Water Res. 34, 2423–2432 (2000). 4. Grandlic, C. J., Palmer, M. W. & Maier, R. M. Optimization of plant growth-promoting bacteria-assisted stabilization of mine tailings. Soil Biol. Biochem. 41, 1734–1740 (2009). g 5. Hong, H., Pruden, A. & Reardon, K. F. Comparison of CE-SSCP and DGGE for monitoring a complex microbial community remediating mine drainage. J. Microbiol. Meth. 69, 52–64 (2007).fl 46. Polprasert, C., Kemmadamrong, P. & Tran, F. Anaerobic baffle reactor (ABR) process for treating a slaughterhouse wastewater. Environ Technol. 13, 857–865 (1992).fl 7. Manariotis, I. D. & Grigoropoulos, S. G. Low-strength wastewater treatment using an anaerobic baffled reactor. Water Environ Res 74, 170–176 (2002). 48. Ma, Q. et al. 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An innovative wood-chip-framework soil infiltrator for treating anaerobic digested swine wastewate analysis of the microbial community. Bioresour Technol. 173, 384–391 (2014). 3. Canion, A. et al. References 22, 656–662 (2010). 21. Lin, Y., Han, X., Lu, H. & Zhou, J. Study of archaea community structure during the biodegradation process of nitrobenzene wastewater in an anaerobic baffled reactor. Int Biodeterior Biodegradation 85, 499–505 (2013). wastewater in an anaerobic baffled reactor. Int Biodeterior Biodegr d l h d d d b fl 2. Nadais, H. et al. Enhancing wastewater degradation and biogas production by intermittent operation of UASB reactors. Energy 36 2164–2168 (2011).fl 3. Lin, Y., Yin, J., Wang, J. & Tian, W. Performance and microbial community in hybrid anaerobic baffled reactor-constructed wetland for nitrobenzene wastewater. Bioresour. Technol. 118, 128–135 (2012).fl 4. Nachaiyasit, S. & Stuckey, D. C. Microbial response to environmental changes in an anaerobic baffled reactor (ABR). Antonie van Leeuwenhoek 67, 111–123 (1995). 5. Peng, J.-f., Song, Y.-h., Wang, Y.-l., Yuan, P. & Liu, R. Spatial succession and metabolic properties of functional microbia communities in an anaerobic baffled reactor. Int Biodeterior Biodegradation 80, 60–65 (2013).fl 26. Grobicki, A. & Stuckey, D. Hydrodynamic characteristics of the anaerobic baffled reactor. Water Res. 26, 371–378 (1992).fl 7. Sarathai, Y., Koottatep, T. & Morel, A. Hydraulic characteristics of an anaerobic baffled reactor as onsite wastewater treatmen system. J Environ Sci. 22, 1319–1326 (2010).f y 28. Ji, J. Y., Zheng, K., Xing, Y. J. & Zheng, P. Hydraulic characteristics and their effects on working performance of compartmentalized anaerobic reactor. Bioresour Technol. 116, 47–52 (2012). Scientific Reports | 6:34640 | DOI: 10.1038/srep34640 12 Author Contributions S.W., L.P. and H.S. conceived and designed the project and experiments. S.W., L.P. and Y.J. performed the experiments. S.W. analyzed the data. P.G. and B.Z. participated in parts of the experiments. The manuscript was written by S.W. and reviewed by all co-authors. Acknowledgements g The authors express their thanks for the supports from the National Natural Science Foundation of China (21525625), the National Basic Research Program (973 Program) of China (2014CB745100), the (863) High Technology Project (2013AA020302). Scientific Reports | 6:34640 | DOI: 10.1038/srep34640 13 www.nature.com/scientificreports/ Scientific Reports | 6:34640 | DOI: 10.1038/srep34640 Additional Information Supplementary information accompanies this paper at http://www.nature.com/srepihi Supplementary information accompanies this paper at http://www.nature.com/srepihi Competing financial interests: The authors declare no competing financial interests. Competing financial interests: The authors declare no competing financial interests. How to cite this article: Wang, S. et al. Evaluation of A Novel Split-Feeding Anaerobic/Oxic Baffled Reactor (A/OBR) For Foodwaste Anaerobic Digestate: Performance, Modeling and Bacterial Community. Sci. Rep. 6, 34640; doi: 10.1038/srep34640 (2016). How to cite this article: Wang, S. et al. Evaluation of A Novel Split-Feeding Anaerobic/Oxic Baffled Reactor (A/OBR) For Foodwaste Anaerobic Digestate: Performance, Modeling and Bacterial Community. Sci. Rep. 6, 34640; doi: 10.1038/srep34640 (2016). This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ © The Author(s) 2016 Scientific Reports | 6:34640 | DOI: 10.1038/srep34640 14
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Rethinking the commemorative landscape in South Africa after the Fall: A pedagogical case study
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  Rethinking the commemorative landscape in South Africa afer the Fall A pedagogical case study1 Samantha Masters ‘I’m not sure that we need statues at all – it’s a colonialist thing, like marking territory.’2 Sethembile Msezane ‘I’m not sure that we need statues at all – it’s a colonialist thing, like marking territory.’2 Sethembile Msezane Memory, heritage and public commemoration through statuary – or its opposite, public denigration through attacks on statuary – have been highly topical issues in the past decade. Tese practices are of course not modern, but have been carried out for centuries, in fact millennia. In 2020, images of the statue of slave merchant Edward Colston in Bristol, UK, being pulled down, jumped on, rolled down the road and thrown into the Bristol harbour amid cheering protestors went viral.3 Tis footage is perhaps to become one of the iconic visuals of this practice. Tis act was ignited by an international wave of anti-racism protest, catalysed by the unwarranted killing of African-American George Floyd by a Minneapolis police ofcer in the US – another iconic, and more tragic, moment of that year. Sporadic attacks on statues in South Africa also occurred in this climate: notably the head of colonialist Cecil John Rhodes at his own monument on the side of Table Mountain was hacked of its bust and stolen on 12/13 July of the same year.4 And few of us living in or connected with South Africa in recent years would not have seen the iconic image of a crane removing 167 RETHINKING THE COMMEMORATIVE LANDSCAPE IN SOUTH AFRICA classics can also be seen as, like Sethembile Msezane’s idea of statues, just a relic of a bygone and ‘colonising’ mentality, the subject should be metamorphosising as well. We should be doing more to frame courses using the questions pertinent to our context; this is one of the ways in which we can actively contribute to the decolonisation of the curriculum and the subject in general. (U)MZANTSI CLASSICS (U)MZANTSI CLASSICS the paint-spattered statue of Rhodes from its vantage point at the foot of Jameson Steps on the University of Cape Town campus on 9 April 2015. Schmahmann (2016: 90) recalls the urgent demands for the removal of Rhodes’ statue one month earlier thus: On March 9, 2015, about a dozen protestors gathered in front of a large and imposing sculpture of mining magnate and politician Cecil John Rhodes – a work centrally placed at the University of Cape Town (UCT) … Calling for its removal as well as an end to the racism they considered operative at the university, the group’s protest culminated in one participant, Chumani Maxwele, tossing a bucket of human excrement at the sculpture. RhodesMustFall, as the movement quickly became known, raised issues of inequality, racism and the need for decolonisation of the university’s structures and curricula. It was about so much more than ‘just a statue’, but the statue and other statuary country-wide became the concrete embodiment and visual reminder of the pervasive and inhumane legacies of colonialism and apartheid.5 A year later, in 2016, I taught a 2nd/3rd-year Ancient Cultures course entitled ‘Rome, Empire and Material Remains’. Tis course aimed to investigate the power of images and material artefacts (coins, statues, buildings and monuments, for example) during the expansion and heyday of the Roman Empire. With issues of memory, commemoration, colonialism and statuary being so topical, this provided an ideal opportunity to use Rome as a lens through which to examine more recent, local and contemporary examples of imperial and political propaganda. Tis included Dutch, British and apartheid-era architecture and statuary in South Africa. It also included examining the Roman practices of veneration and memorialisation of prominent fgures, as well as memory sanctions, or damnatio memoriae. Tese memory sanctions are, in efect, the opposite of remembering; they amount to a conscious forgetting, a type of cultural amnesia, comparable to the damage and removal of statues in the current era. Te link between Roman damnatio memoriae and contemporary statuary is one that other scholars have made6 and ‘thinking through classics’ is an established approach in our feld, not novel by any means. It strikes me, however, that in the changing face of our country, where 168 (U)MZANTSI CLASSICS Te ancient Roman landscape, then, was flled with commemorative buildings, statuary and inscriptions, visible indicators of public benefaction, status, power and prestige. However, statues, monuments and their inscriptions were not necessarily permanent fxtures – though made of stone or bronze and at some point sacrosanct – but were at times mutilated, removed or repurposed. Te urban landscape was hardly fxed, but in a way, metamorphosed – changed its form – as it was regularly ‘updated’ in a variety of ways. So despite making a concerted efort to remember, like us, Romans also grappled with the question of what, or whom, to forget. g pp q g Much has been written about the Roman practice known as damnatio memoriae.8 Te term itself is relatively modern: Omissi (2016: 171) describes how, afer being coined in 1689 (as the title of a dissertation published in Leipzig), the term damnatio memoriae became entrenched in scholarship to refer to ‘a bundle of ancient Roman practices that were directed at convicted traitors, usually afer their death’. It is not a straightforward single practice, but a series of acts overt and subtle, state-decreed and spontaneous, designed to erase or desecrate the memory of public fgures. Tese were mainly, but not exclusively, ‘bad emperors’. Suetonius (Dom. 23) and Pliny (Panegyric 52.4–5) both recount the dramatic attack on Domitian’s portraits and the subsequent decree by the senate to damn his memory. With this directive, his titles would ofcially be stripped, acts annulled, coinage recalled, inscriptions erased, statues melted down or removed, portraits recut into other imperial portraits, or entire heads replaced by new portraits.9 Hackworth Peterson (2011) recounts the Latin words used in the ancient literature to describe this as well as the perceived intentions of these practices. Te ancient combination of the word memoria with verbs like eradere (eradicate), abolere (to abolish), condemnare (to condemn) and damnare (to damn) tells us that the goal clearly was, in her words, a ‘conscious forgetting, analogous to wiping a slate or ancient wax tablet clean’ (Hackworth Peterson 2011: 1). Tis idea of a purposeful eradication from memory is what has been described above as a kind of deliberate cultural amnesia. Public statuary, memorialisation and forgetting: Rome Large-scale public statues are, in addition to being a part of the actual urban landscape or cityscape, also an important part of the historical and political landscape in ancient societies, as well as contemporary ones. How such statuary is treated over time is revealing. Romans spent much efort, expense and energy on the conscious remembering of individuals. Vitruvius (6.3), Pliny (Hist. Nat. 35: 4–11), Polybius (6.53) and Juvenal (8.3, 9, 15, 17–18) tell us that ancestors were commemorated in imagines maiorum, or likenesses of diferent kinds. Wax portraits, likenesses of dead ancestors cast from their actual faces, were stored in the house and paraded through the streets during funerary processions. Ancestors’ portraits were also typically displayed in the house, either in bust form or round bronze or silver shield portraits containing the portrait head of an ancestor, ofen hung on walls. Individuals and families were publicly commemorated through tombs and grand mausolea ofen set up along the main roads in and out of Roman cities, such as the Via Appia (Borg 2019: passim). Tey served as types of ‘billboards’ of remembrance along a highway – the wealthy citizens trying to outdo each other in terms of scale and decoration. Political leaders, and especially emperors of Rome, were immortalised in public statuary and monuments, normally voted by the senate and ofen paid for by wealthy patrons seeking favour with the political elite (Ramage 1983: 202). Hannestad (1988: 47) tells us that to familiarise a growing empire with the image of the emperor, ‘[m]assive circulation of the emperor’s portrait began with Augustus … No [Roman emperor] lef as many likenesses as he did’ (there are about 250 surviving). Tis again makes us think of Msezane’s comment that ‘it’s … like marking territory’: the statue is part of the material footprint of empire. Images of the emperor were, in fact, not just politically important, but were sacrosanct, and it was sacrilege to harm or deface such an image.7 169 RETHINKING THE COMMEMORATIVE LANDSCAPE IN SOUTH AFRICA RETHINKING THE COMMEMORATIVE LANDSCAPE IN SOUTH AFRICA statuary – and the recent RhodesMustFall context as a teaching opportunity. Afer 1994, the new democratically elected South African government revisited the important topic of public memory, and heritage legislation was revisited and revised. Te question of problematic or negative heritage, including public statuary that was part of the old South African colonial and apartheid past, was addressed and an array of bills and acts were proposed and enacted.10 However, a very conservative, ‘circumspect’ approach was adopted (Schmahmann 2016: 93). New heritage policies aimed to address the past, but also sought to be inclusive and reconciliatory. Te policies included the addition of a more representative array of ‘heroes’, particularly from the struggle against the apartheid regime. It allowed for the removal of statuary, but only under particular circumstances, and this would require the consent of the local authority and stringent processes.11 q y g p Tis resulted in only the most ‘problematic’ and contentious monuments or statues of individuals being addressed, such as statues of the architect of apartheid, Hendrik Verwoerd (Coombes 2003: 22). Tese were slowly removed, placed in ‘safe spaces’ or undisclosed locations; in fact, the last known public statue of Verwoerd was, according to news articles, only removed from mid-Vaal in 2011.12 In the spirit of reconciliation, then, most other monuments and statues were allowed to remain. Tese include statues from the Dutch and British colonial eras, and the years aferwards, such as Afrikaner heroes like Paul Kruger. In the spirit of optimism and euphoria for the rainbow nation that swept the country in its early democracy, and in the spirit of forgiveness profered by the Truth and Reconciliation Commission, it can be said that the South African government opted for a kind of cultural amnesty, not the amnesia that appears to have been a key goal of Roman and other memory sanctions. Amnesty and amnesia represent two opposing approaches to memory and the ‘wrongs’ and painful memories of the past.13 Twenty-one years afer the fall of apartheid, with the issue of problematic statuary in South Africa resurfacing in a powerful way, I wanted to investigate my students’ experience of this issue. Comparisons with South African statuary It was possible to use this obvious comparison between such overt types of Roman memory sanction – in particular the violent or symbolic attacks on 170 RETHINKING THE COMMEMORATIVE LANDSCAPE IN SOUTH AFRICA I wanted the students, mostly of the ‘born free’ generation, to explore – empathetically – their own views on the issue of negative heritage (Meskell 2002: 558) and the vandalism of statues, and to understand other perspectives on this topic, thinking through the Roman practice of damnatio memoriae. 171 (U)MZANTSI CLASSICS RETHINKING THE COMMEMORATIVE LANDSCAPE IN SOUTH AFRICA RETHINKING THE COMMEMORATIVE LANDSCAPE IN SOUTH AFRICA watching YouTube videos of statuary being destroyed, I realised that the students, in a reasonably diverse classroom in South Africa in 2016, had very diferent emotional responses to these acts. Te student responses ranged from horror to delight. Some felt extremely strongly about defending the statues, letting them remain as they viewed them as fxtures, and as part of the landscape and ‘of history’. Tey strongly favoured the approach of amnesty, with the addition of more statuary to balance out the biases and tell the neglected histories. Others strongly favoured the swif and dramatic removal of ‘ofensive’ statues, and the forgetting of the individuals who represented the heinous injustices of colonialism and apartheid. In this way they tended in the direction of amnesia. Without considering the identity of the statues, many students had preconceived ideas about statuary per se, as they felt statues are important simply because they were skilfully made with great efort and at great expense. Tese students expressed shock and anger at the act of damaging a piece of ‘art’. Tese students I would describe as those from privileged upbringings, who had attended private or ‘model C’ schools, and had an assumption that ‘art’ has value and that it is, in a way, untouchable (or even sacrosanct). Some students referred to the damage as outright ‘sacrilege’. Others did not have the same awe of the statues. Teir own comments claimed that this had much to do with their background and education and that they were not taught to ‘appreciate art’. For many the initial response to the destruction of the image of a human being, or damage to it, was the difcult element; that it was a human likeness was problematic. Other students felt neutral about damage to the statuary of particular individuals or, on the other hand, their remaining, based on the lapse of time since they were set up (‘Rhodes was long ago’), or their own family history, cultural background and afnity. For example, white middle-class English-speaking students did not care much about the statues of Afrikaner heroes. But neither did they care about the Anglophone statues of Queen Elizabeth or Cecil John Rhodes. However, one of the most interesting outcomes was the discovery that many students did not know the history of their own country and the impact many of the people memorialised in statues had had. Memory and emotion One of the most insightful comments from a member of the class, an isiXhosa student from the Eastern Cape who is the frst member of her family to attend university, was: ‘While I don’t really like the idea of destroying a statue, I can identify with the trashing of statues like those of Rhodes. He was a bigot and a racist and his legacy continues to haunt us. Anyway, where was our moment of catharsis?’ One of the most profound points of comparison between Roman damnatio memoriae, the 2015 Rhodes Must Fall movement, and the removal of the statue of Edward Colston, is not just the causing of damage, but what this student pointed out: catharsis. Te emotional charge with which the damage is inficted in the ancient texts or modern videos recording these moments is palpable. Suetonius (Dom. 23) draws specifc attention to the emotions of those pulling down the statues or images of Domitian.14 Te people are both angry at the tyranny and injustices perpetrated by the emperor and also euphoric at his death. Te acts associated with damnatio memoriae are cathartic and even therapeutic. Pliny (Panegyric 52.4–5) gives an even more dramatic description of the emotional content of the practice of smashing statues or melting them down, again in relation to Domitian’s statues: It was our delight to dash those proud faces to the ground, to smite them with the sword and savage them with the axe, as if blood and agony could follow from every blow. Our transports of joy – so long deferred – were unrestrained; all sought a form of vengeance in beholding those bodies mutilated, limbs hacked in pieces, and fnally that baleful, fearsome visage cast into fre, to be melted down, so that from such menacing terror something for man’s use and enjoyment should rise out of the fames.’ (tr. Radice 1975) Te kind of practices described here and elsewhere draws attention to the emotional satisfaction felt by those carrying out the destruction and defacement of statuary. Tis emotional content and context of these acts is what also emerged very clearly in discussions about the RhodesMustFall movement in the Ancient Cultures course. In reading texts like Suetonius and Pliny, discussing such kinds of overt and violent damnatio memoriae, 172 (U)MZANTSI CLASSICS Many students, however, felt extremely emotional about the statuary – either in favour of attacking and removing these fgures of repression, symbols of past wrongs, or defending them. Te responses were based on a complex intersectionality of background, race and identity, social conscience, family history and personal afront. Tis kind of tension between amnesty and amnesia is what is, in my view, epitomised in the South African discourse around heritage and the Rhodes Must Fall movement. Twenty-four years afer the fall of apartheid, the conservative approach to heritage (amnesty) was no longer palatable to many. Te movements of 2015 demonstrate the ongoing challenges of transformation and reconciliation that are yet to be broached/healed. But is the antagonistic approach that opts for ‘collective amnesia’ any better than amnesty? Schmahmann (2016: 101) laments that ‘[the Rhodes statue’s] removal from campus may be argued to have foreclosed a critical and self-refexive engagement with Rhodes’ legacy and its import on UCT’. Te question I put to my students in the next phase of the exercise is: what can we do with ‘negative heritage’ (Meskell 2002: 558) instead? Tis kind of critical refection opens up a third type of treatment of problematic statuary and heritage: in between amnesia and amnesty is another approach, one that we could term agonistic. Tis term – from the Greek word agon, which means competition or struggle – suggests an approach that does not destroy, damage or ignore but confronts and challenges the heritage landscape, and seeks to frame a variety of issues critically, constructively, using a creative imagination, and even a new vernacular. In this vein, the students were asked to use readings such as an article by Sibusiso Tshabalala and Nadine Botha (2015), ‘8 things to do with unwanted statues and monuments’, as a springboard and come up with solutions that do not involve the two extremes of amnesia (total obliteration) or amnesty (just leave them where they are). RETHINKING THE COMMEMORATIVE LANDSCAPE IN SOUTH AFRICA Tese students acknowledged that they had no traumatic family or community history to call on, many of them being white, and therefore of the group that had beneftted most from the legacy of colonial and/or apartheid practices and policies. 173 RETHINKING THE COMMEMORATIVE LANDSCAPE IN SOUTH AFRICA RETHINKING THE COMMEMORATIVE LANDSCAPE IN SOUTH AFRICA Tis allowed for acknowledgement of how the damage and destruction of statues could both demonstrate unresolved psychological trauma of the South African past, and also be cathartic. At the same time, some of the students advocating defacement or removal (amnesia) recognised how these acts of vengeance or redress could in turn cause psychological trauma: as Vladislavic (2008: 100–102) puts it: ‘People may feel the loss of symbols more acutely than the loss of direct political power or economic status.’ In conclusion, the key goal of the exercise, and so too this chapter, is to recognise the usefulness of employing an ancient lens to look at a contemporary issue, in this case the issue of heritage and memory. Trough refecting on an ancient problem still applicable to modern societies, it was possible to bring about some excellent critical discussion, as well as moments of recognition or even empathy. Te potential for education to efect a paradigm shif, to transform an outlook and bring about personal growth and change, should be harnessed, and while ‘transformation’ through teaching classics may seem like an overly optimistic and idealistic goal, it is one that should be taken seriously. Tere are challenges in teaching classics in a country which is in fux, confronting its past and increasingly being held to account for its stagnant intellectual frameworks and curriculum content. In fnding ways of remaining relevant and useful, seizing all such teaching opportunities should be a default position. Te possibility that classics becomes cast aside as ‘cultural detritus’ can be alleviated somewhat by growing something new and constructive from this fertile substrate. Failing this, one can, in the long run, simply be lef with a stale, bad odour. Insights from this teaching opportunity Tis interactive course led to an excellent, frank and also emotional discussion with the students about a topical issue of local and international relevance. Of those initially ‘defending’ the statues and their right to remain (granting them amnesty), many students were able to view the issue for the frst time from the perspective of those wanting to attack or obliterate them. 174 (U)MZANTSI CLASSICS 4 https://www.news24.com/news24/southafrica/news/cecil-john-rhodes-statue-no-arrests-as- police-probe-who-chopped-of-busts-head-in-cape-town-20200715 5 Rhodes Must Fall had a knock-on efect and spurred several other attacks on statues in South Africa, but also gave impetus to the Fees Must Fall student movement. Te urgency of the issue of public statuary and memorialisation returned to public attention, resulting in a public consultation meeting at Freedom Park on the Transformation of the Heritage Landscape in South Africa (17 April 2015). Draf guidelines and a further white paper on arts, culture and heritage were produced: Heritage Western Cape Draf Guidelines for Public Monuments and Memorials 2015; Revised White Paper on Arts, Culture and Heritage (Second Draf) November 2016. 5 Rhodes Must Fall had a knock-on efect and spurred several other attacks on statues in South Africa, but also gave impetus to the Fees Must Fall student movement. Te urgency of the issue of public statuary and memorialisation returned to public attention, resulting in a public consultation meeting at Freedom Park on the Transformation of the Heritage Landscape in South Africa (17 April 2015). Draf guidelines and a further white paper on arts, culture and heritage were produced: Heritage Western Cape Draf Guidelines for Public Monuments and Memorials 2015; Revised White Paper on Arts, Culture and Heritage (Second Draf) November 2016. 5 Rhodes Must Fall had a knock-on efect and spurred several other attacks on statues in South Africa, but also gave impetus to the Fees Must Fall student movement. Te urgency of the issue of public statuary and memorialisation returned to public attention, resulting in a public consultation meeting at Freedom Park on the Transformation of the Heritage Landscape in South Africa (17 April 2015). Draf guidelines and a further white paper on arts, culture and heritage were produced: Heritage Western Cape Draf Guidelines for Public Monuments and Memorials 2015; Revised White Paper on Arts, Culture and Heritage (Second Draf) November 2016. For example, Platt (2020), Forrest and Johnson (2019) and Freedberg (2003). 6 For example, Platt (2020), Forrest and Johnson (2019) and Freedberg (2003). 9 Ancient Roman memory sanctions can for the sake of scholarly study be categorised into ofcial and unofcial or de facto damnatio memoriae, one ofcially decreed, and one that happens anyway based on a spontaneous emotional response. Tey can happen at roughly the same time, but the decree is not a prerequisite for the actions. (U)MZANTSI CLASSICS In addition, one could diferentiate between overt acts of damnatio memoriae such as the destruction of images of individuals, or statues removed from circulation, statues recut as other people, such as Caligula statues reworked as Augustus or Claudius, the recall of coinage, and the annulment of acts (all of these being applied to the memory of Caligula) and more subtle forms of damnatio memoriae such as the use of counter-messages (Ramage 1983). 10 National Archives and Records Service of South Africa Act (Act 43 of 1996); Legal Deposit Act (Act 54 of 1997); National Library of South Africa Act (Act 92 of 1998); National Heritage Bill (1998); Cultural Institutions Act (Act 119 of 1998); South African Geographical Names Council Act (Act 118 of 1998); National Heritage Council Bill (1999); National Heritage Resources Bill (1999); National Heritage Council Act (Act 11 of 1999); National Heritage Resources Act (Act 25 of 1999); National Council of Library and Information Services Act (Act 6 of 2001); Public Monuments and Memorials Project by SAHRA for DAC 2003 (South African Heritage Resources Agency [SAHRA] management body). 11 National Resources Heritage Act (1999). 12 Te only places where statues of Verwoerd are still accessible are in the Afrikaner enclave of Orania, and at the Voortrekker Monument, where they are available on request. https:// www.news24.com/news24/Archives/City-Press/Voetsek-Verwoerd-20150430; https://mg.co. za/article/2011-06-17-fnding-verwoerd-lifing-the-lid-on-dustbin-of-history/ 12 Te only places where statues of Verwoerd are still accessible are in the Afrikaner enclave of Orania, and at the Voortrekker Monument, where they are available on request. https:// www.news24.com/news24/Archives/City-Press/Voetsek-Verwoerd-20150430; https://mg.co. za/article/2011-06-17-fnding-verwoerd-lifing-the-lid-on-dustbin-of-history/ 13 It is interesting to note that the words ‘amnesia’ and ‘amnesty’ derive from the same Greek prefx and verb – ‘a’ means not and the ‘mnes’ root refers to memory/remembering. While both words therefore mean ‘a forgetting’ of sorts, amnestia (amnesty) comes to mean specifcally ‘a purposeful forgetting of wrong, a political act of forgiveness’ (Plut. Mor. 814b) while amnesia means a ‘forgetting’ or ‘oblivion’. 14 ‘Te senators […] were so overjoyed, that they raced to fll the House, where they did not refrain from assailing the dead emperor with the most insulting and stinging kind of outcries. Tey even had ladders brought and his shields and images torn down before their eyes and dashed upon the ground.’ And then he adds that the senate ‘fnally […] passed a decree that [Domitian’s] inscriptions should everywhere be erased, and all record of him obliterated.’ Suetonius, Dom. Notes 1 Five converging interests have led to the subject of this paper on Roman practices of damnatio memoriae and heritage in South Africa: (1) my interest in ancient material culture in general; (2) my interest in South African heritage and changing heritage practices in our postcolonial, post-apartheid and post-RhodesMustFall context; (3) the urgency to think about our own context as scholars of antiquity, and how to make this feld relevant to our own time and place; (4) the theme of metamorphosis as applied to the changing cityscape and to the ofering of an age-old subject in a ‘reborn’ country that is still trying to re-invent itself; and (5) the possibility to efect change through teaching particular topics in a particular way. 2 https://face2faceafrica.com/article/sethembile-msezane-cecil-rhodes 3 https://www.theguardian.com/uk-news/2020/jun/07/blm-protesters-topple-statue-of- bristol-slave-trader-edward-colston 3 https://www.theguardian.com/uk-news/2020/jun/07/blm-protesters-topple-statue-of- bristol-slave-trader-edward-colston 175 (U)MZANTSI CLASSICS 23 (translation by JC Rolfe 1979). 14 ‘Te senators […] were so overjoyed, that they raced to fll the House, where they did not refrain from assailing the dead emperor with the most insulting and stinging kind of outcries. Tey even had ladders brought and his shields and images torn down before their eyes and dashed upon the ground.’ And then he adds that the senate ‘fnally […] passed a decree that [Domitian’s] inscriptions should everywhere be erased, and all record of him obliterated.’ Suetonius, Dom. 23 (translation by JC Rolfe 1979). 176 References Borg B (2019) In search of deceased senators. In: Roman tombs and the art of commemoration: Borg B (2019) In search of deceased senators. In: Roman tombs and the art of commemoration: Contextual approaches to funerary customs in the second century CE. Cambridge: Cambridge University Press. pp. 1–76 Contextual approaches to funerary customs in the second century CE. Cambridge: Cambridge University Press. pp. 1–76 Bundrick S & Varner E (2001) From Caligula to Constantine: Tyranny & transformation in ( ) g y y f Roman portraiture. Atlanta: Michael C. Carlos Museum Roman portraiture. Atlanta: Michael C. Carlos Museum Calomino D (2016) Defacing the past: Damnation and desecration in imperial Rome. London: Spink Coombes A (2003) History afer apartheid: Visual culture South Africa. Durham, NC: Duke University Press Coombes A (2003) History afer apartheid: Visual culture and public memory in a democrat South Africa. Durham, NC: Duke University Press Coombes A (2003) History afer apartheid: Visual culture and public memory in a democratic South Africa. Durham, NC: Duke University Press South Africa. Durham, NC: Duke University Press Flower H (2006) Te art of forgetting: Disgrace & oblivion in Roman political culture. Chapel Hill: University of North Carolina Press Flower H (2006) Te art of forgetting: Disgrace & oblivion in Roman political culture. Chapel Hill: University of North Carolina Press Forrest B & Johnson J (2019) Confederate monuments and the problem of forgetting. Cultural University of North Carolina Press University of North Carolina Press Forrest B & Johnson J (2019) Confederate monuments and the problem of forgetting. Cultural Geographies 26(1): 127–131 Freedberg D (2003, 16 April) Damnatio memoriae: Why mobs pull down statues. Wall Street Journal (Eastern edn) Hackworth Peterson L (2011) Te presence of ‘damnatio memoriae’ in Roman art. Notes in the History of Art 30(2): 1–8 Hannestad N (1988) Roman art and imperial policy. Aarhus: Aarhus University Press Meskell L (2002) Negative heritage and past mastering in archaeology. Anthropological Quarterly 75(3): 557–574 Omissi A (2016) Damnatio memoriae or creatio memoriae? Memory sanctions as creative processes in the fourth century AD. Te Cambridge Classical Journal 62: 170–199 Platt V (2020, 3 July) Why are people toppling monuments to racism? Scientifc American. https://www.scientifcamerican.com/article/why-people-are-toppling-monuments-to- racism/ Radice B (1975) Letters and panegyricus of Pliny. Cambridge, MA: Harvard University Press Ramage ES (1983) Denigration of predecessor under Claudius, Galba, and Vespasian. Historia Zeitschrif für Alte Geschichte 32(2): 201–214 Schmahmann B (2016) Te fall of Rhodes: Te removal of a sculpture from the University Of Cape Town. Public Art Dialogue 6(1): 90–115 Schmahmann B (2016) Te fall of Rhodes: Te removal of a sculpture from the University Of Cape Town. Public Art Dialogue 6(1): 90–115 Tshabalala S & Botha N (2015) 8 things to do with unwanted statues and monuments. https:// nadinebotha.com/2015/03/31/8-things-to-do-with-unwanted-statues-and-monuments/ Tshabalala S & Botha N (2015) 8 things to do with unwanted statues and monuments. https:// nadinebotha.com/2015/03/31/8-things-to-do-with-unwanted-statues-and-monuments/ Varner E (2004) Mutilation and transformation: Damnatio memoriae and Roman imperial portraiture. Leiden: Brill Vladislavic I (2008) A farm in Elof Street. In: C Basualdo (ed.) William Kentridge: Tapestries. New Haven, CT: Yale University Press 177 CONVERSATION WITH AMY DANIELS a move towards interrogating ideas of beauty and then looking at where those ideas come from and so forth. So my experience of being taught in the past is that the content was decontextualised, it was something that we studied for itself – classics for classics sake. We are very much now aiming to contextualise what we’re teaching and make it relevant to students or to show them a way of seeing it as relevant, of incorporating what they learn in the rest of their studies. In other respects, I suppose, just listening to discussions at conferences, at staf meetings, that kind of thing, there is still a big concern for teaching students the ‘canon’, if that even exists. So, in that sense I can’t say whether or not it has changed. I also came at it afer graduating with my undergrad from having meandered of and looked at classical reception in German literature towards the end of East Germany [laughter] – so very, very focused, all Christa Wolf and others, and then falling back into it with a focus on late antiquity and St Augustine and his Confessions and a more philosophical bent. I can’t really even remember where I started with that one. Conversation with Amy Daniels Grant Parker (GP): Could you tell us a little bit about yourself and your background and what brought you to classics? Amy Daniels (AD): I’m a lecturer of Ancient Cultures and Latin at Stellenbosch University. My falling into classics actually began with a bout of laryngitis where I lost my singing voice and so could not do a BMus. On registration day I was torn between doing a ‘proper’ subject – as my family called it – such as Psychology, and doing Latin, which was my promise to a Latin teacher at my school, Mr Rutter. I ended up sitting next to a classicist on registration who said, ‘Hey, do Ancient Cultures. See what makes Latinists tick.’ So, I got into it not because I knew what it was, but because of a promise I made to a teacher that had never taught me. I stayed, I think because I grew up around classical music and around very Anglocentric children’s books, and that kind of thing, and so the stories I was exposed to would ofen make reference to classical themes: I always knew who Pandora was; I always knew who Hercules was, and so forth. I always knew who Dido and Aeneas were. So, that’s sort of the long and the short of how I got into classics GP: So, between your frst study of classics and 2020, has classics changed? GP: So, between your frst study of classics and 2020, has classics changed? AD: I think it has. When I frst started, some of the notes that we were studying from were still typed on typewriters, and in terms of classics we were told things like, ‘Greek art is beautiful in and of itself,’ and then you just had to study the technical aspects of it. Whereas now I think there has been 178 GP: Well, I’d like to know more about that. I’d like to think about some GP: Well, I’d like to know more about that. I’d like to think about some (U)MZANTSI CLASSICS possibilities. I wonder, for example, if some kind of canon is required for any common language. In an extreme view, I mean, this is something English departments may have faced and perhaps German departments around Goethe and, whoever: that if there’s no canon, whether it’s the Bible or Shakespeare or Milton or whatever you like, if there’s no canon then there’s no koine. Tere’s nothing to talk about with other people that are interested in the same thing. And then, ultimately, what is the same thing? AD: I think your last question is the key: what is the same thing? Is the same thing Shakespeare or is the same thing Elizabethan ideas on masculinity and, I don’t know, race? Is the same thing a text or is it your approach to a text or themes that you’re interested in in a text? Yes, I’m not entirely convinced that a canon is necessary for there to be a koine. I think we should be able to speak with one another whether or not we have studied the same thing. I mean, right now there is nothing more exciting to me than an interdisciplinary discussion on some issue or some theme or some text or whatever the case may be. Yeah. Also, I say this because my own training in my discipline is one that could be described as patchy, but I would like to think of it as diverse and dynamic. So, at the moment, we have had postgraduate students coming to us and asking what they should be reading in terms of canonical texts. And so I’ve been looking at these lists my colleagues have been sending out and I haven’t read half those texts, but I’ve read other works that allow me to bring insights to any discussion we have amongst ourselves as classicists and as Ancient Near Eastern scholars and Semitic scholars in our department. So yes, I don’t know if that adds meaningfully to any type of answer that we could possibly get to. GP: Why do we need to have the answer? GP: Is there a place for the canon today? AD: Tat is something I’ve been wrestling with since I started teaching in 2014. And I have at moments just been so distressed by this – this question – and have asked colleagues what they think and ofen the answer I would get was: ‘Tat’s why you’re here. You need to fgure this out for us.’ ‘I come from the old guard’ is sort of the answer I would get from various people. I suppose, the place of a canon depends on how it is being studied and how it is being taught. And by that, I mean if we are simply going to do philological studies of lines and lines of, I don’t know, Herodotus, Tacitus, whoever, then I don’t actually see any place or value in a South African context because we could be doing that with other texts, texts that perhaps have a more direct infuence on how South Africans are thinking or the questions that we’re grappling with right now. However, if, as I said earlier, we are concerned for bringing ourselves to texts; if we are concerned with bringing our contexts to the texts and vice versa, bringing those texts to ourselves and to our contexts, then perhaps there is a place for a classical canon today in Africa. How you do that I have no idea. AD: Tat is something I’ve been wrestling with since I started teaching in 2014. And I have at moments just been so distressed by this – this question – and have asked colleagues what they think and ofen the answer I would get was: ‘Tat’s why you’re here. You need to fgure this out for us.’ ‘I come from the old guard’ is sort of the answer I would get from various people. GP: Well, I’d like to know more about that. I’d like to think about some 179 (U)MZANTSI CLASSICS (U)MZANTSI CLASSICS Why are we, as in you [chuckle], not able to pose the question, ‘What is a canon and what does the canon mean in Ancient Studies?’ So, let me put it this way: If you were to convene such a conversation – ‘What does, what might the notion of canon mean in Ancient Studies?’ – what would you want to be in that conversation? AD: I think, partly due to feedback from students, and on another level, due to some feedback and discussion from certain colleagues, I would want to know how we defne ‘ancient’ when we think of Ancient Studies because, in some ways, that name has almost become a misnomer: people AD: I think, partly due to feedback from students, and on another level, due to some feedback and discussion from certain colleagues, I would want to know how we defne ‘ancient’ when we think of Ancient Studies because, in some ways, that name has almost become a misnomer: people 180 CONVERSATION WITH AMY DANIELS sign up and they think they’re going to learn about ancient Africa and, by ancient Africa, they exclude – and I have no idea why – Egypt. So, I would want to think about what ‘ancient’ is; at the moment we defne it in our department from at least 3000 BCE or even earlier to the fall of the western Roman Empire. Who came up with that, I’m not quite sure. But I would want to bring that question to the table. I’d also want to know what we mean by ‘ancient studies’; so, at the moment, we mainly have ancient Mediterranean studies, ancient Western Asia and North Africa happening. So, I’d want to get the geography right and I’d want to have African Studies at the table; that’s not really a thing in Africa, funnily enough. I would like to get linguists at the table. I would also want to retain the perspective of those who study ancient Western Asia and North Africa. I’d want them at the table. Ten I would want classicists as well, particularly because of actually how young the infuence of the Graeco-Roman world is in our context. I say young because it comes to us via Europe; even though it takes on a life of its own once it reaches our shores, it still comes to us only in the 17th century. So, it is relevant, but I think that sort of broad spectrum would be helpful. Also, I don’t want my colleagues out of jobs. GP: So– GP: So– AD: See, that’s my problem with canon because where we are now as Ancient Studies in Africa – and by ‘ancient studies’ right now I’m referring to what we have at my institution as Ancient Studies. I think if I were to remodel it, to rethink it or to think of what would be relevant in terms of a possible canon, it stretches so widely and so yes – it’s just so broad that I’m not entirely certain whether we’d want a canon or whether we just want a taste of everything. Even as it stands, the reason I don’t have, perhaps, a massive amount of reading in Graeco-Roman literature is that at the same time I was being trained in that, I was also reading ancient Western Asian wisdom literature and that kind of thing. GP: So, you’re saying the depth and breadth are, in practice, antithetical, but ideally, they should not be? AD: Yes. Tat’s exactly what I’m trying to express. I mean, in any discipline, besides medicine, the ideal of depth of knowledge – I think we specialise 181 (U)MZANTSI CLASSICS so early that it’s not really possible to go deep in equal measure; eventually you’ll have to choose something to investigate further. But ideally, I think we’d want people exposed to more, so the breadth there is important. GP: Could you imagine an anthology of key texts? Because you’ve taught many of them. You’ve read many more than you’ve taught. But if you were to think of what you thought are – if you want to think about the ideal student that read everything she was supposed to and was open to everything equally and came with an inquiring mind and no prejudice, what would you want that student to read? How would you go about compiling that reader? I only suggest this as a kind of project that could instantiate, could give form to the challenges, the ideals you’ve expressed. What would your ideal sourcebook look like? AD: I actually have very little idea. I think also because I come from a background where literature is so important, I’m really tempted to go with texts in translation, be they Te Conversation between a Man and His Ba or Te Patient Suferer of Babylonian literature or Antigone or whatever, but that gives primacy to a very specifc type of reading. GP: So– Are we reading – can we read, I don’t know, rock art? But how would you unify something like that? How would you do these texts – and I use ‘texts’ loosely – justice in a course reader like that? We already struggle to do that at the moment. GP: Te reason I’m asking you is purely selfsh: I’ve faced that exact challenge having signed up to teach exactly such a course before knowing what it should contain. So, yes. I’ve come up with a list of the best texts; the most accessible, productive, interesting texts that I can think of but not yet knowing how hard they will be to scafold – to contextualise. Anyway, I simply say that to indicate that the question you raise is one that I’m grappling with right now. And I don’t think there are pre-cut answers – I don’t think there are predetermined answers … just like I think everyone should be reinventing the canon for themselves, creating their own personalised canon that gives enough to discuss with other people, but is not prescriptive. AD: I do appreciate that idea because I think that’s been my experience of my students. Tey seem to be educated in that way, whereas I was right on the tail-end of an era where for English studies you had to read Chaucer, 182 CONVERSATION WITH AMY DANIELS you had to read Shakespeare, you had to do, I don’t know, some modern American poetry, and a lot of British literature and maybe some JM Coetzee. I still had to read Goethe and Schiller in my undergrad straight out of matric. So, I just see the way my students are able to engage across disciplines with very specifc questions and I think that has to do with context. I mean, if you spoke to a student at a diferent university, they’d be engaging with diferent questions quite possibly. But yes. I quite like this idea of reinventing the canon for yourself or for your discipline or for your little island within your discipline, so that you and your student and whoever are able to converse across boundaries or disciplinary boundaries, as it were. But also actually be comfortable with the discomfort that comes from being in a space where you know a little bit about something but maybe not as much as the person next to you; where you are cultivating curiosity and a skill set that helps you to deal with this new canon of yours or whatever it is – this new set of texts – however they may or may not be unifed. GP: So, is it possible to see a text or a text equivalent as an invitation? As a happy provocation? GP: So, is it possible to see a text or a text equivalent as an invitation? As a happy provocation? AD: Absolutely. And not even always happy. It is always an invitation to think. It is always an invitation to reconsider yourself because ultimately, we bring our thoughts and selves to texts and text equivalents. I’m just going to say ‘text’ in the Derrida sense of texts: you’re a text; I’m a text; everything’s a text. So, yes. It is an invitation to think. It’s an invitation possibly to change. And a text itself, you could see it as a happy provocation; so if you’re excited about that change. But ofen the process of change or the process of thought is a painful one. So, it can be a troubling one. And I think the key is to become ok with being uncomfortable and being troubled, being a little bit out of your depth which is actually why I’ve been teaching what I’ve been teaching for so long. I don’t always see that lightbulb moment in students, but if you’re teaching something that is so far removed from their experience and their thought worlds, they are ofen troubled or confused or may feel alienated from the material that you’re approaching or that you’re presenting to them – presenting them with. And my goal is always to have that experience become something that is ok. Something that could act as a bridge to new understanding or to a whole new world. I suppose what I’m thinking about here is: so, I’ve been looking a lot at my teaching 183 (U)MZANTSI CLASSICS philosophy today, that’s been my project for the day – it’s actually my project for the week. And I looked over my notes from our Oxford conference where I was trying to think about whether a pedagogy of discomfort could lead in some way – because I was just noticing this discomfort amongst students and even more so since I’ve started teaching grammar; they’re really uncomfortable with grammar in the frst year – how that could lead to approaching other things, other subjects, other disciplines with what a Stellenbosch scholar has called ‘critical hope’, building on Freire; critical hope as opposed to naïve hope. So, always being aware that there is hope and, within the realm of understanding, hope of new understanding; hope of building bridges, and so forth. GP: So, for example, much of the debate around monuments involves discomfort, people taking ofence. GP: So, is it possible to see a text or a text equivalent as an invitation? As a happy provocation? In our university, one week afer the next there’s a crisis created by somebody taking ofence, most recently, to somebody that studies hip-hop music using the n-word in a quotation, in a highly contextualised quotation in a class. So, is discomfort the same as taking ofence? Or is there any connection? Is the common denominator the alienation that many students feel coming to a university that looks so much unlike them? GP: So, for example, much of the debate around monuments involves discomfort, people taking ofence. In our university, one week afer the next there’s a crisis created by somebody taking ofence, most recently, to somebody that studies hip-hop music using the n-word in a quotation, in a highly contextualised quotation in a class. So, is discomfort the same as taking ofence? Or is there any connection? Is the common denominator the alienation that many students feel coming to a university that looks so much unlike them? AD: I don’t think they’re entirely disconnected. But for me, being able to engage in a pedagogy of discomfort needs to happen – or to use discomfort productively there needs to be trust frst. So, Christo van der Merwe [Professor Emeritus of the Ancient Studies Department at Stellenbosch University] ofen says: ‘Amy, teaching is about trust. Te students need to trust that you’re never going to test them on anything you haven’t done in class and they need to understand that you trust that they’re not going to cheat. Tey need to trust that you have their best interests at heart.’ So all of it is built on this trust that one is able to build in a lecture setting or in a class setting. And I think that for me is where the discomfort comes in. If there is no trust frst, then discomfort will very quickly lead, I think, to ofence. And ofen – so, I’ve been doing a lot of work on why I’ve been experiencing certain colourised and race discussions as emotionally triggering, triggering to my anxiety, and so forth, and I think it has a lot to do with this trust issue, now that I’m sitting here. Can I trust that what is being said is coming from a place of goodwill and not malice? Now on AD: I don’t think they’re entirely disconnected. CONVERSATION WITH AMY DANIELS the example you mention, I’m a little taken aback by it being within such a highly contextualised situation or context, where I’m assuming there is this trust, in a discussion of a genre that uses the n-word in a way that is trying to, as I understand it, reclaim it. So, I don’t know. It’s a difcult one. I have no idea. As for the monuments: you know, obviously redress is necessary. I think when all of this frst started with Rhodes Must Fall, that was quite a sharp wake-up call for me. As someone who had assimilated a lot of the dominant cultures in these spaces that these discussions were taking place in, from going to the – when was it? – Wednesday night sokkie [dance] in the Neelsie to embracing French existentialism with everything I had in me without even questioning why I was learning that at the southern tip of Africa; being presented with, as I said earlier, these ideas about aesthetics and how the Western ideal is the ideal, I sort of internalised all of that. So, I don’t know if I’m the right person to even comment on monuments and their place. GP: Ok. As we wind up, can I come back – if you will – to Augustine and, I don’t know the nature of your relationship with the Confessions at this point, but if you had any confessions to make of your own [laughter] inspired by Augustine? So, that’s just an invitation to talk about that side of things if you so wish. GP: Ok. As we wind up, can I come back – if you will – to Augustine and, I don’t know the nature of your relationship with the Confessions at this point, but if you had any confessions to make of your own [laughter] inspired by Augustine? So, that’s just an invitation to talk about that side of things if you so wish. AD: I’m not sure about confessions. I think I’ve sort of already started ‘spilling my guts’, in a sense, where I’ve just been unable to fgure out for myself the relevance and value of what I’m engaged in for my context, not as it stands at the moment. Yes, I think I’m just grappling with the moral purposefulness of classics research. GP: Tat seems to me a great place to end. GP: So, is it possible to see a text or a text equivalent as an invitation? As a happy provocation? But for me, being able to engage in a pedagogy of discomfort needs to happen – or to use discomfort productively there needs to be trust frst. So, Christo van der Merwe [Professor Emeritus of the Ancient Studies Department at Stellenbosch University] ofen says: ‘Amy, teaching is about trust. Te students need to trust that you’re never going to test them on anything you haven’t done in class and they need to understand that you trust that they’re not going to cheat. Tey need to trust that you have their best interests at heart.’ So all of it is built on this trust that one is able to build in a lecture setting or in a class setting. And I think that for me is where the discomfort comes in. If there is no trust frst, then discomfort will very quickly lead, I think, to ofence. And ofen – so, I’ve been doing a lot of work on why I’ve been experiencing certain colourised and race discussions as emotionally triggering, triggering to my anxiety, and so forth, and I think it has a lot to do with this trust issue, now that I’m sitting here. Can I trust that what is being said is coming from a place of goodwill and not malice? Now on 184 CONVERSATION WITH AMY DANIELS It’s easier to imagine that what I’m doing is morally defensible when I’m referring to my teaching because there it’s about the people, and again it was the people in this discipline at my institution who drew me to it in the frst place: it was a person at my high school who drew me to Latin; it was just people who were interested in me and in what I was thinking that drew me to this place. And so, I want to pay that forward. Tey were good ancestors to me in classics; I want to be a good ancestor to those who come afer me in whatever discipline. 185
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Fertilizer optimization in rice by eliminating hidden nutrient deficiencies
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py, ssion from al, zhuc, zhang Study on the Effect of zinc asing rice yield. . Liaoning -18 y of the rice 23:241-315. 91. Availability to rice plants 3700;1/1-1/6. 94. Role of wth. Agro Annu (B & A) 1:243- Devi Vs, 1997. The effect on on growth ants. Ann. Plant 28. ake mode and on of silica. Sci trient and Crop nd o11-farm MaiF, Takahashi of rice straw s Soilsci. Plant Y, Nara arian A silicon in pla Rev. Plant Ph 262 Rani YA, Narayan Subbaramam of silicon app and yield of r Physi01.11(2) Takahashi E. 1995 physiological Rice Plant 2:5 copy, to ission from nal, zhuc, zhang 1. Study on the . Effect of zinc easing rice yield. ci. Liaoning 3-18 gy of the rice . 23:241-315. 991. Availability n to rice plants tr. 3700;1/1-1/6. 994. Role of owth. Agro Annu !, (B & A) 1:243- A, Devi Vs, P. 1997. The effect ion on growth plants. Ann. Plant -128. ptake mode and ction of silica. Sci . utrient l and Crop and o11-farm copy, to ission from nal, zhuc, zhang 1. Study on the . Effect of zinc easing rice yield. ci. Liaoning 3-18 gy of the rice . 23:241-315. 991. Availability n to rice plants tr. 3700;1/1-1/6. 994. Role of owth. Agro Annu !, (B & A) 1:243- A, Devi Vs, P. 1997. The effect ion on growth plants. Ann. Plant -128. ptake mode and ction of silica. Sci . utrient l and Crop and o11-farm en vers, or to redistribute to lists, requires prior specific pe ht holder This document is provided strictly for your pe h and educational use. tended than did fly ash and rice straw References straw compost. It has higher SI DongCT, LiuZG, Zou CL, Liang W. y fly ash. availability than these two nutrition of ric of pho- sources (Ma and Takahashi and silicon on nd the 1991). Liaoning Agri light, Silicon application in- Nongye Kexu n of creases both grain and straw Ishizuka Y. 1971. Phys plant. Adv. Ag to the yields of rice under both low pecific pe your pe ences ZG, Zou L, Liang W. utrition of ric nd silicon on aoning Agr ongye Kexu 71. Phys ant. Adv. A ng hidde Chemistry, Cente India field experim vers, or to redistribute to lists, requires prior spe t holder This document is provided strictly for and educational use. tended than did fly ash and rice straw Refere traw compost. It has higher SI DongCT, LiuZ CL fly ash. availability than these two nut of pho- sources (Ma and Takahashi and nd the 1991). Lia light, Silicon application in- No n of creases both grain and straw Ishizuka Y. 197 pla to the yields of rice under both low y and high fertility levels. Basic za- slag, fly ash, and rice straw ompost as sources of Si have Rani AY o- shown positive effects on rice se grain and straw yield, with basic slag giving a better per- formance. Therefore, these Si sources, prefer ably basic slag, se can well be used to increase ho" and sustain rice yields while T 97). solving disposal problems g r atin ural C 41003, pa- mina Agricult atore 64 . This by elim cience and sity, Coimba h of rice n rice b ment of Soil Sc ultural Univers yield growt tion i , Departm du Agricu n with y publish, e publis n-comm hemsel o be be compos The o synth more e oupled assimila ink (Ra 994) b on, cou he incre duction hrough argely a antage ng and of bette osyntha Among S perform tilize deficie M. R. Lath Managem IF copy, to mission from nal, locations- ariankorai avur. The ations be- oil series, conomics ertilization matic ap- ared with ecoinmen- as well as r with bal- achieved y evolved atter yield of ent treatments tter ts" ) Relative yield (%) 100 97 91 61 66 75 92 94 110 96 103 101 py, ssion from al, ocations- riankorai vur. The tions be- oil series, onomics rtilization matic ap- ared with coinmen- s well as with bal- achieved evolved tter yield of nt treatments er " ) Relative yield (%) 100 97 91 61 66 75 92 94 110 96 103 101 12 treat- cated four s were O, 200 kg N nd 36 kg P ), and 50 Zn formed reatments optimum ha~', ex- reatment. mendation omparison Treatment Optimum nutrient treatment (ONT ) ONT + Ca ONT + rig ONT - N ONT - p ONT - K ONT + B ONT + Cu ONT + Fe ONT + Mn ONT + MD D (g to significantly lower relative of balance dry matter yield compared through a sy ONT proaCh was co blanket fertilize such as Ca, Mg, S, Cu, Fe, dations (Table Rice yield ignificant influence on dry profits were hig anced fertiliza greenhouse study confirmed by using the n e with that of the able I). But the other nutri- Mn, Mo, and B, did not show atter yield of sorghum. This N, P, K, and Zn were de- ficient in the experimental oil. In the third phase of the Table I. Relative d sorghum Co 27 for d in the greenhouse stu by Copyright Law, I'n which case no reproduction or redistribution is permitted. republish, to post on servers, or to redistribute to lists, requires prior specific p the publisher or copyright holder This document is provided strictly for your pe non-commercial research and educational use. optimal amount of a nutrient quirements and harvested af- farm trials at t was derived using the nutri- ter 3 wk. The relative dry mat- Ramaptiram and ent sorption curves obtained ter yield (relative to the yield villages in Th by plotting the amount of rill- in the ONT) in each treatment soils of these trient extracted (y axis) was calculated. The exclusion longed to the sam against the amount added (x of N, P, K, and Zn was found the Kalathur. Th by Copyright Law, I'n which case no reproduction or redistribution is permitted. republish, to post on servers, or to redistribute to lists, requires prior specific p the publisher or copyright holder This document is provided strictly for your p non-commercial research and educational use. optimal amount of a nutrient quirements and harvested af- farm trials at t was derived using the nutri- ter 3 wk. The relative dry mat- Ramaptiram an ent sorption curves obtained ter yield (relative to the yield villages in Th by plotting the amount of rill- in the ONT) in each treatment soils of these trient extracted (y axis) was calculated. The exclusion longed to the sam against the amount added (x of N, P, K, and Zn was found the Kalathur. Th f f in the field, were replic e treatment 0 (N, , and 2,24 (P, , an 0,20,35 (K, NZP2K2 + Z T. All the tr ved the of 6.5 kg Zn ONT-Zrt tr nket recomm cluded for co axis) and extrapolating to the point corresponding to three d tion studies indicated that, ents, Zn were found to limit yield a s level of eacl\ of them extracted g times the critical level on the y axis. The results of incuba- (Ta among the nutrients, P, K, and M in the experimental soil as the ma at zero level of incubation that was less than three times that f of the critical level: 10.0 ing so 015en P kg~' (Anonymous 1980), 0.18 meq NH, OAc-K 100 g~' (Anonymous 1980) and 1.20 ing DTPA-Zn kg~' ti (Savithri 1978). Since N was generally found to be limiting h in all soils, it was also ill- ha cluded in the optimum nutri- kg ent treatment (ONT). It con- sisted of those nutrients th were deficient in the soil as a indicated by the incubation study: N = 50 ing kg~', P = 12 ing kg~', K = 0,045 meq 100 g~
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The Role of the Organization Structure in the Diffusion of Innovations
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RESEARCH ARTICLE Introduction Copyright: © 2015 Sáenz-Royo 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. Innovation feeds social and economic progress and drives progress through enhanced efficien- cy [1–3], costs [4–6], and the quality of the products [7]. Innovation includes ideas, values, goods, services, technologies, processes, etc. that emerge and are disseminated both in markets, organizations and within social systems in general. Whether an innovation is effective or not depends on whether it can be assimilated by the socio-economic system it is designed for. This will be more or less complicated, depending on the setting in which it is disseminated. In mar- kets, decisions to adopt the innovation and to commit the resources required to gain access to the product and use it for its intended purpose are normally taken by one and the same person. In organizations, on the other hand, the specialization of duties and distribution of authority and decision-making among various jobs mean that each stage in the innovation assimilation process may be carried out by different people, and this demands a greater level of coordina- tion. The dissemination of innovation in social complex systems can therefore be described as a process of a succession of decisions regarding adoption forming groups of people, or markets, else it can be described as a process of internal diffusion throughout the implementation stage in organisations [8]. The Role of the Organization Structure in the Diffusion of Innovations Carlos Sáenz-Royo1, Carlos Gracia-Lázaro2*, Yamir Moreno2,3,4 1 Centro Universitario de la Defensa. 50090 Zaragoza, Spain, 2 Institute for Biocomputation and Physics of Complex Systems. University of Zaragoza, 50009 Zaragoza, Spain, 3 Departamento de Física Teórica. University of Zaragoza, 50009 Zaragoza, Spain, 4 Complex Networks and Systems Lagrange Lab, Institute for Scientific Interchange, Turin 10126, Italy * cgracia@bifi.es * cgracia@bifi.es a1111 OPEN ACCESS Citation: Sáenz-Royo C, Gracia-Lázaro C, Moreno Y (2015) The Role of the Organization Structure in the Diffusion of Innovations. PLoS ONE 10(5): e0126076. doi:10.1371/journal.pone.0126076 Academic Editor: Zhen Wang, Hong Kong Baptist University, CHINA Received: February 21, 2015 Accepted: March 28, 2015 Published: May 15, 2015 Received: February 21, 2015 Accepted: March 28, 2015 Published: May 15, 2015 Abstract Diffusion and adoption of innovations is a topic of increasing interest in economics, market research, and sociology. In this paper we investigate, through an agent based model, the dynamics of adoption of innovative proposals in different kinds of structures. We show that community structure plays an important role on the innovation diffusion, so that proposals are more likely to be accepted in homogeneous organizations. In addition, we show that the learning process of innovative technologies enhances their diffusion, thus resulting in an im- portant ingredient when heterogeneous networks are considered. We also show that social pressure blocks the adoption process whatever the structure of the organization. These re- sults may help to understand how different factors influence the diffusion and acceptance of innovative proposals in different communities and organizations. The Role of the Organization Structure in the Diffusion of Innovations This paper examines the assimilation of innovation in social systems as a decentralized pro- cess. In such processes, early stage is key to the assimilation of innovation. At this early stage, either members unanimously accept the innovation or they continue with the status quo. When adoption and implementation are produced in separate stages a previous consensus in favour of innovation will simplify the subsequent implementation stage since the people who will need to be involved at a later date are the same ones that previously came out in favour of adopting it. The use of Agent Based Models (ABM) as a methodological tool for the study of social phenomena provides useful insights about the fundamental causal mechanisms at work in social systems [9–11]. The large-scale macroscopic effects of simple forms of microscopic so- cial interaction are very often surprising and generally hard to anticipate, such as the diffusion of information, emergence of norms, coordination of conventions, or participation in collective action. This paper examines the assimilation of innovation in social systems as a decentralized pro- cess. In such processes, early stage is key to the assimilation of innovation. At this early stage, either members unanimously accept the innovation or they continue with the status quo. Competing Interests: The authors have declared that no competing interests exist. The traditional literature on the adoption and dissemination of innovation has been heavily influenced by the epidemiological models for the contagion of diseases and pathologies in human groups, adapted to the social environment (see, e.g., [12], extensions like [13] and the subsequent introduction of the model in complex Networks [14–18]). Our work differs from the models based on epidemiological studies in several respects. First of all, people in the mar- ket o in the organization take binary decisions, positioning themselves either for or against an innovation, with relevant information reaching them from within their group of influence. Their stance is not firm until a favorable consensus is obtained. Meanwhile, the same person can change their position once or various times, i.e., go from being in favor of the innovation to being against it and vice versa while there is someone in their environment that disagrees. Data Availability Statement: All relevant data are within the paper. Data Availability Statement: All relevant data are within the paper. Funding: The authors acknowledge financial support from EU FET project MULTIPLEX (grant 317614), from Spanish Ministerio de Economía y Competitividad (research projects FIS2012-38266- C02-01 and ECO2013-48496-C4-3-R), from Diputación General de Aragón (grupos CREVALOR, FENOL) and from the European Social Fund (CREVALOR). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. 1 / 13 PLOS ONE | DOI:10.1371/journal.pone.0126076 May 15, 2015 PLOS ONE | DOI:10.1371/journal.pone.0126076 May 15, 2015 The model Let I = {1, 2, . . ., N} be the set of nodes in a social system structure: each node represents an in- dividual or a team of several individuals under a common working method (whether subject to discipline or reached by consensus). For each node j 2 I there is, at least, another node i 2 I,i 6¼ j to which it is connected; the graph of relationships is specified by a (N × N) symmetric adja- cency matrix A defined as Aij = 1 whenever i is directly connected to j in the social structure, or Aij = 0 otherwise. We define the influence set of node j as the collection of nodes that are direct- ly connected to it: Hj = {i 2 IjAij = 1}. In the same way, the degree kj of node j is the number of nodes directly connected to it: kj ¼ cardðHjÞ ¼ PN i¼0 Aij. Each node i 2 I has knowledge of each working method. Let Ri t be the performance of node i at time t provided by the conventional method (status quo) and, in the same way, let Ri t be the node i’s performance provided by the new method (innovation). In addition, each node i is characterized by a variable si (the strategy) that takes the value si t ¼ 1 if the node i supports the innovation at time t, or si t ¼ 0 otherwise. Let ai t be the number of nodes connected to i that sup- port the innovation at time t, and let bi t be the number of i’s neighbors that support the status quo: ai t ¼ X N j¼1 sj tAij: bi t ¼ X N j¼1 j1  sj tjAij ¼ ki  ai t ð1Þ ð1Þ Given two nodes (i, j) mutually connected, i having adopted the innovation and j having not (i.e., si t ¼ 1; s j t ¼ 0), we establish that they can interact with each other by exchanging knowl- edge (and exchanging methods) only if the performance difference between the two nodes is contained in an interval : ð2Þ j Ri t  R j t j<  ; ð2Þ resulting in mutual learning. When condition 2 is satisfied, we define the link (i, j) as feasible. This is the case for 2 / 13 PLOS ONE | DOI:10.1371/journal.pone.0126076 May 15, 2015 The Role of the Organization Structure in the Diffusion of Innovations practically the whole range of possible differences in the economic value attributed to innova- tion with respect to the status quo, and for both large and small-sized social structures. The dif- ferences are greater when the relative performance of the innovation on the status quo is not very large. Nevertheless, despite this higher probability of success regarding other topologies, hierarchy is the structure that requires more time to reach a favorable consensus. As is well known, time has a cost (referred to in economy as opportunity cost), established by the profit- ability of its alternative use. On the other hand, the hierarchical appears as a very stable struc- ture, showing a lower sensitivity to changes in the parameters of the model. ER and BA structures give a very low probability of adoption for small innovation performance with re- spect to the status quo and are especially sensitive to changes in the rate of learning and social pressure. However, they present an remarkable advantage in the time needed to achieve favor- able consensus as compared with a hierarchical graph. structures give a very low probability of adoption for small innovation performance with re- spect to the status quo and are especially sensitive to changes in the rate of learning and social pressure. However, they present an remarkable advantage in the time needed to achieve favor- able consensus as compared with a hierarchical graph. In the likelihood of opting for one alternative or another, on the one hand we consider the posi- tions in favor or against the innovation expressed by other people within the radius of influence of the person concerned (the influence is weighted according to the social pressure exercised by the group over the individual according to the culture that is accepted by the members); and on the other, individual value attributed to such innovation. The model presented here allows for interaction between individuals connected to learn at a rate m, which modifies the performance attributed to the status quo and innovation. This single interaction take place if the difference between partners’ performances is less than a threshold . The individual character of the per- formance recognizes the existence of diversity in skills and knowledge of the different people in the group. The skill and knowledge determine the performance of each individual with respect to the two alternatives, influencing its decision. In the more conventional models for dissemi- nating innovation, however, adoption boils down to making decisions concerning use or pur- chase: those who adopt an innovation by, for instance, purchasing a new product for the first time, are then classified as adopters forevermore and only admit peer pressure conducive to the adoption and never in favor of the status quo. Secondly, in this paper the structure of the network articulating the internal workings of the members of the organization or the market has a very important role. The network structure implies that the likelihood of a person being in favor of an innovation will vary depending on their position in the network. Furthermore, the network structure chosen will determine the final outcome of the initiation stage measured by the greater or lesser probability of the consen- sus being in favor of the innovation, and by the time needed to reach such a consensus. In this respect, one relevant factor in this study is the comparison of probabilities and times needed for consensus under various organizational o market structures, including Hierarchical graphs, lattice graphs, Erdös-Rényi (E.R.) networks [19], Scale-free networks [20], Stars and Complete graphs. The results of the simulations show that the hierarchy is the formal structure that gives greater probabilities of reaching a consensus in favor of innovation. PLOS ONE | DOI:10.1371/journal.pone.0126076 May 15, 2015 The model This threshold  introduces a condition for mutual benefit: while high values of  allow altruism (i.e., people with high performance help people with very low performance without expecting anything in return), low values of  only allow win-win relationships. 3 / 13 PLOS ONE | DOI:10.1371/journal.pone.0126076 May 15, 2015 The Role of the Organization Structure in the Diffusion of Innovations Initial conditions The system begins with a number ρ of randomly distributed nodes (the initial adopters) that adopt the innovation from the first moment t = 0, while the other nodes are supporters of the status quo. The performance provided by the status quo is randomly distributed between the nodes according to a gaussian distribution centered in R = 1 with variance σ. Only the initial adopters obtain more performance from the new method, for these nodes the performance provided by the innovation is distributed according to a gaussian centered in R with variance σ, while for the rest of the nodes (i.e., the supporters of the status quo) the efficiency of both methods is the same. Then, the quotient R/R represents the innovation performance value (i.e., the innovation technical superiority). Explicitly, at t = 0, each node i 2 I, with probability ρ/N, adopts the innovation (si t¼0 ¼ 1) and its performances are randomly assigned according to the following distributions: pðRi t¼0 ¼ zÞ ¼ 1 s ffiffiffiffiffiffi 2p p e ðz1Þ2 2s2 ; pðRi t¼0 ¼ zÞ ¼ 1 s ffiffiffiffiffiffi 2p p e ðzRÞ2 2s2 : ð3Þ ð3Þ Otherwise, that is, with probability 1−(ρ/N), node i supports the status quo (si t¼0 ¼ 0) and its performances are given by: pðRi t¼0 ¼ zÞ ¼ 1 s ffiffiffiffiffiffi 2p p e ðz1Þ2 2s2 ; Ri t¼0 ¼ Ri t¼0 : ð4Þ ð4Þ PLOS ONE | DOI:10.1371/journal.pone.0126076 May 15, 2015 Dynamics At each dynamical step, each node chooses (if available) a random feasible neighbor with oppo- site strategy, such that a given node interacts only once at most per step. Then, a time step con- sists of at most N/2 interactions, and each interaction takes place between two nodes (i, j) with different strategies (without loss of generality, si t ¼ 1; s j t ¼ 0). During the process of learning each node shares information on the adopted method, so that a node may improve its perfor- mance on the non-adopted method, provided that the opposite node has greater performance on it. The increment is proportional to the difference in performances: R j t ¼ R j t þ mðRi t  R j t Þ HðRi t  R j t Þ ; Ri t ¼ Ri t þ mðR j t  Ri tÞ HðR j t  Ri tÞ ; ð5Þ ð5Þ where m is a parameter that models the learning ratio by modulating the performance incre- ment resulting from the information exchange: the higher m, the greater the increment in per- formance. H is the Heaviside function that takes the value H(a) = 1 when a > 0, and 0 otherwise. After the learning process, node j imitates the strategy of i (i.e., j adopts the innovation) with probability: P j i t ¼ ð1 þ db j tÞR j t ð1 þ dai tÞRi t þ ð1 þ db j tÞR j t ð6Þ ð6Þ where δ is a parameter that represents the social pressure: the higher the value of δ, the greater the influence exercised by the neighbors. Otherwise, node i imitates j’s strategy, which implies PLOS ONE | DOI:10.1371/journal.pone.0126076 May 15, 2015 4 / 13 The Role of the Organization Structure in the Diffusion of Innovations that the probability for node i to adopt the status quo is given by: that the probability for node i to adopt the status quo is given by: P i j t ¼ 1  P j i t ¼ ð1 þ dai tÞRi t ð1 þ db j tÞR j t þ ð1 þ dai tÞRi t ð7Þ ð7Þ Topologies • Complete graphs are networks in which every pair of nodes are directly connected by a link. The degree of any node is ki = N−1 • Complete graphs are networks in which every pair of nodes are directly connected by a link. The degree of any node is ki = N−1 Topologies In order to model different types of organizations and social system’s structures, we consider several network topologies that represent the nodes and connections of the system being simu- lated. In particular, we have dealt with the following types of graphs: • Hierarchical graphs are networks in which any two nodes are connected by exactly one sim- ple path. In this work, we will use regular hierarchical graphs, in which any intermediate node has a higher-level neighbor (its upper-neighbor) and M lower-level neighbors (its lower- neighbors). Therefore, the degree of an intermediate node i is ki = M+1. In addition, leaf nodes do not have lower-neighbors but only upper-neighbor (kl = 1), and one node (the top- node) does not have upper-neighbor but only lower-neighbors (kb = M). The number of lower-neighbors M is called degree of branching. • In lattice graphs the nodes are disposed in the vertex of a tiling and connected to the k closest nodes. We will use square lattices with constant degree ki = 4, 8 i 2 I. The characteristic dis- tance between two nodes is proportional to the network size. • Erdös-Rényi (ER) networks are random graphs in which the probability p for any pair of nodes to be connected is uniform. They are characterized by a binomial degree distribution Pðki ¼ kÞ ¼ N  1 k ! pkð1  pÞN1k, which tends to a Poisson distribution for large networks Pðki ¼ kÞ ! ðNpÞkeNp k! . [19] • Erdös-Rényi (ER) networks are random graphs in which the probability p for any pair of nodes to be connected is uniform. They are characterized by a binomial degree distribution Pðki ¼ kÞ ¼ N  1 k ! pkð1  pÞN1k, which tends to a Poisson distribution for large networks Pðki ¼ kÞ ! ðNpÞkeNp k! . [19] • Scale-free networks are graphs whose degree distribution follows a power law P(ki = k) = ck−γ, resulting in a small number of highly connected (i.e., very influential) nodes: the hubs. In particular, we will use the Barabási-Albert (hereafter, BA) algorithm [20] to generate the networks. • Stars are particular cases of hierarchical graphs without intermediate nodes. They consist of a central node (the hub, khub = N−1) connected to all other nodes, which have only that con- nection (ki = 1). Results According to section Initial conditions, ρ initial adopters were randomly distributed among a population of N−ρ supporters of the status quo. After that, the dynamics of the adoption pro- cess was run. The simulations were stopped when the number of active nodes (i.e., agents with non-zero probability of changing opinion) vanished. The results shown below were obtained by averaging over a large number (typically 104) of different initial conditions and network realizations. Global consensus is finally reached for all the topologies studied, that is, for each simulation, the innovation is either accepted or rejected by all the agents bringing the system to a frozen state. Consequently, for a given set of parameter values, the fraction of realizations α in which the innovative method has been finally adopted represents the probability of adoption for the 5 / 13 PLOS ONE | DOI:10.1371/journal.pone.0126076 May 15, 2015 The Role of the Organization Structure in the Diffusion of Innovations Fig 1. Acceptance probability versus the performance of the innovation. Fraction of realizations in which the innovative method has been finally adopted as a function of the initial performance of the new method R* for six different types of networks: hierarchical, lattice, Erdös-Rényi, Barabási-Albert, star and complete. Left panels correspond to systems of N = 103 agents while the right panel to N = 104. Upper panels correspond to the case of a number of initial adopters ρ = 10, while the bottom panel to ρ = 100. Other values are R = 1, δ = 0.5, m = 0.5,   R*, σ = N−1. Each point is averaged over 104 network realizations. doi:10.1371/journal.pone.0126076.g001 Fig 1. Acceptance probability versus the performance of the innovation. Fraction of realizations in which the innovative method has been finally Fig 1. Acceptance probability versus the performance of the innovation. Fraction of realizations in which the innovative method has been finally adopted as a function of the initial performance of the new method R* for six different types of networks: hierarchical, lattice, Erdös-Rényi, Barabási-Albert, star and complete. Left panels correspond to systems of N = 103 agents while the right panel to N = 104. Upper panels correspond to the case of a number of initial adopters ρ = 10, while the bottom panel to ρ = 100. Other values are R = 1, δ = 0.5, m = 0.5,   R*, σ = N−1. PLOS ONE | DOI:10.1371/journal.pone.0126076 May 15, 2015 The Role of the Organization Structure in the Diffusion of Innovations Fig 2. Consensus time versus versus the performance of the innovation. Number of steps needed to reach a consensus (either to accept or reject the innovation) as a function of the the initial new method’s performance for the six different topologies considered. By comparing this panels from the curves shown in Fig 1, it can be observed that the values of R* corresponding to the maximum consensus times match the values of R* for which P(acceptance) * 0.5. Other values are N = 1000, R = 1, ρ = 10, δ = 0.5, m = 0.5,   R*, σ = N−1. Each point is averaged over 104 network realizations. Fig 2. Consensus time versus versus the performance of the innovation. Number of steps needed to reach a consensus (either to accept or reject the innovation) as a function of the the initial new method’s performance for the six different topologies considered. By comparing this panels from the curves shown in Fig 1, it can be observed that the values of R* corresponding to the maximum consensus times match the values of R* for which P(acceptance) * 0.5. Other values are N = 1000, R = 1, ρ = 10, δ = 0.5, m = 0.5,   R*, σ = N−1. Each point is averaged over 104 network realizations. doi:10.1371/journal.pone.0126076.g002 (hierarchical and lattice) show higher acceptance probabilities than the complex networks (ER and BA) and, in turn, ER networks show higher success probabilities than BA graphs. In con- clusion, for a given mean connectivity, increasing degree heterogeneity decreases the likelihood that the proposal will be accepted. Furthermore, comparing the two top panels, it can be ob- served that the size of the system does not have a significant influence on the acceptance proba- bility. Nevertheless, in heterogeneous and star graphs, large system sizes show lower ratios of acceptance. Regarding the effect of the seed size, as can be seen by comparing the curves in the bottom panel which those shown in the up-left panel, the increase of the number of initial adopters has a positive influence on the success probability. Comparing the three panels, we see that it is the amount of initials adopters ρ, not its fraction, that determines the success prob- ability. PLOS ONE | DOI:10.1371/journal.pone.0126076 May 15, 2015 Results Each point is averaged over 104 network realizations. doi:10.1371/journal.pone.0126076.g001 innovative method or technology. In Fig 1, we plot this fraction α versus the average initial per- formance of the new method R, where each point represents the numerical result averaged over 104 different network realizations. Without loss of generality, the average initial perfor- mance of the status quo was fixed to R = 1, therefore, R/R = R, which means that R repre- sents the improvement in performance of the new method. Different symbols and curves refer to the different structures studied: hierarchical, lattice, ER, BA, star and complete. Other pa- rameters are indicates in the figure. Firstly, to study the influence of the degree distribution on the dynamics, let us focus our at- tention in the first four topologies: hierarchical, lattice, ER and BA, whose networks realiza- tions were constructed with the same mean connectivity hki * 4. As shown, the regular graphs 6 / 13 PLOS ONE | DOI:10.1371/journal.pone.0126076 May 15, 2015 The Role of the Organization Structure in the Diffusion of Innovations Fig 3. Acceptance probability versus learning ratio and social pressure. Fraction of realizations in which the innovation has been adopted P (acceptance) versus the learning ratio m (left panel) and versus the social pressure parameter δ (right panel) for the six different types of networks studied. The value of R* is chosen so that P(acceptance) * 0.5 for m = 0.5, being R* = 1.55, 2.2, 3.3, 4.5, 35, 155 for the hierarchical, lattice, Erdös-Rényi, Barabási- Albert, star and complete graphs respectively. Other values are N = 1000, R = 1, ρ = 10, δ = 0.5,   R*, σ = N−1. Each point is averaged over 104 different realizations. See the main text for further details. Fig 3. Acceptance probability versus learning ratio and social pressure. Fraction of realizations in which the innovation has been adopted P (acceptance) versus the learning ratio m (left panel) and versus the social pressure parameter δ (right panel) for the six different types of networks studied. The value of R* is chosen so that P(acceptance) * 0.5 for m = 0.5, being R* = 1.55, 2.2, 3.3, 4.5, 35, 155 for the hierarchical, lattice, Erdös-Rényi, Barabási- Albert, star and complete graphs respectively. Other values are N = 1000, R = 1, ρ = 10, δ = 0.5,   R*, σ = N−1. Each point is averaged over 104 different realizations. See the main text for further details. doi:10.1371/journal.pone.0126076.g003 the system shared the same opinion about the innovation. For each structure of the networks of contacts we see how a peak in the transition time is revealed, signaling the existence of a phase transition between the two different final states. In fact, by comparing these results with those shown in the upper-left panel of Fig 1, it can be observed that the values of the innovation performance R corresponding to the maximum consensus time (the peaks of Fig 2) match the values of R for which the acceptance probability has intermediate values (transitions in Fig 1). PLOS ONE | DOI:10.1371/journal.pone.0126076 May 15, 2015 This is due to the fact that achieving a critical mass of adopters in early stages is key to the success of the innovation. This result has important implications because it indicates that the efforts required to spread an innovation are independent of the size of the system. (hierarchical and lattice) show higher acceptance probabilities than the complex networks (ER and BA) and, in turn, ER networks show higher success probabilities than BA graphs. In con- clusion, for a given mean connectivity, increasing degree heterogeneity decreases the likelihood that the proposal will be accepted. Furthermore, comparing the two top panels, it can be ob- served that the size of the system does not have a significant influence on the acceptance proba- bility. Nevertheless, in heterogeneous and star graphs, large system sizes show lower ratios of acceptance. Regarding the effect of the seed size, as can be seen by comparing the curves in the bottom panel which those shown in the up-left panel, the increase of the number of initial adopters has a positive influence on the success probability. Comparing the three panels, we see that it is the amount of initials adopters ρ, not its fraction, that determines the success prob- ability. This is due to the fact that achieving a critical mass of adopters in early stages is key to the success of the innovation. This result has important implications because it indicates that the efforts required to spread an innovation are independent of the size of the system. Each panel of Fig 2 represents, for each of the different topologies considered, the time needed to reach global consensus, that is, the number of dynamical steps until all the agents of 7 / 13 PLOS ONE | DOI:10.1371/journal.pone.0126076 May 15, 2015 This is because, while for low values of R the proposal is rejected in the initial phases and for high values of R it is accepted in most interactions, for values of R close to the transition the probability for individuals to accept the proposal takes intermediate values, causing fluctua- tions which in turn slow down the convergence to the final state. Regarding the effect of the learning process on the opinion dynamics, the left panel of Fig 3 represents the acceptance probability P(acceptance) versus the learning ratio parameter m for the six different topologies considered. According to the results showed in Fig 1, the initial performance of the innovation R for each topology is chosen so that P(acceptance) * 0.5 for m = 0.5, being R = 1.55, 2.2, 3.3, 4.5, 35, 155 for the hierarchical, lattice, Erdös-Rényi, Barabási-Albert, star and complete graphs respectively. As shown, the more information ex- change, the greater the likelihood of acceptance of the innovation. The first four kinds of net- works (hierarchical, lattice, ER and BA) were made up with the same mean connectivity hki * 4. Among these topologies, regular networks (hierarchical and lattice) show smoother transitions than complex graphs (ER and BA), which means that degree heterogeneity in- creases the sensitivity to the learning process, while regular structures are more robust. Fur- thermore, star structures are less sensitive to the learning ratio. On the other hand, with respect to the influence of social pressure on the dynamics, the right panel of Fig 3 represents, for the 8 / 13 PLOS ONE | DOI:10.1371/journal.pone.0126076 May 15, 2015 The Role of the Organization Structure in the Diffusion of Innovations Fig 4. Acceptance probability versus the interaction threshold. Fraction of realizations in which the innovation has been adopted as a function of the performance difference threshold  beyond which two nodes do not interact with each other by exchanging knowledge and exchanging methods, for the six different topologies studied and different values of the initial performance of the innovation R* = 2, 3, 5, 10, 150. Other values are N = 1000, R = 1, ρ = 10, δ = 0.5, m = 0.5, σ = N−1. Each point is averaged over 104 network realizations. doi:10 1371/journal pone 0126076 g004 Fig 4. Acceptance probability versus the interaction threshold. PLOS ONE | DOI:10.1371/journal.pone.0126076 May 15, 2015 Influence of the connectivity on the acceptance probability. Fraction of realizations in which the innovative method has been adopted versus the initial performance of the innovation R* for different values of the mean connectivity hki (left and center panel) and for different values of the degree of branching M (right panel). Left panel corresponds to Barabási-Albert networks, center panel to Erdös-Rényi graphs and right panel to hierarchical structure. Other values are N = 1000, R = 1, ρ = 10, δ = 0.5, m = 0.5,   R*, σ = N−1. Each point is averaged over 104 network realizations. doi:10.1371/journal.pone.0126076.g005 function centered at R−R = R−1, which means that, although individual performances (Ri; R i ) may vary due to the learning process, the threshold  has no influence on the final state provided that the minimum trust principle is satisfied at the initial state. Regarding the effect of connectivity on the opinion dynamics, the left and center panels of Fig 5 show the acceptance probability as a function of the initial performance of the innovative method for different values of the mean connectivity hki. The left panel corresponds to Bara- bási-Albert graphs and the center panel to Erdös-Rényi networks. As shown, increased connec- tivity hinders the diffusion of the innovation, which is a consequence of the fact that social pressure increases with increasing the number of contacts and therefore, in the first states, the probability for an agent to accept the innovation. In the same way, the right panel of Fig 5 stud- ies the influence of the degree of branching M (i.e., the number of lower-neighbors of an inter- mediate node) on the acceptance probability in the hierarchical structures. The curves show the fraction of realizations in which the innovative method has been adopted as a function of the initial new method’s performance R for different values of M. As illustrated in the figure, increasing the degree of branching implies a decrease in the probability of the new method being adopted, as a consequence of the increase in social pressure caused by the increase of contacts. Fraction of realizations in which the innovation has been adopted as a function of the performance difference threshold  beyond which two nodes do not interact with each other by exchanging knowledge and exchanging methods, for the six different topologies studied and different values of the initial performance of the innovation R* = 2, 3, 5, 10, 150. Other values are N = 1000, R = 1, ρ = 10, δ = 0.5, m = 0.5, σ = N−1. Each point is averaged over 104 network realizations. doi:10.1371/journal.pone.0126076.g004 doi:10.1371/journal.pone.0126076.g004 same initial performance values R, the fraction of realizations in which the innovation has been adopted as a function of the social pressure parameter δ for the different social structures considered. As can be observed, social pressure makes it harder for the innovation to spread re- gardless of the structure of the network, which is a consequence of the fact that, according to Eqs 6–7, the imitation probability decreases with increasing social pressure. Let us now focus on the impact of restrictions on interactions between agents. According to formula Eq (2), two agents can interact with each other by exchanging knowledge and methods only if a principle of minimal trust is satisfied, namely, if the absolute value of the performance difference between both agents is lower than a threshold . Fig 4 represents the probability of acceptance of the innovation as a function of the parameter ; each panel plots the results for a given value of the innovation performance value R, while each symbol represents each of the different topologies considered. As can be observed, all the curves are consistent with a step 9 / 13 PLOS ONE | DOI:10.1371/journal.pone.0126076 May 15, 2015 The Role of the Organization Structure in the Diffusion of Innovations Fig 5. Influence of the connectivity on the acceptance probability. Fraction of realizations in which the innovative method has been adopted versus the initial performance of the innovation R* for different values of the mean connectivity hki (left and center panel) and for different values of the degree of branching M (right panel). Left panel corresponds to Barabási-Albert networks, center panel to Erdös-Rényi graphs and right panel to hierarchical structure. Other values are N = 1000, R = 1, ρ = 10, δ = 0.5, m = 0.5,   R*, σ = N−1. Each point is averaged over 104 network realizations. Fig 5. Discussion Although the main aim of this work is to study the dynamics of the diffusion of innovations, this paper can be useful for understanding the adoption as a problem of opinion formation in human groups. The diffusion of innovations in markets takes time because not all individuals adopt at the same time, where adoption means that individuals purchase or use the innovation. Within the organization, when the adoption of an innovation involves the generalized use of it among all members the diffusion process will be affected by how the collective decision process is structured and managed. The literature on public opinion [21–23] describe this forming as the result of a process of influences of some people over others, using unidirectional means of influence (for example, mass media) or multiple directional ones (for example, social 10 / 13 PLOS ONE | DOI:10.1371/journal.pone.0126076 May 15, 2015 The Role of the Organization Structure in the Diffusion of Innovations networks). In some scenarios all individuals have the same capacity to exert influence while in others there are opinion leaders with a greater level of influence than anyone else [24]. According to this approach, this paper belongs to the studies that analyze the dissemination process of an opinion, using computer simulation of mathematical models of interpersonal in- fluences in networks with nodes and lines of communication linking these nodes. In the con- text of our work the opinion-formation ends up building a consensus, whether favorable or not, around an innovation that arises at some particular points in the organization. Each per- son in the organization has attached a likelihood of accepting the innovation which increases with the positive externalities (which are attributed to network effects [25], coordination games [26], learning from others [27], social pressure [28] and trust [29]) resulting from the pressure to adopt a favorable opinion exerted by the members that had opted for that favorable position previously, capability to learn about de alternatives, and with economic value of the innovation. Our paper uses the same methodology of simulating mathematical models of interpersonal in- fluences as [30] on public opinion formation. The authors assume that some individuals have different influence than the others (opinion leaders and followers); the probability of staying to one opinion is either zero or one; no change of opinion is contemplated; and the networks that determine the mutual influences are formed at random. Discussion In our study, all individuals are equal (although the model can incorporate influential asymmetries); the probabilities of supporting one opinion or another are between zero and one; people can change their status, either for or against, between one iteration and the next; the relative value of the innovation is included as a determining factor for the likelihood of support; people learn from others about economic value of alternatives giving heterogeneity; and the networks in which diffusion occurs respond to different structures commonly found in the market o real organizations as business firms. So far organizations and organization structures are viewed as institutions for solving coordina- tion and motivation problems [31], and as tools for creating, transferring and using knowledge [32]. Our paper also demonstrates the relevance of the formal structure of a social system in en- abling the assimilation of proposals for change and innovative initiatives, i.e. as determinant of social systems’ innovation capacity. PLOS ONE | DOI:10.1371/journal.pone.0126076 May 15, 2015 References 1. Adler PS, Clark KB. Behind the learning curve: A sketch of the learning process. Management Science. 1991; 37(3):267–281. doi: 10.1287/mnsc.37.3.267 2. Rogers EM. Diffusion of Innovations. New York: Free Press; 1962. 2. Rogers EM. Diffusion of Innovations. New York: Free Press; 1962. 3. Balasubramanian N, Lieberman MB. Industry learning environments and the heterogeneity of firm per- formance. Strategic Management Journal. 2010; 31(4):390–412. 4. Yelle LE. The learning curve: Historical review and comprehensive survey. Decision Sciences. 1979; 10(2):302–328. doi: 10.1111/j.1540-5915.1979.tb00026.x 5. Zangwill WI, Kantor PB. Toward a theory of continuous improvement and the learning curve. 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Discovery and diffusion of knowledge in an endogenous social network. American Journal of Sociololgy. 2005; 110(4):937–976. doi: 10.1086/426555 11. Castellano C, Fortunato S, Loreto V. Statistical physics of social dynamics. Reviews of Modern Phys- ics. 2009; 81(2):591–646. doi: 10.1103/RevModPhys.81.591 12. Bass FM. A new product growth for model consumer durables. Management Science. 1969; 15 (5):215–227. doi: 10.1287/mnsc.15.5.215 13. Mahajan V, Muller E. and Wind Y. New-product diffusion models. Mahajan V., Muller E. and Wind Y Ed- itors. Springer; 2000. 14. Boccaletti S, Latora V, Moreno Y, Chavez M, Hwang DU. Physics Reports. 2006; 424:175–308. doi: 10.1016/j.physrep.2005.10.009 15. Sanz J, Xia C-Y, Meloni S, and Moreno Y. Dynamics of Interacting Diseases. Physical Review X. 2014; 4:041005. doi: 10.1103/PhysRevX.4.041005 16. Moore C, Newman ME. Epidemics and percolation in small-world networks. Physical Review E. 2000; 61(5):5678–5682. doi: 10.1103/PhysRevE.61.5678 17. Newman ME. Spread of epidemic disease on networks. Physical review E. 2002; 66(1):016128. doi: 10.1103/PhysRevE.66.016128 18. Dodds PS, Watts DJ. A generalized model of social and biological contagion. Summary and concluding remarks The diffusion of innovation, that is, the study of patterns of how new ideas or technologies spread throughout a community is a topic of interest in many fields, including economics, soci- ology, market research and politics. In this paper we have studied the probability for a proposal to be accepted by different collectives. Different communities are modeled through different topologies of the contact network, and the process is studied through an agent based model whose inter individual interactions mimic both the learning process and the acceptance or re- jection of the proposal. Our results show that the structure of the network of contacts has a strong influence on the innovation diffusion, being more difficult for a proposal to be accepted when the connectivity of agents is heterogeneously distributed. We have shown that the learning process plays a posi- tive role in the diffusion, being heterogeneous structures more sensitive to the lack of informa- tion exchange. We have also studied the effect of social pressure on the acceptance dynamics, showing that social pressure hinders innovation spreading irrespective of the collective struc- ture. Finally, we have shown that networks with high average connectivity obstruct the diffu- sion of innovation. These results are of interest for understanding how different factors influence the diffusion and acceptance of a technological, technical or legislative proposal in different communities. PLOS ONE | DOI:10.1371/journal.pone.0126076 May 15, 2015 11 / 13 The Role of the Organization Structure in the Diffusion of Innovations Author Contributions Conceived and designed the experiments: CSR CGL YM. Performed the experiments: CSR CGL YM. Analyzed the data: CSR CGL YM. Contributed reagents/materials/analysis tools: CSR CGL YM. Wrote the paper: CSR CGL YM. PLOS ONE | DOI:10.1371/journal.pone.0126076 May 15, 2015 PLOS ONE | DOI:10.1371/journal.pone.0126076 May 15, 2015 References Journal of Theoretical Bi- ology. 2005; 232(4):587–604. doi: 10.1016/j.jtbi.2004.09.006 PMID: 15588638 19. Erdös P, Rényi A. On random graphs. Publicationes Mathematicae Debrecen. 1959; 6:29 20. Barabási AL, Albert R. Emergence of scaling in random networks. Science. 1999; 286(5439):509–512. doi: 10.1126/science.286.5439.509 PMID: 10521342 21. Katz E, Lazarsfeld PF. Personal influence: The part played by people in the flow of mass communica- tions. New Brunswick, NJ: Transaction Publishers; 2006. 22. 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Journal of Personality and Social Psychology. 1996; 70(6): 1218–1230. doi: 10.1037/0022- 3514.70.6.1218 27. Salganik MJ, Dodds PS, Watts DJ. Experimental study of inequality and unpredictability in an artificial cultural market. Science. 2006; 311(5762):854–856. doi: 10.1126/science.1121066 PMID: 16469928 28. Cialdini RB, Goldstein NJ. Social Influence: Compliance and Conformity. Annual Review of Psycholo- gy. 2004; 55:591–621. doi: 10.1146/annurev.psych.55.090902.142015 PMID: 14744228 29. Moldoveanu MC, Baum JA. I Think You Think I Think You’re Lying: The Interactive Epistemology of Trust in Social Networks. Management Science. 2011; 57(2):393–412. doi: 10.1287/mnsc.1100.1279 30. Watts DJ, Dodds PS. Influentials, networks, and public opinion formation. Journal of consumer re- search. 2007; 34(4):441–458. doi: 10.1086/518527 31. Milgrom P, Roberts J. The economics of modern manufacturing: Technology, strategy, and organiza- tion. The American Economic Review. 1990; 80:511–528. 32. 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Morphological variability is greater at developing than mature mouse neuromuscular junctions
Journal of anatomy
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O R I G I N A L P A P E R O R I G I N A L P A P E R Aleksandra M. Mech1 | Anna-Leigh Brown1  | Giampietro Schiavo1,2,3  | James N. Sleigh1,2 James N. Sleigh1,2 1Department of Neuromuscular Diseases, UCL Queen Square Institute of Neurology, University College London, London, UK 2UK Dementia Research Institute, University College London, London, UK 3Discoveries Centre for Regenerative and Precision Medicine, University College London Campus, London, UK Journal of Anatomy. 2020;237:603–617. Abstract Abstract The neuromuscular junction (NMJ) is the highly specialised peripheral synapse formed between lower motor neuron terminals and muscle fibres. Post-synaptic acetylcho- line receptors (AChRs), which are found in high density in the muscle membrane, bind to acetylcholine released into the synaptic cleft of the NMJ, thereby enabling the conversion of motor action potentials to muscle contractions. NMJs have been studied for many years as a general model for synapse formation, development and function, and are known to be early sites of pathological changes in many neuromus- cular diseases. However, information is limited on the diversity of NMJs in different muscles, how synaptic morphology changes during development, and the relevance of these parameters to neuropathology. Here, this crucial gap was addressed using a robust and standardised semi-automated workflow called NMJ-morph to quantify features of pre- and post-synaptic NMJ architecture in an unbiased manner. Five wholemount muscles from wild-type mice were dissected and compared at imma- ture (post-natal day, P7) and early adult (P31−32) timepoints. The inter-muscular variability was greater in mature post-synaptic AChR morphology than that of the pre-synaptic motor neuron terminal. Moreover, the developing NMJ showed greater differences across muscles than the mature synapse, perhaps due to the observed distinctions in synaptic growth between muscles. Nevertheless, the amount of nerve to muscle contact was consistent, suggesting that pathological denervation can be reliably compared across different muscles in mouse models of neurodegeneration. Additionally, mature post-synaptic endplate diameters correlated with fibre type, in- dependently of muscle fibre diameter. Altogether, this work provides detailed infor- mation on healthy pre- and post-synaptic NMJ morphology from five anatomically and functionally distinct mouse muscles, delivering useful reference data for future comparison with neuromuscular disease models. Correspondence James N. Sleigh, Department of Neuromuscular Diseases, UCL Queen Square Institute of Neurology, University College London, London, UK. Email: j.sleigh@ucl.ac.uk Funding information Medical Research Council, Grant/Award Number: MR/S006990/1; Wellcome Trust, Grant/Award Number: 107116/Z/15/Z; Horizon 2020 Framework Programme, Grant/Award Number: 739572; UK Dementia Research Institute Foundation, Grant/Award Number: UKDRI-1005 Morphological variability is greater at developing than mature mouse neuromuscular junctions Aleksandra M. Mech1 | Anna-Leigh Brown1  | Giampietro Schiavo1,2,3  | James N. Sleigh1,2 Funding information Medical Research Council, Grant/Award Number: MR/S006990/1; Wellcome Trust, Grant/Award Number: 107116/Z/15/Z; Horizon 2020 Framework Programme, Grant/Award Number: 739572; UK Dementia Research Institute Foundation, Grant/Award Number: UKDRI-1005 Received: 8 April 2020  |  Revised: 4 May 2020  |  Accepted: 7 May 2020 Received: 8 April 2020  |  Revised: 4 May 2020  |  Accepted: 7 May 2020 Received: 8 April 2020  |  Revised: 4 May 2020  |  Accepted: 7 May 2020 DOI: 10.1111/joa.13228 604  |     1 | INTRODUCTION of fast twitch fibr 604 MECH et al. of fast twitch fibres is shorter than slow twitch, and they lie on a spectrum of decreasing times from IIa to IIb. Type IIa fibres are ox- idative and most similar to slow twitch fibres, while type IIx and IIb are both glycolytic, with IIb fibres being required for the short- est and most intense movements. There are conflicting reports as to size categories of each fibre subtype, which is probably due to their diameters fluctuating between types of muscle; for example, slow twitch fibres are larger than fast twitch in the rat soleus mus- cle, which is composed of 86%–93% slow twitch fibres, but smaller in the rat extensor digitorum longus (EDL) muscle that is made up of only 2%–3% slow twitch fibres (Alnaqeeb and Goldspink, 1987). Nevertheless, slow twitch fibres are more often reported as being smaller than fast twitch, with type IIb being the largest (Stifani, 2014). In addition to intrinsic disparities, muscle fibre types are in- nervated by distinct classes of alpha motor neuron (Stifani, 2014), the firing patterns of which appear to impact the contractile proper- ties of fibres (Pette and Vrbová, 1985), while the fibre also appears to be able to retrogradely influence the identity of the innervating neuron (Chakkalakal et al., 2010). The motor neurons that innervate slow twitch fibres generally have smaller cell bodies and axons, re- quire a lower threshold to stimulation, and have slower conduction speeds, whereas fast-fatiguable motor neurons innervating type IIb fibres are at the other end of the spectra for these characteristics (Stifani, 2014). The neuromuscular junction (NMJ) is a peripheral synapse formed between lower motor neurons and skeletal muscle fibres that has been studied in many different species, including humans (Nyström, 1968; Kuno et al., 1971; Cramer and Van Essen, 1995; Prakash et al., 1996; Campbell and Ganetzky, 2012; Sakowski et al., 2012; Hammond et al., 2016; Jones et al., 2017; Grice et al., 2018). Upon action potential-mediated depolarisation of the pre-synaptic neuro- nal membrane, the neurotransmitter acetylcholine (ACh) is released into the NMJ synaptic cleft, where it binds to ACh receptors (AChRs) found in high density on post-synaptic muscle membranes in direct apposition to motor nerve terminals. This results in sodium ion in- flux into the muscle, post-synaptic membrane depolarisation and, if a threshold is reached, excitation−contraction coupling and muscle contraction. 604  |     1 | INTRODUCTION of fast twitch fibr Due to its experimental accessibility and large size, the NMJ has been used for many years as a model for pre- and early post-natal synapse development (Sanes and Lichtman, 1999). During this time, the mammalian NMJ undergoes a series of growth and maturation processes that refine the synaptic architecture and en- hance neurotransmission efficiency to facilitate motor performance (Shi et al., 2012). At birth, mammalian NMJs are poly-innervated, meaning that the endplate is contacted by more than one motor nerve ending, and in some instances more than 10 different axons (Tapia et al., 2012). During early post-natal development, equating to less than 2 weeks in mice, asynchronous axon withdrawal occurs and NMJs become mono-innervated. Known as synapse elimination, this process is thought to refine peripheral nerve architecture, and is driven by ac- tivity, inter-nerve competition and reciprocal nerve−muscle−glia sig- nalling (Favero et al., 2012; Turney and Lichtman, 2012; Smith et al., 2013b). This is accompanied by significant changes in the post-syn- apse to maintain the close proximity of motor terminals to AChRs. For example, the density and number of AChRs increase, and the morphology of AChR clusters transforms from a simple, plaque- like shape to a complex, multi-perforated, pretzel-like structure (Marques et al., 2000). This is, at least in part, due to remodelling of the post-synaptic membrane to create junctional folds, enlarging the contact area within the synaptic cleft. Moreover, the molecular com- position of the membrane also changes to assure correct functioning of the mature synapse; for instance, the foetal γ AChR subunit is exchanged for the adult ε subunit (Missias et al., 1996). The size of NMJs has been reported to correlate with fibre di- ameter in several different species and muscles (Harris, 1954; Nyström, 1968; Kuno et al., 1971; Balice-Gordon and Lichtman, 1990; Sieck et al., 2012). Moreover, manipulations of muscle fibre size can cause equivalent changes in the NMJ (Balice-Gordon et al., 1990). However, the relationship between size of NMJs and fibres may only be observable when fibre classes are individually assessed (Waerhaug and Lømo, 1994; Prakash et al., 1995a) or within certain types of muscle (Jones et al., 2016). Indeed, similar to muscle fibre diameters, inconsistencies in morphological features of NMJs asso- ciated with the distinct fibre types have been reported. Funding information Journal of Anatomy. 2020;237:603–617.   |  603 wileyonlinelibrary.com/journal/joa This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2020 The Authors. Journal of Anatomy published by John Wiley & Sons Ltd on behalf of Anatomical Society K E Y W O R D S epitrochleoanconeus, fast twitch, flexor digitorum brevis, lumbricals, morphology, motor neuron, muscle fibre type, neuromuscular junction, NMJ-morph, slow twitch, synapse, transversus abdominis |  wileyonlinelibrary.com/journal/joa rodent NMJ (Gonzalez-Freire et al., 2014), while axotomy causes NMJ dismantling prior to axonal degeneration (Miledi and Slater, 1970). Consequently, it is of considerable importance to be able to accurately assess the integrity of neuromuscular synapses in models of pathology. Pathological NMJ phenotypes (e.g. denervation counts and neurofilament accumulation) are often assessed by eye, possibly reducing objectivity and limiting inter-study comparisons. However, this issue has been tackled by the Gillingwater laboratory, who have developed a free, ImageJ-based methodology called NMJ-morph for comparative analyses of neuromuscular synapses (Jones et al., 2016), which has been successfully used to evaluate NMJs in patients with cancer cachexia (Boehm et al., 2020). By using thresholded, binary images, data on 20 NMJ morphological variables can be quickly and reliably obtained, with the proviso that several are not independent (e.g. nerve terminal perimeter and area). Operator-independent in- formation on the pre-synaptic nerve terminal, post-synaptic AChRs and overlapping features can be objectively generated for accurate cross-muscle, cross-study and even cross-laboratory comparisons. rodent NMJ (Gonzalez-Freire et al., 2014), while axotomy causes NMJ dismantling prior to axonal degeneration (Miledi and Slater, 1970). Consequently, it is of considerable importance to be able to accurately assess the integrity of neuromuscular synapses in models of pathology. Pathological NMJ phenotypes (e.g. denervation counts and neurofilament accumulation) are often assessed by eye, possibly reducing objectivity and limiting inter-study comparisons. However, this issue has been tackled by the Gillingwater laboratory, who have developed a free, ImageJ-based methodology called NMJ-morph for comparative analyses of neuromuscular synapses (Jones et al., 2016), which has been successfully used to evaluate NMJs in patients with cancer cachexia (Boehm et al., 2020). By using thresholded, binary images, data on 20 NMJ morphological variables can be quickly and reliably obtained, with the proviso that several are not independent (e.g. nerve terminal perimeter and area). Operator-independent in- formation on the pre-synaptic nerve terminal, post-synaptic AChRs and overlapping features can be objectively generated for accurate cross-muscle, cross-study and even cross-laboratory comparisons. (DSHB), Iowa City, IA, USA, supernatant] and 1/25 mouse pan anti- synaptic vesicle 2 (SV2, DSHB, supernatant). 1/1,000 Alexa Fluor 555 α-bungarotoxin (α-BTX; Life Technologies, B35451) was used to identify post-synaptic AChRs. 2.1 | Animals Wild-type mice on a C57BL/6J background were killed at P7 and P31−32 for post-natal developmental and early adult timepoints, respectively. Muscles were harvested from three males and three females at each age, weighing an average of 4.0 g at P7 and 17.0 g at P31−32 (Table  S1). P7 animals were taken from a single litter, whereas P31−32 mice came from two different litters. All muscles were dissected from the left side of the body, except for lumbricals of the forepaw, which were taken from the right. Mouse handling and experiments were performed under license from the UK Home Office in accordance with the Animals (Scientific Procedures) Act (1986) and approved by the University College London – Queen Square Institute of Neurology Ethics Committee. For clarity, ‘endplates’, ‘AChRs’ and ‘AChR clusters’ are slightly different post-synaptic structures of decreasing size. Endplates in- clude the post-synaptic staining, along with associated perforations and other areas devoid of α-BTX that occur within the perimeter of staining. Hence, endplate area is a good proxy for overall NMJ size. AChRs represent the footprint of post-synaptic staining alone (i.e. excluding α-BTX-negative areas). AChR clusters include only the α-BTX staining found in discrete groupings. 604  |     1 | INTRODUCTION of fast twitch fibr For instance, using both light and electron microscopy, the average area of NMJs in predominantly slow twitch soleus muscles of the rat was shown to be larger than in the fast twitch EDL (McArdle and Sansone, 1977; Wood and Slater, 1997). Contrasting with this, NMJs innervating type I fibres in rat diaphragm muscle were shown to be less than half the size of those innervating type II fibres (Prakash et al., 1996). However, correcting for fibre diameter, the NMJ size does not vary quite so much across the fibre types and muscles, although type I fibre NMJs are larger than fast twitch NMJs in rat diaphragm muscle (Prakash et al., 1995b, 1996; Prakash and Sieck, 1998). This suggests that features other than fibre diameter contribute to NMJ size dif- ferences between muscle fibre types, for example, the innervating motor neuron and its firing properties (Waerhaug and Lømo, 1994). Mammalian muscles are composed of two main classes of force-generating (extrafusal) muscle fibre, ‘slow twitch’ (type I) and ‘fast twitch’ (type II), which have differing anatomical, physical and metabolic properties (Schiaffino and Reggiani, 2011). Slow twitch fi- bres generate energy through oxidative metabolism and can be con- tinuously used for long periods of time (i.e. hours), therefore they function primarily to maintain body posture. Based on differential expression of myosin heavy chain (MHC) genes, fast twitch fibres can be further subdivided into three principal categories: type IIa (expressing MYH2); IIx (expressing MYH1); and IIb (expressing MYH4; Talbot and Maves, 2016). The time to contraction and time to fatigue The NMJ is an early and important site of pathology in many dif- ferent neuromuscular conditions, including amyotrophic lateral scle- rosis (Williamson et al., 2019), spinal muscular atrophy (Murray et al., 2008) and Charcot-Marie-Tooth disease (Spaulding et al., 2016). Furthermore, aging appears to result in structural alterations at the MECH et al. 605 2.3 | Neuromuscular junction imaging and analysis Neuromuscular junctions were imaged using an LSM 510 laser- scanning microscope (Zeiss, Oberkochen, Germany) with Argon/2 and HeNe543 lasers for neurons (labelled green; 2H3/SV2) and post-synaptic AChRs (labelled red; α-BTX), respectively. Z-stacks with a 1-µm interval were taken using a Plan-Apochromat 63 ×/1.4 oil immersion objective. Images of P31−32 samples were taken with 0.7 × zoom, and images of P7 samples with 2 × zoom. ImageJ soft- ware (https://imagej.nih.gov/ij/) combined with the NMJ-morph workflow (Jones et al., 2016) was used to describe 19 morphological variables of NMJs across all five muscles. The resolution of analysed images was 512 × 512 pixels, corresponding to 101.02 × 101.02 µm at P31−32 and 35.71 × 35.71 µm at P7. For all variables, 20 en face NMJs with clearly visible pre-synaptic axons and terminals were as- sessed (resulting in analysis of 1200 NMJs in total). A 20th variable of poly-innervation percentage was visually assessed at 100 NMJs per muscle (resulting in analysis of 6000 NMJs in total), as previously outlined (Sleigh et al., 2014b). In the case of lumbrical and flexor digitorum brevis (FDB) muscles, where more than one muscle was processed, NMJs were assessed across all muscles (as were muscle fibres, see below). All analyses were performed on maximum inten- sity Z-stack projections, and no samples were excluded from analy- ses once imaged. Of several available thresholding methods, three provided the most accurate binary representation of NMJs. ‘Huang’, ‘Yen’ or manual method were chosen based on how well they resem- bled the original image. The ‘Huang’ method was used for 56% and 79% of NMJs from P31−32 and P7, respectively, the ‘Yen’ method for 42% and 1%, and the manual method for 2% and 20%. Here, the same software was used to assess morphology of neuromuscular synapses at two different timepoints, represent- ing a developing (postnatal day 7, P7) and a mature (P31−32) NMJ. Performing analyses in five different wholemount muscles with varying fibre type populations, this novel morphometric approach was used to assess the influence of post-natal maturation, fibre type and fibre diameter on pre- and post-synaptic NMJ morphology, as well as post-natal synaptic development/growth. 2.7 | Clustering analysis Data were assumed to be normally distributed unless evidence to the contrary could be provided by the D'Agostino and Pearson omnibus normality test. GraphPad Prism 8 (version 8.4.0) was used for all statistical analyses. Means  ±  SEM are plotted and, when possible, individual data points generated from each mouse (i.e. the mean values from 20 NMJs or 30 fibres). In all bar charts, muscles are plotted on the x-axes in ascending order of the per- centage of fast twitch fibres (Table  1). Moreover, the muscle colour scheme is maintained throughout all figures and supple- mentary figures [i.e. yellow, transversus abdominis (TVA); cyan, hindpaw lumbricals; purple, epitrochleoanconeus (ETA); red, FDB; green, forepaw lumbricals]. Individual morphological variables were statistically analysed using a repeated-measures one-way analysis of variance (anova), because the five dissected muscles were all taken from the same six mice per timepoint, i.e. the mus- cles were matched. If the anova indicated a significant difference, Bonferroni's multiple comparisons test was performed to assess significance between pairwise combinations of all five muscles. Correlation was assessed using Pearson's product moment cor- relation. When performing 20 associated tests (i.e. the number of tests used per timepoint), a value of P  <  0.00256 was used to determine significance and thereby maintain an α of 0.05. To calculate the percentage difference in morphological variables between timepoints, the mean value at P31−32 was divided by the mean at P7 and multiplied by 100. Two-way anovas were performed for each morphological trait (test variables being age and muscle), followed by Sidak's multiple comparisons test to as- sess whether there was a difference between timepoints in each muscle. Morphological variables were converted to z-scores, and samples clustered by Euclidean distance and hierarchal clustering. The com- plete linkage method was used to find breaks. Euclidean distance between two samples is the square root of the sum of squared differences between all z-score-scaled morphological variables. Complete clustering is a distance metric that defines the distance between clusters as the maximum possible distance between points in a cluster. Heat maps and clustering were created using the pheat- map package (https://CRAN.R-proje​ct.org/packa​ge=pheatmap). microscopy on an inverted LSM780 laser-scanning microscope (Zeiss) with a 20 × objective. Using ImageJ, fibre diameters were determined by taking five width measurements at different points along individual fibres. Damaged fibres were avoided, as were end- ings that were patently larger than the rest of the fibre. Thirty fi- bres were measured per muscle per animal to generate mean values (resulting in analysis of 900 fibres in total). Analysed fibres were randomly sampled and very unlikely to be those in which NMJ mor- phology was assessed. Principal component analysis (PCA) was used for dimensionality reduction of the datasets of P7 and P31−32 NMJ morphological variables by collapsing the variables into new ‘principal components’ created through linear combinations of the original datasets. Data. table (https://CRAN.R-proje​ct.org/packa​ge=data.table) and ti- dyverse (Wickham et al., 2019) packages were used to load the mor- phological data into R (version 3.6.2). PCA plots and loading analysis were created using the PCAtools package (https://github.com/kevin​ bligh​e/PCAtools) and ggplot2 (Wickham, 2016). MECH et al. 606 oscope 2.6 | Principal component analysis See also Figure 1. 2.2 | Tissue preparation and immunohistochemistry After imaging P31−32 NMJs for morphological analyses, microscope slides were immersed in phosphate-buffered saline to facilitate coverslip removal. Muscles were then teased using forceps to sepa- rate and aid identification of individual fibres, before re-mounting in Fluoromount-G on new microscope slides. Muscles were imaged at a resolution of 1024 × 1024 by differential interference contrast Muscles were dissected (Murray et al., 2014; Sleigh et al., 2014a; Tarpey et al., 2018) and stained as previously described (Sleigh et al., 2014b, 2017). The following antibodies were used to co- visualise axons and pre-synaptic nerve terminals: 1/250 mouse anti-neurofilament [2H3, Developmental Studies Hybridoma Bank 3.1 | Wholemount immunohistochemical analyses of mouse muscles and neuromuscular junctions Five mouse muscles from diverse body positions with varying in- nervating nerves and fast twitch fibre percentages (Table  1), all of which are also found in humans, were selected for morpho- logical analysis of NMJs (Figure 1). The lumbricals of the forepaw mediate metacarpophalangeal joint flexion (i.e. paw clasping), are innervated by the median/ulnar nerve, and are composed of ~100% fast twitch muscle fibres (Figure 1a; Russell et al., 2015). Consisting of ~90% fast twitch fibres and innervated by the radial nerve, the ETA is located on the medial surface of the upper fore- limb and contributes to forearm supination (i.e. outward rotation of the palm; Figure 1b; Bradley et al., 1989). Innervated by lower TA B LE 1 Characteristics of the five muscles used in this study Muscle Body position Innervating nerve % Fast twitch Animal Reference Epitrochleoanconeus (ETA) Forelimb Radial 90 Mouse Bradley et al. (1989) Flexor digitorum brevis (FDB) Hindlimb Medial plantar 95.6 Mouse Tarpey et al. (2018) Forepaw lumbricals Forelimb Median/ulnar 100 Mouse Russell et al. (2015) Hindpaw lumbricals Hindlimb Medial/lateral plantar 90 Mouse Smith et al. (2013a) Transversus abdominis (TVA) Thorax Lower intercostal 68 Rat Delp and Duan (1996) TA B LE 1 Characteristics of the five muscles used in this study TA B LE 1 Characteristics of the five muscles used in this study |  607 MECH et al. FIGURE 1 Dissection of five different muscles for wholemount assessment of neuromuscular junction (NMJ) morphology. The forepaw lumbricals (green, a), epitrochleoanconeus (ETA; purple, b), transversus abdominis (TVA; yellow, c), hindpaw lumbricals (cyan, di) and flexor digitorum brevis (FDB; red, d) are five thin and flat muscles that can be dissected from mice (i and ii) and immunohistochemically analysed as wholemount preparations (iii) to assess pre- (SV2/2H3, green) and post-synaptic (alpha bungarotoxin, α-BTX, magenta) NMJ morphology (iv). Representative staining of hindpaw lumbrical NMJs can be found in Figure 2(a). Imaged muscles were taken from a P31 mouse. Individual muscles in panel i are highlighted by arrowheads coloured the same as muscles in the mouse schematic on the left; this colour scheme is maintained in all figures and supplementary figures. White arrows in panel ii identify innervating nerves. Scale bars: 1 mm (iii); 20 µm (iv). The mouse schematic was created with BioRender (https://biore​nder.comwileyonlinelibrary.com]). See also Table 1 [Colour figure can be viewed at wileyonlinelibrary.com] MECH et al. 607 FIGURE 1 Dissection of five different muscles for wholemount assessment of neuromuscular junction (NMJ) morphology. The forepaw lumbricals (green, a), epitrochleoanconeus (ETA; purple, b), transversus abdominis (TVA; yellow, c), hindpaw lumbricals (cyan, di) and flexor digitorum brevis (FDB; red, d) are five thin and flat muscles that can be dissected from mice (i and ii) and immunohistochemically analysed as wholemount preparations (iii) to assess pre- (SV2/2H3, green) and post-synaptic (alpha bungarotoxin, α-BTX, magenta) NMJ morphology (iv). Representative staining of hindpaw lumbrical NMJs can be found in Figure 2(a). Imaged muscles were taken from a P31 mouse. Individual muscles in panel i are highlighted by arrowheads coloured the same as muscles in the mouse schematic on the left; this colour scheme is maintained in all figures and supplementary figures. White arrows in panel ii identify innervating nerves. Scale bars: 1 mm (iii); 20 µm (iv). The mouse schematic was created with BioRender (https://biore​nder.comwileyonlinelibrary.com]). See also Table 1 [Colour figure can be viewed at wileyonlinelibrary.com] FI G U R E 2 Post-synaptic morphology of mature neuromuscular junctions (NMJs) is more variable between muscles than pre-synaptic. (a) Five different wholemount muscles were dissected from P31−32 mice, and immunohistochemically processed to visualise and assess pre- (SV2/2H3, green) and post-synaptic (α-bungarotoxin, α-BTX, magenta) morphology at mature NMJs. The image is of a P31 hindpaw lumbrical muscle. Scale bars: 20 µm. TA B LE 1 Characteristics of the five muscles used in this study (b–d) Of 20 morphological variables analysed using repeated-measures one-way anovas (Table S3), several different pre-synaptic (b), post-synaptic (c) and overlapping (d) morphologies varied between muscles (only significant data are presented, i.e. Bonferroni-corrected P < 0.00256). *P < 0.05, **P < 0.01, ***P < 0.001 Bonferroni's multiple comparisons test. Means ± SEM are plotted (n = 6), as well as individual data points generated from each mouse (i.e. the mean values from 20 NMJs). Lumb. (fore), lumbricals of the forepaw; Lumb. (hind), lumbricals of the hindpaw. See also Tables 3 and S3 [Colour figure can be viewed at wileyonlinelibrary.com] MECH et al. MECH et al. 608 FIGURE 3 The developing neuromuscular junction (NMJ) shows greater inter-muscular variability in morp FIGURE 3 The developing neuromuscular junction (NMJ) shows greater inter-muscular variability in morphology than mature synapses. (a) Five different wholemount muscles were dissected from P7 mice, and immunohistochemically processed to visualise and assess pre- (SV2/2H3, green) and post-synaptic (α-bungarotoxin, α-BTX, magenta) morphology at developing NMJs. The image is of a P7 transversus abdominis (TVA) muscle, and the arrow identifies a poly-innervated NMJ. Scale bars: 20 µm. (b–d) Of 20 morphological variables analysed using repeated-measures one-way anovas (Table S3), several different pre-synaptic (b), post-synaptic (c) and overlapping (d) morphologies varied between muscles (only significant data are presented, i.e. Bonferroni-corrected P < 0.00256). *P < 0.05, **P < 0.01, ***P < 0.001 Bonferroni's multiple comparisons test. Means ± SEM are plotted (n = 6), as well as individual data points generated from each mouse (i.e. the mean values from 20 NMJs). Lumb. (fore), lumbricals of the forepaw; Lumb. (hind), lumbricals of the hindpaw. See also Tables 3 and S3 [Colour figure can be viewed at wileyonlinelibrary.com] FIGURE 3 The developing neuromuscular junction (NMJ) shows greater inter-muscular variability in morphology than mature synapses. (a) Five different wholemount muscles were dissected from P7 mice, and immunohistochemically processed to visualise and assess pre- (SV2/2H3, green) and post-synaptic (α-bungarotoxin, α-BTX, magenta) morphology at developing NMJs. The image is of a P7 transversus abdominis (TVA) muscle, and the arrow identifies a poly-innervated NMJ. Scale bars: 20 µm. (b–d) Of 20 morphological variables analysed using repeated-measures one-way anovas (Table S3), several different pre-synaptic (b), post-synaptic (c) and overlapping (d) morphologies varied between muscles (only significant data are presented, i.e. Bonferroni-corrected P < 0.00256). *P < 0.05, **P < 0.01, ***P < 0.001 Bonferroni's multiple comparisons test. TA B LE 1 Characteristics of the five muscles used in this study (b) Of 20 morphological variables analysed using repeated-measures one-way anovas (Table S2), only post-synaptic features varied between muscles (only significant data are presented, i.e. Bonferroni-corrected P < 0.00256). *P < 0.05, **P < 0.01, ***P < 0.001 Bonferroni's multiple comparisons test. Means ± SEM are plotted (n = 6), as well as individual data points generated from each mouse (i.e. the mean values from 20 NMJs). Lumb. (fore), lumbricals of the forepaw; Lumb. (hind), lumbricals of the hindpaw. See also Tables 2 and S2 [Colour figure can be viewed at wileyonlinelibrary.com] FI G U R E 2 Post-synaptic morphology of mature neuromuscular junctions (NMJs) is more variable between muscles than pre-synaptic. (a) Five different wholemount muscles were dissected from P31−32 mice, and immunohistochemically processed to visualise and assess pre- (SV2/2H3, green) and post-synaptic (α-bungarotoxin, α-BTX, magenta) morphology at mature NMJs. The image is of a P31 hindpaw lumbrical muscle. Scale bars: 20 µm. (b) Of 20 morphological variables analysed using repeated-measures one-way anovas (Table S2), only post-synaptic features varied between muscles (only significant data are presented, i.e. Bonferroni-corrected P < 0.00256). *P < 0.05, **P < 0.01, ***P < 0.001 Bonferroni's multiple comparisons test. Means ± SEM are plotted (n = 6), as well as individual data points generated from each mouse (i.e. the mean values from 20 NMJs). Lumb. (fore), lumbricals of the forepaw; Lumb. (hind), lumbricals of the hindpaw. See also Tables 2 and S2 [Colour figure can be viewed at wileyonlinelibrary.com] 608  |     MECH et al. intercostal nerves, the TVA is a postural muscle also involved in respiration that is found in the anterior abdominal wall consist- ing of ~68% fast twitch fibres (Figure 1c; Delp and Duan, 1996; consists of ~96% fast twitch fibres and is innervated by the medial plantar nerve (Tarpey et al., 2018), whereas the hindpaw lumbricals are ~90% fast twitch and innervated by both medial and lateral FIGURE 3 The developing neuromuscular junction (NMJ) shows greater inter-muscular variability in morphology than mature synapses. (a) Five different wholemount muscles were dissected from P7 mice, and immunohistochemically processed to visualise and assess pre- (SV2/2H3, green) and post-synaptic (α-bungarotoxin, α-BTX, magenta) morphology at developing NMJs. The image is of a P7 transversus abdominis (TVA) muscle, and the arrow identifies a poly-innervated NMJ. Scale bars: 20 µm. preparations without the need for sectioning (Figure  1a–di–ii). This permits visualisation of the entire innervation pattern of these muscles using combined neuron-specific SV2/2H3 antibod- ies and AChR-targeting α-BTX (Figure 1a–diii), and accurate evalu- ation of NMJ structure (Figure 1a–div; Sleigh et al., 2014a). 3.2 | Mature post-synaptic neuromuscular junction morphology varies more than pre-synaptic between muscles Muscles were dissected from wild-type mice aged P31−32, a time- point chosen to represent a mature, early adult NMJ. To confirm this, NMJ development was assessed by calculating the percentage of synapses innervated by more than one motor neuron (i.e. poly- innervation), which is a key feature of immature NMJs (Sanes and Lichtman, 1999). As expected, all muscles had undergone the process of synapse elimination to become mono-innervated (Table 2), while post-synapses displayed clear pretzel-like structures (Figures 1a–div and 2a). The foetal to adult AChR subunit switch is also usually com- plete by this stage (Missias et al., 1996). Together, this suggests that analysis at P31−32 allows assessment of relatively well-developed NMJs across all five muscles. FIGURE 4 Principal components analysis (PCA) of developing and mature neuromuscular junction (NMJ) morphology. Two-dimensional PCA representation of NMJs as defined by 20 morphological variables from each animal, colour-coded by muscle type. Inverted triangles and squares identify P7 and P31−32 data, respectively. The first principal component (PC1, x-axis) accounts for 90.5% of the variability, and PC2 (y-axis) 4.9%. Each datum represents the centre point of the map positions of averages taken from 20 NMJs for each muscle from each animal. Note the ability of PC1 to distinguish between timepoints and PC2 to generally separate muscle types. Lumb. (fore), lumbricals of the forepaw; Lumb. (hind), lumbricals of the hindpaw. See also Figure S1 [Colour figure can be viewed at wileyonlinelibrary.com] FI G U R E 5 Post-natal neuromuscular junction (NMJ) development varies between muscles. (a and b) To assess the change in morphological variables from P7 (clear bars) to P31−32 (hatched bars), values from the five muscles at both timepoints were analysed using two-way anovas (age and muscle as the test variables; Table S4). Of the 20 variables, three pre-synaptic (a) and five post-synaptic (b) displayed significant interactions (#Bonferroni-corrected P < 0.00256), suggesting that NMJs mature differentially between muscles. Means ± standard error of the mean are plotted (n = 6). **P < 0.01, ***P < 0.001 Sidak's multiple comparisons test. Lumb. (fore), lumbricals of the forepaw; Lumb. (hind), lumbricals of the hindpaw. See also Table S4 [Colour figure can be viewed at wileyonlinelibrary.com] FI G U R E 5 Post-natal neuromuscular junction (NMJ) development varies between muscles. TA B LE 1 Characteristics of the five muscles used in this study Means ± SEM are plotted (n = 6), as well as individual data points generated from each mouse (i.e. the mean values from 20 NMJs). Lumb. (fore), lumbricals of the forepaw; Lumb. (hind), lumbricals of the hindpaw. See also Tables 3 and S3 [Colour figure can be viewed at wileyonlinelibrary.com] consists of ~96% fast twitch fibres and is innervated by the medial plantar nerve (Tarpey et al., 2018), whereas the hindpaw lumbricals are ~90% fast twitch and innervated by both medial and lateral plantar nerves (Smith et al., 2013a). All five muscles possess the major advantage that they are thin and flat, and can, therefore, be dissected and immunohistochemically processed as wholemount intercostal nerves, the TVA is a postural muscle also involved in respiration that is found in the anterior abdominal wall consist- ing of ~68% fast twitch fibres (Figure 1c; Delp and Duan, 1996; Murray et al., 2014). Situated in the hindpaw, the FDB and lum- bricals aid metatarsophalangeal joint extension (i.e. paw opening) and flexion (i.e. paw clasping), respectively (Figure 1d). The FDB 609 MECH et al. MECH et al. FIGURE 4 Principal components analysis (PCA) of developing and mature neuromuscular junction (NMJ) morphology. Two-dimensional PCA representation of NMJs as defined by 20 morphological variables from each animal, colour-coded by muscle type. Inverted triangles and squares identify P7 and P31−32 data, respectively. The first principal component (PC1, x-axis) accounts for 90.5% of the variability, and PC2 (y-axis) 4.9%. Each datum represents the centre point of the map positions of averages taken from 20 NMJs for each muscle from each animal. Note the ability of PC1 to distinguish between timepoints and PC2 to generally separate muscle types. Lumb. (fore), lumbricals of the forepaw; Lumb. (hind), lumbricals of the hindpaw. See also Figure S1 [Colour figure can be viewed at wileyonlinelibrary.com] 3.2 | Mature post-synaptic neuromuscular junction morphology varies more than pre-synaptic between muscles See also Figure S3 and Table S7 [Colour figure can be viewed at wileyonlinelibrary.com] NMJs found in a diverse set of wholemount muscles display signif- icant variation in post-synaptic, but not pre-synaptic, architecture. NMJ-morph was then used to assess pre-synaptic, post-synaptic and overlapping features of NMJ morphology (Figure 2; Tables 2 and S2). As a relative measure of the range in morphology observed be- tween muscles, the fold difference between the maximum and min- imum values for each variable were calculated (Table 2). Assessing inter-muscle variability in the pre-synapse, the number of terminal branches showed the greatest fold difference (2.21), while com- plexity displayed the least (1.14). In the post-synapse, the number of AChR clusters showed the greatest fold distinction (2.15) and compactness the smallest (1.10). In the two combined pre- and post-synaptic variables of synaptic contact area and overlap, max- imum to minimum fold differences of 1.37 and 1.22 were observed, respectively. For efficiency, 20 NMJs per muscle per timepoint were anal- ysed rather than the suggested 30 (Jones et al., 2016). Nonetheless, graphical evidence is provided that there is little to no distinction in NMJ variables when 20–50 NMJs were observed (Jones et al., 2016). Consistent with this, intra-muscular morphological variation between the six mice dissected here was low. This is also perti- nent to the point that several paw muscles were combined in the presented analyses, because subtle distinctions in morphologies have been reported between the first and fourth lumbricals of the hindpaw and the second and fourth lumbricals of the forepaws (Jones et al., 2016). Muscles were combined because comparative denervation analyses have already been performed in this manner in mouse models of neuromuscular disease (Sleigh et al., 2014b; 2020). As 20 separate repeated-measures anovas were conducted, the Bonferroni correction was applied to determine that a P-value of less than 0.00256 for each test is required to maintain an α of 0.05. With this stringent P-value, no significant differences were observed in pre-synaptic or combined morphological variables (Table  S2). The lack of difference in the overlap between pre- and post-synaptic staining indicates that despite morphological variety between mus- cles, the level of nerve to muscle interaction remains fairly constant, suggesting that pathological denervation assessed in different mus- cles can be reliably compared. In contrast, six post-synaptic variables displayed significant differences between muscles (Figure 2b). 3.2 | Mature post-synaptic neuromuscular junction morphology varies more than pre-synaptic between muscles The TVA had the largest reported values for most post-synaptic variables and forepaw lumbricals the smallest. These data indicate that mature 3.2 | Mature post-synaptic neuromuscular junction morphology varies more than pre-synaptic between muscles (a and b) To assess the change in morphological variables from P7 (clear bars) to P31−32 (hatched bars), values from the five muscles at both timepoints were analysed using two-way anovas (age and muscle as the test variables; Table S4). Of the 20 variables, three pre-synaptic (a) and five post-synaptic (b) displayed significant interactions (#Bonferroni-corrected P < 0.00256), suggesting that NMJs mature differentially between muscles. Means ± standard error of the mean are plotted (n = 6). **P < 0.01, ***P < 0.001 Sidak's multiple comparisons test. Lumb. (fore), lumbricals of the forepaw; Lumb. (hind), lumbricals of the hindpaw. See also Table S4 [Colour figure can be viewed at wileyonlinelibrary.com] 610  |     MECH et al. FI G U R E 6 Fibre diameter differs between mature muscles, but does not influence neuromuscular junction (NMJ) morphology. (a) Representative differential interference contrast images of teased muscle fibres from the five wholemount muscles. Scale bars: 100 µm. (b) Fibre diameters are not equal across muscles; however, no significant correlation between fibre diameter and any of the 20 NMJ morphological variables was observed (Table S7). P < 0.001 repeated-measures one-way anova. *P < 0.05, **P < 0.01, ***P < 0.001 Bonferroni's multiple comparisons test. Means ± SEM are plotted (n = 6), as well as individual data points generated from each mouse (i.e. the mean values from 30 fibres). Lumb. (fore), lumbricals of the forepaw; Lumb. (hind), lumbricals of the hindpaw. See also Figure S3 and Table S7 [Colour figure can be viewed at wileyonlinelibrary.com] MECH et al. MECH et al. 610 FI G U R E 6 Fibre diameter differs between mature muscles, but does not influence neuromuscular junction (NMJ) morphology. (a) Representative differential interference contrast images of teased muscle fibres from the five wholemount muscles. Scale bars: 100 µm. (b) Fibre diameters are not equal across muscles; however, no significant correlation between fibre diameter and any of the 20 NMJ morphological variables was observed (Table S7). P < 0.001 repeated-measures one-way anova. *P < 0.05, **P < 0.01, ***P < 0.001 Bonferroni's multiple comparisons test. Means ± SEM are plotted (n = 6), as well as individual data points generated from each mouse (i.e. the mean values from 30 fibres). Lumb. (fore), lumbricals of the forepaw; Lumb. (hind), lumbricals of the hindpaw. | TA B LE 2 P31−32 NMJ morphological variables Variable Mean Maximum Minimum Fold difference Poly-innervation (%) 0.30 0.50 0.17 2.94 Nerve terminal perimeter (µm) 203.80 270.39 147.61 1.83 Nerve terminal area (µm2) 146.62 169.33 120.11 1.41 # Terminal branches 31.32 44.09 19.98 2.21 # Branch points 25.22 28.84 20.80 1.39 Total branch length (µm) 106.28 129.27 82.31 1.57 Average branch length (µm) 4.61 6.03 3.80 1.59 Complexity 4.74 4.97 4.37 1.14 Axon diameter (µm) 1.48 1.64 1.29 1.27 AChR perimeter (µm) 164.56 222.39 118.45 1.88 AChR area (µm2) 198.64 254.43 150.07 1.70 Endplate diameter (µm) 27.14 35.28 22.92 1.54 Endplate perimeter (µm) 74.04 92.07 62.22 1.48 Endplate area (µm2) 296.98 407.30 214.33 1.90 # AChR clusters 3.06 4.25 1.98 2.15 AChR cluster area (µm2) 101.52 114.73 83.94 1.37 Compactness (%) 68.57 71.02 64.60 1.10 Fragmentation 0.43 0.58 0.31 1.86 Synaptic contact area (µm2) 123.34 139.75 102.20 1.37 Overlap (%) 63.36 68.57 56.11 1.22 Mean values for all NMJ variables were calculated for each muscle per animal from 20 NMJs, the means of which (n = 6) were then recorded for each muscle. The plotted mean, maximum and minimum values for each NMJ variable were determined from these calculated values. The fold difference was calculated by dividing the maximum value by the minimum. Variables are divided into pre-synaptic at the top, post-synaptic in the middle, and combined pre- and post-synaptic at the bottom. See also Figure 2 and Table S2. AChR, acetylcholine receptor. lumbricals, with the forepaw lumbricals showing intermediate val- ues. The number of branch points displayed the greatest fold dif- ference between maximum and minimum values (3.30), while axon diameter displayed the least (1.20; Table 3). neuromuscular contacts. It has been suggested that synapse elimina- tion generally occurs along a proximal-to-distal gradient, with NMJs further from the thorax taking longer to undergo this important developmental process (Bixby and van Essen, 1979). However, the TVA, which is the most proximal muscle assessed here, showed the highest percentage of poly-innervated NMJs (~36%). Moreover, the three paw muscles showed very similar degrees of poly-innervation (18%–19%) despite the FDB and hindpaw lumbricals being more dis- tal than forepaw lumbricals. These data indicate that at P7 there is no body-wide location effect on synapse elimination. 3.3 | Intra-muscular variation in synaptic architecture is greater at immature synapses To determine whether developing NMJs display similar distinctions in NMJ structure, morphological analysis was also performed at P7. Poly-innervation counts confirmed that the process of synapse elimination was not yet complete in any of the five muscles (Table 3), indicating that the P7 timepoint allows analysis of immature MECH et al. 611 means of which (n = 6) were then recorded for each muscle. The plotted mean, maximum and Mean values for all NMJ variables were calculated for each muscle per animal from 20 NMJs, the MECH et al. 612 TA B LE 3 P7 NMJ morphological variables TA B LE 3 P7 NMJ morphological variables Variable Mean Maximum Minimum Fold difference Poly-innervation (%) 19.90 35.50 8.83 4.02 Nerve terminal perimeter (µm) 96.92 142.96 52.91 2.70 Nerve terminal area (µm2) 35.39 52.27 17.63 2.96 # Terminal branches 51.05 78.25 28.57 2.74 # Branch points 35.38 58.31 17.68 3.30 Total branch length (µm) 55.66 85.26 28.80 2.96 Average branch length (µm) 1.31 1.55 1.15 1.35 Complexity 4.68 5.42 3.96 1.37 Axon diameter (µm) 0.84 0.91 0.76 1.20 AChR perimeter (µm) 116.44 142.25 81.63 1.74 AChR area (µm2) 66.62 100.06 31.66 3.16 Endplate diameter (µm) 15.48 21.63 12.33 1.75 Endplate perimeter (µm) 42.31 54.55 33.06 1.65 Endplate area (µm2) 85.59 121.07 46.33 2.61 # AChR clusters 3.92 4.56 3.02 1.51 AChR cluster area (µm2) 25.38 38.70 15.10 2.56 Compactness (%) 76.22 82.94 68.72 1.21 Fragmentation 0.54 0.60 0.47 1.28 Synaptic contact area (µm2) 27.77 42.53 11.88 3.58 Overlap (%) 42.11 46.45 37.31 1.24 Mean values for all NMJ variables were calculated for each muscle per animal from 20 NMJs the Mean values for all NMJ variables were calculated for each muscle per animal from 20 NMJs, the Mean values for all NMJ variables were calculated for each muscle per animal from 20 NMJs, the means of which (n = 6) were then recorded for each muscle. The plotted mean, maximum and minimum values for each NMJ variable were determined from these calculated values. The fold difference was calculated by dividing the maximum value by the minimum. Variables are divided into pre-synaptic at the top, post-synaptic in the middle, and combined pre- and post-synaptic at the bottom. See also Figure 3 and Table S3. component is as different from each other as possible, and thus gen- erates additional information about the original dataset. Together, these analyses of the developing NMJ indicate that there is more variation between muscles at P7 than at the ma- ture adult synapse; the post-synapse displays a comparably large amount of inter-muscle variation at both timepoints, while the pre-synapse shows greater heterogeneity between muscles during immaturity. Neuromuscular junctions from both P7 and P31−32 were plot- ted along the two most important PCs (Figure 4). PC1 on the x-axis accounts for 90.5% of the variation in the dataset and clearly sep- arated the two timepoints, with some muscles within timepoints also diverging (e.g. P7 FDB and TVA). PC2 on the y-axis explains only 4.9% of the variation within the dataset, but roughly separated NMJs across different muscles, with little to no separation of time- points. Distribution of muscles across PC1 and PC2 indicates that at P31−32, the forepaw lumbricals are most different from the TVA, while FDB and hindpaw lumbricals are most similar (Figure 4). This is corroborated by clustering analysis (Figure S1a) and pairwise sig- nificance testing between all muscles for all variables (Table S2). At P7, the FDB and hindpaw lumbricals are again most similar, while the TVA and ETA are most dissimilar to the FDB and hindpaw lumbricals (Figure 4). This is again supported by clustering analysis (Figure S1b) and pairwise significance testing (Table  S3). Generally speaking, NMJs at maturity were more variable than developing NMJs (greater spread of datapoints), especially along PC2, with the TVA showing | Nonetheless, regional distinctions in poly-innervation have been reported (Bixby and van Essen, 1979), so perhaps additional factors, such as muscle fibre type (Lee, 2019), contribute to the observed results (see below). The developing post-synapse showed a similar number of sig- nificant differences between muscles as the mature NMJ, with all variables showing distinctions, except for AChR perimeter, the number of AChR clusters and fragmentation (Figure 3c). Similar to pre-synaptic morphology, the TVA and ETA generally had the largest post-synaptic structures, followed by the forepaw lumbricals, then the FDB and hindpaw lumbricals. AChR area showed the greatest fold difference between muscles (3.16), while compactness showed the smallest (1.21; Table 3). The NMJ-morph software was used to assess pre-synaptic, post-synaptic and overlapping morphologies at the developing NMJ (Figure 3; Tables 3 and S3). Unlike at mature synapses, all but two variables, average branch length and axon diameter, showed signifi- cant distinctions between muscles at P7 (Figure 3b; Table S3). Similar to several pre-synaptic features at P31−32, most motor terminal variables for the TVA and ETA were larger than FDB and hindpaw Finally, consistent with the myriad of pre- and post-synaptic distinctions between muscles, the synaptic contact area showed significant variation (Figure 3d), and a fold difference between maxi- mum and minimum values of 3.58 (Table 3). Repeating the finding at P31−32, the percentage overlap between pre- and post-synapse did not differ (1.24-fold difference), once again confirming the possibil- ity of cross-muscle denervation analysis. 612  | Mean values for all NMJ variables were calculated for each muscle per animal from 20 NMJs, the Endplate area added most to PC1, with nerve terminal area, AChR perimeter and AChR cluster area also contributing. The endplate area strongly re- flects the overall size of the NMJ because it includes the area con- tained within the perimeter of α-BTX staining. Analysis of NMJs in nine different muscles from wild-type CD1 mice aged 6 weeks cor- roborates this finding that NMJ size drives the majority of variation between different muscles (Jones et al., 2016). The number of termi- nal branches and AChR perimeter contributed the most to PC2, with much smaller influences from poly-innervation and compactness. These differences in development/growth rate perhaps reflect the function of each muscle and its relative importance during early post-natal life. The TVA and ETA are the two muscles with the larg- est NMJs at both timepoints and that display the least change from P7 to P31−32. These two muscles are therefore perhaps some of the most well developed during the first week of life in mice, at least in terms of the size of pre- and post-synaptic structures (keeping in mind that poly-innervation is rife in the TVA at P7). The TVA has a role in respiration (Mesquita Montes et al., 2016), so has a clear need to be functional from birth. Furthermore, feeding behaviours of pups are likely to be more dependent on postural muscles and those that mediate larger movements of the limbs than perhaps those that facilitate finer movements of the paws. Being able to breathe and feed immediately after birth are clearly two of the most important early mouse behaviours, perhaps accounting for why the TVA and ETA muscles appear most well developed. These results indicate that the rates of NMJ development and growth are driven by their functional importance at birth and during the first weeks of life. Finally, an Eigencor plot was created, which depicts Pearson product moment correlation coefficients (r) between PCs and the four metadata variables of percentage fast twitch muscle fi- bres, animal weight, sex and age (Figure  S1d). PC1 correlated significantly with age (P < 0.001, r = 0.87), which is unsurprising as this PC clearly separated developing and mature NMJs, and weight (P < 0.001, r = −0.84), which is obviously impacted by age (Table S1). muscles is likely to differ, such that differences at P7 become smaller by P31−32. Supporting this idea is the observation that synapse elimination does not occur to the same extent by P7 in all five mus- cles. To assess morphological development and growth at the NMJ, the percentage change over time was calculated for each muscle, and two-way anovas performed on all 20 NMJ morphological prop- erties, with age and muscle being the two statistical test variables (Table S4). All morphological variables except for complexity showed a significant difference between ages. There is also a significant difference between muscles for most variables, except for aver- age branch length, axon diameter, AChR cluster area, compactness and overlap. Similarly, individual muscles show differences in most variables between timepoints, with at least one muscle displaying a difference in every variable (Table  S4). Additionally, several dif- ferent pre- and post-synaptic variables show a significant interac- tion (Figure 5), indicating that the amount of change occurring at the NMJ during post-natal development is muscle-specific, i.e. NMJs across different muscles do not develop and grow at the same rate between P7 and P31−32. Indeed, lumbricals of the hindpaw and FDB showed the greatest average change per morphological vari- able (260% and 303%, respectively), while the ETA (168%) and TVA (170%) the smallest (percentage values include poly-innervation; Table S4). the greatest irregularity at P31−32 and the ETA at P7. These pat- terns were corroborated by the clustering analyses (Figure S1a,b). This variability may be due to the constituent fibres of the imaged muscles; for instance, the TVA, which has the greatest variability at maturity, has the highest percentage of slow twitch fibres (~32%); thus, if a fibre/NMJ is selected at random, it has more chance of being slow-twitch than in the other muscles. This pattern is not ob- served at P7, but muscle fibre types are not yet fully determined at this age in mice (Schiaffino and Reggiani, 2011). That being said, the 100% fast twitch forepaw lumbrical muscle shows similar variability at P31−32, if not more, to the remaining three muscles. The variabil- ity may thus instead be linked to additional properties of the muscle, for example, differences in fibre or overall muscle sizes. To find which features contributed most to PC1 and PC2, a loading plot analysis was performed (Figure S1c), which shows how strongly each morphological variable drives each PC. Nevertheless, samples within timepoints did not appear to cluster by weight (Figure S1a,b), indicating that small differences in body weight at a particular timepoint do not greatly impact NMJ morphology. Age (r = 0.42) and weight (r = −0.41) also correlated with PC2 (P < 0.05), although less so. However, muscle fibre type correlated most significantly with PC2 (P < 0.001, r = −0.60), which indicates that fibre type percentage is possibly driving distinctions in NMJ morphology between the assessed muscles. No correla- tions were observed between sex and PCs, suggesting that up to the age of P31−32, there are no major differences between NMJs of male and female mice, which was corroborated by the clustering analyses (Figure S1a,b). Nonetheless, as the two sexes diverge in muscle mass and strength later in life, this may not continue to be observed. 3.6 | Fast twitch fibres are more likely to be innervated by smaller neuromuscular junctions A possible cause for the observed inter-muscular differences in NMJ morphology is the relative abundance of fast and slow twitch fibres. Indeed, NMJ size has been previously reported to be related to mus- cle fibre type (Prakash et al., 1996; Wood and Slater, 1997), and PC2, which separated NMJs across muscles, correlated significantly with the percentage of fast twitch muscle fibres (Figure S1d). Thus, all 3.4 | Neuromuscular junction endplate area is a powerful discriminator of developmental stage and muscle type To better understand which morphological variables changed most throughout NMJ development, a PCA was performed. PCA is an ex- ploratory data analysis tool widely used for dimensionality reduction because of its ability to condense large datasets in an interpretable way. The technique functions by collapsing the existing variables into new principal components (PCs) that are created through lin- ear combinations of the original dataset. During the creation of PCs, the variance between components is maximised so that each new MECH et al. 613 3.7 | Muscle fibre size does not dictate mature neuromuscular junction morphology Moving on to the influence of fibre type on additional NMJ vari- ables, no significant correlations were observed between the per- centage of fast twitch muscle fibres and any of the pre-synaptic or overlapping morphologies at either timepoint (Table S5). In contrast, a significant correlation was observed between muscle fibre type and endplate diameter at P31−32 (Figure S2). There was an inverse correlation, indicating that smaller endplate diameters are perhaps linked with fast twitch muscle fibres, although this pattern was not observed at P7. Correlation between fibre type percentages and the rate of NMJ development/growth between P7 and P31−32 in all 20 morphological variables was also assessed, but no significant asso- ciations were observed (Table S6). This suggests that fibre type has little to no influence on change in NMJ morphology over the first month of post-natal development. Instead, perhaps one or a com- bination of the myriad transmembrane receptors, secreted ligands and other trans-synaptic proteins found at the NMJ are driving these inter-muscle distinctions, several of which are known to be involved in the developmental process of synapse elimination (English, 2003; Singhal and Martin, 2011). Neuromuscular junction size has been reported to correlate with muscle fibre size in several different species (Harris, 1954; Nyström, 1968; Kuno et al., 1971; Balice-Gordon and Lichtman, 1990; Sieck et al., 2012), which may be contributing to the presented findings. The five wholemount muscles were therefore re-mounted after teasing so that the influence of muscle fibre diameters on NMJ struc- ture could be assessed (Figure 6a). Only muscles from the P31−32 timepoint were used because the sole morphological correlation with fibre type was identified at that age (Figure S2). Significant dif- ferences in fibre diameter between several muscles were observed (Figure 6b), with the ETA having the largest fibres and the forepaw lumbricals the smallest (2.05-fold difference). When fibre diameters were correlated with the percentage of fast twitch fibres, no signifi- cant relationship was found (P = 0.483, r = −0.419, data not shown). This indicates that fibre type does not impact fibre diameter across the five wholemount muscles that were analysed. This contrasts with the general assumption that fast twitch fibres have larger diam- eters (Stifani, 2014), and indicates that subsets of muscles may dis- play idiosyncratic fibre features, perhaps linked with overall size and thickness of the muscle, as has been observed previously (Alnaqeeb and Goldspink, 1987). across all five muscles. Furthermore, the percentage of fast twitch fibres reported for the TVA was taken from rat, because mouse data were unavailable. Not all muscles show similar slow and fast twitch fibre type percentages between these two rodents, for example, the plantaris is 0% slow twitch in mouse and ~8% in rat, with rat muscles reported to show a relatively ‘slower’ phenotype than mouse mus- cles (Bloemberg and Quadrilatero, 2012). The 68% fast twitch fibres in the TVA may thus be an underestimate for mice. Nevertheless, several muscles between the two species are comparable, for ex- ample, tibialis anterior, EDL and masseter (Schiaffino, 1974; Gorza, 1990; Bloemberg and Quadrilatero, 2012). Furthermore, given that the TVA is at one end of the fibre type spectrum of the muscles ana- lysed in this study, the impact on correlations of the mouse TVA hav- ing a different fast twitch percentage from rat may be less than if the TVA was found within the spectrum. In relation to this and the ap- plicability of the findings of this study for human muscles, fibre type percentages are also only infrequently conserved between mice and humans (Schiaffino and Reggiani, 2011), and there are major distinc- tions in NMJ morphology (Jones et al., 2017). NMJ variables at P7 and P31−32 were correlated with previously reported (Table 1) fast twitch muscle fibre percentages (Table S5). First, assessing synapse elimination, no correlations were ob- served between the percentage of fast twitch fibres and poly-inner- vation at both timepoints. This contrasts with findings of Lee (2019), who showed a significant correlation between slow twitch fibres and poly-innervation across EDL, soleus and sternomastoid muscles of P9 wild-type mice. This was directly corroborated by observations of individual fibres within the soleus muscle indicating a greater degree of poly-innervation at NMJs on slow fibres (Lee, 2019). The conflict between the results obtained here and the previous study could be due to several reasons, including the use of different time- points and muscles. Several additional publications found no link be- tween muscle fibre type and synapse elimination, albeit in different species (mouse vs. rabbit) and using different methods (direct vs. in- direct; Bixby and van Essen, 1979; Soha et al., 1987; Cramer and van Essen, 1995). Nevertheless, assessing neuronal inputs at the NMJ in combination with immunofluorescent fibre typing is a powerful tool to better understand the importance of fibre type on synapse elimination, and should therefore be used in the future to address this within the five wholemount muscles. 3.5 | Morphological change over neuromuscular junction development differs between muscles As there is more structural variety between muscles at develop- ing NMJs than mature synapses, the rate of maturation across the 614 MECH et al. 3.7 | Muscle fibre size does not dictate mature neuromuscular junction morphology Consistent with PC2 of the PCA, muscle fibre type may indeed influence aspects of mature NMJ morphology, with smaller end- plate diameters being significantly associated with fast twitch fibres (Figure  S2). Although all other parameters at both timepoints did not significantly correlate with fibre type, many showed the same general trend as endplate diameters, i.e. larger features of NMJ mor- phology tended to be observed in muscles with lower percentages of fast twitch fibres, and this was particularly true for the post-syn- aptic architecture. There are studies in individual muscles that both support (McArdle and Sansone, 1977; Wood and Slater, 1997) and contrast (Prakash et al., 1996) with this finding. Nevertheless, the fibre type percentages used here were drawn from several different published works, which is likely to impact consistency and accuracy To determine whether fibre size influences synaptic architecture, fibre diameters were correlated with all NMJ morphological variables at P31−32 (Table S7). No significant associations were observed, sug- gesting that fibre diameter does not play a major role in determining synaptic structure, with the caveat that individual measured fibres were not the same as those from which NMJs were studied. Similar to the poly-innervation phenotype, future work is therefore needed to immunohistochemically co-assess fibre types, their diameters and MECH et al. 615 AUTHOR CONTRIBUTIONS JNS conceived the experiments; AMM, JNS performed the research; AMM, ALB, JNS analysed the data; GS provided expertise and dis- cussion; AMM, JNS wrote the manuscript with input from all au- thors. All authors approved submission of this work. associated NMJ morphologies within the five muscles to address this more directly. This will be difficult to perform for all fibre subtypes, as many MHC-specific antibodies work only on non-fixed muscles (Rossor et al., 2020), while fixed samples are needed for SV2/2H3 staining; that being said, co-staining of fixed muscle with type I and IIa fibre markers has been reported (Sieck et al., 2012; Lee, 2019). ACKNOWLEDGEMENTS The authors would like to thank Dr. David Villarroel-Campos (University College London) for critical comments on the manu- script, and the Schiavo and Prof. Linda Greensmith laborato- ries (University College London) for providing such a supportive, stimulating and collaborative environment in which to work. This work was supported by the Medical Research Council Career Development Award (MR/S006990/1) [JNS], a Wellcome Trust Senior Investigator Award (107116/Z/15/Z) [GS], the European Union's Horizon 2020 Research and Innovation programme under grant agreement 739572 [GS], and the UK Dementia Research Institute Foundation award (UKDRI-1005) [GS]. The graphical ab- stract was created with BioRender. When normalised to muscle fibre size, it has been shown that slow twitch NMJs in rat diaphragm muscle cover a relatively greater area than fast twitch NMJs (Prakash et al., 1995b, 1996; Prakash and Sieck, 1998). To determine whether a similar pattern is observed in mice, the ratios of P31−32 endplate diameters and endplate areas to fibre diameters were calculated for each muscle and correlated to fibre type (Figure S3). These two morphological variables were chosen because endplate diameter significantly correlated with fibre type, while endplate area drove most NMJ variation in PC1 and is arguably the best proxy for overall NMJ size. While significant dif- ferences were observed between muscles (Figure 3a,c), no correla- tions between fibre type and relative endplate diameter or relative endplate area were observed (Figure S3b,d). This suggests that the pattern of NMJ size to fibre type ratio observed in rat diaphragm is not replicated in these mouse muscles. ORCID ORCID Anna-Leigh Brown  https://orcid.org/0000-0003-1493-208X Giampietro Schiavo  https://orcid.org/0000-0002-4319-8745 James N. Sleigh  https://orcid.org/0000-0002-3782-9045 DATA AVAILABILITY STATEMENT The data that support the findings of this study are available from the corresponding author upon reasonable request. The data that support the findings of this study are available from the corresponding author upon reasonable request. Using an ImageJ-based workflow, morphometric data on mouse NMJs from five different wholemount muscles were generated at early post-natal and adult timepoints. Developing NMJs were shown to have much greater inter-muscle variability in pre- and post-synaptic structures than mature NMJs, which only showed distinctions in their post-synapse. These differences appear to be independent of muscle fibre diameter, at least at P31−32. Moreover, NMJ growth was significantly different between mus- cles over this early post-natal period. It is well known that NMJ size and morphology are predictive of function, and that muscle fibre types differ in their functional requirements of neurotrans- mission (Kuno et al., 1971; Harris and Ribchester, 1979; Waerhaug and Lømo, 1994; Ribchester et al., 2004; Jones et al., 2016). Hence, NMJ morphologies at both timepoints were correlated with fast twitch muscle fibre percentages. Pre-synaptic neuronal structures did not associate with fibre type; however, there was a possible link between post-synaptic endplate diameter and fast twitch fibre percentage. In the future, it will be important to indepen- dently verify the fibre type composition of the muscle analysed here and to extend these analyses into additional wholemount and thicker muscles of varying sizes, including smaller predominantly slow twitch muscles and larger mainly fast twitch muscles. This study also provides useful, non-biased, baseline data on the pre- and post-synaptic morphology of mouse NMJs in five anatomi- cally and functionally diverse muscles at two different timepoints. These data can be used by the scientific community in future cross-study, comparative work assessing pathological changes oc- curring at the NMJ in mouse models of neuromuscular disease. CONFLICT OF INTERESTS The authors have no competing interest to declare. The authors have no competing interest to declare. and muscle. Frontiers in Aging Neuroscience, 6, 208. Prakash, Y.S., Miller, S.M., Huang, M. and Sieck, G.C. 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Additional supporting information may be found online in the Talbot, J. and Maves, L. (2016) Skeletal muscle fiber type: using insights from muscle developmental biology to dissect targets for susceptibil- ity and resistance to muscle disease. Wiley Interdisciplinary Reviews: Developmental Biology, 5, 518–534. Supporting Information section. Supporting Information section. How to cite this article: Mech AM, Brown A-L, Schiavo G, Sleigh JN. Morphological variability is greater at developing than mature mouse neuromuscular junctions. J. Anat. 2020;237:603–617. https://doi.org/10.1111/joa.13228 Tapia, J.C., Wylie, J.D., Kasthuri, N. et al (2012) Pervasive synaptic branch removal in the mammalian neuromuscular system at birth. Neuron, 74, 816–829. How to cite this article: Mech AM, Brown A-L, Schiavo G, Sleigh JN. Morphological variability is greater at developing than mature mouse neuromuscular junctions. J. Anat. 2020;237:603–617. https://doi.org/10.1111/joa.13228 Tarpey, M.D., Amorese, A.J., Balestrieri, N.P. et al (2018) Characterization and utilization of the flexor digitorum brevis for assessing skeletal muscle function. Skeletal Muscle, 8, 14.
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Influence of the Ambient Storage of LiNi0.8Mn0.1Co0.1O2 Powder and Electrodes on the Electrochemical Performance in Li-ion Technology
Batteries
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Citation: de Meatza, I.; Citation: de Meatza, I.; Landa-Medrano, I.; Sananes-Israel, S.; Eguia-Barrio, A.; Bondarchuk, O.; Lijó-Pando, S.; Boyano, I.; Palomares, V.; Rojo, T.; Grande, H.-J.; et al. Influence of the Ambient Storage of LiNi0.8Mn0.1Co0.1O2 Powder and Electrodes on the Electrochemical Performance in Li-ion Technology. Batteries 2022, 8, 79. https://doi.org/ 10.3390/batteries8080079 Academic Editor: Torsten Brezesinski Received: 5 July 2022 Accepted: 25 July 2022 Published: 28 July 2022 Keywords: NMC811; aging; lithium-ion cells; cathodes; transition metal oxides Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Influence of the Ambient Storage of LiNi0.8Mn0.1Co0.1O2 Powder and Electrodes on the Electrochemical Performance in Li-ion Technology Iratxe de Meatza 1,2,* , Imanol Landa-Medrano 1 , Susan Sananes-Israel 1, Aitor Eguia-Barrio 1, Oleksandr Bondarchuk 3, Silvia Lijó-Pando 1, Iker Boyano 1, Verónica Palomares 2 , Teófilo Rojo 2, Hans-Jürgen Grande 1,4 and Idoia Urdampilleta 1 1 CIDETEC, Basque Research and Technology Alliance (BRTA), 20014 Donostia-San Sebastian, Spain; ilanda@cidetec.es (I.L.-M.); ssananes@cidetec.es (S.S.-I.); aeguia@cidetec.es (A.E.-B.); slijo@cidetec.es (S.L.-P.); iboyano@cidetec.es (I.B.); hgrande@cidetec.es (H.-J.G.); iurdampilleta@cidetec.es (I.U.) 1 CIDETEC, Basque Research and Technology Alliance (BRTA), 20014 Donostia-San Sebastian, Spain; ilanda@cidetec.es (I.L.-M.); ssananes@cidetec.es (S.S.-I.); aeguia@cidetec.es (A.E.-B.); slijo@cidetec.es (S.L.-P.); iboyano@cidetec.es (I.B.); hgrande@cidetec.es (H.-J.G.); iurdampilleta@cidetec.es (I.U.) 2 Department of Organic and Inorganic Chemistry, Universidad del País Vasco (UPV/EHU), P.O. Box 664, 48080 Bilbao, Spain; veronica.palomares@ehu.eus (V.P.); teo.rojo@ehu.eus (T.R.) 3 International Iberian Nanotechnology Laboratory, Av. Mestre José Veiga, s/n, 4715-330 Braga, Portugal; alex.bondarchuk@inl.int 4 POLYMAT U i it f th B C t UPV/EHU A d T l 72 y ( ); g ( J ); p ( ) 2 Department of Organic and Inorganic Chemistry, Universidad del País Vasco (UPV/EHU), P.O. Box 664, 48080 Bilbao, Spain; veronica.palomares@ehu.eus (V.P.); teo.rojo@ehu.eus (T.R.) 3 I i l Ib i N h l L b A M J é V i / 4715 330 B P l y ( ) g ( J ) p ( ) 2 Department of Organic and Inorganic Chemistry, Universidad del País Vasco (UPV/EHU), P.O. Box 664, 48080 Bilbao, Spain; veronica.palomares@ehu.eus (V.P.); teo.rojo@ehu.eus (T.R.) 3 International Iberian Nanotechnology Laboratory, Av. Mestre José Veiga, s/n, 4715-330 Braga, Portugal; alex.bondarchuk@inl.int 4 4 POLYMAT, University of the Basque Country, UPV/EHU, Avda. Tolosa 72, 20018 Donostia-San Sebastian, Spain 4 POLYMAT, University of the Basque Country, UPV/EHU, Avda. Tolosa 72, 4 POLYMAT, University of the Basque Country, UPV/EHU, Avda. Tolosa 72, 20018 D ti S S b ti S i * Correspondence: imeatza@cidetec.es batteries batteries batteries batteries Abstract: Nickel-rich LiNi0.8Mn0.1Co0.1O2 (NMC811) is one of the most promising Li-ion battery cathode materials and has attracted the interest of the automotive industry. Nevertheless, storage conditions can affect its properties and performance. In this work, both NMC811 powder and electrodes were storage-aged for one year under room conditions. The aged powder was used to prepare electrodes, and the performance of these two aged samples was compared with reference fresh NMC811 electrodes in full Li-ion coin cells using graphite as a negative electrode. The cells were subjected to electrochemical as well as ante- and postmortem characterization. The performance of the electrodes from aged NM811 was beyond expectations: the cycling performance was high, and the power capability was the highest among the samples analyzed. Materials characterization revealed modifications in the crystal structure and the surface layer of the NMC811 during the storage and electrode processing steps. Differences between aged and fresh electrodes were explained by the formation of a resistive layer at the surface of the former. However, the ageing of NMC811 powder was significantly mitigated during the electrode processing step. These novel results are of interest to cell manufacturers for the widespread implementation of NMC811 as a state-of-the-art cathode material in Li-ion batteries. 1. Introduction It is already 30 years since the commercialization of the first Li-ion battery allowed the revolution of portable devices that have become an indivisible part of our day-to-day [1]. Laptops, cell phones and other electronic devices would not be as powerful if fed by other energy storage systems such as nickel-cadmium or nickel metal hydride batteries [2]. Nevertheless, Li-ion batteries are nowadays facing a more ambitious challenge: they have been nominated to enable the wide implementation of electric vehicles in our society [3]. Even though their driving range is approximately 400 km for most of the models and most of them are still not able to be charged fast, they fulfill the requirements of a wide sector of the population [4]. In fact, electric vehicle sales have significantly grown in recent years, and their growth is expected to be more prominent in the future [5]. For that aim, it is 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/). https://www.mdpi.com/journal/batteries Batteries 2022, 8, 79. https://doi.org/10.3390/batteries8080079 Batteries 2022, 8, 79 2 of 18 necessary to increase the energy density of the cells, which are the building blocks of the battery packs powering these electric vehicles [6]. necessary to increase the energy density of the cells, which are the building blocks of the battery packs powering these electric vehicles [6]. y p p g From a materials point of view, anodes in lithium-ion batteries principally consist of graphite as the active material [7]. However, silicon is being included in the formulation, in small fractions, to increase the capacity of the negative electrodes [8]. Indeed, the capacity is 10-fold higher than that of graphite, and the substitution of 10% of the graphite in the electrode by silicon would theoretically double the specific capacity (from ~360 mAh/g of graphite to ~680 mAh/g of 10% Si + 90% graphite) [9]. Moreover, research in anodes is not limited to the adoption of Si in the formulation: lithium metal is the next step on the ladder leading to the increase in energy density [10]. On the cathode side, lithium-ion cells based on the layered LiNixCoyMnzO2 (NMC, with x + y + z = 1) cathode material have gained much interest and have already been implemented in some electric vehicles [11]. 1. Introduction Moreover, it has been accepted that as a result of the increase of Ni content in the material, its capacity, as well as its energy density, are increased [12], even though this is usually calculated using a fixed cut-off voltage for the comparison of the different NMC samples, which, in turn, have different Li+ extractions ratios for the same cut-off voltage [13]. Thus, the original LiNi0.3Co0.3Mn0.3O2 (NMC111) has now been overtaken by LiNi0.5Co0.3Mn0.2O2 (NMC532), LiNi0.6Co0.2Mn0.2O2 (NMC622), LiNi0.8Co0.1Mn0.1O2 (NMC811) and ultimately LiNi0.9Co0.05Mn0.05O2 (NMC90505) [14]. While the latter is still beyond state-of-the-art, NMC811 is targeted as the next NMC material for the automotive industry [15]. Never- theless, the main trade-off of the extra capacity provided by higher nickel content is the decrease in the stability of the NMC811 towards oxidation upon charge [16]. In particular, the occurrence of irreversible reactions at higher potentials with the increase of Ni leads to the formation of an insulating layer at the surface of the NMC particles, the increase of the cell impedance, the evolution of oxygen, the loss of active material and, due to all these factors, the decrease of cycle life of the cells [14]. Thus, material developers are currently focused on optimizing the characteristics and testing conditions of these materials to obtain a successful balance between high energy density and acceptable cycling performance. The increase of Ni (or, to be more accurate, the decrease of Co) in the material presents another difficulty from a structural point of view. In the crystal structure of NMC, Co3+ is placed in the center of the hexagon, surrounded by Ni and Mn atoms [17]. The de- crease of Co content in the material favors the mixing of Ni and Li upon delithiation in a phenomenon known as cation mixing [18]. This occurs because the sizes of Ni2+ (0.69 Å) and Li+ (0.76 Å) are similar [19]. Thus, Li cations are trapped at the atomic positions where nickel ions should be located. Doping of the NMC has been suggested as a possible solution to minimize cation mixing [20]. Other alternatives involve the overlithiation of NMC or the decrease of its nickel content [17]. Finally, stability issues with high-nickel NMCs are not limited to the cycling stage. It has been observed that these materials are sensitive to exposure to ambient moisture, with higher instability caused by the increase of Ni content [21]. 2.1. Electrode Preparation NMC811 (T81R from Targray) was used as the active material in the positive elec- trode. The other two materials in the positive electrode were PVDF (Polyvinylidene fluoride, PVDF Solef® 5130) as the binder and carbon black (C-NERGY Super C65 from IMERYS Carbon & Graphite) as the conductive additive. A PVDF in N-methyl pyrroli- done (NMP) solution was initially prepared. Then, C65 was added while stirring. Finally, NMC811 was included in the solution, and the slurry was cast onto aluminum foil (ac- quired from Hydro, 20 µm thick) at Cidetec’s electrode manufacturing line. The loading of the coating was 20 mg/cm2 (4.0 mAh/cm2 calculated using 190 mAh/g as the capacity of NMC811, based on the datasheet of the material). After drying, the electrodes were calendered to 3.0 g/cm3 (37% porosity) at room temperature. The electrode formulation was 95% NMC811, 3% PVDF and 2% C65. The positive electrodes consisting of the aged NMC were prepared at a laboratory scale due to the low amount of material subjected to aging, which was not enough to work at the pilot plant scale. The slurry preparation method was the same as for the rest of the samples, but the coating of the current collector was conducted using a laboratory-scale doctor blade. The mass loading and the electrode density were the same for these electrodes and the electrodes prepared at the coating line. Despite different coating scales, FE-SEM imaging displayed no remarkable difference in the morphology of these electrodes (Figure S1). The negative electrode consisted of graphite (MEG-2C, SGL Carbon) as the active material, carbon black (C-NERGY Super C45 from IMERYS Carbon & Graphite) as the conductive additive, carboxymethylcellulose (CMC, Walocel 2000 from DOW) as a disperser and thickener and Styrene-Butadiene Rubber (SBR, BM-451B from ZEON) as the binder. CMC was initially dispersed in water, and C45 was added to this dispersion under stirring. Later, graphite and SBR were subsequently added to the solution. The slurry was cast onto copper foil (Furukawa, 8 µm thick), dried and calendered at Cidetec’s pilot plant facilities at room temperature. The electrode loading and final densities were 13 mg/cm2 (4.4 mAh/cm2, based on the datasheet of the material) and 1.45 g/cm3 (35% porosity), respectively. The electrode formulation was 94% graphite, 2% C45, 2% CMC and 2% SBR. 1. Introduction Indeed, the analysis of NMC stability towards ambient storage has been a research topic that has attracted the interest of many research groups in recent years. Jung et al. analyzed the influence of ambient storage on NMC811 using electrochemical cycling, Raman spectroscopy and X-ray photoelectron spectroscopy [22]. They observed the formation of a significant amount of nickel carbonate on the surface of the aged NMC811, which compromised the cycling stability of the cells based on this electrode. The formation of carbonates upon ambient storage was further analyzed by Sicklinger et al. [23]. In their work, NMC111 and NMC811 were aged in the same conditions and the surface contami- nants formed (carbonates and hydroxides) were quantified by means of thermogravimetric analysis coupled with mass spectroscopy. Interestingly, they demonstrated that the impuri- ties formed could be partially removed from the aged samples when they were subjected to a thermal treatment. In a recent study, Busà et al. [12] exposed NMC811 electrodes to different degrees of humidity. They evidenced that after 28 days of air exposure, the formation of lithium hydroxide and carbonate on the NMC surface significantly limited Batteries 2022, 8, 79 3 of 18 the cycling stability of the cells due to the decrease in the electronic and ionic conductivity of the aged samples. In all these works, aging was performed in the electrodes, while the influence of processing ambient-stored NMC811 powder was not discussed. Furthermore, it is not trivial for the industry to investigate the conditions in which high-nickel NMC powder and electrodes can be stored. This has motivated the present study. In this work, NMC811 was exposed to ambient air for 1 year, and electrodes were prepared by processing this powder. In parallel, electrodes were also prepared with fresh NMC811. Furthermore, electrodes prepared with fresh-NMC were also ambient-air exposed for 1 year. To sum up, this study compared fresh-NMC electrodes (fresh), NMC electrodes subjected to ambient storage (aged-electrodes) and electrodes prepared with aged NMC powder (aged-NMC). These electrodes were subjected to galvanostatic cycling, power tests, electrochemical impedance spectroscopy (EIS) and different materials characterization techniques. 2.3. Assembly of Coin Cells Ø = 17.7 and 16.6 mm disks were punched with a high precision disk cutter from the graphite and NMC coatings, respectively. These electrodes were dried for 16 h at 120 ◦C under a vacuum (10 mbar) prior to the assembly of the coin cells (CR2025, Hohsen). On the other hand, the covers of the coin cells were cleaned with ethanol in an ultrasonic bath for 15 min and dried at 60 ◦C for 1 h. The coin cells were assembled with a separator (18.92 mm diameter glass fiber, Albet GF 52) and 100 µL of electrolyte solution 1 mol/L Lithium hexafluorophosphate in (1:1 vol.%) ethylene carbonate:dimethyl carbonate + 2 wt.% vinylene carbonate–99.9% (1 M LiPF6 in EC:DMC (1:1) + 2% VC) purchased from Solvionic was added. Cell assembly was conducted in dry-room facilities (dew point = −40 ◦C). 2.4. Electrochemical Testing The cells were tested at 25 ± 1 ◦C inside a room controlled with air conditioning. All the cells were initially formed at C/20 C-rate between 4.2 and 2.8 V. A constant voltage (CV) step until C/40 was included. Then, three cells per type of positive electrode were subjected to each of the two different test plans. The first one consisted of a charge to 30% of State of Charge (SOC) and an impedance scan. Later, they were cycled at C/3 until reaching 80% of State of Health (SOH). In this case, the charge consisted of the CV step (to C/10) following the constant current step. During this galvanostatic cycling, periodical checkup tests were conducted. These tests consisted of a single cycle at C/20 used to perform a differential capacity analysis (dQ/dV) and a subsequent charge to 50% of SOC. At this stage of charge, a discharge pulse at 1C (0.19 A/gNMC) was applied for 30 s in order to calculate the DC resistance on the basis of Ohm’s law. Finally, cells were charged to 100% SOC and C/3 galvanostatic cycling was continued. After reaching 80% SOH, the cells were charged to 30% SOC and subjected to EIS analysis. This electrochemical testing protocol was also followed and described in previous work [24]. After EIS experiments, the cells were subjected to postmortem analysis. The second testing protocol consisted of a power test. The cells were charged to 50% of SOC and subjected to different discharge C-rates: 1C, 3C, 5C, 8C, 10C, 12C, 15C, 17C and 20C. These pulses were limited by time (10 s) and voltage (2.8 V). Galvanostatic experiments were conducted using a BaSyTec CTS Battery Test System. Furthermore, electrochemical impedance spectroscopy measurements were performed us- ing a Potentiostat/Galvanostat PGSTAT30 equipped with a Frequency Response Analyzer (FRA2, N4L). Impedance spectra were fitted using Zview software version 3.5f (Scribner). Spectra were obtained between 106 and 10−2 Hz with a perturbation amplitude of 10 mV at the beginning- and end-of-life of the cells. 2.2. Aging of the Electrodes/Materials Two different aging procedures were analyzed in the current study. On the one hand, NMC powder was stored under ambient air for 1 year at ~55% relative humidity and 15–25 ◦C and processed later to obtain electrodes with aged NMC (hereafter referred to as Aged-NMC). On the other hand, a coating prepared with fresh NMC was subjected to ex- posure to ambient air for one year, and after this period, electrodes were cut, vacuum dried (120 ◦C and 10 mbar for 16 h) and tested (hereafter Aged-electrode). The cells aged under these conditions were compared with NMC electrodes not subjected to aging (hereafter fresh). The same NMC811 batch was used during all the studies. NMC “fresh” powder Batteries 2022, 8, 79 4 of 18 and NMC fresh electrodes were stored and sealed in a dry room (dew point = −40 ◦C) to avoid aging from the ambient humidity. and NMC fresh electrodes were stored and sealed in a dry room (dew point = −40 ◦C) to avoid aging from the ambient humidity. 2.5. Postmortem Characterization After reaching 80% SOH, setting the cells at 30% SOC and conducting the EIS analyses, the cells were introduced in an Ar-filled glove box (MBraun) and disassembled inside ([O2] < 0.1 ppm, [H2O] < 0.5 ppm). The electrodes were thoroughly cleaned inside the glove box using dimethyl carbonate to remove the salts from the electrolyte. Afterwards, they were dried and sealed in bags to minimize their exposition to ambient air before their corresponding analysis. The positive electrodes were analyzed by means of X-ray diffrac- tion (XRD), field emission scanning electron microscopy (FE-SEM) and X-ray photoelectron spectroscopy (XPS). The crystal structure of the NMC electrodes after cycling, as well as before and after the aging protocols in electrode and powder forms, was analyzed by XRD using a Bruker D8 Discover diffractometer (Cu Kα radiation, λ = 0.154 nm) equipped with a LynxEye PSD detector. The diffractograms were recorded between 2θ = 10◦and 85◦ at 0.003◦s−1, while the obtained data were fitted by means of the Pattern Matching Batteries 2022, 8, 79 5 of 18 PSD 5 of 18 PSD method using the FULLPROF program [25]. The morphology of the same electrodes and powders was characterized by field emission scanning electron microscopy (FE-SEM, ULTRA plus ZEISS). The samples were prepared in the lab and stuck to the holder using carbon adhesive tape. FE-SEM micrographs at different magnifications (from ×200 to ×20,000) were obtained. In addition, the composition of the sample was confirmed using energy-dispersive X-ray spectroscopy (EDX). The XPS spectra of the same samples were collected at pass energies of 200 eV and 20 eV for survey spectra and individual elements, respectively. Charge neutralization was achieved with both low-energy electron and argon ion flood guns (0.5 eV, 100 µA and 70 µA current, respectively) during XPS measurements. The XPS spectra were peak-fitted using Avantage processing software (Thermo Fisher Scientific—East Grinstead, United Kingdom). The Lorentzian/Gaussian line shape and “Smart” background subtraction were used for peak fitting. Quantification was carried out using sensitivity factors provided by the Avantage’s library. Elemental composition depth profiling was carried out by means of the ion source (MAGCIS, Thermo Fisher Scientific) operated in monoatomic mode with beam energy 500 eV, current 0.9 µA. The sputtering rate established for Ta2O5 film was 0.045 nm/s. The Ar+ beam was raster-scanned over a 2 mm × 2 mm area. 3.1.1. Galvanostatic Cycling 1.1. Galvanostatic Cycling Li-ion cells consisting of fresh, aged-electrode (electrode exposed to calendar aging) and aged-NMC (electrodes prepared with NMC powder exposed to calendar aging) cath- odes were subjected to galvanostatic cycling at C/3 to analyze the discharge capacity and the capacity retention. The evolution of these two parameters upon the cycling life of the cells is shown in Figure 1. In addition, the Coulombic efficiency of these cells is shown in Figure S2. Li-ion cells consisting of fresh, aged-electrode (electrode exposed to calendar aging) and ged-NMC (electrodes prepared with NMC powder exposed to calendar aging) cathodes ere subjected to galvanostatic cycling at C/3 to analyze the discharge capacity and the capac- y retention. The evolution of these two parameters upon the cycling life of the cells is shown Figure 1. In addition, the Coulombic efficiency of these cells is shown in Figure S2. igure 1. Galvanostatic cycling experiments conducted at C/3 current rate: (a) discharge capacity er gram of NMC and (b) capacity retention during the cycle life of the cells. Figure 1. Galvanostatic cycling experiments conducted at C/3 current rate: (a) discharge capacity per gram of NMC and (b) capacity retention during the cycle life of the cells. gure 1. Galvanostatic cycling experiments conducted at C/3 current rate: (a) discharge capacity r gram of NMC and (b) capacity retention during the cycle life of the cells. Figure 1. Galvanostatic cycling experiments conducted at C/3 current rate: (a) discharge capacity per gram of NMC and (b) capacity retention during the cycle life of the cells. The capacity provided by the cells with the different positive electrodes is shown in igure 1a, while the corresponding SOH evolution is represented in Figure 1b. The dis- harge capacity achieved by the cells with the aged-electrodes was lower from the begin- ing-of-life (BOL) of these cells. In fact, they could only provide 166.4 ± 0.6 mAh/g and ompleted 213 cycles before reaching 80% of SOH. In any case, the unsuccessful electro- hemical performance provided by these cells was expected based on the works reported n the literature [22]⁠. On the other hand, the results obtained with the cells using the aged- NMC electrodes outperformed those with the aged-electrodes in terms of capacity reten- on and discharge capacity. 2.5. Postmortem Characterization btained data were fitted by means of the Pattern Matching method using the FULLPROF rogram [25]⁠. The morphology of the same electrodes and powders was characterized by eld emission scanning electron microscopy (FE-SEM, ULTRA plus ZEISS). The samples ere prepared in the lab and stuck to the holder using carbon adhesive tape. FE-SEM mi- ographs at different magnifications (from ×200 to ×20000) were obtained. In addition, the omposition of the sample was confirmed using energy-dispersive X-ray spectroscopy DX). The XPS spectra of the same samples were collected at pass energies of 200 eV and 0 eV for survey spectra and individual elements, respectively. Charge neutralization was hieved with both low-energy electron and argon ion flood guns (0.5 eV, 100 μA and 70 A current, respectively) during XPS measurements. The XPS spectra were peak-fitted us- g Avantage processing software (Thermo Fisher Scientific—East Grinstead, United King- om). The Lorentzian/Gaussian line shape and “Smart” background subtraction were used r peak fitting. Quantification was carried out using sensitivity factors provided by the vantage’s library. Elemental composition depth profiling was carried out by means of the n source (MAGCIS, Thermo Fisher Scientific) operated in monoatomic mode with beam nergy 500 eV, current 0.9 μA. The sputtering rate established for Ta2O5 film was 0.045 m/sec. The Ar+ beam was raster-scanned over a 2 mm × 2 mm area. 2 mm × 2 mm area. m/sec. The Ar beam w 3.1. Electrochemical Experiments 1. Electrochemical Experiments 3.1. Electrochemical Experiments 1. Electrochemical Experiments 3.1.1. Galvanostatic Cycling 1.1. Galvanostatic Cycling The lues obtained by the fitting of the spectra are compiled in Table S1. igure 2. Nyquist diagrams of a representative cell per type of positive electrode: fresh (black lines), ged-NMC (blue lines) and aged-electrode (red lines). Continuous and dashed lines are used for the pectra obtained at the beginning and the end of life, respectively. The equivalent circuits for the itting of the spectra are also included. Figure 2. Nyquist diagrams of a representative cell per type of positive electrode: fresh (black lines), aged-NMC (blue lines) and aged-electrode (red lines). Continuous and dashed lines are used for the spectra obtained at the beginning and the end of life, respectively. The equivalent circuits for the fitting of the spectra are also included. gure 2. Nyquist diagrams of a representative cell per type of positive electrode: fresh (black lines), ed-NMC (blue lines) and aged-electrode (red lines). Continuous and dashed lines are used for the ectra obtained at the beginning and the end of life, respectively. The equivalent circuits for the ting of the spectra are also included. Figure 2. Nyquist diagrams of a representative cell per type of positive electrode: fresh (black lines), aged-NMC (blue lines) and aged-electrode (red lines). Continuous and dashed lines are used for the spectra obtained at the beginning and the end of life, respectively. The equivalent circuits for the fitting of the spectra are also included. ure 2. Nyquist diagrams of a representative cell per type of positive electrode: fresh (black lines), ed-NMC (blue lines) and aged-electrode (red lines). Continuous and dashed lines are used for the ctra obtained at the beginning and the end of life, respectively. The equivalent circuits for the ing of the spectra are also included. Figure 2. Nyquist diagrams of a representative cell per type of positive electrode: fresh (black lines), aged-NMC (blue lines) and aged-electrode (red lines). Continuous and dashed lines are used for the spectra obtained at the beginning and the end of life, respectively. The equivalent circuits for the fitting of the spectra are also included. Fitting of the spectra showed that the ohmic resistance (Rohm) was very similar for the cells at their BOL. Thus, the differences obtained in the electrochemical experiments uld not be ascribed to differences in the casing or the contacts. It was also similar to the OL, with a slightly lower value for the cells with aged-electrodes due to their lower cycle e. 3.1.1. Galvanostatic Cycling 1.1. Galvanostatic Cycling In contrast, those made of aged-electrodes were the cells with the highest double layer sistance (Rdl) at both the BOL and EOL. This suggests the formation of a resistive layer the interfaces of the particles during storage-aging as reported by other authors [12]⁠. n the other hand, the cells with the aged-NMC electrodes had the lowest interface re- tance at BOL and EOL. Thus, the electrodes processed with the aged-NMC had lower uble layer resistance than those processed with fresh NMC both before and after being bjected to galvanostatic cycling. Finally, the resistances attributed to charge transfer (Rct d Rct-2) were also determined. On the one hand, the cells with the aged-electrodes and e fresh electrodes had similar values at their BOL. This suggests that storage-aging af- cted the particle surface, while the core of the particles remained initially unaffected. As Fitting of the spectra showed that the ohmic resistance (Rohm) was very similar for all the cells at their BOL. Thus, the differences obtained in the electrochemical experiments could not be ascribed to differences in the casing or the contacts. It was also similar to the EOL, with a slightly lower value for the cells with aged-electrodes due to their lower cycle life. In contrast, those made of aged-electrodes were the cells with the highest double layer resistance (Rdl) at both the BOL and EOL. This suggests the formation of a resistive layer in the interfaces of the particles during storage-aging as reported by other authors [12]. On the other hand, the cells with the aged-NMC electrodes had the lowest interface resistance at BOL and EOL. Thus, the electrodes processed with the aged-NMC had lower double layer resistance than those processed with fresh NMC both before and after being subjected to galvanostatic cycling. Finally, the resistances attributed to charge transfer (Rct and Rct-2) were also determined. On the one hand, the cells with the aged-electrodes and the fresh electrodes had similar values at their BOL. This suggests that storage-aging affected the particle surface, while the core of the particles remained initially unaffected. As can be seen by the EOL, the cells with the aged-electrodes had the highest charge transfer resistance, while those with fresh electrodes provided a lower value. In contrast, the cells assembled with the aged-NMC had the lowest charge transfer resistance both at the BOL and EOL. 3.1.1. Galvanostatic Cycling 1.1. Galvanostatic Cycling In fact, they achieved 313 cycles before their end-of-life (EOL) The capacity provided by the cells with the different positive electrodes is shown in Figure 1a, while the corresponding SOH evolution is represented in Figure 1b. The discharge capacity achieved by the cells with the aged-electrodes was lower from the beginning-of-life (BOL) of these cells. In fact, they could only provide 166.4 ± 0.6 mAh/g and completed 213 cycles before reaching 80% of SOH. In any case, the unsuccessful electrochemical performance provided by these cells was expected based on the works reported in the literature [22]. On the other hand, the results obtained with the cells using the aged-NMC electrodes outperformed those with the aged-electrodes in terms of capacity retention and discharge capacity. In fact, they achieved 313 cycles before their end-of-life (EOL) and, interestingly, provided higher discharge capacity in the first cycles than the cells consisting of the fresh coatings (189.9 ± 0.9 and 178.6 ± 0.6 mAh/g, respectively). The latter, however, exhibited longer cyclability as they completed 379 cycles above 80% SOH. The complementary electrochemical experiments facilitated a better perspective of th biliti f th ll ith th diff t iti l t d Fi tl th ll The complementary electrochemical experiments facilitated a better perspective of the capabilities of the cells with the different positive electrodes. Firstly, the cells were Batteries 2022, 8, 79 6 of 18 the 6 of 18 the subjected to EIS at BOL (just after formation) and EOL (after reaching 80% SOH). Note that EIS experiments were conducted after setting the cells at 30% SOC. The corresponding Nyquist plots, as well as the equivalent circuits used to fit the spectra, are shown in Figure 2. In addition, the assignment of the different features was reported in previous work [24] and is further discussed in the Supporting Information together with the Bode diagrams (Figure S3). The values obtained by the fitting of the spectra are compiled in Table S1. EIS at BOL (just after formation) and EOL (after reaching 80% SOH). Note that EIS exper ments were conducted after setting the cells at 30% SOC. The corresponding Nyquist plots, well as the equivalent circuits used to fit the spectra, are shown in Figure 2. In addition, e assignment of the different features was reported in previous work [24]⁠ and is further scussed in the Supporting Information together with the Bode diagrams (Figure S3). 3.1.1. Galvanostatic Cycling 1.1. Galvanostatic Cycling Error bars indicate the standard deviation between the different cells with the same cathodes. The experiments, consisting of a 1C discharge pulse of 30 s, evidenced that the DC re- nce was higher for the cells with aged-electrodes. This is in good agreement with the s of the galvanostatic cycling and the higher AC resistance determined by the EIS ex- ments. Furthermore, the DC resistance was very similar for the cells with the fresh and ged-NMC electrodes during all their cycle life. Thus, the increase of the DC resistance cell was discarded as the main reason for the lower cycling performance of the cells aged-NMC. As mentioned, each of the DC resistance measurements was coupled with 0 galvanostatic cycle. By analyzing the capacity obtained throughout the voltage pro- is possible to assign the plateaus to the different electrochemical reactions. The evolu- of the voltage and the area of the signals obtained during the cycle life of the cells is l to extract information on their aging processes. This technique is known as differen- pacity analysis (dQ/dV)[26]. The dQ/dV of cells with different cathodes during all their life are shown in Figure S4, while those from the 25th cycle (BOL) and the last dQ/dV The experiments, consisting of a 1C discharge pulse of 30 s, evidenced that the DC resistance was higher for the cells with aged-electrodes. This is in good agreement with the results of the galvanostatic cycling and the higher AC resistance determined by the EIS experiments. Furthermore, the DC resistance was very similar for the cells with the fresh and the aged-NMC electrodes during all their cycle life. Thus, the increase of the DC resistance of the cell was discarded as the main reason for the lower cycling performance of the cells with aged-NMC. As mentioned, each of the DC resistance measurements was coupled with a C/20 galvanostatic cycle. By analyzing the capacity obtained throughout the voltage profile, it is possible to assign the plateaus to the different electrochemical reactions. The evolution of the voltage and the area of the signals obtained during the cycle life of the cells is useful to extract information on their aging processes. This technique is known as differential capacity analysis (dQ/dV) [26]. 3.1.1. Galvanostatic Cycling 1.1. Galvanostatic Cycling Based on the conclusions obtained from the comparison of the aged and the fresh electrodes, a similar charge transfer resistance could be expected for these cells since the storage-aging should only affect the surface of the particles. The use of ambient aged NMC powder Batteries 2022, 8, 79 7 of 18 7 of 18 impacted the charge transfer resistance of these electrodes. This can be associated with the crystalline structure and will be discussed in Section 3.2.1 (XRD). ted with the crystalline structure and will be discussed in Section 3.2.1 (XRD). In addition, checkup experiments consisting of a C/10 galvanostatic cycle and a DC tance measurement were conducted every 25 cycles of the cycling performance ex- impacted the charge transfer resistance of these electrodes. This can be associated with the crystalline structure and will be discussed in Section 3.2.1 (XRD). ted with the crystalline structure and will be discussed in Section 3.2.1 (XRD). n addition, checkup experiments consisting of a C/10 galvanostatic cycle and a DC ance measurement were conducted every 25 cycles of the cycling performance ex- y In addition, checkup experiments consisting of a C/10 galvanostatic cycle and a DC resistance measurement were conducted every 25 cycles of the cycling performance experiments discussed above. The evolution of this resistance during the cycle life of the cells is shown in Figure 3. tance measurement were conducted every 25 cycles of the cycling performance ex ments discussed above. The evolution of this resistance during the cycle life of the is shown in Figure 3. re 3. DC resistance evolution during the cycle life of the cells with different cathodes: fresh k squares), aged-NMC (blue triangles) and aged-electrode (red circles). Error bars indicate the dard deviation between the different cells with the same cathodes. Figure 3. DC resistance evolution during the cycle life of the cells with different cathodes: fresh (black squares), aged-NMC (blue triangles) and aged-electrode (red circles). Error bars indicate the standard deviation between the different cells with the same cathodes. 3. DC resistance evolution during the cycle life of the cells with different cathodes: fresh squares), aged-NMC (blue triangles) and aged-electrode (red circles). Error bars indicate the rd deviation between the different cells with the same cathodes. Figure 3. DC resistance evolution during the cycle life of the cells with different cathodes: fresh (black squares), aged-NMC (blue triangles) and aged-electrode (red circles). 3.1.1. Galvanostatic Cycling 1.1. Galvanostatic Cycling The dQ/dV of cells with different cathodes during all their cycle life are shown in Figure S4, while those from the 25th cycle (BOL) and the last dQ/dV completed before reaching 80% SOH (EOL) are shown in Figure 4. leted before reaching 80% SOH (EOL) are shown in Figure 4. Positive and negative values in the Y-axis of the dQ/dV correspond to the charge and discharge reactions, respectively. Four main features can be observed in each of them, which are assigned to different electrochemical processes in the literature [26]. Starting from the charge reaction, the first peak (c1) is associated with the lithiation of the graphite anode. The other three peaks (c2, c3 and c4) are useful for analyzing the reactions in the NMC, even if graphite also contributes to these signals [27]. Subsequently, c2 is ascribed to the transition from the initial hexagonal phase (H1) to monoclinic (M) upon delithiation. In c3, NMC undergoes the transition from M to a second hexagonal phase (H2). The last peak (c4) is associated with the transition from H2 to a third hexagonal phase (H3). This process is usually associated with the degradation of the battery, particularly for high-nickel NMC. Finally, the vertical line at 4.2 V is related to the CV step of the charge: the longer the line, the higher the charge capacity that was obtained in this step. In the subsequent discharge, H3→H2, H2→M and M→H1 in combination with graphite delithiation reactions occur in d4, d3 and d2, while d1 is exclusively associated with the delithiation of graphite. dQ/dV Batteries 2022, 8, 79 8 of 18 8 of 18 in the BOL (Figure 4a) shows that the reactions occur at the same voltage regardless of the positive electrode. The size of the peaks was smaller for the cells with the aged-electrode, in good agreement with the lower capacity obtained. On the other hand, the cells with the fresh and aged-NMC electrodes were overlapped. The discharge capacities in these cycles were 193.6 ± 0.6 and 193.1 ± 1.3 mAh/g, respectively. 3.1.1. Galvanostatic Cycling 1.1. Galvanostatic Cycling The subsequent discharge capacities at C/3 were 175.6 ± 0.1 and 177.7 ± 1.8 mAh/g, respectively: the capacity of the cells was higher for the cells with the aged-NMC when the C-rate was increased from C/20 to C/3, which is in good agreement with the lower charge transfer and double layer resistances of the electrodes in these cells (determined by EIS experiments). W 8 of 18 Figure 4. dQ/dV of the cells (a) at the beginning and (b) end of life performed at C/20 between 2.8 and 4.2 V at C/20. Included cells with fresh (black line), aged-NMC (blue line) and aged-electrode (red line) cathodes. Main features of the scan are labelled as c1, c2, c3, c4, d1, d2, d3 and d4. Dashed lines are included to evidence the overpotential generated for the main signals. Figure 4. dQ/dV of the cells (a) at the beginning and (b) end of life performed at C/20 between 2.8 and 4.2 V at C/20. Included cells with fresh (black line), aged-NMC (blue line) and aged-electrode (red line) cathodes. Main features of the scan are labelled as c1, c2, c3, c4, d1, d2, d3 and d4. Dashed lines are included to evidence the overpotential generated for the main signals. gure 4. dQ/dV of the cells (a) at the beginning and (b) end of life performed at C/20 between 2.8 nd 4.2 V at C/20. Included cells with fresh (black line), aged-NMC (blue line) and aged-electrode ed line) cathodes. Main features of the scan are labelled as c1, c2, c3, c4, d1, d2, d3 and d4. Dashed nes are included to evidence the overpotential generated for the main signals. Figure 4. dQ/dV of the cells (a) at the beginning and (b) end of life performed at C/20 between 2.8 and 4.2 V at C/20. Included cells with fresh (black line), aged-NMC (blue line) and aged-electrode (red line) cathodes. Main features of the scan are labelled as c1, c2, c3, c4, d1, d2, d3 and d4. Dashed lines are included to evidence the overpotential generated for the main signals. igure 4. dQ/dV of the cells (a) at the beginning and (b) end of life performed at C/20 between 2.8 nd 4.2 V at C/20. Included cells with fresh (black line), aged-NMC (blue line) and aged-electrode ed line) cathodes. Main features of the scan are labelled as c1, c2, c3, c4, d1, d2, d3 and d4. 3.1.1. Galvanostatic Cycling 1.1. Galvanostatic Cycling Dashed nes are included to evidence the overpotential generated for the main signals. Figure 4. dQ/dV of the cells (a) at the beginning and (b) end of life performed at C/20 between 2.8 and 4.2 V at C/20. Included cells with fresh (black line), aged-NMC (blue line) and aged-electrode (red line) cathodes. Main features of the scan are labelled as c1, c2, c3, c4, d1, d2, d3 and d4. Dashed lines are included to evidence the overpotential generated for the main signals. Positive and negative values in the Y-axis of the dQ/dV correspond to the charge and discharge reactions, respectively. Four main features can be observed in each of them, which are assigned to different electrochemical processes in the literature [26]⁠. Starting rom the charge reaction, the first peak (c1) is associated with the lithiation of the graphite node. The other three peaks (c2, c3 and c4) are useful for analyzing the reactions in the NMC, even if graphite also contributes to these signals [27]. Subsequently, c2 is ascribed o the transition from the initial hexagonal phase (H1) to monoclinic (M) upon delithia- ion. In c3, NMC undergoes the transition from M to a second hexagonal phase (H2). The ast peak (c4) is associated with the transition from H2 to a third hexagonal phase (H3). This process is usually associated with the degradation of the battery particularly for The dQ/dV at the EOL of the cells is shown in Figure 4b. In addition, dashed lines were included in the figure to compare the voltage at which each of the features occurs compared to those in Figure 4a. Upon charge, c1 and c2 exhibited an increased voltage during their cycle life. Interestingly, the associated d1 signal upon discharge occurred at a lower voltage. A c1-d1 redox pair was exclusively ascribed to the delithiation/lithiation of graphite, while the other contributions were influenced by the reaction of NMC811. Thus, the increased overpotential of c1 and decreased overpotential of d1 could be associated with the slippage of the graphite and the NMC811 electrodes [28]. The lower overpotential for the cells cycled for a larger number of cycles suggests that the mismatch was increased with the number of cycles. The area of the c1-d1 peaks was slightly decreased when the Batteries 2022, 8, 79 9 of 18 9 of 18 curves at BOL and EOL were compared. 3.1.1. Galvanostatic Cycling 1.1. Galvanostatic Cycling However, this decrease was significantly lower than for the rest of the cells, indicating that the delithiation and lithiation of the anodes were not significantly affected even at the EOL of the cells. In contrast, the magnitude of the rest of the redox pairs except c3-d3 was decreased. In addition, d4 also almost maintained the original area by the EOL. Based on the discussion in Figure 4a, the decrease of the c2-d2 and c4-d4 peaks points out that the H1 →M and H2 →H3 transitions (and the subsequent M →H1) occur to a lower extent as a result of the EOL of the cells. It is difficult to evaluate the evolution of the c3-d3 redox pair as the signals are wider and might also partially include the H1 →M transition, which should really occur at a lower potential. Nevertheless, it is not likely that M →H2 and H2 →M are bottleneck reactions for charge and discharge, respectively. One of the most meaningful features in the charge profile by the EOL of the cells appeared at 4.2 V: as mentioned, the vertical line observed was due to the CV step included by the end of the charge. The presence of this charge step might be the reason for the very small differences observed between the d4 signals at the BOL and the EOL; once the CV step was completed, there were similar amounts of NMC811 in the H3 phase to subsequently obtain a similar H3 →H2 reaction capacity. The very different size of the c4 feature at BOL and EOL, in combination with the similar size of d4, confirms that a significant fraction of the charge is occurring at this CV step. Again, it is difficult to compare d3 at the BOL and EOL, while the area d2 is lower by the end of life. Considering both the slight voltage shift for c2 and the influence of the CV step, it can be deduced that, apart from the possible loss of active material, the overpotential caused by the increase of both the charge transfer and the double layer resistances might also be influencing the cycle life of the cells. Indeed, these dQ/dV were conducted on galvanostatic cycles performed at C/20 while regular cycling consisted of C/3 cycles; at the latter C-rate, the influence of overpotential was increased. 3.1.2. Power Tests The response of the cells to high currents was explored by conducting discharge power tests. After formation, the cells were set at 50% SOC and 1C, 3C, 5C, 8C, 10C, 12C, 15C, 17C and 20C discharges were conducted with 10 s and 2.8 V time and voltage cut-offs. Cells were charged to 50%SOC after each discharge pulse. The galvanostatic curves for the different samples are shown in Figure 5. 10 of 18 gure 5. Power tests conducted on the lithium-ion cells with the NMC811 electrodes subjected to fferent aging protocols. Figure 5. Power tests conducted on the lithium-ion cells with the NMC811 electrodes subjected to different aging protocols. ure 5. Power tests conducted on the lithium-ion cells with the NMC811 electrodes subjected to erent aging protocols Figure 5. Power tests conducted on the lithium-ion cells with the NMC811 electrodes subjected to different aging protocols. As could be expected based on the impedance analyses previously discussed, the cells th the aged-electrodes showed the worst power capability among those included in this udy. In fact, they were the sole type of cell unable to resist 8C pulses without reaching the scharge cut-off limit Again the power tests were in good agreement with the EIS results As could be expected based on the impedance analyses previously discussed, the cells with the aged-electrodes showed the worst power capability among those included in this study. In fact, they were the sole type of cell unable to resist 8C pulses without reaching the discharge cut-off limit. Again, the power tests were in good agreement with the EIS Batteries 2022, 8, 79 10 of 18 10 of 18 results of the cells with the fresh and aged-NMC electrodes. The latter, which had the lower interface resistance, could resist pulses up to 10C. Moreover, none of the cells with the fresh electrodes could be subjected to the same current without reaching the 2.8 V cut-off. j g To sum up the electrochemical experiments, cells assembled with the aged-electrodes provided the worst electrochemical performance in all the experiments conducted. They provided the lowest discharge capacity, capacity retention and power capability. This is in good agreement with the highest AC and DC resistances observed for these cells. The most unforeseen performance was obtained with the cells with aged-NMC. 3.2.1. X-ray Diffraction XRD analysis of the fresh and aged NMC was conducted to obtain information on the evolution of the crystal structure upon aging. The diffractograms of the fresh and aged NMC powders are shown in Figure S5. They were fitted with the R −3 m hexagonal space group (JCPDS No. 00-85-1968, Li0.89Ni1.01O2) [29]. Both a and c unit cell parameters were very similar before and after the ambient storage period (a = 2.8721(1) and c = 14.2082(8) Å for the fresh NMC and a = 2.8737(5) and c = 14.2063(8) Å for the aged NMC) evidencing that the aging procedure did not affect the crystal structure in the bulk of the NMC particles. XRD analyses were repeated on the electrodes after processing the aged and fresh NMC, and the diffractograms obtained are shown in Figure 6. The diffractogram of the aged-electrode is also included. 11 of 18 6. X-ray diffraction patterns of the fresh (black line), aged-NMC (blue line) and aged-elec- (red line) coatings. (003)/(104) peak intensity ratios, theoretical reflections for NMC, graphite uminum and unit cell parameters for the fitting of each diffractogram are included. Figure 6. X-ray diffraction patterns of the fresh (black line), aged-NMC (blue line) and aged-electrode (red line) coatings. (003)/(104) peak intensity ratios, theoretical reflections for NMC, graphite and aluminum and unit cell parameters for the fitting of each diffractogram are included. 6. X-ray diffraction patterns of the fresh (black line), aged-NMC (blue line) and aged-elec- ed line) coatings. (003)/(104) peak intensity ratios, theoretical reflections for NMC, graphite minum and unit cell parameters for the fitting of each diffractogram are included. Figure 6. X-ray diffraction patterns of the fresh (black line), aged-NMC (blue line) and aged-electrode (red line) coatings. (003)/(104) peak intensity ratios, theoretical reflections for NMC, graphite and aluminum and unit cell parameters for the fitting of each diffractogram are included. Aluminum from the current collector (Fm −3 m, cubic, JCPDS No. 03-65-2869) and ite from the graphitic fraction in the carbon black (R −3 m, hexagonal, JCPDS No. 5918) were included in the fitting together with the NMC phase. The unit cell pa- ers obtained are shown in the inset in Figure 6. The a and c cell parameters of the in the fresh and aged electrodes were similar, in good agreement with the results sed for the NMC samples before processing. 3.2. Materials Characterization 3.2. Materials Characterization 3.1.2. Power Tests They exhibited the lowest interface and charge transfer resistances, and their discharge capacity was higher during the first cycles, particularly with increasing C-rate. Their capacity retention, however, was lower than for the cells with fresh electrodes. In order to shed some light on the reasons leading to these controversial results, the electrodes and NMC powders were subjected to different materials characterization techniques. These experiments will be discussed in the following section. 3.2.2. Field Emission Scanning Electron Microscopy and Energy-Dispersive X-ray Spectroscopy The morphology and chemical composition of the NMCs and the electrodes subjected to different aging protocols, as well as the different electrodes after the C/3 cycling test, were analyzed by means of SEM and EDX. Even if these techniques are useful in other studies, they did not provide relevant information to the current one. The SEM images obtained are shown in Figure S7. In addition, electrodes did not show significant morphological differences either before or after their cycling life. However, it was interesting to observe that the carbon fraction was very similar for all samples (~30%). This evidences that even if a resistive layer, possibly made of carbonate [32], could be formed during the aging of the electrode, it is difficult to characterize it by means of this technique. For that aim, surface-characterization techniques, such as XPS, were used. 3.2.1. X-ray Diffraction Regarding the electrodes processed Aluminum from the current collector (Fm −3 m, cubic, JCPDS No. 03-65-2869) and graphite from the graphitic fraction in the carbon black (R −3 m, hexagonal, JCPDS No. 01-73-5918) were included in the fitting together with the NMC phase. The unit cell parameters obtained are shown in the inset in Figure 6. The a and c cell parameters of the NMC in the fresh and aged electrodes were similar, in good agreement with the results discussed for the NMC samples before processing. Regarding the electrodes processed Batteries 2022, 8, 79 11 of 18 11 of 18 with aged-NMC powder, a parameter was similar to those from the aged and fresh elec- trodes, but c was higher for the electrode prepared with aged-NMC. This means that the interlayer distance in the NMC crystal structure was enlarged when the aged-NMC was processed, which in literature is associated with cation mixing [30]. Furthermore, cation mixing is evidenced in the (003)/(104) peaks intensity ratio [31]. Fresh, aged-electrode and aged-NMC coatings subjected to XRD showed I003/I104 values of 1.79, 1.72 and 1.12, respectively. Values below 1.2, such as that of the aged-NMC electrode evidence cation mixing [29]. The increase of the interlayer distance is in good agreement with the lower impedance and higher initial discharge capacity observed for the cells with the electrodes made of aged NMC powder, as lithium ions should move more easily in the interlayer space. This characteristic could also allow a better response to high C-rates. Nevertheless, cation mixing is also associated with lower irreversibility towards crystal structure transitions, which ultimately leads to lower cycling performance [7]. As a result of the EOL of the cells, the crystal structure of the NMCs was not significantly different. The diffractograms of these samples are shown in Figure S6. 3.2.2. Field Emission Scanning Electron Microscopy and Energy-Dispersive X-ray Spectroscopy 3.2.3. X-ray Photoelectron Spectroscopy (XPS) In good agreement with the former work, Ni3+/Ni was 0.48 and 0.40 for the fresh and aged NMC samples, respectively. Thus, some Ni3+ undergoes reduction to Ni2+ upon ambient storage. This is analyzed in detail below. sociated with the presence of carbonate like species. The area of these signals was higher for the aged NMC, suggesting that the exposition of NMC to ambient air resulted in the formation of carbonates on the surface of this material [12]⁠. These carbonate-like species could not be found in the XRD analyses. There are two possibilities to explain this fact: (i) the carbonate layer was thin, and the total amount of this compound was negligible com- The O1s spectra of the electrodes under study in this work are displayed in Figure 8. Moreover, the same region of the scan is also shown for the postmortem cathodes in Figure S8. The information regarding the signals and the fitting of the spectra of the pristine and postmortem electrodes is compiled in Tables S2 and S3, respectively. he carbonate layer was thin, and the total amount of this compound was negligible com- pared with the total NMC and (ii) this carbonate was amorphous and, therefore, not de- ectable by XRD. On the other hand, the O1s spectra could be deconvoluted into two dif- erent features with maximum values at 529.2 and 531.7 eV [22]⁠. The latter is associated with O=C bonds, and the larger area of this signal for the aged NMC confirms the for- mation of a native carbonate layer at the NMC particle surface [34]⁠. Regarding the signal at 529.2 eV, it is attributed to O-Metal bonds [22]⁠. Again, the area of this feature is higher or the aged NMC. The interpretation of this fact is more challenging as the main contrib- utor to this feature should be the metal-oxygen bond coming from the NMC itself. How- ever, metal carbonates and single-metal oxides (NixOy, MnxOy, CoxOy) segregated from the active material NMC can contribute to this signal [35]⁠. Given the fact that the Li/Ni ratio calculated from the atomic ratio obtained in the fitting of the XPS spectra) was lower for p p p p y Opposite to the NMC powders, the O1s spectra of the surface of the processed elec- trodes did not evidence the presence of O-M. 3.2.3. X-ray Photoelectron Spectroscopy (XPS) Given the fact that the Li/Ni ratio (calculated from the atomic ratio obtained in the fitting of the XPS spectra) was Batteries 2022, 8, 79 12 of 18 r that 12 of 18 r that lower for the aged NMC (Li/Ni = 7 and Li/Ni = 5 for the fresh and the aged samples, respectively) and that the carbonates are mainly lithium carbonates, not contributing to the 529 eV component of the O1s, it can be speculated that aging leads to the segregation of metal oxides to the NMC surface. That would explain why the 529 eV component is stronger for the aged NMC. y p py ( ) The same samples previously subjected to XRD, SEM and EDX experiments were also analyzed by XPS. The C1s and O1s regions of the scan conducted on the fresh and aged NMC powder are shown in Figure 7. The peak area for the deconvoluted features is ncluded in Table S2. Figure 7. XPS spectra of the NMC powders: C1s region of (a) fresh and (b) aged NMC powder, and O1s region (c) of the fresh and (d) aged NMC. Figure 7. XPS spectra of the NMC powders: C1s region of (a) fresh and (b) aged NMC powder, and O1s region (c) of the fresh and (d) aged NMC. igure 7. XPS spectra of the NMC powders: C1s region of (a) fresh and (b) aged NMC powder, and O1s region (c) of the fresh and (d) aged NMC. Figure 7. XPS spectra of the NMC powders: C1s region of (a) fresh and (b) aged NMC powder, and O1s region (c) of the fresh and (d) aged NMC. C1s regions could be deconvoluted into three different contributions, which were as- signed to C-C (284.9 eV), C–OR (286.4 eV) and C=O (289.6 eV) bonds [33]⁠. The presence of he former might be ascribed to the carbon coating layer that is usually added to enhance he conductive properties of the NMC. On the other hand, the other two features are as- sociated with the presence of carbonate-like species. The area of these signals was higher Finally, Ni3+/Ni (Ni3+ fraction of the overall Ni, the latter calculated by the sum of Ni3+ and Ni2+) was determined for the two NMC powders following the methodology recently described elsewhere [21]. 3.2.3. X-ray Photoelectron Spectroscopy (XPS) The same samples previously subjected to XRD, SEM and EDX experiments were also analyzed by XPS. The C1s and O1s regions of the scan conducted on the fresh and aged NMC powder are shown in Figure 7. The peak area for the deconvoluted features is included in Table S2. C1s regions could be deconvoluted into three different contributions, which were assigned to C-C (284.9 eV), C–OR (286.4 eV) and C=O (289.6 eV) bonds [33]. The presence of the former might be ascribed to the carbon coating layer that is usually added to enhance the conductive properties of the NMC. On the other hand, the other two features are associated with the presence of carbonate-like species. The area of these signals was higher for the aged NMC, suggesting that the exposition of NMC to ambient air resulted in the formation of carbonates on the surface of this material [12]. These carbonate-like species could not be found in the XRD analyses. There are two possibilities to explain this fact: (i) the carbonate layer was thin, and the total amount of this compound was negligible compared with the total NMC and (ii) this carbonate was amorphous and, therefore, not detectable by XRD. On the other hand, the O1s spectra could be deconvoluted into two different features with maximum values at 529.2 and 531.7 eV [22]. The latter is associated with O=C bonds, and the larger area of this signal for the aged NMC confirms the formation of a native carbonate layer at the NMC particle surface [34]. Regarding the signal at 529.2 eV, it is attributed to O-Metal bonds [22]. Again, the area of this feature is higher for the aged NMC. The interpretation of this fact is more challenging as the main contributor to this feature should be the metal-oxygen bond coming from the NMC itself. However, metal carbonates and single-metal oxides (NixOy, MnxOy, CoxOy) segregated from the active material NMC can contribute to this signal [35]. 3.2.3. X-ray Photoelectron Spectroscopy (XPS) In addition, the feature ascribed to carbonate (C=O) with a maximum at 531.5 eV could be deconvoluted into two signals: the first one at 531.5 eV and the second at 532.5 eV, ascribed to C-O bonds. Interestingly, a small feature was also detected at 533.5 eV in the fresh sample. This is also associated with the presence of carbonate groups [33]. Thus, all of them will be quantified and discussed together in the following lines (hereafter referred to as carbonate-based compounds). Nevertheless, the presence of distinct carbonate-based compounds on the surface of the samples evi- dences the influence of the aging protocols on the chemical characteristics of the NMC coatings. This also explains the different double-layer resistances measured on the pristine cells by means of EIS. The area of the carbonate signal was increased in the following order: fresh ≈aged-NMC < aged-electrode. Thus, the formation of carbonate-like species on the surface of the NMC powder upon ambient storage previously discussed is in good agreement with the results observed at the electrode scale for the fresh and aged-electrode samples. Interestingly, the signal was very similar for the fresh electrodes and the electrodes made of aged-NMC. The fitting of the C1s region also supports this, as the areas of the Batteries 2022, 8, 79 13 of 18 13 of 18 signals at 287.0 eV and 288.9 eV (ascribed to C-O and C=O bonds, respectively) were similar for the fresh and aged-NMC electrodes and slightly higher for the aged-electrode. In a recent study, Wood et al. [36] reported the removal of surface impurities of different NMCs in water solutions. It is likely that the same occurred when the aged-NMC was processed in NMP. In this context, the key question is what occurs with the carbonate dissolved in the NMP. Three facts should be considered. Firstly, the overall amount of carbonate generated in the aging of NMC was very low as its presence could not be determined by bulk char- acterization techniques such as XRD or EDX. Indeed, its concentration should be several orders of magnitude lower than other electronic insulating components of the electrode, i.e., the PVDF binder. Secondly, a fraction of the carbonate dissolved in NMP could be lost as CO2 facilitated by the vigorous stirring during the NMC electrode slurry processing. 3.2.3. X-ray Photoelectron Spectroscopy (XPS) In fact, the native carbonate layer in NMC electrodes was reported to be removed and evolved as CO2 after cell assembly, just charging the cell to 3.9 V [34]. Additionally, thirdly, the rest of the carbonate could be homogeneously distributed throughout the electrode, minimizing its impact in the impedance of the NMC electrode. nd that the carbonates are mainly lithium carbonates, not contributing to the 529 eV ponent of the O1s, it can be speculated that aging leads to the segregation of metal o o the NMC surface. That would explain why the 529 eV component is stronger fo ged NMC. Finally, Ni3+/Ni (Ni3+ fraction of the overall Ni, the latter calculated by the sum o nd Ni2+) was determined for the two NMC powders following the methodology rec described elsewhere [21]⁠. In good agreement with the former work, Ni3+/Ni was 0.48 .40 for the fresh and aged NMC samples, respectively. Thus, some Ni3+ undergoes re ion to Ni2+ upon ambient storage. This is analyzed in detail below. The O1s spectra of the electrodes under study in this work are displayed in Figu Moreover, the same region of the scan is also shown for the postmortem cathodes in ure S8. The information regarding the signals and the fitting of the spectra of the pri nd postmortem electrodes is compiled in Tables S2 and S3, respectively. Figure 8. XPS spectra of the NMC811 coatings: O1s region of the (a) fresh, (b) aged-NMC and aged-electrode coatings. Figure 8. XPS spectra of the NMC811 coatings: O1s region of the (a) fresh, (b) aged-NMC and (c) aged-electrode coatings. igure 8. XPS spectra of the NMC811 coatings: O1s region of the (a) fresh, (b) aged-NMC an ged-electrode coatings. Figure 8. XPS spectra of the NMC811 coatings: O1s region of the (a) fresh, (b) aged-NMC and (c) aged-electrode coatings. Batteries 2022, 8, 79 14 of 18 14 of 18 Finally, the signals associated with the O-Metal bonds of the aged and the fresh elec- trodes could not be found at the electrode surface. In order to find this feature, the depth profile of the coatings was conducted on the three samples. The corresponding spectra, shown in Figure S9, show a similar O-M signal in the internal layers of the coatings, evidenc- ing that the bulk composition was similar for the different samples. In parallel, Ni3+/Ni was calculated for the different samples in the depth profile analyses. 3.2.3. X-ray Photoelectron Spectroscopy (XPS) The evolution of the Ni3+ with the depth for the three samples is shown in Figure S10. Interestingly, both the fresh and aged-electrode showed a marked difference between the surface coating and the first internal layer, evidencing that these aging conditions only affected the surface of the NMC particles (in good agreement with EIS results). Nevertheless, Ni3+/Ni increase in the aged-NMC electrode was gradual. This suggests that the processing of the aged NMC also affected the oxidation state of the internal layers of this transition metal oxide. This supports the hypothesis of the reduction of some Ni3+ to Ni2+ in the bulk of the material, which would facilitate the cation mixing determined by means of XRD fitting. Furthermore, the negative Ni3+/Ni value observed for the aged-NMC electrode suggests the presence of Ni-F bonds. The electrodes consisted of NMC811, PVDF and conductive C, and the sole chance for the origin of this negative value should be the interaction between the binder and the active material. Thus, it can be concluded that processing the electrodes with aged powder led to a stronger attachment between these two components. In a recent study, Lin et al. [37] evidenced that the presence of a nickel valence gradient from the particle surface (Ni2+ rich) to the core (Ni2+ poor) enhances the thermal and cycling stability of NMC811. They evidenced that this gradient mitigated the formation of micro-cracks during the charge–discharge cycles, which cause a decrease in the electronic conductivity and contribute to the capacity decay. In our study, we have observed decreased DC and AC resistances with the aged-NMC electrodes, in which this nickel valence gradient has been detected. Nevertheless, the cycle life of these cells was found to be shorter than for those consisting of fresh electrodes. It must be mentioned that, in our case, this gradient was not obtained on purpose, and it was caused together with detrimental issues such as cation mixing. In any case, this gradient could be the reason behind the unexpectedly high electrochemical performance of the aged-NMC electrodes. p g Finally, the signals in the O1s and C1s regions of the postmortem electrodes (Figure S8) evidenced the presence of similar amounts of carbonate for aged-NMC and fresh elec- trodes. The area of this feature was lower for the aged-electrode. 3.2.3. X-ray Photoelectron Spectroscopy (XPS) This indicates that most of this carbonate was formed upon cycling, with a higher presence of this component for the electrodes with higher cycle life. In these samples, the O1s spectra could be de- convoluted in two main peaks: the first at ~531.5 eV and the second at ~533.4 eV. The area of these two contributions was similar (~1:1 ratio). Based on the above discussion, the abundance of C=O and C-O bonds was similar. In addition, the area of the signal at ~290 eV in the C1s region, attributed to C-F bonds, decreased in the following order: aged-electrode > aged-NMC > fresh. Furthermore, the area of the signal at 587.5 eV in the F1s spectra (Figure S8), associated with the F-C bonds, decreased following the same trend. The increase of these signals could be associated with the formation of a thicker cathode electrolyte interphase (CEI) that would lead to an increased double layer resistance (in good agreement with the EIS results). In fact, this would enhance the overpotential of the charge and discharge reactions, which was observed as one of the causes promoting the EOL of the cells. 4. Conclusions As expected, the highest capacity retention was obtained with the fresh electrodes (379 cycles at SOH ≥80%). However, the capacity retention and power capability with the aged-NMC samples were well beyond the expectations: they achieved 313 cycles at SOH ≥80% and did not reach the cut-off voltage unless they were subjected to 10C pulses (the limit for fresh electrodes was 8C). Furthermore, the AC and DC resistances for the cells consisting of the latter cathodes were surprisingly low, and the capacity in the formation Batteries 2022, 8, 79 15 of 18 15 of 18 cycle was higher (189.9 ± 0.9 and 178.6 ± 0.6 mAh/g for cells with aged-NMC and fresh electrodes, respectively). XRD of the pristine electrodes revealed an increased interlayer distance and cation mixing after the processing of the aged NMC811. On the other hand, XPS analyses revealed: (i) the formation of a carbonate layer at the NMC particle surface, (ii) the redistribution of these carbonates upon the material processing, (iii) the stronger binder-NMC interactions after processing this aged NMC811 and (iv) the formation of a buffer region with higher Ni2+ fraction in these electrodes, in good agreement with the cation mixing evidenced by XRD. To the best of our knowledge, the latter three effects have not been previously discussed in the literature. It was determined that the storage of NMC811 electrodes might become a critical step in the production chain of battery manufacturers to avoid the formation of a resistive layer. On the other hand, storage-aging of NMC811 powder is not such a critical step; even though the material properties were affected, the electrochemical performance was in the same range as that obtained with fresh electrodes due to material washing during electrode manufacturing. The removal of the contaminants generated on the surface of the NMC particles might be advisable [23], but results in this work show that, during the electrode processing, their impact might be mitigated. This is a significant breakthrough, as most of the literature related to calendar aging of NMC811 points out the importance of avoiding the exposition of NMC811 to ambient air. In any case, lithium-ion cell manufacturers should thoroughly design their warehousing protocols to avoid the inconveniences described in this manuscript. Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/batteries8080079/s1, Figure S1. 4. Conclusions Cross section SEM images obtained from (a–d) fresh and (e,f) aged-NMC coatings at different magnifications: ×1000 (a,e), ×1500 (b,f), ×3500 (c,g) and ×10,000 (d,h); Figure S2. Coulombic efficiency values obtained in the galvanostatic cycling experiments conducted at the C/3 current rate. Zoom-in of the first 10 cycles is provided in the inset figure; Figure S3. Bode diagrams recorded at the beginning- and end-of-life of a cell; Figure S4. DVA of the cells with the (a) fresh, (b) aged-NMC and (c) aged-electrode cathodes during all their cycle life; Figure S5. XRD patterns of the fresh (black line) and aged (blue line) NMC powders; Figure S6. XRD patterns of the postmortem fresh (black line) and aged-NMC (blue line) and aged-electrode cathodes; Figure S7. SEM images were obtained from (a) fresh and (b) aged NMC powder. Micrographs at 5K magnification of (c) fresh, (d) aged-NMC and (e) aged-electrode coatings. Micrographs at 20K magnification of (f) fresh, (g) aged-NMC and (h) aged-electrode coatings. Micrographs at 10K magnification of (i) fresh, (j) aged-NMC and (k) aged-electrode postmortem (PM) electrodes; Figure S8. XPS spectra of the postmortem electrodes. (a) O1s, (b) C1s, and (c) F1s regions of the postmortem fresh cathodes. (d) O1s, (e) C1s, and (f) F1s regions of the postmortem aged-NMC cathodes. (g) O1s, (h) C1s, and (i) F1s regions of the postmortem aged-electrode cathodes; Figure S9. Depth profile XPS spectra of the different coatings. O1s regions of the (a) fresh, (b) aged-NMC and (c) aged-electrode coatings; Figure S10. Ni3+/Ni total fraction with the depth of the sample obtained by the methodology described in reference [21] for the fresh (black squares), aged-electrode (red circles) and aged-NMC (blue triangles) coatings; Table S1. Resistance values obtained from the fitting of the impedance spectra of the cells consisting of fresh-electrode, aged-NMC and aged-electrode cathodes and graphite anodes at the beginning (BOL) and end of life (EOL); Table S2. Signals from the XPS spectra of the fresh and aged NMC powder and results from the fittings, beam energy (BE) of these signals, full width at half maximum (FWHM) of the fittings, area calculated from the fittings and atomic percentage calculated from this area; Table S3. 4. Conclusions Signals from the XPS spectra of the fresh and aged-NMC and aged-electrode coatings before electrochemical tests and results from the fittings, beam energy (BE) of these signals, full width at half maximum (FWHM) of the fittings, area calculates from the fittings and atomic percentage calculated from this area; Table S4. Signals from the XPS spectra of the fresh and aged-NMC and aged-electrode electrodes obtained from disassembled cells after electrochemical tests and results from the fittings, beam energy (BE) of these signals, full width at half maximum (FWHM) of the fittings, area calculates from the fittings and atomic percentage calculated from this area. References [38–41] are cited in the supplementary materials. Batteries 2022, 8, 79 16 of 18 Author Contributions: I.d.M.: Conceptualization, Funding acquisition, Project administration, Su- pervision, Writing—review and editing. I.L.-M.: Conceptualization, Data curation, Formal analysis, Investigation, Writing—original draft. S.S.-I.: Conceptualization, Project administration, Supervision, Writing—review and editing. A.E.-B.: Conceptualization, Methodology, Supervision, Writing— review and editing. O.B.: Data curation, Formal analysis, Writing—review and editing. S.L.-P.: Methodology, Validation, Supervision. I.B.: Methodology, Validation, Supervision. V.P.: Supervision; Visualization; Writing—review and editing. T.R.: Supervision; Visualization; Writing—review and editing. H.-J.G.: Supervision; Writing—review and editing. I.U.: Funding acquisition, Resources, Supervision, Writing—review and editing. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by European Union’s Horizon 2020 research and innovation programme [No. 814389 (SPIDER project)]; and the CDTI—Ministerio De Ciencia e Innovación’s ‘CERVERA Centros Tecnológicos’ program [CER-20191006 (ALMAGRID project)]. V.P. and T.R. also wish to thank the funding from Gobierno Vasco/Eusko Jaurlaritza (IT-1226-19). Institutional Review Board Statement: Not applicable. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study are available on request from the corresponding author. The raw data are not publicly available due to confidentiality provisions in project consortium agreements. Acknowledgments: Authors acknowledge Carmen Palacios for the SEM micrographs, Enara Bueses, Iñigo Arzac and Andoni Contreras for the electrode manufacturing at CIDETEC’s pilot plant and Lara Liébana for assembling the coin cells. Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. References 1. Zhang, H.; Armand, M.; Rojo, T. Innovative Polymeric Materials for Better Rechargeable Batteries: Strategies from CIC Energigune. J. Electrochem. Soc. 2019, 166, A679–A686. [CrossRef] 2. Winter, M.; Barnett, B.; Xu, K. Before Li Ion Batteries. Chem. Rev. 2018, 118, 11433–11456. [CrossRef] [PubMed] 3. Chen, S.; Dai, F.; Cai, M. Opportunities and Challenges of High-Energy Lithium Metal Batteries for Electric Vehicle Applications. ACS Energy Lett. 2020, 5, 3140–3151. [CrossRef] [ ] [ ] 3. 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The Epidemiology and Detectability of Asymptomatic Plasmodium Vivax and Plasmodium Falciparum Infections in Low, Moderate and High Transmission Settings in Ethiopia&amp;nbsp;
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The Epidemiology and Detectability of Asymptomatic Plasmodium Vivax and Plasmodium Falciparum Infections in Low, Moderate and High Transmission Settings in Ethiopia Elifaged Hailemeskel Armauer Hansen Research Institute https://orcid.org/0000-0002-1757-8023 Surafel K Tebeje Armauer Hansen Research Institute Sinknesh Behaksra Armauer Hansen Research Institute Girma Shumie Armauer Hansen Research Institute Getasew Shitaye Department of Biomedical Sciences, School of Medical Sciences, Bahir Dar University Migbaru Keffale Armauer Hansen Research Institute Wakweya Chali Armauer Hansen Research Institute Abrham Gashaw Armauer Hansen Research Institute Temesgen Ashine Armauer Hansen Research Institute Chris Drakeley London School of Hygiene & Tropical Medicine Teun Bousema Department of Microbiology, Radboud University Medical Center Endalamaw Gadisa Armauer Hansen Research Institute Fitsum G Tadesse (  fitsum.girma@ahri.gov.et ) Malaria and Neglected Tropical Diseases Directorate, Armauer Hansen Research Institute, PO Box 1005, Addis Ababa, Ethiopia https://orcid.org/0000-00031931-1442 Research Keywords: Plasmodium infection, elimination, asymptomatic, transmission, nPCR, detectability, density distribution Posted Date: October 13th, 2020 DOI: https://doi.org/10.21203/rs.3.rs-90711/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published on January 22nd, 2021. See the published version at https://doi.org/10.1186/s12936-021-03587-4. Page 1/11 Abstract Background: As countries move to malaria elimination, detecting and targeting asymptomatic malaria infections might be needed. Here, we investigated the epidemiology and detectability of asymptomatic Plasmodium falciparum and P. vivax infections in different transmission settings in Ethiopia. Method: A total of 1093 dried blood spot (DBS) samples were collected from afebrile and apparently healthy individuals across ten study sites in Ethiopia from 2016 to 2020. Of these, 862 were from community and 231 from school based cross-sectional surveys. Malaria infection status was determined by microscopy or rapid diagnostics tests (RDT) and 18S rRNA based nested PCR (nPCR). The annual parasite index (API) was used to classify endemicity as low (API>0 and<5), moderate (API ≥5 and <100) and high transmission (API≥100) and detectability of infections was assessed in these settings. Results: In community surveys, the overall prevalence of asymptomatic Plasmodium infections by microscopy/RDT, nPCR and all methods combined was 12.2% (105/860), 21.6% (183/846) and 24.1% (208/862), respectively. The proportion of nPCR positive infections that was detectable by microscopy/RDT was 48.7% (73/150) for P. falciparum and 4.6% (2/44) for P. vivax. Compared to low transmission settings, the likelihood of detecting infections by microscopy/RDT was increased in moderate (Adjusted odds ratio [AOR]: 3.4; 95% confidence interval [95%CI]:1.6-7.2, P=0.002) and high endemic settings (AOR=5.1; 95%CI=2.6-9.9, P<0.001). After adjustment for site and correlation between observations from the same survey, the likelihood of detecting asymptomatic infections by microscopy/RDT (AOR per year increase = 0.95, 95%CI=0.9-1.0, P=0.013) declined with age. Conclusion: Conventional diagnostics missed nearly half of the asymptomatic Plasmodium reservoir detected by nPCR. The detectability of infections was particularly low in older age groups and low transmission settings. These findings highlight the need for sensitive diagnostic tools to detect the entire parasite reservoir and potential infection transmitters. Introduction Following considerable successes in the control of malaria in the last two decades, progress plateaued or stalled in many settings in Africa [1]. Ethiopia runs a successful malaria control program [2] that makes it one of the four countries (together with India, Rwanda, and Pakistan) that continues to maintain the declining trend in malaria burden [3]. As a result, the country is on track for a 40% reduction in incidence (together with Rwanda, Zambia, and Zimbabwe) and malaria mortality rates (together with Zambia) by 2020 [1]. To guide elimination efforts that currently targets 239 selected districts, the National Malaria Control Program (NMCP) of Ethiopia stratified the country into four strata using district level annual parasite index (API) data from 2017 [4] as malaria-free (API, 0), low (API, 0–5), moderate (API, 5–100), and high (API, ≥ 100) [4]. Despite its value, the adopted stratification lacks granularity and is not able to capture relevant spatial and temporal heterogeneities in low endemic settings [5, 6]. The unique epidemiology of malaria transmission in Ethiopia; the presence of strictly seasonal transmission in some settings and perennial transmission elsewhere, as well as different levels of co-endemicity of Plasmodium falciparum and P. vivax [2], calls for the use of tailored approaches to characterize the epidemiology of malaria. District level stratification that relies on malaria incidence data has limitations in settings where case numbers are extremely low. Incidence data are also sensitive to changes in care seeking behavior, rates of testing of suspected cases, and reporting completeness [7]. Screening approaches to determine the prevalence of (often asymptomatic) infections that are present in communities have great potential to define transmission intensity [8]. However, parasite prevalence estimates are greatly affected by parasite density distributions in communities that determine the detectability of infections by different diagnostics. Malaria elimination efforts may benefit from targeting all infections present in communities, irrespective of clinical presentation [9–11]. There is a growing body of evidence on the public health importance of asymptomatic malaria infections and their contribution to onwards malaria transmission in high [12, 13] and low transmission settings [13, 14]. Importantly, most asymptomatic infections detected in community surveys are of low parasite density and the proportion of all infections that are submicroscopic varies between settings [15]. Previous studies in Ethiopia detected a significant burden of asymptomatic P. falciparum and P. vivax infections [16–19]. These studies used different diagnostic techniques and sampling designs, making it difficult to compare parasite prevalence estimates or diagnostic performance indicators across settings. The aim of the present study was to understand the epidemiology of asymptomatic Plasmodium infections in different settings in Ethiopia and their detectability by microscopy, rapid diagnostics test (RDT) and molecular methods. Methods And Materials Study areas The study was conducted in ten districts (woredas) encompassing different transmission settings (Fig. 1). Malaria transmission is highly heterogeneous in Ethiopia and transmission intensity varies spatially and temporally [20]. Study sites representing low (n = 2), moderate (n = 4), and high (n=4) transmission settings as per the national stratification were selected from five administrative regions (Fig. 1). Low transmission settings include Gomma and Babile districts from Oromia region. Moderate transmission settings include Bahir Dar Zuria and North Achefer districts from Amhara region and Arba Minch Zuria from the Southern region and Mao Komo from Benishangul region. High transmission districts were from Gambela (Lare and Abobo), Amhara (Jawi), and Benishangul (Meng) regions. Study population and sample collection Samples were collected in community and school-based cross-sectional surveys from 2016–2020. Specifically, community-based surveys were conducted at Abobo, Lare, Mao-komo, Menge, and Gomma districts in 2016, Babile district in 2018, and Arba Minch Zuria district in 2020. School based surveys were conducted at North Achefer, Bahir Dar Zuria, and Jawi districts in 2017. For the school-based surveys, students were randomly selected from elementary school students stratified by age as described before [21] following protocols developed by Brooker and colleagues [22]. Page 2/11 Prior to recruitment of participants for community surveys, sensitization was undertaken by teams that involve study team members, village-based health extension workers, malaria focal person of the district, local administrators, and elderly. The study purpose, procedure, risk, and benefit were explained in local language. After this first step, volunteer community members were invited to join the study upon obtaining informed written consent and enrolled in the study on first come, first served basis. Finger prick blood samples (~300µL) collected from all participants were used to diagnose malaria using RDT (First Response® malaria Antigen pLDH/HRP2 P.f and Pan Combo Card Test, Premier Medical Corporation Ltd, Dist. Valsad, India) or thin and thick blood films, and to prepare dried blood spots (DBS) on 3MM Whatman filter papers (Whatman, Maidstone, UK). Malaria was diagnosed using RDT at Abobo, Lare, Mao-Komo, Menge, and Gomma districts whilst microscopy was used at the school surveys, Arba Minch Zuria and Babile districts. Detailed clinical and socio-demographic data were captured using a pretested semi-structured interview-based questionnaire. Axillary body temperature was measured for all participants. If a participant was found febrile (axillary temperature ≥37.5oC) or reports history of fever in the past 48 hours, malaria status was checked using RDT and treated immediately when found positive following the national treatment guideline [23]. DBS were air dried, protected from direct sunlight, and enclosed in zip locked plastic bags individually with self-indicating silica gel (Loba Chemie, Mumbai, India). Samples were transported at ambient temperature and stored at -20˚C until further use. Giemsastained thick and thin smears were read independently by two experienced malaria microscopists. A third expert microscopist was consulted in case of discordant results. Thick smear slides were declared negative if no parasites were detected after observing 100 fields under oil immersion (100X magnification). Species specific detection of Plasmodium parasites by 18S rRNA based nested polymerase chain reaction Genomic DNA was extracted from 6mm diameter DBS punches using Chelex-Saponin extraction method [24] In brief, DNA was eluted after an overnight lysis in 0.5% saponin (SIGMA)/PBS (SIGMA) buffer and washing step followed by boiling at 97 ˚C in 150 µL of 6% Chelex (Bio Rad) in DNase/RNase free water (SIGMA). From the final eluate, 80 µL was transferred into a new plate and stored at -20oC until further use. Plasmodium species identification was done by nested polymerase chain reaction (nPCR) that targeted the small subunit 18S rRNA gene as described before [25]. A positive control (for P. falciparum NF54 culture from Radboudumc, Nijmegen, The Netherlands; for P. vivax the malaria reference laboratory positive controls from the London School of Hygiene and Tropical Medicine, London, UK) and negative controls (PCR grade water) were run in every reaction plate. Amplified products were visualized using UV transilluminator (Bio Rad, USA) after electrophoresis using 2% agarose gels (SIGMA, ALDRICH) stained with Ethidium Bromide (Promega, Madison, USA). Statistical analysis For the school surveys, sample size was calculated based on protocols by Brooker and colleagues [22] for the original study that aimed at assessing longitudinal evaluation of parasite prevalence in school children [21]. For this study, 70.0% (231/330) of the students were successfully sampled. For the community surveys, we expected an overall prevalence of 6.8% asymptomatic Plasmodium infections based on previous observations [26-37] with a precision of 5%. Based on previous experience, a minimum of 75 samples for the school surveys and 114 for the community samples was targeted across the study sites [21]. Data was double entered into excel, compiled, checked for consistency, and analyzed using Stata version 15 (Stata corporation; College Station, TX, USA) and GraphPad Prism 5.3 (GraphPad Software Inc., CA, USA). Proportions were compared between categories using Fisher’s exact test and Pearson’s chisquared test where it was appropriate. Equality tests on unmatched data such as age between school and community surveys were tested by two-sample Wilcoxon rank-sum (Mann-Whitney) test. Generalized Estimating Equation (GEE) was used to allow parameter estimates and standard errors adjusted for clustering across the study sites; exchangeable correlation matrix and robust standard errors were used. Sample characteristics such as age, gender, and transmission intensity were tested in the model for their association with infection prevalence and roles as potential confounders. A 5% level of significance was considered in all cases. Results Characteristics of study participants A total of 1093 individuals, 231 from school (3 schools; 75–80 per school survey) and 862 from community surveys (7 surveys; 114–161 per study site) participated in the study. None of the participants was febrile at the time of sampling. Female participants constituted 43.5% (372/855) of community and 51.8% (118/228) of school surveys (P= 0.026). The overall median age of the participants was 16 years (Interquartile range [IQR]: 11–35). As expected, participants from the school surveys were younger (median age, 12; IQR, 11–14) than community surveys (median age, 23; IQR, 10–38; P < 0.001). Results are presented separately for community and school surveys, focusing on community surveys for the main comparisons (Table 1). Within the community surveys, participants from low (median age, 30; IQR, 18–45; n=232) and moderate (median age, 30; IQR, 12–42; n = 272) endemic settings were older than participants from high endemic settings (median age, 13; IQR, 8-28; n = 318; P < 0.001). Prevalence of asymptomatic malaria infection across the study sites In the community surveys, the overall prevalence of asymptomatic Plasmodium infections was 12.2% (105/860) by microscopy/RDT and 21.6% (183/846) by nPCR (Table 1); 24.1% (208/862) of participants were parasite positive by either nPCR and/or microscopy/RDT. When considering infecting Plasmodium species by nPCR, 16.4% (139/846) of samples were P. falciparum positive; 3.7% (31/846) were P. vivax and 1.5% (13/846) were mixed P. vivax and P. falciparum. Although the school surveys were from high and moderate transmission sites, there was overall lower Plasmodium infection prevalence in the school surveys than in the community surveys as measured by all methods combined (11.3% vs 24.1%; χ2= 17.9, P < 0.001) Page 3/11 Among the school surveys, the overall prevalence of asymptomatic malaria was 0.4% (1/231) by microscopy/RDT whilst 11.3% (26/231) were parasite positive either by nPCR or both methods combined. Of these nPCR positive malaria infections from the school surveys, 2.6% (6/231) were due to P. falciparum, 5.2% (12/231) were due to P. vivax, and 3.5% (8/231) were due to mixed P. falciparum and P. vivax species infections (Additional table 1 and Table 2). Across the community surveys, in high transmission settings, nPCR-based prevalence of malaria infection ranged from 17.6% (n/N) at Meng to 46.1% (47/102) at Lare district. In the moderate transmission sites, the nPCR-based prevalence was 29.3% (34/116) at Mao-Komo and 12.4% (20/161) at Arbaminch Zuria district. In low transmission sites, the overall nPCR infection prevalence was 9.4% (22/234). The overall microscopy/RDT based prevalence was 23.1% (81/351), 7.9% (22/277), and 0.9% (2/232), in high, moderate, and low transmission settings, respectively (Table 1). Among the community samples, the prevalence of Plasmodium infections detected by all methods combined was substantially higher for the high transmission settings (36.7%, 129/351; 95% CI, 31.9–41.9; P<0.001) compared to moderate (20.6%, 57/277; 95%CI, 16.2–25.8) and low transmission settings (9.4%, 22/234; 95%CI, 6.3–13.9). Moreover, the burden of asymptomatic Plasmodium infection was higher in the 5–15 age groups as measured by microscopy/RDT (20.7%, 50/241, P < 0.001) and nPCR (26.7%, 62/232, P = 0.008) (Table 1) as compared to under-five children and adults older than 15 years (Table 1). Detectability of asymptomatic Plasmodium infections in different endemicities Among community samples, microscopy/RDT detected 44.2% (80/181) of nPCR detected Plasmodium infections (Agreement=86.9%, κ=0.526, Table 2). All, but 8 RDT positive P. falciparum and 1 microscopy positive P. vivax sample, were also nPCR positive (Additional table 2). The likelihood that Plasmodium infected individuals (i.e. individuals who were parasite positive by any diagnostic method) were detected by RDT was increased for individuals living in higher transmission settings (AOR= 5.1, 95%CI = 2.6–9.9, P < 0.001) and individuals living in moderate transmission (AOR: 3.4, 95% CI=1.6-7.2, P = 0.002) compared to low transmission settings (Additional table 3, Fig.2). Age was an important predictor of asymptomatic malaria positivity by microscopy/RDT. After adjusting for site and correlation between observations from the same survey, a 5% decline in detection using microscopy/RDT was observed for every year increase of age from those that tested positive by all methods (AOR = 0.95, 95%CI = 0.9–1.0, P = 0.013). The parasite species composition and detectability varied between transmission settings (Fig. 2). Among the Plasmodium species detected in the community samples, the majority were attributable to P. falciparum (77.4%, 161/208) when all samples were combined. Of the nPCR detected P. falciparum-mono species infections (n=139) and mixed-species infections (n=13), microscopy/RDT successfully detected P. falciparum in 48.7% (73/150) of infections (Table 2). Of the nPCR detected P. vivax-mono infections (n = 31) and mixed-species infections (n = 13), microscopy/RDT successfully detected P. vivax in 4.6% (2/44) of infections (Table 2). Discussion This study describes the prevalence and detectability of asymptomatic Plasmodium infections in ten different transmission settings by nPCR and conventional diagnostics (i.e. microscopy/RDT). More asymptomatic infections were detected in high transmission settings by both methods. The detectability of asymptomatic Plasmodium infections using microscopy/RDT relative to nPCR increased as transmission intensity increases. As a result, most infections in low transmission settings were not detectable by microscopy/RDT. In Ethiopia, several cross-sectional studies have documented asymptomatic parasite carriage using conventional and molecular methods [33-35, 38, 39]. The current multi-site study allowed an assessment of factors influencing the prevalence of infections as well as their detectability by microscopy-RDT. The prevalence of asymptomatic Plasmodium infections in the current study was in the same range as other reports from high [34, 38] and moderate [27] transmission settings in Ethiopia and elsewhere [29, 35, 40, 41]. Consistent with other studies [39, 42, 43], the current study observed that microscopy/RDT detected fewer asymptomatic infections as compared to PCR. The proportion of Plasmodium infections that was detectable by microscopy/RDT increased with increasing in transmission intensity. Whilst this trend has been reported in meta-analyses for P. falciparum [40, 44, 45], it is striking that this trend is also apparent in the current study within one country affected by both P. falciparum and P. vivax. Moreover, the effect size was comparatively large with approximately 5-fold higher detectability of infections in high endemic settings compared to low endemic settings. The trend of increasing detectability with increasing transmission intensity may be attributable to the fact that asymptomatically infected individuals have higher average parasite densities in high transmission settings [44, 46]. Moreover, in low endemic settings individuals will receive fewer infectious bites with, due to the absence of super-infections, lower parasitemia over the course of infection [40, 47]. Low genetic diversity of the parasite population in low transmission settings may also contribute to rapidly acquired immunity to the specific clones [48], further limiting parasite density. An impact of immunity on parasite density and the detectability of infections is also illustrated by the negative impact of increasing age on the detectability on infections in line with the current study [49]. Lower parasite densities in P. vivax compared to P. falciparum [50, 51] also results in a low detectability of P. vivax infections by microscopy/RDT. This low density in P. vivax is mainly attributable to the parasite’s preference to infect reticulocytes [52, 53] that typically constitute less than 1% of the total erythrocyte population [54] and also to the early acquisition of immunity [53]. These findings have implications for estimates of the relative burden of P. falciparum and P. vivax infections. The introduction of sensitive molecular tools may thus improve the detection of P. vivax infections substantially. Since treatment strategies differ for P. falciparum and P. vivax, this is relevant for public health interventions. Page 4/11 Although RDT and microscopy were used separately in the study sites due to logistics reasons, the prevalence measured by conventional RDT and microscopy was assumed to be comparable [41]. Nine samples that were declared microscopy/RDT positive were negative by nPCR while seven samples that were detected P. falciparum positive by RDT were P. vivax positive by nPCR. False RDT positivity might be due to the presence of parasite antigens after adequate clearance of parasites which might explain the variation between RDT positivity and PCR negative detection among asymptomatic malaria infections [55, 56]. Hence, there is a possibility that RDT can be positive for lingering antigens of P. falciparum while missing the low-density P. vivax infection from the same patient. Conclusion Conventional diagnostics missed nearly half of the asymptomatic malaria reservoir detected by nPCR. Moreover, the detectability of asymptomatic Plasmodium infections in all endemic sites might reflect the long persistence of these infections from weeks up to months in high [57] as well as in low transmission settings [58, 59] even in the presence of effective control and elimination interventions. As these infections can have relevance for onward malaria transmission [13, 44, 60], a detailed understanding of the distribution, detectability, and contribution to the infectious reservoir of asymptomatic infections will greatly improve our ability to target all relevant infections. The wide scale presence of low-density infections calls for more in-depth studies on understanding parasite density oscillations, their relevance for malaria symptoms, and onward transmission to mosquitoes. Declarations Ethical statement The study protocol was approved by the Ethiopian National Research Ethics Review Committee (3.10/016/20), and the institutional ethics review boards of the Department of Biochemistry (Ref.No.SOM/DRERC/BCH005/2009) and the College of Natural Sciences (Ref.No. SOM/DRERC/BCH005/2009) at Addis Ababa University, and the Armauer Hansen Research Institute (P035/17, P024/17 and P032/18). Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Competing interests The authors declared that they have no competing interests. Funding The study was supported by the Armauer Hansen Research Institute ((via its core funding from Norwegian Agency for Development Cooperation and Swedish International Development Cooperation); the Netherlands organization for international cooperation in higher education (Nuffic) [grant number NFPPhD.14/150] to FGT; the European Research Council [ERC-2014-StG 639776] to TB; the Bill & Melinda Gates foundation [INDIE; OP P1173572] to TB, FGT and CD. The funders have no role in the in the design of the study, collection, analysis, interpretation of the data and in writing the manuscript. Author’s contributions FGT, EH, EG, CD and TB conceived the study, contributed to data analysis and critically commented on the manuscript. EH analyzed the data. EH and SKT drafted the manuscript. EH, SK, WC, GS and MK conducted the laboratory study. WC, MK, EH, SKT, AG, GS, TA collected blood samples. All authors read and approved the final manuscript. Acknowledgements We thank all the study participants for their willingness and local facilitators of the study sites for their support during the sample collection. We also thank the WHO certified microscopists (Tewabech Lema and Tsehay Orlando) at Adama Malaria Center for their support. We appreciate the regional and district health officers for their collaboration. The malaria team members and researchers at AHRI (Tizita Tsegaye, Tadele Emiru, Temesgen Tafesse, Mikiyas Gebremichael, Misgana Muluneh, Endashaw Esayas, Tsegaye Hailu, Haile Abera, Demekech Damte, Tiruwork Fanta, Senya Asfer, and Eyuel Asemahegn) played an important role in making the study successful. We are indebted to the drivers of AHRI for their support during the field sample collection. References 1. 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PMI: Malaria Operational Plan FY 2019 Addis Ababa: USAID, Ethiopia; 2019. 2019. https://reliefweb.int/sites/reliefweb.int/files/resources/fy-2019ethiopia-malaria-operational-plan.pdf Tables Table 1. Community-based prevalence of asymptomatic Plasmodium infection using nPCR and microscopy/RDT Attributes Gender Age group (years) Category Parasite prevalence by nPCR, P-value Parasite prevalence by microscopy/RDT, % (n/N) [95% CI] % (n/N) [95% CI] Male 22.5 (106/472) [18.9-26.5] 13.5(65/483) [10.7-16.8] Female 20.2 (74/367) [16.4-24.6] ≤5 21.5 (14/65) [13.1-33.3] 5-15 26.7 (62/232) [21.4-32.8] 0.442 10.7(40/372) [7.9-14.3] 20.7 (50/241) [16.1-26.3] <0.001 ≥15 16.9 (87/513) [13.9-20.5] 5.6 (29/515) [3.9-7.9] Study sites (n/N) Lare 46.1 (47/102) [36.6-55.8] 35.9 (41/114) [27.6-45.2] High transmission Abobo 34.2 (39/114) [26.1-43.4] 23.7 (28/118) [16.9-32.3] Meng 17.6 (21/119) [11.8-25.6] 10.1 (12/119) [5.8-16.9] Low transmission Mamo-Komo 29.3 (34/116) [21.7-38.3] 16.4 (19/116) [10.7-24.3] Arba Minch zuria 12.4 (20/161) [8.1-18.5] 1.8 (3/161) [0.6-5.6] Babile 15.4 (18/117) [9.9-23.2] 1.7 (2/117) [0.4-6.6] Gomma 3.4 (4/117) [1.3-8.8] 0.0 (0/115 [NA] <0.001 Transmission intensity <0.001 High 31.9 (107/335) [6.3-13.9] 23.1(81/351) [18.9-27.8] Moderate 19.5 (54/277) [15.2-24.6] 7.9 (22/277) [5.3-11.8] Low 9.4 (22/234) [6.3-13.9] 0.9 (2/232) [0.2-3.4] <0.001 Overall Prevalence (n/N) ------- 21.6 (183/846) [18.9-24.5] <0.001 12.2 (105/860) [10.1-14.6] Note: CI= confidence interval, API= Annual Parasite Index /1000 people. Age was missed for 40 samples. Gender was missed for seven samples. Page 8/11 0.232 10.6 (7/66) [5.1-20.7] 0.008 Moderate transmission P-value Table-2. Species specific asymptomatic malaria infection prevalence and detectability across the study sites using nPCR and microscopy/RDT from 20162020 Sample source and API Study sites Microscopy/RDT nPCR % [n/N] % [n/N] Pf Pv Mixed Proportion Pf Proportion of infec detected by micros (%) Pv Mixed Proportion (%) of Pv Any Pf 56.5 (%) of Pv Community- Lare* 33.3 0.0 2.6 7.3 41.2 0.9 3.9 10.6 57.4 [38/114] [0/114] [3/114] [3/41] [42/102] [1/102] [4/102] [5/47] [27/47] 17.8 0.0 5.9 25.0 28.1 3.5 2.6 17.9 58.9 [21/118] [0/118] [7/118] [7/28] [32/114] [4/114] [3/114] [7/39] [23/39] [20/35] 10.1 0.0 0.0 0.0 13.5 3.4 0.8 23.8 42.9 47.1 [12/119] [0/119] [0/119] [0/12] [16/119] [4/119] [1/119] [5/21] [9/21] [8/17] 20.2 0.0 2.8 12.3 26.9 2.7 2.4 15.9 55.1 55.1 [71/351] [0/351] [10/351] [10/81] [90/335] [9/335] [8/335] [17/107] [59/107] [54/98] Maokomo 16.4 [19/116] 0.0 0.0 0.0 18.9 9.5 0.8 35.3 50.0 65.2 [0/116] [0/116] [0/19] [22/116] [11/116] [1/116] [12/34] [17/34] [15/23] AMZ 0.0 0.6 1.2 50.0 3.1 2.5 45.0 10.0 13.3 [0/161] [1/161] [2/161] [3/6] 6.8 [11/161] [5/161] [4/161] [9/20] [2/20] [2/15] 6.8 0.4 0.72 13.6 11.9 5.8 1.8 38.9 35.2 44.7 [19/277] [1/277] [2/277] [3/22] [33/277] [16/277] [5/277] [21/54] [19/54] [17/38] 0.00 0.0 0.0 0.0 2.6 0.0 25.0 0.0 0.0 [0/115] [0/115] [0/115] [0/115] [3/117] [0/117] [1/4] [0/2] [0/1] 1.7 0.0 0.0 0.0 0.0 27.8 15.4 15.4 [2/117] [0/117] [0/117] [0/2] 11.1 [13/117] [5/117] [0/117] [5/18] [2/18] [2/13] 0.9 0.0 0.0 0.0 6.8 2.6 0.0 27.3 10.0 14.3 [2/232] [0/232 [0/232] [0/2] [16/234] [6/234] [0/234] [6/22] [2/20] [2/14] Grand Total 10.7 0.1 12.4 16.4 1.5 24.0 44.2 48.7 [92/860] [1/860] 1.4 [12/860] [13/105] [139/846] [31/846] [13/846] [44/183] [80/181] [73/150 Jawi 1.3 0.0 0.0 0.0 5.3 10.7 8.00 77.8 5.6 10.0 [1/75] [0/75] [0/75] [0/1] [4/75] [8/75] [6/75] [14/18] [1/18] [1/10] 0.0 0.0 0.0 0.0 5.00 2.5 75.0 0.0 0.0 [0/80] [0/80] [0/80] [0/80] 2.50 [2/80] [4/80] [2/80] [6/8] [0/8] [0/4] N. Achefer 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 [0/76] [0/76] [0/76] NA [0/76] [0/76] NA [0/76] NA NA Total 0.4 0.0 0.0 0.0 2.6 5.2 3.5 76.9 3.8 7.1 [1/231] [0/231] [0/231] [0/1] [6/231] [12/231] [8/231] [20/26] [1/26] [1/14] (API ≥100) Abobo¥ Meng Total Community – (API≥5& <100) Total Low Gomma§ (API>0 &<5) Babile Total Community School (API ≥100) School (API≥5& <100) School BDZ 0.8 [1/117] 4.3 3.7 [26/46 57.1 Note: AMZ= Arba Minch Zuria, BDZ= Bahir Dar Zuria, N. Achefer= North Achefer, Pf= P. falciparum, Pv= P. vivax, mixed= Pf+ Pv, Any= all species detected. Proportion of Pv is calculated together with mixed infections. API= annual parasite index /1000 people. * 12 DBS samples (11 Pf and 1 mixed infection positive by RDT) were missed from Lare for PCR. ¥ 4 DBS samples (3 Pf and 1 mixed infection positive by RDT) were missed from Abobo for PCR. § microscopy was not done for 2 samples that were Pf positive by PCR. Figures Page 9/11 Figure 1 Location of study sites and their Annual Parasite Index (API) as per the stratification by National Malaria Control Program based on 2017 data: BA= Babile, GO=Gomma, NA= North Achefer, AMZ= ArbaMinch Zuria, BDZ= Bahir Dar Zuria, MK= Mao-komo, AB= Abobo, LA= Lare, JA= Jawi, ME= Meng [61]. Note: The designations employed and the presentation of the material on this map do not imply the expression of any opinion whatsoever on the part of Research Square concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. This map has been provided by the authors. Page 10/11 Figure 2 Community and school- based surveys asymptomatic malaria infection prevalence and detectablity using nPCR and microscopy/RDT: black circles represent parasite prevalence by nPCR (x-axis) and proportion all infections detected by microscopy/RDT (left y-axis); white circles indicate parasite prevalence by microscopy/RDT (right y-axis). School surveys were (N. Achefer) North Achefer, BDZ (Bahir Dar Zuria) and Jawi. Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. Additionaltable3.docx Additionaltable2.docx Additionaltable1.docx Page 11/11
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Oligometastatic Malignant Solid Neoplasm
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Qeios · Definition, February 2, 2020 Open Peer Review on Qeios Open Peer Review on Qeios Oligometastatic Malignant Solid Neoplasm National Cancer Institute National Cancer Institute Qeios ID: CRAQSD · https://doi.org/10.32388/CRAQSD Source National Cancer Institute. Oligometastatic Malignant Solid Neoplasm. NCI Thesaurus. Code C161019. Code C161019. A malignant solid neoplasm that has metastasized to a limited number of sites— existing in a state intermediate between localized disease and widespread metastatic disease. Qeios ID: CRAQSD · https://doi.org/10.32388/CRAQSD 1/1
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Open access journal of surgery
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Editorial either directly from the spinal cord or indirectly from muscle. EMG is considered to evaluate nerve root integrity. In the earlier times of nascent spine surgery procedures, it was not possible to detect a neurological iatrogenic injury until the patient woke up from anesthesia. Now a day’s majority of spinal surgeons use intraoperative neurophysiological monitoring during procedures at cord level especially in deformity correction. The aim behind this is to limit the risk of accidental injury to the neural structures. The purpose of IONM (Intra – operative Neuromonitoring) is to reduce the risk to the patient of iatrogenic damage to the Spinal Cord, and/or to provide functional guidance to the surgeon and anesthesiologist. Neuromonitoring is helpful in spine surgeries to detect injury in conditions where one would expect a neurological deterioration during positioning of the patient, during operative maneuvers and also when corrections are attempted during kyphosis or scoliosis surgeries. To accomplish these objectives, a subordinate of the surgical team with special expertise in neurophysiology obtains and co-interprets triggered and spontaneous electrophysiologic signals from the patient periodically or continuously throughout the course of the operation. In general, a trained neurophysiologist attaches a computer system to the patient using stimulating and recording electrodes. The primary objective is to identify and prevent the development of a new neurologic deficit or worsening of a preexisting neurologic injury in a patient who is undergoing surgery. The intention behind spinal cord monitoring is to prevent intraoperative injury that results in irreversible paraplegia or Tetraplegia. As the IONM is done in anaesthetized patient, IONP is used to determine neurological status of patient during surgery. It is done by evaluating & recording the responses that are produced by the patient’s nervous system to various types of stimulations that are given by the monitoring devise & thus monitoring the neural pathway. As the patient is given anaesthesia, the Neuromonitoring is connected to the area of interest to be evaluated intra-op. These are connected to a computer devise where the signals are recorded time to time. These recording are started before the surgery and referred as baseline recordings and are continuously monitored throughout the surgery. Any substantial change or variations from the baseline values are used to determine whether significant neurological injury has occurred, The term significant change is used to refer to the degree of changes seen in the neurophysiologic recordings. Editorial Volume 5 Issue 1 - July 2017 DOI: 10.19080/OAJS.2017.05.555651 Open Access J Surg Copyright © All rights are reserved by Sohael M Khan Editorial Volume 5 Issue 1 - July 2017 DOI: 10.19080/OAJS.2017.05.555651 Open Access J Surg Copyright © All rights are reserved by Sohael M Khan Neuromonitoring in Spine Surgeries Sohael M Khan1*, Pradeep K Singh2, Kedar Phadke3, Shashank Jain4 and Pratik Patil4 1Assistant Professor and Consultant Spine Division, Department of Orthopaedics, Jawaharlal Nehru Medical College, India 2Chief Spine Surgeon, Department of Spine Surgery, Dr H L Hiranandani Hospital, India 3Chief Spine Surgeon, Department of Spine Surgery, Vadodara Institute of Orthopaedics, India 4Junior Resident, Department of Orthopaedics, Jawaharlal Nehru Medical College, India Submission: July 04, 2017; Published: July 06, 2017 *Corresponding author: Sohael M Khan, Assistant Professor and Consultant Spine Division, Department of Orthopaedics, J Medical College, Wardha, Maharashtra, India. Editorial Changes that are termed significant have been shown to correlate well with intraoperative injury to the nervous system. It is also possible that these changes, which are seen intra-operative, can arise from other technical errors like anaesthesia parameters or technical issues with the monitoring system. The main role of neurophysiologic monitoring team is to determine whether or not the significant changes noted intra-operatively are truly There a various modalities of Neuromonitoring used. The most common ones are SSEP (somatosensory evoked potential), transcranial electric motor evoked potentials (TcMEPs), and electromyography (EMG). SSEP’s help in evaluating dorsal column sensory pathway (TcMEPs) allow for a functional evaluation of the motor pathway, which includes the anterior or ventral corticospinal tracts of the spinal cord, plexus, and the peripheral nerve. Intermittently its applied transcranial electrical stimuli result in evoked potentials that can be recorded Open Access J Surg 5(1): OAJS.MS.ID.555651 (2017) 001 Open Access Journal of Surgery related to surgical procedure or they are due to technical error. The aim of the team is to inform the surgeon if there are any changes from the base line recording. decision is made based on an understanding of the type of surgery the patient is to undergo, the types of intraoperative injuries that may occur, and the mechanisms of how these injuries occur in surgery. By planning ahead with these issues in mind, the team can also attempt to anticipate the type of changes that might be expected to occur and the risky periods during surgery when these changes would likely occur. Ideally, the team would prospectively plan for interventions to reduce intraoperative neurologic injury [1]. Key to the success of intraoperative neurophysiologic monitoring is a good understanding of the capabilities and limitations of the neurophysiologic tests being monitored. These limitations should be understood not only by the intraoperative neurophysiologist, but also by the anesthesiologist and surgeon. For seamless integration of intraoperative neuro- physiologic monitoring into the intraoperative team, it is imperative that a good established working relationship eventually develops between the intraoperative neurophysiology team, anesthesiologist, and surgeon. This relationship allows for rapid communication between teams and a quick resolution of issues, optimizing the benefits of intraoperative neuro- physiologic monitoring for the patient. Synchronization of efforts among the surgical, anesthetic, and Neuromonitoring teams allows for smooth progress of surgery and minimizes conflict. Conclusion However, in today’s scenario Spine Surgery and Spinal cord injury are managed meticulously. Realizing the need of awareness of Spinal Cord the International Spinal Cord Society has decided to observe 5th September every year as SCI day [1]. Hence, the spine patients need more precise care and should have some knowledge regarding the same. Therefore, Neuromonitoring in spine surgery is becoming a need of the day. Neuromonitoring is an instrumental technique that provides information about iatrogenic neurologic injury. Precise understanding and high quality of medical practice of intra operative Neuromonitoring will be critical thus helping the surgeons to avoid injury to the cord and post - operative neurodeficit. Important considerations are choice of anesthetic technique and maintenance of blood pressure. Because inhalational agents tend to produce dose dependent changes in latency and amplitude of evoked potentials, their use should be avoided or minimized when Neuromonitoring is being used One of the first issues to tackle when planning for intra- operative neurophysiologic monitoring is to define the types of neurophysiologic tests to perform on a particular patient undergoing surgery. This This work is licensed under Creative Commons Attribution 4.0 Licens DOI: 10.19080/OAJS.2017.05.555651 References 1. Chhabra HS, Batra S (2016) Spinal Cord Injury and its Impact on the patient, Family and the society. Int J Recent Surg Med Sci 2(1):1-4. This work is licensed under Creative Commons Attribution 4.0 Licens DOI: 10.19080/OAJS.2017.05.555651 Your next submission with Juniper Publishers will reach you the below assets • Quality Editorial service • Swift Peer Review • Reprints availability • E-prints Service • Manuscript Podcast for convenient understanding • Global attainment for your research • Manuscript accessibility in different formats ( Pdf, E-pub, Full Text, Audio) • Unceasing customer service Track the below URL for one-step submission https://juniperpublishers.com/online-submission.php our next submission with Juniper Publishers will reach you the below assets this article: Sohael M K, Pradeep K S, Kedar P, Shashank J, Pratik P. Neuromonitoring in Spine Surgeries. Open Access J Surg. 2017; 5( : 10.19080/OAJS.2017.05.555651. 002
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miRNA Temporal Analyzer (mirnaTA): a bioinformatics tool for identifying differentially expressed microRNAs in temporal studies using normal quantile transformation
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Cer et al. GigaScience 2014, 3:20 http://www.gigasciencejournal.com/content/3/1/20 TECHNICAL NOTE Open Access miRNA Temporal Analyzer (mirnaTA): a bioinformatics tool for identifying differentially expressed microRNAs in temporal studies using normal quantile transformation Cer et al. Cer et al. GigaScience 2014, 3:20 http://www.gigasciencejournal.com/content/3/1/20 Cer et al. GigaScience 2014, 3:20 http://www.gigasciencejournal.com/content/3/1/20 Open Access Regina Z Cer1,2*, J Enrique Herrera-Galeano1,2, Joseph J Anderson3, Kimberly A Bishop-Lilly1,2 and Vishwesh P Mokashi1 Regina Z Cer1,2*, J Enrique Herrera-Galeano1,2, Joseph J Anderson3, Kimberly A Bishop-Lilly1,2 and Vishwesh P Mokashi1 * Correspondence: regina.cer@med.navy.mil 1Biological Defense Research Directorate, Naval Medical Research Center-Frederick, 8400 Research Plaza, Fort Detrick, MD 21702, USA 2Henry M. Jackson Foundation for the Advancement of Military Medicine, 6720-A Rockledge Drive, Suite 100, Bethesda, MD 20817, USA Full list of author information is available at the end of the article Abstract Background: Understanding the biological roles of microRNAs (miRNAs) is a an active area of research that has produced a surge of publications in PubMed, particularly in cancer research. Along with this increasing interest, many open-source bioinformatics tools to identify existing and/or discover novel miRNAs in next-generation sequencing (NGS) reads become available. While miRNA identification and discovery tools are significantly improved, the development of miRNA differential expression analysis tools, especially in temporal studies, remains substantially challenging. Further, the installation of currently available software is non-trivial and steps of testing with example datasets, trying with one’s own dataset, and interpreting the results require notable expertise and time. Subsequently, there is a strong need for a tool that allows scientists to normalize raw data, perform statistical analyses, and provide intuitive results without having to invest significant efforts. Findings: We have developed miRNA Temporal Analyzer (mirnaTA), a bioinformatics package to identify differentially expressed miRNAs in temporal studies. mirnaTA is written in Perl and R (Version 2.13.0 or later) and can be run across multiple platforms, such as Linux, Mac and Windows. In the current version, mirnaTA requires users to provide a simple, tab-delimited, matrix file containing miRNA name and count data from a minimum of two to a maximum of 20 time points and three replicates. To recalibrate data and remove technical variability, raw data is normalized using Normal Quantile Transformation (NQT), and linear regression model is used to locate any miRNAs which are differentially expressed in a linear pattern. Subsequently, remaining miRNAs which do not fit a linear model are further analyzed in two different non-linear methods 1) cumulative distribution function (CDF) or 2) analysis of variances (ANOVA). After both linear and non-linear analyses are completed, statistically significant miRNAs (P < 0.05) are plotted as heat maps using hierarchical cluster analysis and Euclidean distance matrix computation methods. Conclusions: mirnaTA is an open-source, bioinformatics tool to aid scientists in identifying differentially expressed miRNAs which could be further mined for biological significance. It is expected to provide researchers with a means of interpreting raw data to statistical summaries in a fast and intuitive manner. Keywords: microRNA, miRNA Temporal Analyzer, mirnaTA, Time series, Differential expression, DE, Quantile normalization, Linear model, Normal quantile transformation © 2014 Cer et al.; licensee BioMed Central Ltd. © 2014 Cer et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. miRNA Temporal Analyzer (mirnaTA): a bioinformatics tool for identifying differentially expressed microRNAs in temporal studies using normal quantile transformation Regina Z Cer1,2*, J Enrique Herrera-Galeano1,2, Joseph J Anderson3, Kimberly A Bishop-Lilly1,2 and Vishwesh P Mokashi1 Findings Development of mirnaTA The miRNA Temporal Analyzer (mirnaTA) is a by- product of a recent study where human peripheral blood mononuclear cells (hPBMCs) were exposed to bacterial pathogens and assayed at three different time points. Briefly, total RNA was extracted, miRNA sequencing li- braries were constructed, sequencing was performed on the MiSeq sequencer, and miRNA sequences were iden- tified from sequencing reads using a standard procedure (see in Additional file 1: Figure S1). Then, mirnaTA was used to identify differentially expressed miRNAs for further validation in the laboratory [Cer et al., unpublished data]. mirnaTA performs a number of distinct steps (Figure 1): (i) normalization of the raw count data into quantiles using Normal Quantile Transformation (NQT), (ii) ana- lysis of NQT data to locate any miRNA species which are either increasing or decreasing linearly using linear regression model, (iii) further analysis of miRNA species that did not fit in linear model by two different methods: (a) normal distribution function known as cumulative distribution function (CDF) if the number of time points is = < 3 or (b) analysis of variances (ANOVA) if the num- ber of time points is >3, (iv) generation of heat maps for any miRNAs that are differentially expressed with statis- tical significance (P < 0.05), and (v) providing intuitive HTML output formats. Some of these steps are explored in more detail below. MicroRNAs (miRNAs) are short single-stranded non- coding RNAs approximately 19–22 nucleotide long, which are critical regulators of gene expression and have been implicated in a wide range of physiological processes, such as apoptosis and growth, as well as pathological processes, including inflammatory responses, cancer, neurodegenera- tive and cardiovascular diseases [1-7]. This rapid growth is evident by the exponentially increasing number of miR- NAs reported in the recent Release 21 (June 2014) of miR- Base [8,9] which contains 35,828 mature miRNA products in 223 different species. Accompanying this growth is the development of many miRNA discovery bioinformatics tools including, but not limited to miRscan [10], miR- Finder [11], miRDeep [12] and miRanalyzer [13], to help researchers identify miRNAs from existing miRNA data- bases and/or predict novel miRNAs from NGS data. In recent years, the number of miRNA experiments in- volving multiple time-series has largely increased [14]. Input file requirement A d l i Any datasets resulting from RNA-seq experiments, array- based miRNA profiling or RT-PCR experiments, can be used as input data for the mirnaTA as long as the tab- delimited input files contain two columns, miRNA name and sequence read count of the miRNA. The number of time points that can be submitted to mirnaTA is a mini- mum of 2 to a maximum of 20. mirnaTA is able to handle to three replicates as follows: if the correlation coefficient (r) value between two replicates is more than or equal to 0.70, the count values from replicates are averaged and used in the study. Incremental correlation is applied for each additional replicate (see User Guide available at [25] for more details). If the r value is less than 0.7, the repli- cate data is not used in the calculations. The correlation coefficient, r value 0.7 was chosen arbitrarily as it was deemed reasonable for replicates to be considered suffi- ciently correlated. However, users are able to change this to any value although we do not recommend to use any value below 0.7. Despite the fact that there are several publications dealing with the statistical analysis of time-series expres- sion data, there are significant computational challenges in making sense of the massive and complex datasets produced by time-series experiments. There are a num- ber of differential expression (DE) tool packages including STEM [17], MaTSE [18], Linear Models for Microarray Data (LIMMA) [19], Significance Analysis of Microarrays (SAM) [20], Extraction of Differential Gene Expression (EDGE) [21], and Bayesian Estimation of Temporal Regu- lation (BETR) [22]. In miRNA research in particular, the two most commonly used DE tools are edgeR [23] and DEseq [24]. However, learning to use these tools requires the expertise of bioinformaticians as they often come with several package dependencies and not every research la- boratory employs these specialists nor has advanced com- puting infrastructure. There is a crucial need for a simple tool that would allow any bench scientist to analyze the differential expression of miRNAs or other subjects in temporal studies. Findings This is not surprising since biological processes are often dynamic, and therefore, a cross-sectional study based on a single time point would not provide important informa- tion that time-course studies can provide. Particularly in miRNA studies, researchers often implement multiple- time points to capture how certain miRNAs may display transient expression changes over the course of treatment or infection over time. For instance, Jayaswal et al. carried out a drug study involving a multiple myeloma cell line, U266, and consisting of six time points—0, 2, 4, 8, 24, and 48 hours with two biological replicates per time point for both miRNA and mRNA [15]. In another study by Li Z et al., miRNA expression profiles were assessed in the mouse livers in a time-course experiment at days 1, 3, 7, 15, 30 and 120 post treatment [16]. Abstract This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Cer et al. GigaScience 2014, 3:20 http://www.gigasciencejournal.com/content/3/1/20 Cer et al. GigaScience 2014, 3:20 http://www.gigasciencejournal.com/content/3/1/20 Page 2 of 7 Page 2 of 7 Data normalization by NQT Data normalization, a critical step of DE analysis, attempts to remove technical variability while preserving biological significance. Although there is no consensus on the best method for the normalization of microRNA sequencing Page 3 of 7 Cer et al. GigaScience 2014, 3:20 http://www.gigasciencejournal.com/content/3/1/20 Figure 1 An overview of the mirnaTA workflow using a three time point example. mirnaTA converts the tab-delimited, matrix raw data into quantiles using Normal Quantile Transformation (NQT). Then, using a linear regression model, mirnaTA identifies miRNA species which either increase or decrease linearly. Any miRNA species with P < 0.05 are considered to be statistically significant (shown in the black box). miRNAs that did not fit into a linear model with statistical significance are further analyzed using either cumulative distribution function (CDF) or analysis of variance (ANOVA) depending on the number of time points. Figure 1 An overview of the mirnaTA workflow using a three time point example. mirnaTA converts the tab-delimited, matrix raw data into quantiles using Normal Quantile Transformation (NQT). Then, using a linear regression model, mirnaTA identifies miRNA species which either increase or decrease linearly. Any miRNA species with P < 0.05 are considered to be statistically significant (shown in the black box). miRNAs that did not fit into a linear model with statistical significance are further analyzed using either cumulative distribution function (CDF) or analysis of variance (ANOVA) depending on the number of time points. medium computational complexity [28]. Briefly, raw data is normalized using NQT as follows: random numbers are drawn from the normal distribution based on the number of observations (here the number of miRNA species) and these numbers are sorted (Equation 1). Then quantiles are assigned according to each rank (Equations 2–3). All the equations included here are R language commands unless or otherwise specified: data, when seven commonly used normalization methods, namely, global normalization, Lowess normalization, trimmed mean method (TMM), quantile normalization, scaling normalization, variance stabilization (VSN) and in- variant method (IN), were evaluated, the Lowess normalization and the quantile normalization were found to be the best approaches [26]. It should be noted that edgeR uses TMM and DEseq uses a negative binomial (NB) model. mirnaTA is different in that it implements quantile normalization (also referred here as (NQT)) chosen for its robustness and reduced bias. Data normalization by NQT Quantile normalization is a rank-based procedure which scales data within each quantile separately and has been shown to have bias near zero relative to qRT–PCR [27]. Another factor considered in choosing quantile-based scaling is its rn ¼ sort rnorm no  of  miRNAs     ð1Þ qt ¼ rank x ð Þ=length x ð Þ ð2Þ nqt ¼ rn rank qt ð Þ ½  ð3Þ ð3Þ Cer et al. GigaScience 2014, 3:20 http://www.gigasciencejournal.com/content/3/1/20 Page 4 of 7 Cer et al. GigaScience 2014, 3:20 http://www.gigasciencejournal.com/content/3/1/20 Cer et al. GigaScience 2014, 3:20 http://www.gigasciencejournal.com/content/3/1/20 Identification of other differentially expressed miRNA species using a non-linear analysis Identification of other differentially expressed miRNA species using a non-linear analysis The remaining miRNA species that did not fit in a linear model are further analyzed depending on the number of time points in the experiment: (a) the cumulative distri- bution function (CDF) is applied when the number of time points is = <3 (Equation 9), and (b) without the as- sumption of equal variances for all groups, the analysis of variance (ANOVA) [29] is applied when the number of time points is >3. Similar to linear analysis criteria, any miRNAs with P <0.05 are considered to be statistically significant. Conclusions mirnaTA is an open-source bioinformatics tool that can be run in Linux, Mac or Windows with Perl and R pack- age dependencies. While there are many other time-series analysis tools available, the main advantages of mirnaTA are: cdf pval ¼ 1−pnormððas:numeric tpn ð Þ −as:numeric tp1 ð ÞÞ; mean ¼ 0; sd ¼ 1; lower:tail ¼ TRUE; log:p ¼ FALSEÞÞ where tp1 ¼ nqt data f or time point 1 and tpn ¼ nqt data f or time point 2 or time point 3 ð9Þ (1). Simplicity: Even though it is a command line program and users need to be aware of corrections for multiple testing, they need to provide only one input file and enter one line of Perl syntax, and do not need to have a strong statistical or computational background. ð9Þ (2). Reliability: The statistically significant miRNA species identified by linear regression model have met very stringent criteria, and therefore could be further examined for confirmation or validation in wet laboratories. anovapval ¼ oneway:test y∼x ð Þwhere y ¼ nqt data and x ¼ time points divided into two groups ð10Þ Identification of differentially expressed miRNAs by a linear regression model y = the number of time points. For example, to run mir- naTA for a dataset with three time points, two replicates and correlation coefficient value of 0.85 (default is 0.7), the syntax would be “perl nmrc_mirnata.pl -i example_ datasets/3tp2rep_dataset -n 2 –t 3 –c 0.85”. To demon- strate the functionality of mirnaTA, a comprehensive User Guide with examples using different datasets and expected results are provided at our SourceForge [25] page and also archived in GigaDB [30]. Using a linear regression, quantile transformed data is analyzed to identify any miRNA species which display either increasing or decreasing expression. Using NQT data over different time points, the corresponding P- values, intercept, and slope values are calculated (Equations 4–8), and any miRNAs with P <0.05 are con- sidered to be statistically significant: lmod ¼ lm y ∼x ð Þ where x ¼ time; y ¼ NQT data ð4Þ s ¼ summary lmod ð Þ ð5Þ lmod ¼ lm y ∼x ð Þ where x ¼ time; y ¼ NQT data ð4Þ s ¼ summary lmod ð Þ ð5Þ Visualizations of mirnaTA outputs p mirnaTA provides a rich source of HTML format data, including raw data, quantile transformed data, a heat map of differentially expressed miRNAs (P < 0.05) which were identified to be linearly increasing or decreasing using a linear regression model, as well as a heat map of differentially expressed miRNAs (P < 0.05) which were identified to be increasing or decreasing using either a cumulative distribution function (CDF) or an analysis of variance (ANOVA), if present. Also included are several intermediate files including a list of significant miRNA species with their P-values, slope, and intercept values (Figure 2). All these information is integrated into an HTML interface which can be viewed in any web browser. It should be noted that the heat map text files may also be visualized in other software packages such as PermutMatrix [31]. p ¼ as:characterð pf ðs$ fstatistic 1½ ; s$ fstatistic 2½ ; s$ fstatistic 3½ ; lower:tail ¼ FALSEÞÞ ð6Þ intercept ¼ s$coefficients 1½  ð7Þ slope ¼ s$coefficients 2½  ð8Þ d ifi i f h diff i ll d iRNA Availability and requirements (4). Wide applicability: The mirnaTA can be used to analyze any kind of data (gene, weather data, weight, etc.), and does not have to be restricted to miRNA data. All a user has to do is to replace the miRNA name in the first column with another subject of study. Availability and requirements Project name: miRNA Temporal Analyzer (mirnaTA) Project home page: http://sourceforge.net/projects/mirnata Operating system (s): Linux, Mac and Windows Programming language: Perl and R Other Requirements: None License: The GNU Lesser General Public License, version 3.0 (LGPL-3.0) A i i b d i N In summary, although mirnaTA does not yet provide researchers with “a push-button” solution for differential expression analysis, it gives them the power to use a simple Perl syntax to not only identify differentially expressed miRNAs, but also automatically generate intui- tive graphical outputs. This simple and automatic imple- mentation of previously detached concepts in mirnaTA could be instrumental in saving a significant amount of time for many basic research scientists. Any restrictions to use by non-academics: None Syntax for running mirnaTA All a user has to do is to replace the miRNA name in the first column with another subject of study. Syntax for running mirnaTA (3). Practicality: The mirnaTA only requires a simple input file which can be prepared by anyone, takes up to three replicates and 20 time points, and generates publication quality graphics in PNG format. The syntax to run mirnaTA is simple, as follows: perl nmrc_mirnata.pl -i < input_file > −n < x > −t < y>, where nmrc_mirnata.pl is the wrapper Perl script, < input_file > is a tab-delimited, matrix file, x = the number of replicates, Cer et al. GigaScience 2014, 3:20 http://www.gigasciencejournal.com/content/3/1/20 Cer et al. GigaScience 2014, 3:20 Page 5 of 7 Figure 2 mirnaTA Outputs. (A) A Portable Network Graphic (PNG) image showing the raw data (before normalization) vs. NQT data (after normalization). (B) A heat map of differentially expressed miRNAs with statistical significance (P < 0.05) which were identified to be linearly increasing or decreasing using linear regression model. (C) One of the many intermediate data files (shown here is a text file of P-values, intercept and slope data of significant miRNAs). (D) A heat map of differentially expressed miRNAs with statistical significance (P < 0.05) which were identified to be increasing or decreasing using either cumulative distribution function (CDF) of the normal distribution or analysis of variance (ANOVA). Note that all these images and text files are available for viewing on web browsers by opening ‘mirnata.html’ file in ‘output_files’ directory. Figure 2 mirnaTA Outputs. (A) A Portable Network Graphic (PNG) image showing the raw data (before normalization) vs. NQT data (after normalization). (B) A heat map of differentially expressed miRNAs with statistical significance (P < 0.05) which were identified to be linearly increasing or decreasing using linear regression model. (C) One of the many intermediate data files (shown here is a text file of P-values, intercept and slope data of significant miRNAs). (D) A heat map of differentially expressed miRNAs with statistical significance (P < 0.05) which were identified to be increasing or decreasing using either cumulative distribution function (CDF) of the normal distribution or analysis of variance (ANOVA). Note that all these images and text files are available for viewing on web browsers by opening ‘mirnata.html’ file in ‘output_files’ directory. (4). Wide applicability: The mirnaTA can be used to analyze any kind of data (gene, weather data, weight, etc.), and does not have to be restricted to miRNA data. Cer et al. GigaScience 2014, 3:20 http://www.gigasciencejournal.com/content/3/1/20 Cer et al. GigaScience 2014, 3:20 http://www.gigasciencejournal.com/content/3/1/20 Cer et al. GigaScience 2014, 3:20 http://www.gigasciencejournal.com/content/3/1/20 Author details 1 1Biological Defense Research Directorate, Naval Medical Research Center-Frederick, 8400 Research Plaza, Fort Detrick, MD 21702, USA. 2Henry M. Jackson Foundation for the Advancement of Military Medicine, 6720-A Rockledge Drive, Suite 100, Bethesda, MD 20817, USA. 3Chem Bio Research 1Biological Defense Research Directorate, Naval Medical Research Center-Frederick, 8400 Research Plaza, Fort Detrick, MD 21702, USA. 2Henry M. Jackson Foundation for the Advancement of Military Medicine, 6720-A Rockledge Drive, Suite 100, Bethesda, MD 20817, USA. 3Chem Bio Research Center of Excellence, Defense Threat Reduction Agency, 2800 Bush River Road E2800-b198, Aberdeen Proving Ground, MD 21010, USA. 21. Leek JT, Monsen E, Dabney AR, Storey JD: EDGE: extraction and analysis of differential gene expression. Bioinformatics 2006, 22(4):507–508. Center of Excellence, Defense Threat Reduction Agency, 2800 Bush River Road E2800-b198, Aberdeen Proving Ground, MD 21010, USA. 22. Aryee MJ, Gutierrez-Pabello JA, Kramnik I, Maiti T, Quackenbush J: An improved empirical bayes approach to estimating differential gene expression in microarray time-course data: BETR (Bayesian Estimation of Temporal Regulation). BMC Bioinformatics 2009, 10:409. Received: 8 April 2014 Accepted: 30 September 2014 Published: 13 October 2014 23. Robinson MD, McCarthy DJ, Smyth GK: edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 2010, 26(1):139–140. Authors’ contributions RZC wrote Perl and R scripts, packaged the workflow, released code and prepared the manuscript. JEH wrote custom R functions and oversaw R statistical analyses. JJA tested the package and provided patches. KAB tested the package and edited the manuscript. VPM oversaw the project and gave scientific advice. All authors read, contributed and approved the final manuscript. 39(Web Server issue):W132–W138. 14. Barrett T, Wilhite SE, Ledoux P, Evangelista C, Kim IF, Tomashevsky M, Marshall KA, Phillippy KH, Sherman PM, Holko M, Yefanov A, Lee H, Zhang N, Robertson CL, Serova N, Davis S, Soboleva A: NCBI GEO: archive for functional genomics data sets–update. Nucleic Acids Res 2013, 41(Database issue):D991–D995. 15. Jayaswal V, Lutherborrow M, Ma DD, Hwa Yang Y: Identification of microRNAs with regulatory potential using a matched microRNA-mRNA time-course data. Nucleic Acids Res 2009, 37(8):e60. Competing interests The authors declare that they have no competing interests. The authors declare that they have no competing interests. 13. Hackenberg M, Rodriguez-Ezpeleta N, Aransay AM: miRanalyzer: an update on the detection and analysis of microRNAs in high-throughput sequencing experiments. Nucleic Acids Res 2011, 39(Web Server issue):W132–W138. References 1. Kim VN, Han J, Siomi MC: Biogenesis of small RNAs in animals. Nat Rev Mol Cell Biol 2009, 10(2):126–139. 1. Kim VN, Han J, Siomi MC: Biogenesis of small RNAs in animals. Nat Rev Mol Cell Biol 2009, 10(2):126–139. 24. Anders S, Huber W: Differential expression analysis for sequence count data. Genome Biol 2010, 11(10):R106. 2. Xiao C, Rajewsky K: MicroRNA control in the immune system: basic principles. Cell 2009, 136(1):26–36. 2. Xiao C, Rajewsky K: MicroRNA control in the immune system: basic principles. Cell 2009, 136(1):26–36. 25. SourceForge: http://sourceforge.net/projects/mirnata. 26. Garmire LX, Subramaniam S: Evaluation of normalization methods in mammalian microRNA-Seq data. RNA 2012, 18(6):1279–1288. 3. Tili E, Michaille JJ, Calin GA: Expression and function of micro-RNAs in immune cells during normal or disease state. Int J Med Sci 2008, 5(2):73–79. 27. Bullard JH, Purdom E, Hansen KD, Dudoit S: Evaluation of statistical methods for normalization and differential expression in mRNA-Seq experiments. BMC Bioinformatics 2010, 11:94. 4. Matsushima K, Isomoto H, Inoue N, Nakayama T, Hayashi T, Nakayama M, Nakao K, Hirayama T, Kohno S: MicroRNA signatures in Helicobacter pylori-infected gastric mucosa. Int J Cancer 2011, 128(2):361–370. 4. Matsushima K, Isomoto H, Inoue N, Nakayama T, Hayashi T, Nakayama M, Nakao K, Hirayama T, Kohno S: MicroRNA signatures in Helicobacter pylori-infected gastric mucosa. Int J Cancer 2011, 128(2):361–370. 28. McCormick KP, Willmann MR, Meyers BC: Experimental design, preprocessing, normalization and differential expression analysis of small RNA sequencing experiments. Silence 2011, 2(1):2. 28. McCormick KP, Willmann MR, Meyers BC: Experimental design 5. Wang WX, Wilfred BR, Madathil SK, Tang G, Hu Y, Dimayuga J, Stromberg AJ, Huang Q, Saatman KE, Nelson PT: miR-107 regulates granulin/progranulin with implications for traumatic brain injury and neurodegenerative disease. Am J Pathol 2010, 177(1):334–345. 5. Wang WX, Wilfred BR, Madathil SK, Tang G, Hu Y, Dimayuga J, Stromberg AJ, Huang Q, Saatman KE, Nelson PT: miR-107 regulates granulin/progranulin with implications for traumatic brain injury and neurodegenerative disease. Am J Pathol 2010, 177(1):334–345. 29. Dalgaard P: Statistics and Computing: Introductory Statistics with R. New York: Springer Science; 2002. 6. Garza-Manero S, Pichardo-Casas I, Arias C, Vaca L, Zepeda A: Selective distribution and dynamic modulation of miRNAs in the synapse 6. Garza-Manero S, Pichardo-Casas I, Arias C, Vaca L, Zepeda A: Selective distribution and dynamic modulation of miRNAs in the synapse 30. Abbreviations ANOVA A l i ANOVA: Analysis of variance; CDF: Cumulative distribution function; DE: Differential expression; miRNA: microRNA; NGS: Next-generation sequencing; NQT: Normal quantile transformation; PNG: Portable network graphics; TMM: Trimmed mean method. 11. Huang TH, Fan B, Rothschild MF, Hu ZL, Li K, Zhao SH: MiRFinder: an improved approach and software implementation for genome-wide fast microRNA precursor scans. BMC Bioinformatics 2007, 8:341. 12. An J, Lai J, Lehman ML, Nelson CC: miRDeep*: an integrated application tool for miRNA identification from RNA sequencing data. Nucleic Acids Res 2013, 41(2):727–737. Acknowledgements mirnaTA was developed as a part of a study supported by the Defense Threat Reduction Agency (DTRA) project CBM.DIAGB.03.10.NM.028. JJA and VPM are military service members or employees of the U.S. Government and this work was prepared as part of their official duties. Title 17 U.S.C. §105 provides that ‘Copyright protection under this title is not available for any work of the United States Government’. Title 17 U.S.C. §101 defines a U.S. Government work as a work prepared by a military service member or employee of the U.S. Government as part of that person’s official duties. The opinions or assertions contained herein are the private ones of the author (s) and are not to be construed as official or reflecting the views of either the 16. Li Z, Branham WS, Dial SL, Wang Y, Guo L, Shi L, Chen T: Genomic analysis of microRNA time-course expression in liver of mice treated with genotoxic carcinogen N-ethyl-N-nitrosourea. BMC Genomics 2010, 11:609. 17. Ernst J, Bar-Joseph Z: STEM: a tool for the analysis of short time series gene expression data. BMC Bioinformatics 2006, 7:191. 18. Craig P, Cannon A, Kukla R, Kennedy J: MaTSE: the gene expression time-series explorer. BMC Bioinformatics 2013, 14:S1. 19. Smyth GK: Linear models and empirical bayes methods for assessing differential expression in microarray experiments. Stat Appl Genet Mol Biol 2004, 3:Article 3. Epub 2004 Feb 12. g Department of the Navy or the Department of Defense, nor the U.S. Government. 20. Grace C, Nacheva EP: Significance Analysis of Microarrays (SAM) offers clues to differences between the genomes of adult philadelphia positive ALL and the lymphoid blast transformation of CML. Cancer Inform 2012, 11:173–183. Additional file and its possible role in Alzheimer’s disease. Brain Res 2013, 1584:80–93. and its possible role in Alzheimer’s disease. Brain Res 2013, 1584:80–93. 7. Ono K, Kuwabara Y, Han J: MicroRNAs and cardiovascular diseases. FEBS J 2011, 278(10):1619–1633. Additional file 1: Figure S1. Detailed steps for generating input files for mirnaTA. FASTQ files generated from any NGS sequencing platform are converted into FASTA files. Artificially introduced 3′ adapter sequences are trimmed, and post-trimmed reads that are a minimum of 15 base pairs are filtered against contaminants. Reads that do not match to contaminants are screened for mature miRNA species (black box) which are further analyzed for statistical significance using mirnaTA. 8. Ambros V, Bartel B, Bartel DP, Burge CB, Carrington JC, Chen X, Dreyfuss G, Eddy SR, Griffiths-Jones S, Marshall M, Matzke M, Ruvkun G, Tuschl T: A uniform system for microRNA annotation. RNA 2003, 9(3):277–279. 9. Kozomara A, Griffiths-Jones S: miRBase: integrating microRNA annotation and deep-sequencing data. Nucleic Acids Res 2011, 39(Database issue):D152–D157. 10. Lim LP, Lau NC, Weinstein EG, Abdelhakim A, Yekta S, Rhoades MW, Burge CB, Bartel DP: The microRNAs of Caenorhabditis elegans. Genes Dev 2003, 17(8):991–1008. Availability of supporting data Archival version of the supporting files, user guide and additional datasets used in the paper are hosted in the GigaScience GigaDB database [30], and for the most up to date versions please see the source forge page: http://sourceforge.net/projects/mirnata. Page 6 of 7 References Cer RZ, Herrera-Galeano JE, Anderson JJ, Bishop-Lilly KA, Mokashi VP: Software and Supporting Material for: “mirnaTA: a Bioinformatics Tool for Page 7 of 7 Page 7 of 7 Cer et al. GigaScience 2014, 3:20 http://www.gigasciencejournal.com/content/3/1/20 Cer et al. GigaScience 2014, 3:20 http://www.gigasciencejournal.com/content/3/1/20 Identifying Differentially Expressed microRNAs in Temporal Studies using Normal Quantile Transformation (NQT)”. GigaScience Database; 2014. http://dx.doi.org/10.5524/100107. 31. Caraux G, Pinloche S: PermutMatrix: a graphical environment to arrange gene expression profiles in optimal linear order. Bioinformatics 2005, 21(7):1280–1281. doi:10.1186/2047-217X-3-20 Cite this article as: Cer et al.: miRNA Temporal Analyzer (mirnaTA): a bioinformatics tool for identifying differentially expressed microRNAs in temporal studies using normal quantile transformation. GigaScience 2014 3:20. Identifying Differentially Expressed microRNAs in Temporal Studies using Normal Quantile Transformation (NQT)”. GigaScience Database; 2014. http://dx.doi.org/10.5524/100107. 31. Caraux G, Pinloche S: PermutMatrix: a graphical environment to arrange gene expression profiles in optimal linear order. Bioinformatics 2005, 21(7):1280–1281. doi:10.1186/2047-217X-3-20 Cite this article as: Cer et al.: miRNA Temporal Analyzer (mirnaTA): a bioinformatics tool for identifying differentially expressed microRNAs in temporal studies using normal quantile transformation. GigaScience 2014 3:20. 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https://europepmc.org/articles/pmc3066643?pdf=render
English
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STOX1: Key Player in Trophoblast Dysfunction Underlying Early Onset Preeclampsia with Growth Retardation
Journal of pregnancy
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Hindawi Publishing Corporation Journal of Pregnancy Volume 2011, Article ID 521826, 7 pages doi:10.1155/2011/521826 Hindawi Publishing Corporation Journal of Pregnancy Volume 2011, Article ID 521826, 7 pages doi:10.1155/2011/521826 Hindawi Publishing Corporation Journal of Pregnancy Volume 2011, Article ID 521826, 7 pages doi:10.1155/2011/521826 Marie van Dijk and Cees B. M. Oudejans Department of Clinical Chemistry, VU University Medical Center, de Boelelaan 1117, 1081 HV Amsterdam, The Netherlands Department of Clinical Chemistry, VU University Medical Center, de Boelelaan 1117, 1081 HV Amsterdam, The Netherlands Department of Clinical Chemistry, VU University Medical Center, de Boelelaan 1117, 1081 HV Amsterdam, The f Clinical Chemistry, VU University Medical Center, de Boelelaan 1117, 1081 HV Amsterdam, The Netherlands Correspondence should be addressed to Marie van Dijk, m.vdijk@vumc.nl Correspondence should be addressed to Marie van Dijk, m.vdijk@vumc.nl Received 14 September 2010; Accepted 2 November 2010 Academic Editor: Jeffrey Keelan Copyright © 2011 M. van Dijk and C. B. M. Oudejans. 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. Currently, only two preeclampsia susceptibility genes (ACVR2A, STOX1) have been identified within confirmed regions with significant genome-wide linkage, although many genetic screens in multiple populations have been performed. In this paper, we focus on the STOX1 gene. The epigenetic status of this gene is discussed explaining the maternal transmission of the STOX1 susceptibility allele observed in preeclamptic families. The known upstream regulation and downstream effector genes of the transcription factor STOX1 are described. Finally, we propose a model in which we combine the cell type-specific and allele- specific effects of STOX1. This includes intrinsic effects (differential CpG island methylation) and extrinsic effects (regulation of effector genes). 1. Outline of This Paper gestation onwards, and which consist of de novo hyperten- sion (diastolic blood pressure >90 mm Hg with increment >20 mm Hg from first trimester diastolic blood pressure) and proteinuria (>300 mg per 24 h) as defined by the Royal College of Obstetricians and Gynaecologists [8]. Although the symptoms occur in the mother, it is now well established that the placenta is the driving force; the only cure currently available is delivery of the baby and thus removal of the placenta [9]. The importance of the STOX1 preeclampsia susceptibility gene, as discovered in 2005 in Dutch females [1], was initially challenged [2, 3]. However, the latest data including those from independent groups not only confirm the role of STOX1 in trophoblast dysfunction underlying preeclampsia [4–6] but also indicate a role for its paralog, STOX2 [7]. Rather than providing a complete overview of all aspects of preeclampsia, we provide a focused overview by summarizing and discussing the latest data on the role of the STOX1 as a key player in trophoblast dysfunction underlying familial early-onset preeclampsia with growth retardation. We propose a model in which we combine the cell type- and allele-specific (epi)genetic effects of STOX1 and suggest future directions of research. In first trimester placenta, insufficient spiral artery remodeling is the fetal pathophysiological origin of preeclampsia [10]. In a normal pregnancy, extravillous trophoblasts from the fetal placenta invade the maternal decidua up to one-third of the myometrium (see also Figure 1). These trophoblasts are thereby transforming the maternal spiral arteries by replacing smooth muscle and elastic tissue with fibrinoid material changing them from low-capacity high-resistance into high-capacity low-resistance vessels. In preeclampsia, this extravillous trophoblast-directed spiral artery remodeling is incomplete leading to a decrease in placental blood flow, which subsequently leads to a response from the mother increasing Review Article STOX1: Key Player in Trophoblast Dysfunction Underlying Early Onset Preeclampsia with Growth Retardation Marie van Dijk and Cees B. M. Oudejans 2. Introduction YY: placenta homozygous for the Y-allele; HH: placenta homozygous for the H-allele; uu: both alleles are unmethylated; mm: both alleles are methylated; um: imprinting (only one allele is methylated). her blood pressure. This in turn leads to maternal systemic failure giving rise to the maternal symptoms [10]. phenotypically homogenous as defined by familial early- onset preeclampsia with an abnormal placental development complicated by IUGR [13, 14]. 2. Introduction Preeclampsia is a pregnancy-associated disease occurring in 5–8% of pregnancies and a major cause of maternal and fetal morbidity and mortality. The disease is characterized by maternal symptoms which may occur from 20 weeks 2 Journal of Pregnancy Villous stroma Decidua Spiral artery Endovascular extravillous trophoblast Interstitial extravillous trophoblast Column extravillous trophoblast Villous cytotrophoblast Syncytiotrophoblast Proliferative Invasive YY HH Villous stroma Syncytiotrophoblast Villous cytotrophoblast Column extravillous trophoblast uu and mm uu↓and mm↑ uu and mm uu↓and mm↑ um um um um uu uu Interstitial/ endovascular extravillous trophoblast Figure 1: Model of a villus with an extravillous trophoblast column invading the maternal decidua. Below this model, showing different cell types within and originating from a villus, a table shows the STOX1 methylation pattern observed or hypothesized to be found in the different cell types. YY: placenta homozygous for the Y-allele; HH: placenta homozygous for the H-allele; uu: both alleles are unmethylated; mm: both alleles are methylated; um: imprinting (only one allele is methylated). Villous stroma Decidua Spiral artery Endovascular extravillous trophoblast Interstitial extravillous trophoblast Column extravillous trophoblast Villous cytotrophoblast Syncytiotrophoblast Interstitial extravillous trophoblast Endovascular extravillous trophoblast Villous stroma Syncytiotrophoblast Invasive Figure 1: Model of a villus with an extravillous trophoblast column invading the maternal decidua. Below this model, showing different cell types within and originating from a villus, a table shows the STOX1 methylation pattern observed or hypothesized to be found in the different cell types. YY: placenta homozygous for the Y-allele; HH: placenta homozygous for the H-allele; uu: both alleles are unmethylated; mm: both alleles are methylated; um: imprinting (only one allele is methylated). Figure 1: Model of a villus with an extravillous trophoblast column invading the maternal decidua. Below this model, showing different cell types within and originating from a villus, a table shows the STOX1 methylation pattern observed or hypothesized to be found in the different cell types. YY: placenta homozygous for the Y-allele; HH: placenta homozygous for the H-allele; uu: both alleles are unmethylated; mm: both alleles are methylated; um: imprinting (only one allele is methylated). Figure 1: Model of a villus with an extravillous trophoblast column invading the maternal decidua. Below this model, showing different cell types within and originating from a villus, a table shows the STOX1 methylation pattern observed or hypothesized to be found in the different cell types. 3. Preeclampsia Is a Heterogeneous Disease Interestingly, as a comparable function to STOX1, which shows a gain-of-function as discussed below, is suggested, and STOX2 is downregulated in preeclampsia samples, compatible with a loss-of-function, strong favor of genetic but placental origin of preeclampsia. The placental origin of severe early-onset preeclampsia has an important implication in genetic screens; the fetal genotype directs the maternal phenotype [29]. It is there- fore essential to realize that research on genetic linkage in familial early-onset preeclampsia should focus on the placental genotype rather than the maternal genotype. For case-control studies, the correction needed and/or allowed is that preeclamptic females born from unaffected and affected mothers should be considered controls and cases, respectively. The importance of the criteria mentioned above can immediately be seen in studies that have tried to confirm the STOX1 susceptibility allele in their population [2–4]. Firstly, the preeclamptic women were not clearly defined, the populations consisted of a combination of mild and severe, early- and late-onset preeclampsia, and not always with proof of familial inherited disease. Secondly, as the studies were performed using the maternal genotype, these patients can only be informative if their mother had developed preeclampsia as well. In one study, also performed in a Dutch population, they did take both criteria into account by performing a transmission distortion test (TDT) with correction for grandmaternal origin, and a significant mater- nal transmission of the STOX1 Y153H allele was detected in preeclamptic families when looking across three generations [4]. Recently, another study has been published looking at the STOX1 Y153H SNP in the Norwegian population. Again no significant transmission of the H-allele was found when the preeclampsia patients were analyzed in total. But, when looking at recurrent preeclampsia patients, a trend towards significance was detected in H-allele transmission when also corrected for the potential of a parent-of-origin effect [7]. However, these data were obtained in the maternal instead of the placental genotype, thereby diluting the true maternal transmission as not all of these mothers were also born out of a preeclamptic pregnancy. This study, however, also looked at expression of different genes in decidua from preeclampsia, IUGR, preeclampsia complicated by IUGR, and uncom- plicated pregnancies; no differences were found in STOX1 expression between these groups. 3. Preeclampsia Is a Heterogeneous Disease Interestingly, a significant difference was observed for STOX2 gene expression when healthy decidua samples were compared to decidua samples that suffered from preeclampsia complicated by IUGR, where the preeclampsia samples showed a lower expression of STOX2 compared to controls [7]. Although the analysis was performed in term decidua samples which contain mainly maternal cells, they do consist of approximately 20% placental extravillous trophoblasts. The STOX2 gene on chromosome 4q35 is located in close proximity to a chromosomal area (4q31-4q32) that has been implicated in preeclampsia in different populations (Finland, Australia, and New Zealand) as well [19, 20, 30]. No function has been allocated to this paralog of STOX1 but as it has a complemented with data from WTCCC3 (2225 cases, 2500 controls) (http://www.wtccc.org.uk). By design, most cases are from nonfamilial forms; the outcome of these case- control studies is therefore bound to be different from, but complementary to, the family-based linkage studies. These studies will predominantly identify maternal susceptibility alleles associated with low to moderate risks and high population frequencies and associated with a low familial component and with the less fatal forms (late-onset without growth retardation). Placental susceptibility alleles with high risks, low population frequencies, and representing the strongly fatal forms (early-onset with growth retardation) with high familial components have been and will be identified by the linkage studies of affected families. 3. Preeclampsia Is a Heterogeneous Disease It is well established that preeclampsia is a multifactorial disease in which both fetal, that is, placental and maternal, factors are contributing [8]. Different combinations of factors can then lead to differences in disease severity, time of onset, and the occurrence of additional complications, for example, IUGR (Intra-Uterine Growth Restriction). Some of these factors will also run in specific families as there is also a strong genetic component found in the occurrence of preeclampsia [11]. Although already three pathways are known to be involved in preeclampsia, that is, PlGF-sFLT-sENG, TGFβ-NOS, and COMT-2ME [9, 12], these are all second-order factors that give rise to the maternal symptoms. The first-order causative factors, both placental and maternal, are still largely unknown. One of the important starting points in the identification of these first-order factors is to subcategorize the disease to yield phenotypic homogenous patient populations. The Dutch patients associated with STOX1 turned out to be To identify factors involved in preeclampsia, multiple genetic screens (genome-wide scans, variance-components linkage analysis, and association analysis) have been performed in multiple patient populations (families, case control) [13– 20]. This has yielded limited results; only two susceptibility genes have emerged, namely, ACVR2A [21] and STOX1 [1], both within confirmed regions with significant genome-wide linkage and both involving normal variations (SNPs) in line with the common variant-common disease hypothesis [22]. Both of these genes were originally identified in familial forms of preeclampsia. Additional susceptibility genes and alleles of the same type as found in ACVR2A and STOX1 (common polymorphisms) can also be identified by power- ful approaches using genome-wide case-control association analyses. One example, the data are not in the public domain yet, is the ongoing GenPE study by the Wellcome Trust Case Control Consortium (WTCCC2, 2000 cases, 2500 controls) 3 Journal of Pregnancy strong favor of genetic but placental origin of preeclampsia. The placental origin of severe early-onset preeclampsia has an important implication in genetic screens; the fetal genotype directs the maternal phenotype [29]. It is there- fore essential to realize that research on genetic linkage in familial early-onset preeclampsia should focus on the placental genotype rather than the maternal genotype. For case-control studies, the correction needed and/or allowed is that preeclamptic females born from unaffected and affected mothers should be considered controls and cases, respectively. 3. Preeclampsia Is a Heterogeneous Disease The importance of the criteria mentioned above can immediately be seen in studies that have tried to confirm the STOX1 susceptibility allele in their population [2–4]. Firstly, the preeclamptic women were not clearly defined, the populations consisted of a combination of mild and severe, early- and late-onset preeclampsia, and not always with proof of familial inherited disease. Secondly, as the studies were performed using the maternal genotype, these patients can only be informative if their mother had developed preeclampsia as well. In one study, also performed in a Dutch population, they did take both criteria into account by performing a transmission distortion test (TDT) with correction for grandmaternal origin, and a significant mater- nal transmission of the STOX1 Y153H allele was detected in preeclamptic families when looking across three generations [4]. Recently, another study has been published looking at the STOX1 Y153H SNP in the Norwegian population. Again no significant transmission of the H-allele was found when the preeclampsia patients were analyzed in total. But, when looking at recurrent preeclampsia patients, a trend towards significance was detected in H-allele transmission when also corrected for the potential of a parent-of-origin effect [7]. However, these data were obtained in the maternal instead of the placental genotype, thereby diluting the true maternal transmission as not all of these mothers were also born out of a preeclamptic pregnancy. This study, however, also looked at expression of different genes in decidua from preeclampsia, IUGR, preeclampsia complicated by IUGR, and uncom- plicated pregnancies; no differences were found in STOX1 expression between these groups. Interestingly, a significant difference was observed for STOX2 gene expression when healthy decidua samples were compared to decidua samples that suffered from preeclampsia complicated by IUGR, where the preeclampsia samples showed a lower expression of STOX2 compared to controls [7]. Although the analysis was performed in term decidua samples which contain mainly maternal cells, they do consist of approximately 20% placental extravillous trophoblasts. The STOX2 gene on chromosome 4q35 is located in close proximity to a chromosomal area (4q31-4q32) that has been implicated in preeclampsia in different populations (Finland, Australia, and New Zealand) as well [19, 20, 30]. No function has been allocated to this paralog of STOX1, but as it has a high sequence similarity to STOX1, some of its functions are potentially comparable. 6. Epigenetics of STOX1 Although a parent-of-origin effect was already noticed in the first microsatellite analysis [14], as well as maternal transmission of the STOX1 Y153H susceptibility allele by sequencing the DNA of multiple generations [1], no (differential) methylation could be detected in the CpG island located within the promoter region of STOX1 [2, 31], excluding the existence of imprinting of this gene controlled by the CpG island in the promoter. Recently, however, we identified and analyzed another CpG island located in intron 1 of STOX1 [32]. Methylation of this intronic CpG region leads to reduced expression in common with the normal association between hypermethylation and downregulation of expression. Secondly, in specific cells, this region is subject to differential and allele-specific methylation. In column extravillous trophoblast samples, the methylated allele is paternal in origin. No allele-specific methylation could be detected in early placenta samples consisting of multiple cell types, but a significant increase in methylation was seen in samples homozygous for the STOX1 Y153H preeclampsia susceptibility allele [32]. As column extravillous trophoblasts are formed from villous cytotrophoblasts, we hypothesize that these cells are paternally imprinted as well. In cell types, where methylation is independent of parental origin, the Y153H genotype has the potential to direct the level of methylation [32]. How these cell type and allelic differences can be integrated with the function of STOX1 in the placenta will be discussed in the model proposed below. The intraintronic gene of CTNNA3, LRRTM3, is also an effector gene of STOX1 [33]. Although LRRTM3 is also expressed in placenta, most studies performed on its function are done in brain tissue and cells. This interest comes from the finding that LRRTM3 promotes APP (Amyloid Precursor Protein) processing by BACE1, leading to an increase in Amyloid beta production [34]. Amyloid beta is found to be accumulated in brains of Alzheimer’s disease patients. STOX1 was found to be able to transactivate LRRTM3 in brain and placenta and showed upregulated expression in brains affected by Alzheimer’s disease [33]. A successful genome-wide approach to identify STOX1 target genes has been described by Rigourd and cowork- ers [5]. By stable transfection of STOX1 in the JEG-3 choriocarcinoma cell line as a model for trophoblasts, they identified and confirmed by quantitative RT-PCR five genes upregulated (PTGDS, ALOX5, TNFSF10, beta-ARRESTIN, and TMEM45a), while four genes were found to be down- regulated upon STOX1 overexpression (ANXA3, ZNF22, APOA2, and FAM43A) [5]. 6. Epigenetics of STOX1 Furthermore, by analyzing the promoter regions of genes affected by STOX1 in the microarray analysis, the most significant promoter binding site being located more than once in each promoter was found to be the FOX transcription factor binding site FKHD. As the STOX1 DNA-binding domain shows great similarity to FOX transcription factors, promoters containing multiple FKHD sites were chosen for chromatin immunoprecipitation experiments followed by quantitative PCR. Promoter regions of six genes (COL6A3, S100A4, ARRDC3, TNFSF10, EBI3, and PLCB1) were found to be significantly enriched in cells overexpressing STOX1 [5]. The microarray data of this study were also compared to published transcriptomic data com- paring normal and preeclamptic placentas. The correlation between these studies was found to be highly significant looking at genes affected by STOX1- and preeclampsia- modified genes [5]. Interestingly, the microarray data by Rigourd and coworkers has subsequently been used in the study identifying downregulation of STOX2 in preeclamptic deciduas complicated by IUGR. Their data also found 5. Familial Preeclampsia Susceptibility Genes The CTNNA3 gene was one of the original candidate effector genes based on its chromosomal location in close proximity to STOX1, its function in cell-cell adhesion, and its upregulation found in a preliminary microarray analysis done in STOX1-transfected SGHPL-5 cells, an extravillous trophoblast cell line. By studying both SGHPL-5 cells and villous explants, we showed that by binding of STOX1 to the CTNNA3 promoter this increased the expression of αT- catenin, the protein product of CTNNA3, and component of the cell-cell adhesion complex. This in turn leads to reduced trophoblast invasion and maintains proliferation [6]. Furthermore, villous explants homozygous for the STOX1 H-allele associated with preeclampsia have a reduced trophoblast outgrowth and an induced responsiveness of CTNNA3 expression upon STOX1 expression [6]. In other words, the genotype linked to preeclampsia, that is, STOX1 Y153H, has a demonstrated effect on extravillous trophoblast invasion, central in the etiology of preeclampsia. H-allele gives a higher binding affinity as does the Y-allele in one of the STOX1 downstream effector genes, CTNNA3 [6]. The CTNNA3 gene was one of the original candidate effector genes based on its chromosomal location in close proximity to STOX1, its function in cell-cell adhesion, and its upregulation found in a preliminary microarray analysis done in STOX1-transfected SGHPL-5 cells, an extravillous trophoblast cell line. By studying both SGHPL-5 cells and villous explants, we showed that by binding of STOX1 to the CTNNA3 promoter this increased the expression of αT- catenin, the protein product of CTNNA3, and component of the cell-cell adhesion complex. This in turn leads to reduced trophoblast invasion and maintains proliferation [6]. Furthermore, villous explants homozygous for the STOX1 H-allele associated with preeclampsia have a reduced trophoblast outgrowth and an induced responsiveness of CTNNA3 expression upon STOX1 expression [6]. In other words, the genotype linked to preeclampsia, that is, STOX1 Y153H, has a demonstrated effect on extravillous trophoblast invasion, central in the etiology of preeclampsia. this would indicate that these two genes have similar functions but opposite effects in placental function. 5. Familial Preeclampsia Susceptibility Genes As already mentioned above, only two preeclampsia sus- ceptibility genes have been identified so far, ACVR2A [21] and STOX1 [1]. ACVR2A, located on 2q22, was originally identified in an Australian/New Zealand population of preeclamptic pedigrees [21, 23]. Also, in a Norwegian case- control study, SNPs (Single Nucleotide Polymorphisms) within this gene were found to give significant linkage [24]. ACVR2A is a receptor for the cell-signaling protein Activin A. Activin A has a major role in promoting decidualization of endometrial stromal cells [25] and in the regulation of trophoblast differentiation and invasion into the decidua [26]. Secondly, multiple studies have found increased Activin A in serum of preeclamptic women [27], making its receptor ACVR2A a very interesting protein in the development of preeclampsia. No functional studies have yet been published looking at the significance of the intronic SNPs of ACVR2A found in preeclamptic patients [21, 23, 24]. Although intronic SNPs do not appear to be relevant, they should not be discarded as it can be envisaged that these SNPs are within areas controlling ACVR2A transcription or are located within novel yet to be identified transcripts. Our identification of the STOX1 gene was performed in a Dutch population consisting of affected siblings and their relatives [13]. This identification was preceded by microsatellite marker analysis which revealed a parent-of- origin effect; the alleles shared between affected preeclampsia sisters were always maternal in origin [14]. By sequencing the complete coding region with linkage on chromosome 10q22, the STOX1 Y153H common polymorphism was identified, with maternal transmission in three generations [1]. The preeclamptic families with linkage to 10q22 and associated with STOX1 were a phenotypically homogenous patient cohort suffering from familial severe early-onset preeclamp- sia complicated by IUGR. This indicates that the disease in these families has a fetal, that is, placental, origin. In this respect, discordant monozygotic twins provide excellent evidence for the placental origin of early-onset preeclampsia; the consistent observation in monozygotic parous twins lacking concordance for preeclampsia implicates that fetal, that is, placental, contributions are essential [28]. Therefore, the discordance seen in monozygous parous twins is in 4 Journal of Pregnancy H-allele gives a higher binding affinity as does the Y-allele in one of the STOX1 downstream effector genes, CTNNA3 [6]. 8. An Integrated Model of STOX1 in Placental Cells It has become clear that processes in the early placenta in which STOX1 is involved are cell type dependent and allele dependent. This includes intrinsic effects (differential CpG island methylation) and extrinsic effects (regulation of effector genes) [6, 32]. The cell type-specific effects on expression observed can be combined with the methylation pattern seen in a model depicted in Figure 1. The model shows the methylation status of the different cell types within the early placenta in combination with the Y153H allele carried. For simplicity, only the homozygous alleles are shown. It can be seen that, within the villus, stromal cells and syncytiotrophoblasts are not subject to imprinting, but can be either methylated or unmethylated [32]. However, their methylation status is dependent on the Y153H allele carried, with the placentas homozygous for the H-allele showing a higher level of methylation than the placentas homozygous for the Y-allele [32]. Although syncytiotrophoblasts in our model are not subject to imprinting, this cannot be ruled out as syncytiotrophoblasts, like extravillous trophoblasts, are originating from villous cytotrophoblasts. The villous cytotrophoblasts are hypothesized to be imprinted within the villus, but as this is only a small population of cells it does not change the overall methylation status observed in early placenta samples. The proliferative column extravillous trophoblasts are proven to be paternally imprinted, but as methylation is less than 50% [32] differentiation of the proliferative extravillous trophoblasts into invasive extrav- illous trophoblasts is hypothesized to be subject to loss of imprinting. Early placenta column extravillous trophoblasts homozygous for the H-allele show a higher binding affinity to the CTNNA3 effector gene than placentas homozygous for the Y-allele [6]. This is followed by a trend towards higher expression of CTNNA3 mRNA in these cells. This is in agreement with the proposed model as these cells are not subject to differences in methylation level caused by parental independent H-allele directed methylation. This is not the case in the villus part of the early placenta. Although placentas homozygous for the H-allele still will have a higher binding affinity, this is not translated in an upregulated expression of CTNNA3; there is even a trend towards downregulated expression in these placentas [6]. 9. Conclusions and Future Directions This paper has been intended to provide an overview of the data available from successful genetic screens to identify preeclampsia susceptibility genes and subsequent studies on these genes, that is, ACVR2A and STOX1, in other populations. These two genes are a first step on defining preeclampsia; different genes affect different pathways con- verging to a final common endophenotype: hypertension with proteinuria. As most data available is on the transcrip- tion factor STOX1, it has been the main focus of this paper to discuss the epigenetic status of STOX1, to explain the maternal transmission seen in preeclamptic families, and its upstream regulation and downstream effector genes in placenta and brain. Finally, we propose an integrated model of the cell type-specific and allele-specific effects on both differential methylation as the expression of effector genes of STOX1. 7. STOX1 Is a Transcription Factor Three STOX1 isoforms are known to be expressed with iso- form A being the full-length protein. Although also isoforms B and C contain the complete (B) or part (C) of the winged helix DNA-binding domain, they both do not contain a nuclear export signal (NES), only a nuclear localization signal (NLS), keeping them confined to the nucleus and nucleolus, respectively. The general focus therefore is on isoform A as this is the only isoform to contain the regulatory domains needed for its functioning as discussed below. The STOX1 winged helix DNA-binding domain shows great similarity to the binding domain seen in the family of FOX transcription factors [1]. Its upstream regulation mechanism is also similar as seen in multiple members of the FOX transcription factor family, the PI3K-Akt pathway [6]. When phosphorylated by Akt, the STOX1 protein is inhibited from entering the nucleus and subsequently degraded by ubiquitination. When phosphorylation does not take place, STOX1 functions as a transcription factor in the nucleus with binding to DNA. The Y153H SNP is located within the winged helix binding domain and affects an amino acid with high molecular effect [1]. By chromatin immunoprecipitation assays, it was established that in the extravillous trophoblast cell line SGHPL-5, the 5 Journal of Pregnancy paternal imprinting in villous cytotrophoblasts [35]. Upon differentiation into proliferative extravillous trophoblasts the expression of CTNNA3 becomes biallelic and is absent after further differentiation into invasive extravillous tro- phoblasts. significant correlation between the genes that were up- or downregulated in JEG-3 cells overexpressing STOX1 and the decidua samples of preeclamptic women [7]. Therefore, although JEG-3 cells were used with STOX1 overexpression to induce effects, the outcomes are comparable to physio- logical changes found in preeclampsia samples, justifying the microarray results obtained. 8. An Integrated Model of STOX1 in Placental Cells This effect can be explained using the model; a vast amount of cells within the villus have a methylation status that is dependent on the allele carried, giving placentas homozygous for the H-allele an overall downregulated expression caused by the higher level of methylation. Furthermore, of interest is the observation that CTNNA3 itself is also subject to STOX1, however, is only one of the genes that give susceptibility for preeclampsia. It is therefore needed that more genes are identified in other populations to have a complete understanding of the origin of the disease. For new identifications to be successful, it is essential to subcategorize the disease to yield phenotypic homogenous patient popula- tions and to bear in mind that for preeclampsia originating in the early placenta it is the genotype of the children born from these pregnancies that provide information. Secondly, as STOX1 is a transcription factor, there are many more genes regulated by STOX1 which need to be identified in a genome-wide manner either by microarrays or by chromatin immunoprecipitation experiments followed by deep sequencing. This will provide a complete overview of STOX1 function in placenta, brain, and other organs, in both health and disease. Finally, by identifying which other genes are involved in preeclampsia, especially the early-onset form with growth retardation, a complete genotype-phenotype correlation can be envisaged. For this to succeed, functional studies on genes associated with preeclampsia should not only focus on one gene but combine data that is and becomes available. In this respect, it is very interesting to investigate the combined action of the two genes discussed in this paper, STOX1 and ACVR2A, using a physiological ex vivo placental explant model which takes into account the different cell types involved [36]. 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Effectiveness of interventions to promote healthy diet in primary care: systematic review and meta-analysis of randomised controlled trials
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Nawaraj Bhattarai, A Toby Prevost, Alison J Wright, Judith Charlton, Caroline Rudisill and Martin C Gulliford Effectiveness of interventions to promote healthy diet in primary care: systematic review and meta-analysis of randomised controlled trials Nawaraj Bhattarai, A Toby Prevost, Alison J Wright, Judith Charlton, Caroline Rudisill and Martin C Gulliford Effectiveness of interventions to promote healthy diet in primary care: systematic review and meta-analysis of randomised controlled trials Article (Published version) (Refereed) Article (Published version) (Refereed) Original citation: Bhattarai, Nawaraj, Prevost, A Toby, Wright, Alison J., Charlton, Judith, Rudisill, Caroline and Gulliford, Martin C. (2013) Effectiveness of interventions to promote healthy diet in primary care: systematic review and meta-analysis of randomised controlled trials. BMC public health, 13 (1). p. 1203. ISSN 1471-2458 DOI: 10.1186/1471-2458-13-1203 © 2013 2013 The Authors, licensee BioMed Central Ltd. © CC BY 2.0 This version available at: http://eprints.lse.ac.uk/55376/ Available in LSE Research Online: January 2014 LSE has developed LSE Research Online so that users may access research output of the School. Copyright © and Moral Rights for the papers on this site are retained by the individual authors and/or other copyright owners. Users may download and/or print one copy of any article(s) in LSE Research Online to facilitate their private study or for non-commercial research. You may not engage in further distribution of the material or use it for any profit-making activities or any commercial gain. You may freely distribute the URL (http://eprints.lse.ac.uk) of the LSE Research Online website. Original citation: Bhattarai, Nawaraj, Prevost, A Toby, Wright, Alison J., Charlton, Judith, Rudisill, Caroline and Gulliford, Martin C. (2013) Effectiveness of interventions to promote healthy diet in primary care: systematic review and meta-analysis of randomised controlled trials. BMC public health, 13 (1). p. 1203. ISSN 1471-2458 Abstract Background: A diet rich in fruit, vegetables and dietary fibre and low in fat is associated with reduced risk of chronic disease. This review aimed to estimate the effectiveness of interventions to promote healthy diet for primary prevention among participants attending primary care. Methods: A systematic review of trials using individual or cluster randomisation of interventions delivered in primary care to promote dietary change over 12 months in healthy participants free from chronic disease or defined high risk states. Outcomes were change in fruit and vegetable intake, consumption of total fat and fibre and changes in serum cholesterol concentration. Results: Ten studies were included with 12,414 participants. The design and delivery of interventions were diverse with respect to grounding in behavioural theory and intervention intensity. A meta-analysis of three studies showed an increase in fruit consumption of 0.25 (0.01 to 0.49) servings per day, with an increase in vegetable consumption of 0.25 (0.06 to 0.44) serving per day. A further three studies that reported on fruit and vegetable consumption together showed a pooled increment of 0.50 (0.13 to 0.87) servings per day. The pooled effect on consumption of dietary fibre, from four studies, was estimated to be 1.97 (0.43 to 3.52) gm fibre per day. Data from five studies showed a mean decrease in total fat intake of 5.2% of total energy (1.5 to 8.8%). Data from three studies showed a mean decrease in serum cholesterol of 0.10 (−0.19 to 0.00) mmol/L. Conclusion: Presently-reported interventions to promote healthy diet for primary prevention in primary care, which illustrate a diverse range of intervention methods, may yield small beneficial changes in consumption of fruit, vegetables, fibre and fat over 12 months. The present results do not exclude the possibility that more effective intervention strategies might be developed. Keywords: Diet, Health promotion, Primary care, Systematic review, Meta-analysis intake of fruit, and 59% of the recommended intake of vegetables [2]. A higher intake of dietary fibre is associated with lower risk of all-cause mortality [3], as well as lower incidence of colorectal cancer [4] and stroke [5]. The esti- mated mean fibre intake for American adults is 15.9 gram per day, lower than the recommended intake of at least 25–38 gram per day [6]. RESEARCH ARTICLE Open Access * Correspondence: nawaraj.bhattarai@glasgow.ac.uk 1Department of Primary Care and Public Health Sciences, King’s College London, London, UK 3Health Economics and Health Technology Assessment Unit, Institute of Health and Wellbeing, University of Glasgow, 1 Lilybank Gardens, Glasgow G12 8RZ, UK Full list of author information is available at the end of the article Abstract Cardiovascular diseases and dia- betes are associated with obesity and high dietary intakes of fat and sugars [7] but a typical American diet includes 280% of the recommended intake of calories from solid fats and sugars [2]. Obesity imposes a significant burden of morbidity and mortality on populations. The health care costs associated with obesity are substantial and the © 2013 Bhattarai et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Effectiveness of interventions to promote healthy diet in primary care: systematic review and meta-analysis of randomised controlled trials Nawaraj Bhattarai1,3*, A Toby Prevost1, Alison J Wright1, Judith Charlton1, Caroline Rudisill2 and Martin C Gulliford1 © 2013 Bhattarai et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. DOI: 10.1186/1471-2458-13-1203 © 2013 2013 The Authors, licensee BioMed Central Ltd. © CC BY 2.0 © 2013 2013 The Authors, licensee BioMed Central Ltd. © CC BY 2.0 This version available at: http://eprints.lse.ac.uk/55376/ LSE has developed LSE Research Online so that users may access research output of the School. Copyright © and Moral Rights for the papers on this site are retained by the individual authors and/or other copyright owners. Users may download and/or print one copy of any article(s) in LSE Research Online to facilitate their private study or for non-commercial research. You may not engage in further distribution of the material or use it for any profit-making activities or any commercial gain. You may freely distribute the URL (http://eprints.lse.ac.uk) of the LSE Research Online website. Bhattarai et al. BMC Public Health 2013, 13:1203 http://www.biomedcentral.com/1471-2458/13/1203 Background Comparators included usual care or no intervention. We excluded those trials comparing one type of diet promotion intervention with another only because our aim was to estimate the effect size differ- ence between a diet promotion intervention and the existing usual care or no intervention; we did not aim to compare any two methods of diet promotion interven- tions. A minimum follow- up period of 12 months after randomisation was required. Only English language publi- cations were included. vast majority of the costs are attributable to treating health consequences of obesity including type 2 diabetes, cancer and cardiovascular diseases [8]. There is evidence for the effectiveness of primary care-based interventions to promote physical activity [9], alcohol reduction [10] and smoking cessation [11]. The regularity of patient consultations in primary care [12], and the value that patients place on medical advice [13], offer opportunities for general practitioners to play important roles in promoting health and preventing disease. This may include the provision of advice on healthy eating. Several randomised trials have evaluated the potential to modify patients dietary habits through primary care based interventions. However, earlier system- atic reviews of the effectiveness of dietary interventions for primary prevention are limited in their applicability to primary care by the inclusion of studies set in work places, shopping centres and churches. Some reviews have included non-randomised studies [14] and trials with short follow-up, as well as participants with estab- lished medical conditions, or patients with defined high- risk status [14-17], with the possibility of diet restrictions and which would limit the participation in diet promotion intervention. Recent work on understanding the effectiveness of interventions to increase healthy diet has focused on the importance of using behaviour science theory to understand determinants of behaviour. Use of specific intervention techniques including setting goals, moni- toring behaviour, and reviewing progress towards goals in the light of feedback may be key to dietary behaviour change [18,19]. The effectiveness of behavioural interven- tions may also depend on factors such as the frequency of contacts, the type of professional involved, and whether delivered individually or in a group setting. We report a systematic review and meta-analysis of randomised controlled trials of primary care-based diet promotion interventions for primary prevention in adults with minimum 12 months follow up. Background An increase in intake of fruit and vegetables of one por- tion per day (80 g/day) may be associated with a 10% rela- tive reduction in risk of ischaemic heart disease and 6% reduction in stroke, with between 1% and 6% reduction in risk of certain cancers [1]. However, a typical American diet includes only includes 42% of the recommended daily Bhattarai et al. BMC Public Health 2013, 13:1203 http://www.biomedcentral.com/1471-2458/13/1203 Page 2 of 14 in primary care, including dietary counselling, motiv- ational interviews, advice for behaviour change, computer- delivered dietary information, reminder telephone calls and postal newsletters. Primary care in this context refers to interventions delivered through the first point of contact in a health care system, where the service provider acts as the principal source of advice to patients, rather than through specialist referral. Dietary promotion intervention in this context means any methods which are used to promote healthy diet, including healthy eating advice and counselling, telephone calls, group lectures or use of any other dietary education materials in- cluding posters, booklets and guidelines. We excluded multifaceted interventions including those with physical activity promotion along with diet promotion and we did not set any threshold amount of physical activity for exclusion. Target populations included the general popula- tion of adults aged 16 years or over, including both men and women. We excluded studies in pregnant women, patients at high risk of, or diagnosed with, cardiovascu- lar diseases, type 2 diabetes, cancer or other chronic conditions, as well as studies in first or second degree relatives of affected individuals. We also excluded studies that included participants at high risk of colorectal cancer (because of adenomatous polyps) [20] or breast cancer (with mammographic abnormalities) [21,22]. In order to focus on a population approach to primary prevention, we excluded trials which included participants who were pregnant, or with existing chronic conditions, or at high risk of diseases such as colorectal or breast cancer, or with participants who were relatives of family members with chronic health problems linked to diet. Such high risk participants or those with established chronic conditions linked with diet, may be more motivated to make dietary behaviour changes which may apparently show higher effectiveness of interventions promoting healthy diet in primary care. Also, there may be possibilities of diet restrictions which may limit the participation in diet promotion intervention and may apparently show lower effectiveness. Background The aim was to quantify whether diet promotion for primary prevention in primary care is effective in sustained dietary modifi- cations over at least one year. We also aimed to charac- terise existing interventions in terms of their theoretical basis and intervention techniques employed, and explore whether these were related to intervention effectiveness. Eligibility criteria The review included reports of randomised or cluster controlled trial study designs. Outcome measures included fruit and vegetable intake (servings/day), fat (% of total energy intake), fibre consumption (gram per day) and change in serum cholesterol level (mg/dl or mmol/l). Interventions included any diet promotion intervention Methodological quality assessment Methodological quality assessment NB appraised each study for methodological quality using a standard guidance and checklist [25]. We assessed the methodological quality and risk of bias in terms of ran- domisation, allocation concealment, blinding, loss to fol- low up and outcome assessment tool validity. Search methods, study selection and data extraction search terms “dietary intervention AND primary care”, “diet promotion intervention AND primary care”, “diet advice AND primary Care”, “counselling AND diet AND primary care”, “diet promotion AND primary care”, “diet advice AND behaviour change”, “advice in primary care AND behaviour AND diet”, “nutritional counselling AND primary care”, “lifestyle counselling AND cardiovascular risk AND primary care”, “fruit AND vegetable AND primary care”, “nutritional counselling AND general practice”, “dietary intervention AND general practice”, “dietary intervention AND primary care practice”, “nutritional advice AND general practice”, “primary care and diet modification”, “fruit and vegetables consumption AND general practice” “primary care AND fiber consump- tion” and “fruit and vegetable consumption AND diet intervention”. We also reviewed reference lists of relevant articles and previous systematic reviews. The search was carried out initially in September 2012 and again in March 2013. NB carried out initial screening of title and abstracts against inclusion criteria and retrieved those potentially eligible. NB and MCG independently assessed the re- trieved full text articles and any differences were reviewed and agreed. NB extracted data concerning participants, interventions, and outcomes in a tabular form designed for this review. AJW and NB extracted data on the nature of each intervention, including total number of contacts with participants, mode(s) of administration and intervention techniques used [23] and coded the extent to which intervention was based on psychological theories of the determinants of behaviour change, using a published coding scheme [24]. NB and MCG cross checked the extracted data. We used random effects meta-analysis to pool the estimates from individual studies. We used the I2 stat- istic to describe the variation in effect size attributable to heterogeneity among studies; higher values suggest- ing greater heterogeneity. We also constructed funnel plots to assess for the publication bias for the studies included in the review. We used the metan command in STATA version 12 for the analysis. Search methods, study selection and data extraction We searched Medline, PsycINFO, EMBASE, Centre for Reviews and Dissemination, and the Cochrane Library, with no restrictions in the date and year, using the combined Bhattarai et al. BMC Public Health 2013, 13:1203 http://www.biomedcentral.com/1471-2458/13/1203 Page 3 of 14 in mg/dl, the conversion factor of 38.6598 was used to express them as mmol/L. search terms “dietary intervention AND primary care”, “diet promotion intervention AND primary care”, “diet advice AND primary Care”, “counselling AND diet AND primary care”, “diet promotion AND primary care”, “diet advice AND behaviour change”, “advice in primary care AND behaviour AND diet”, “nutritional counselling AND primary care”, “lifestyle counselling AND cardiovascular risk AND primary care”, “fruit AND vegetable AND primary care”, “nutritional counselling AND general practice”, “dietary intervention AND general practice”, “dietary intervention AND primary care practice”, “nutritional advice AND general practice”, “primary care and diet modification”, “fruit and vegetables consumption AND general practice” “primary care AND fiber consump- tion” and “fruit and vegetable consumption AND diet intervention”. We also reviewed reference lists of relevant articles and previous systematic reviews. The search was carried out initially in September 2012 and again in March 2013. NB carried out initial screening of title and abstracts against inclusion criteria and retrieved those potentially eligible. NB and MCG independently assessed the re- trieved full text articles and any differences were reviewed and agreed. NB extracted data concerning participants, interventions, and outcomes in a tabular form designed for this review. AJW and NB extracted data on the nature of each intervention, including total number of contacts with participants, mode(s) of administration and intervention techniques used [23] and coded the extent to which intervention was based on psychological theories of the determinants of behaviour change, using a published coding scheme [24]. NB and MCG cross checked the extracted data. Identification of trials We screened titles and abstracts of 2,932 papers and identified 49 full text articles. We further identified 3 full text articles from cross-checking references of 49 full text articles. We then identified 10 trials [27-36] for inclusion in this study after excluding trials not meeting the eligibility criteria. The details are shown in the flow diagram (Figure 1). Study and participants characteristics We present the study and participant characteristics in Table 1. Ten studies included in the systematic review were published between 1988 and 2006. These studies were conducted in samples representing the primary care general population in Japan [27] (1 study), USA [28,31,32,34-36] (6 studies), Italy [30] (1 study) and UK [29,33] (2 studies).The randomised study sample size varied among studies and ranged from 213 to 3,179 participants with 12,414 participants randomised in total. Participants were men and women, but three studies [28,31,36] included women only, with the age ranging from 18 to 79 years. Participants were generally healthy without established chronic diseases. Participants in one study [28] were postmenopausal women consuming at least 36% energy from fat; participants in another study [31] had serum cholesterol values of 200 mg/dl (5.17 mmol/l) or more but as this is close to the population mean value, this study was not excluded as being directed at high risk individuals. We present the study and participant characteristics in Table 1. Ten studies included in the systematic review were published between 1988 and 2006. These studies were conducted in samples representing the primary care general population in Japan [27] (1 study), USA [28,31,32,34-36] (6 studies), Italy [30] (1 study) and UK [29,33] (2 studies).The randomised study sample size varied among studies and ranged from 213 to 3,179 participants with 12,414 participants randomised in total. Participants were men and women, but three studies [28,31,36] included women only, with the age ranging from 18 to 79 years. Participants were generally healthy without established chronic diseases. Participants in one study [28] were postmenopausal women consuming at least 36% energy from fat; participants in another study [31] had serum cholesterol values of 200 mg/dl (5.17 mmol/l) or more but as this is close to the population mean value, this study was not excluded as being directed at high risk individuals. Intervention and control characteristics For each trial, we extracted the intervention effects at 12 months. We estimated the intervention effect as the difference in the change in mean outcome values (follow up value minus baseline value) between the intervention group and control group. If the baseline data were not reported in the trials, we used the difference in the mean outcomes between groups at follow up. If not supplied, we followed standard procedure [26] to derive standard error (SE) for each measure. Where fruit and vegetable consumption was expressed in grams, the conversion factor of one serving = 80 gm was used to express them as servings and where the serum cholesterol was expressed The diet promotion interventions in the trials varied in number of contacts with participants, mode of delivery and behaviour change techniques employed (Table 2). The number of scheduled contacts for intervention with the participants in the intervention groups ranged between one and twenty. Most involved at least one face to face contact, but two [32,34] involved only a combination of telephone calls and mailed intervention materials. Of the interventions using face-to-face sessions, only one [28] was solely delivered in a group format, while the others used a combination of group and individual contacts. Bhattarai et al. BMC Public Health 2013, 13:1203 http://www.biomedcentral.com/1471-2458/13/1203 Page 4 of 14 Figure 1 Study selection flow diagram. Figure 1 Study selection flow diagram. Many of the interventions also involve printed mate- rials. The interventions involved between two and eight intervention techniques. Interventions that involved a greater number of contacts with participants did not necessarily employ a greater number of techniques. In each study, the control group was not enrolled in any intervention, but four had minimal interventions: dietary guidelines [28], standard health education from leaflets [29], a non-personalised conversation without diet counselling [30] and breast self-examination counselling [31] (Table 2). Four [31,32,34,35] of the ten interventions were expli- citly described as being based on at least one psycho- logical theory of behaviour change. While none of the four reports explicitly linked all components of the inter- vention to all the relevant constructs of the theoretical model(s) upon which they claimed to be based, they all explicitly linked at least one intervention technique to at least one determinant of behaviour specified by relevant psychological theory. Intervention and control characteristics In three [31,32,34] of the interventions, the intervention was tailored for partici- pants according to how they varied on a psychological construct specified by a theory. In each trial, previously validated self-administered food frequency questionnaires, or modified simpler versions, were used to measure the study outcomes. Diet intakes were estimated using the average of the diet consump- tion in the previous 24 hours to 1 month, collected using food frequency questionnaires. No on 8 2 oval our) on of theory techniques CT tech used 0 2 theory Methodological quality of included studies The funnel plots and Egger’s test for potential publication bias were not informative as insufficient studies were identified for each outcome. Table 3 presents a summary Number of practices Participants randomised (% Female) Eligible age range (mean) years Ethnicity and socioeconomic status Diet assessment tool One group general practice 437 randomised 368 participated (49) 25-60 (41.7) Social class 1 or 2: controls, 30% men, 24% women; intervention 39% men, 43% women. Self-administered food frequency questionnaire 28 physician practices within 6 clinics 2121 (68) 26% > 65 years White: 91%; Some college education: 73%. Family income below $25000 per year: 28%. Telephone interview administered food frequency questionnaire University clinical centres in three states 2208 (100) 50-79 (60) White (55%), Black (28%), Hispanic (16%); <High School (11%), High School (20%), Post high school with no college degree (35%), graduate /post graduate (33%) Self-administered food frequency questionnaire Three rural Virginia physician practices 754 (64) 18-72 (46.34) White: 61%, African American: 37%; 8th grade: College degree: 24%; Income < $10,000: 14.69%, ≥$41,000:19%. Telephone interview administered fat and fibre behaviour questionnaire One clinic 213 (100) 20-40 (33.4) 76% White, 13.5% Black, 4% Hispanic, 5.5% Asian, 1.5% other; 85% completed college Telephone interview administered food frequency questionnaire, based on 24 hr diet recall on each of three days Health maintenance organisation 1459 (50) 18-69 (45.8) White (85.9%), Black (4.5%), Asian (5.8%), Hispanic (3.0%), Other (0.8%); Household income < $25,000 12.2%, ≥$70,000 21.7%. Telephone interview administered Food Frequency Questionnaire (FFQ) and Diet Habits Questionnaire 8 family practices 956 (50) 35-59 (47.3) Non-manual occupation, intervention 60%, control 49%; rented accommodation intervention 11%, control 25%. Self-administered food frequency questionnaire. 33 general practitioners 3179 (50) 18-65 (44.5) Not reported Family physician administered food frequency questionnaire Health maintenance organization (HMO) 616 (100) 40-70 (53.8) Minority groups: 7%; College graduates: 40% Self-administered fat and fibre behaviour questionnaire (FFBQ) Not reported 550 (68) 40-69 (56) Not reported Self-administered diet history questionnaire (DHQ) aire. aire and t omote dietary proving health, ging social norm her fibre foods, ng positive for n of booklet taking 2 weeks later, a ed by physician ants who had ntion. theory consequences of behaviour usual care 3. provide information regarding others’ approval 5. goal setting (behaviour) 8. Barrier identification and problem solving? 9. Set graded tasks 19. Provide feedback on performance 21. provide information on how to do the behaviour 27. sed g, Yes – stage change from the TTM nt on effort/ owards r and on l behaviour pt self monitoring de information o perform the 8 s – stage ange from e TTM Methodological quality of included studies Use of follow up prompts sessions in groups 6 weeks, bi-weekly y for 9 months and 18 months. Group periences. None N/A 5. goal setting – behaviour 8 2 Not counselled, but given Dietary Guidelines for Americans 8. problem solving 12. prompt rewards contingent on effort/ success towards behaviour and on successful behaviour 16. prompt self monitoring 21. provide information on how to perform the behaviour 22. model/demonstrate the behaviour 26. prompt practice 29. plan social support usual care p behaviour 22. model/demonstrate the behaviour 26. prompt practice 29 plan social support 21. provide information on how to perform the behaviour 2 22. model/demonstrate the behaviour No intervention until after end of study ention) tting – behaviour 8 m solving pt rewards nt on effort/ owards r and on l behaviour pt self monitoring de information o perform the without the use of a brochure. No dietary advice, however advised on Breast Self Examination(BSE) e y ed iew . on, nd he Non rint ng ed d e Soc the Non onsistent w ***4 techn p rmation sequences ur 7 2 Usual care (No intervention) – behaviour asks edback on on how to ehaviour monstrate (?) w-up - behaviour 3 or 4*** 2 or 3** No intervention until after end of study behaviour 22. model/demonstrate the behaviour 26. prompt practice 29 plan social support 21. provide information on how to perform the behaviour 2 22. model/demonstrate the behaviour N/A new onstr ons in ce of ventio elp m h be Standard health education leaflet, Guide to healthy eating 10 prompt review of behavioural goals 16. or 17.(For some) self- monitoring – not quite clear if this was of the behaviour or of weight. 21. instruction on how to perform the behaviour 1. provide information about the consequences of behaviour 2 0 A simpler and non personalized conversation without the use of a brochure. 21. provide instruction on how to perform the behaviour ersonal self and 5. goal setting – behaviour 7 3 No dietary advice, however advised on Breast Self Examination(BSE) 8. barrier identification and problem solving 9. set graded tasks 10. prompt review of behavioural goals 19. provide feedback on performance 21. provide instruction on how to perform the behaviour 37. motivational interviewing 5. goal setting – behaviour 3 2 No intervention 19. provide feedback on performance 21. provide instruction on how to do the behaviour e following four intervention techniques: prompt specific goal setting, prompt review of behavioural s overweight, otherwise three techniques. http://www.biomedcentral.com/1471 2458/13/1203 p p p Standard health education leaflet, Guide to healthy eating review of l goals or some) self- – not quite was of the or of weight. ion on how to e behaviour nformation consequences ur 2 0 A simpler and non personalized conversation without the use of a brochure. e instruction on rform the ng – behaviour 7 3 No dietary advice however about the consequences of behaviour non personalized conversation without the use of a brochure. 21. provide instruction on how to perform the behaviour cognitive TTM Yes – personal barriers, self efficacy and stage of change 5. goal setting – behaviour 7 3 No dietary advice, however advised on Breast Self Examination(BSE) 8. barrier identification and problem solving 9. set graded tasks 10. prompt review of behavioural goals 19. provide feedback on performance 21. provide instruction on how to perform the behaviour 37. motivational interviewing N/A 5. goal setting – behaviour 3 2 No intervention 19. provide feedback on performance 21. provide instruction on how to do the behaviour control theory (out of the following four intervention techniques: prompt specific goal setting, prompt review of behavioural es used if participant was overweight, otherwise three techniques. without the use of a brochure. No dietary advice, however advised on Breast Self Examination(BSE) e y ed iew . n, nd he Non rint ng ed d e Soc theo Non onsistent w ***4 techn men d ding Participation at 12 months Outcome assessm validity reported l. Nurse assessment was blinded 448/550 (81%) Yes stated 1,141/2,208 (52%) Yes stated Intervention 86%; control 74% Yes ome assessors and participants d to be blinded 2,977/3,179 (93%) Yes al. Clinic staff conducting data ction were blinded Intervention 89%; control 85% Yes stated 1,205/1,459 (83%) Partial stated 329/368 (89%) Not stated stated 516/754 (68%) Yes 1,818/2,121 (86%) Yes stated 177/213 (83%) Yes Bhattarai et al. BMC Public Health 2013, 13:1203 http://www.biomedcentral.com/1471-2458/13/1203 Bhattarai et al. BMC Public Health 2013, 13:1203 http://www.biomedcentral.com/1471-2458/13/1203 Page 10 of 14 Vegetable consumption was increased by 0.25 (0.06 to 0.44, p = 0.01) servings per day, with less evidence of hetero- geneity among studies was I2 = 58.4%, p = 0.091 (Figure 2). Three studies [30-32] reporting intervention effects on consumption of fruit and vegetable combined together showed a pooled effect of 0.50 (0.13 to 0.87, 0.008) servings per day. p There was evidence of substantial heterogeneity (I2 = 90.5%; p < 0.001) (Figure 2). The sum- mary measure had a conservatively wide 95% confidence interval (0.13 to 0.87) for the pooled effect by use of the random effects model. of the methodological quality of included studies. In general, description of randomisation method, blinding of the outcome assessment and allocation concealment were poor in the trials. None of the studies reported what steps were taken to ensure intervention fidelity [37]. Only one study assessed the psychological con- structs targeted by the intervention, and briefly reported a mediation analysis to see if the intervention worked by changing the targeted beliefs. Therefore, the ability of the studies to test the psychological mechanisms of the interventions’ effects on dietary intakes was seriously compromised [24]. Intervention effect on fibre intake Six studies [27,29,33-36] reported the intervention effect on fibre intake, but two [34,35] of these studies were not included in the meta-analysis. The estimated inter- vention effects in the study [35] reporting the fibre intake in grams per 1000 kcal was 0.32 (SE 0.19) grams per 1000 kcal, whereas in the study [34] reporting the fibre intake score, the estimated intervention effect was −0.04 (SE 0.04), with a negative score indicating increased Fruit and vegetable intake Pooled analysis of three studies [27-29], which reported intervention effects on consumption of fruits and vege- tables separately, showed a mean difference of 0.25 (95% confidence interval (CI) 0.01 to 0.49, p = 0.04) serving per day for fruit consumption. There was evidence of ap- preciable heterogeneity (I2 = 88.0%, p < 0.001) (Figure 2). Figure 2 Individual study and pooled effects of diet promotion on intakes of fruit, vegetables, and combined fruit and vegetable at 12 months. ES = effect size, 95% CI = 95% confidence intervals. Figure 2 Individual study and pooled effects of diet promotion on intakes of fruit, vegetables, and combined fruit and vegetable at 12 months. ES = effect size, 95% CI = 95% confidence intervals. Bhattarai et al. BMC Public Health 2013, 13:1203 http://www.biomedcentral.com/1471-2458/13/1203 Bhattarai et al. BMC Public Health 2013, 13:1203 http://www.biomedcentral.com/1471-2458/13/1203 Bhattarai et al. BMC Public Health 2013, 13:1203 http://www.biomedcentral.com/1471-2458/13/1203 Page 11 of 14 0.19 mmol/L, p = 0.049) more mean decrease in serum cholesterol level in people receiving the diet promotion intervention compared to the comparators (heterogeneity among studies I2 = 3%, p = 0.36) (Figure 3C). fibre intake. Pooled analysis of the remaining four studies showed evidence of increase of 1.97 (0.43 to 3.52, p = 0.012) grams of fibre consumption per day. There was some evi- dence of heterogeneity (I2 = 70.4%, p = 0.017) (Figure 3A). The impact of intervention characteristics on observed effects A descriptive analysis was implemented because the small number of included studies meant that it was not possible to conduct meta-regression analyses to formally test the impact of intervention characteristics on observed effect sizes. For fruit and vegetable intake, assessed either singly or in combination, and for serum cholesterol, effect sizes did not clearly increase with increasing participant contact. In contrast, for fibre and fat intake, the interventions with the most contacts clearly had the largest effects. None of the interventions that contributed data to pooled effect size estimates for fruit, vegetable or fibre intake were based on theory. However, for fruit and vegetable consumption con- sidered together, interventions based on theory appeared to have larger effects than those not based on theory. In contrast, the most effective interventions for reducing fat intake were not based on theory, nor was there any clear benefit of using theory for reducing serum choles- terol. Fruit and vegetable intake There was no clear relationship between the total number of intervention techniques used in an interven- tion and the effect sizes observed. Intervention effects on fat intake Seven studies reported the effects of intervention on fat intake, but two [32,34] of these studies were not included in the meta-analysis. The estimated intervention effect in one [32] of these studies was −0.1(SE0.02) and in the other [34] study was −0.06 (SE 0.041), both expressed in scores scales with negative score indicating decreased fat intake. Pooled analysis from five studies [28,29,31,35,36] showed a mean decrease of 5.16% (95% CI −8.81 to −1.52, p = 0.005) in fat intake, expressed as percentage of total energy intake per day. There was heterogeneity between studies (I2 = 98.8%, p < 0.001) with one study [28] show- ing a very large change (Figure 3B). Intervention effects on cholesterol Dietary cholesterol intake was evaluated in one study [28] with a 79.2 mg (95% CI 61.9 to 96.5) more decrease in dietary cholesterol per day when compared to those not receiving the diet promotion intervention. Three studies [29,31,33] analysed the intervention effects in the serum cholesterol level. There was a 0.10 mmol/L (0 to Figure 3 Individual study and pooled effects of diet promotion on fiber and fat intake and serum cholesterol level at 12 months. ES = effect size, 95%CI = 95% confidence intervals. Figure 3 Individual study and pooled effects of diet promotion on fiber and fat intake and serum cholesterol level at 12 months. ES = effect size, 95%CI = 95% confidence intervals. Bhattarai et al. BMC Public Health 2013, 13:1203 http://www.biomedcentral.com/1471-2458/13/1203 Bhattarai et al. BMC Public Health 2013, 13:1203 http://www.biomedcentral.com/1471-2458/13/1203 Page 12 of 14 Page 12 of 14 assessors and trial personnel may have introduced bias in trials thereby overestimating the effectiveness of interven- tion. We restricted the inclusion of trials investigating the effects of multi-factorial interventions to avoid the potential confounding effect of other health promoting interventions. However, assessing effectiveness of only diet promotion interventions may have overestimated the effects in practice where diet promotion may run simultaneously with other health promotion activities. Where reported, most of the trial participants were white and had some college education, while few reported socio- economic status. We cannot confirm whether our results can be applied to populations with different ethnic, edu- cational and socio-economic characteristics. We do not know whether it is cost-effective to implement diet pro- motion interventions with similar intensity as reported in the trials in the primary care general population. Par- ticipants with unknown previous exposures to health campaigns and media may increase the selection bias. We may have missed some unpublished or published trials. Discussion The results of this systematic review of randomised con- trolled trials suggest that moderately sustained but small effects on diet can be achieved through diet promotion interventions in primary care. The heterogeneity in observed effect sizes suggests that these interventions, despite all being delivered or deliverable in primary care, varied a great deal in their impact on behaviour. The studies employed a range of behaviour change techniques in- cluding one-to-one counselling along with variety of media with variable theoretical under-pinning. Incomplete report- ing makes it difficult to establish how interventions were intended to achieve their effects. No study reported monitoring treatment fidelity, so it is unclear whether interventions were delivered as their designers planned. If interventions failed to change behaviour, was this because the components of the intervention were not effective? or because these were not successfully delivered? Comparison with other studies A Cochrane systematic review [17] reported that diet promotion intervention in health care setting increased fruit and vegetable intake by 1.88 (95% CI1.07 to 2.70) servings per day; and irrespective of setting fruit intake alone increased by 0.67 (95% CI 0.007 to 1.28) servings per day and vegetable intake alone by 0.92 (95% CI0.34 to 1.49) servings per day, fibre intake increased by 6.51 (95% CI2.20 to 10.82) grams per day, in health care setting total dietary fat intake expressed as percentage of total calories fell by 5.38% (95% CI −7.84 to −2.92) and total blood cholesterol level reduced by 0.11 mmol/l (95% CI – 0.19 to −0.03). Effect sizes were generally con- siderably smaller in our study compared to the Cochrane review. The Cochrane review included studies with partic- ipants with chronic conditions, as well as participants at high risk of colorectal cancer and breast cancer. Studies were carried out in faith and work settings as well as in primary care. These differences in participant characteris- tics, resulting from differences in inclusion/exclusion cri- teria between our study and Cochrane review may explain these differences in results. Another review [16] published in 1998, reported 5.5% reduction in total blood cholesterol level; expressed in a different measurement unit than our study and we could not compare the changes in blood cholesterol with our results. We acknowledge several limitations. Most of the included trials used self-reported measures of dietary change, and there are possibilities that intervention effects may have suffered responder bias. Furthermore, we cannot rule out the possibility of contamination in open trials which could have resulted in the exchange of information on diet mod- ifications and may have modified outcomes in controls. Dietary interventions cannot be completely blinded; how- ever, outcome assessors can be blinded to avoid the chance of selection bias in trials. Not blinding outcome Strengths and limitations Our review has several strengths compared with earlier reviews [14-17]. We estimated the long-term effective- ness of dietary interventions in primary care using only randomised controlled trials or cluster randomised trials with at least 12 months follow-up, where interventions were delivered mostly by health care professionals. In order to focus on a population approach to primary pre- vention, we did not include trials which included partic- ipants with existing chronic conditions or at high risk of diseases such as colorectal or breast cancer and partici- pants with relatives or family members with chronic health problems linked to diet. We did not include trials implemented in the workplace or faith settings where characteristics of participants may be different from those attending the primary health care. We did not restrict the inclusion of trials on the basis of percentage loss to follow up which limited the evidence to trials with more adherent participants and we included two trials [29,34] in our review which were not included in the Cochrane review. The restriction of studies with more than 20% loss to follow up may introduce bias in the findings limited to trials with more adherent participants. We also explored the existing interventions in terms of behaviour change techniques used. Author details 1 1Department of Primary Care and Public Health Sciences, King’s College London, London, UK. 2Department of Social Policy, London School of Economics and Political Science, London, UK. 3Health Economics and Health Technology Assessment Unit, Institute of Health and Wellbeing, University of Glasgow, 1 Lilybank Gardens, Glasgow G12 8RZ, UK. 21. Boyd NF, Martin LJ, Beaton M, Cousins M, Kriukov V: Long-term effects of participation in a randomized trial of a low-fat, high-carbohydrate diet. Cancer Epidemiol Biomarkers Prev 1996, 5(3):217–222. 22. Lee-Han H, Cousins M, Beaton M, McGuire V, Kriukov V, Chipman M, Boyd N: Compliance in a randomized clinical trial of dietary fat reduction in patients with breast dysplasia. Am J Clin Nutr 1988, 48(3):575–586. Authors’ contributions MCG conceived the study. All authors contributed to the design of the study. NB carried out the search, extracted the data, performed the analysis and prepared the first draft. AJW and NB extracted the data in terms of the behavioural theories and coded the interventions. MCG and NB cross checked the extracted data and analysis. All authors contributed to the revisions, read and approved the final manuscript. 14. Ammerman AS, Lindquist CH, Lohr KN, Hersey J: The efficacy of behavioral interventions to modify dietary fat and fruit and vegetable intake: a review of the evidence. Prev Med 2002, 35(1):25–41. 14. Ammerman AS, Lindquist CH, Lohr KN, Hersey J: The efficacy of behavioral interventions to modify dietary fat and fruit and vegetable intake: a review of the evidence. Prev Med 2002, 35(1):25–41. 15. Pomerleau JL K, Knai C, McKee M: Interventions designed to increase adult fruit and vegetable intake can be effective- a systematic review of literature. J Nutr 2005, 135:2486–2495. 16. Tang JL, Smith GD, Armitage JM, Lancaster T, Silagy CA, Fowler GH, Neil HAW, Ebrahim S: Systematic review of dietary intervention trials to lower blood total cholesterol in free-living subjectsCommentary: Dietary change, cholesterol reduction, and the public health—what does meta-analysis add? Bmj 1998, 316(7139):1213–1220. Competing interests h h d l h Competing interests The authors declare that they have no competing interests. 12. Royal College of General Practitioners: Healthy people: promoting health and preventing disease. London: Royal College of General Practitioners; 2009. 13. Boyce T, Peckham S, Hann A, Trenholm S: A proactive approach. Health promotion and ill health prevention. London: The King’s Fund; 2010. Acknowledgements This study was supported by the UK National Prevention Research Initiative whose funding partners include the Alzheimer’s Research Trust; Alzheimer’s Society; Biotechnology and Biological Sciences Research Council; British Heart Foundation; Cancer Research UK; Chief Scientist Office, Scottish Government Health Directorate; Department of Health; Diabetes UK; Economic and Social Research Council; Engineering and Physical Sciences Research Council; Health & Social Care Research & Development Office for Northern Ireland; Medical Research Council; The Stroke Association; Welsh Assembly Government; and World Cancer Research Fund. 17. Rees K, Dyakova M, Ward K, Thorogood M, Brunner E: Dietary advice for reducing cardiovascular risk. Cochrane Database Syst Rev 2013. Mar 28(3): CD002128. doi:10.1002/14651858.CD002128.pub4. 18. Michie S, Abraham C, Whittington C, McAteer J, Gupta S: Effective techniques in healthy eating and physical activity interventions: a meta-regression. Health Psychol 2009, 28(6):690–701. This research was supported by the National Institute for Health Research (NIHR) Biomedical Research Centre at Guy’s and St Thomas’ NHS Foundation Trust and King’s College London. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR or the Department of Health. 19. Dombrowski SU, Sniehotta FF, Avenell A, Johnston M, MacLennan G, Araújo-Soares V: Identifying active ingredients in complex behavioural interventions for obese adults with obesity-related co-morbidities or additional risk factors for co-morbidities: a systematic review. Health Psychol Rev 2010, 6(1):7–32. 20. Smith-Warner SA, Elmer PJ, Tharp TM, Fosdick L, Randall B, Gross M, Wood J, Potter JD: Increasing vegetable and fruit intake: randomized intervention and monitoring in an at-risk population. Cancer Epidemiol Biomarkers Prev 2000, 9(3):307–317. Conclusions 9. Orrow G, Kinmonth A-L, Sanderson S, Sutton S: Effectiveness of physical activity promotion based in primary care: systematic review and meta-analysis of randomised controlled trials. BMJ 2012, 344:e1389. 10. Kaner EF, Beyer F, Dickinson HO, Pienaar E, Campbell F, Schlesinger C, Heather N, Saunders J, Burnand B: Effectiveness of brief alcohol interventions in primary care populations. Cochrane Database Syst Rev. 2007 Apr 18(2): CD004148. doi: 10.1002/14651858.CD004148.pub3. 10. Kaner EF, Beyer F, Dickinson HO, Pienaar E, Campbell F, Schlesinger C, Heather N, Saunders J, Burnand B: Effectiveness of brief alcohol interventions in primary care populations. Cochrane Database Syst Rev. 2007 Apr 18(2): CD004148. doi: 10.1002/14651858.CD004148.pub3. 11. Stead LF, Bergson G, Lancaster T: Physician advice for smoking cessation. Cochrane Database Syst Rev 2008, 16(2):CD000165. 11. Stead LF, Bergson G, Lancaster T: Physician advice for smoking cessation. Cochrane Database Syst Rev 2008, 16(2):CD000165. 11. Stead LF, Bergson G, Lancaster T: Physician advice for smoking cessation. Cochrane Database Syst Rev 2008, 16(2):CD000165. 12. Royal College of General Practitioners: Healthy people: promoting health and preventing disease. London: Royal College of General Practitioners; 2009. 13. Boyce T, Peckham S, Hann A, Trenholm S: A proactive approach. Health promotion and ill health prevention. London: The King’s Fund; 2010. 14. Ammerman AS, Lindquist CH, Lohr KN, Hersey J: The efficacy of behavioral inter entions to modif dietar fat and fr it and egetable intake a re ie Conclusions Our review suggests that diet promotion interventions in the primary care population yield modest positive effects on diet intake over one year. Opportunities to promote dietary change may be taken by general practice based health care staff during patient general practice Bhattarai et al. BMC Public Health 2013, 13:1203 http://www.biomedcentral.com/1471-2458/13/1203 Page 13 of 14 visits. Our findings should be interpreted with caution because of some of the limitations of the review and the included studies. The present studies may offer limited potential to inform the science of dietary behaviour change in primary care. For example, it is unclear whether inter- ventions are ineffective or whether these are difficult to deliver in routine practice, possibly requiring greater training to deliver consistently. Our findings raise ques- tions concerning whether a brief single diet counselling can be effective as more intensive diet promotion inter- ventions; whether diet promotion interventions will have the same effect in minority ethnic groups and in a deprived general population; and whether the limited effects sizes are of long-term benefit in relation to resources used. 5. Chen GC, Lv DB, Pang Z, Dong JY, Liu QF: Dietary fiber intake and stroke risk: a meta-analysis of prospective cohort studies. Eur J Clin Nutr 2013(67):96–100. 110.1038/ejcn.2012.1158. 6. King DE, Mainous Iii AG, Lambourne CA: Trends in Dietary Fiber Intake in the United States, 1999–2008. J Acad Nutr Diet 2012, 112(5):642–648. 7. Hooper L, Summerbell CD, Higgins JPT, Thompson RL, Capps NE, Smith GD, Riemersma RA, Ebrahim S: Dietary fat intake and prevention of cardiovascular disease: systematic review. Bmj 2001, 322(7289):757–763. 7. Hooper L, Summerbell CD, Higgins JPT, Thompson RL, Capps NE, Smith GD, Riemersma RA, Ebrahim S: Dietary fat intake and prevention of cardiovascular disease: systematic review. Bmj 2001, 322(7289):757–763. 8. McCormick B, Stone I, Corporate Analytical T: Economic costs of obesity and the case for government intervention. Obes Rev 2007, 8:161–164. 9. Orrow G, Kinmonth A-L, Sanderson S, Sutton S: Effectiveness of physical activity promotion based in primary care: systematic review and meta-analysis of randomised controlled trials. BMJ 2012, 344:e1389. 10. Kaner EF, Beyer F, Dickinson HO, Pienaar E, Campbell F, Schlesinger C, Heather N, Saunders J, Burnand B: Effectiveness of brief alcohol interventions in primary care populations. Cochrane Database Syst Rev. 2007 Apr 18(2): CD004148. doi: 10.1002/14651858.CD004148.pub3. 8. McCormick B, Stone I, Corporate Analytical T: Economic costs of obesity and the case for government intervention. Obes Rev 2007, 8:161–164. 2. U.S. Department of Agriculture, U.S. Department of Health and Human Services: Dietary Guidelines for Americans. 7th edition. Washington, DC: U.S. Government Printing Office; 2010. References Takahashi Y, Sasaki S, Okubo S, Hayashi M, Tsugane S: Blood pressure change in a free-living population-based dietary modification study in Page 14 of 14 Bhattarai et al. BMC Public Health 2013, 13:1203 http://www.biomedcentral.com/1471-2458/13/1203 Bhattarai et al. BMC Public Health 2013, 13:1203 http://www.biomedcentral.com/1471-2458/13/1203 Japan. J Hypertens 2006, 24(3):451–458. 410.1097/1001. hjh.0000209980.0000236359.0000209916. Japan. J Hypertens 2006, 24(3):451–458. 410.1097/1001. y hjh.0000209980.0000236359.0000209916. 28. Coates RJ, Bowen DJ, Kristal AR, Feng Z, Oberman A, Hall WD, George V, Lewis CE, Kestin M, Davis M, et al: The women’s health trial feasibility study in minority populations: changes in dietary intakes. Am J Epidemiol 1999, 149(12):1104–1112. 29. Roderick P, Ruddock V, Hunt P, Miller G: A randomized trial to evaluate the effectiveness of dietary advice by practice nurses in lowering diet related coronary heart disease risk. Br J Gen Pract 1997, 47(414):7–12. 30. Sacerdote C, Fiorini L, Rosato R, Audenino M, Valpreda M, Vineis P: Randomized controlled trial: effect of nutritional counselling in general practice. Int J Epidemiol 2006, 35(2):409–415. 31. Stevens VJ, Glasgow RE, Toobert DJ, Karanja N, Smith KS: One-year results from a brief, computer-assisted intervention to decrease consumption of fat and increase consumption of fruits and vegetables. Prev Med 2003, 36(5):594–600. 32. Kristal AR, Curry SJ, Shattuck AL, Feng Z, Li S: A randomized trial of a tailored, self-help dietary intervention: the puget sound eating patterns study. Prev Med 2000, 31(4):380–389. 33. Baron JA, Gleason R, Crowe B, Mann JI: Preliminary trial of the effect of general practice based nutritional advice. Br J Gen Pract 1990, 40:137–141. 34. Fries E, Edinboro P, McClish D, Manion L, Bowen D, Beresford SAA, Ripley J: Randomized trial of a low-intensity dietary intervention in rural residents: The Rural Physician Cancer Prevention Project. Am J Prev Med 2005, 28(2):162–168. 35. Beresford SAA, Curry SJ, Kristal AR, Lazovich D, Feng Z, Wagner EH: A dietary intervention in primary care practice :The Eating Patterns Study. Am J Public Health 1997, 87:610–616. 36. Gann PH, Chatterton RT, Gapstur SM, Liu K, Garside D, Giovanazzi S, Thedford K, Van Horn L: The effects of a low-fat/high-fiber diet on sex hormone levels and menstrual cycling in premenopausal women. Cancer 2003, 98(9):1870–1879. 37. Bellg AJ, Borrelli B, Resnick B, Hecht J, Minicucci DS, Ory M, Ogedegbe G, Orwig D, Ernst D, Czajkowski S: Enhancing treatment fidelity in health behavior change studies: best practices and recommendations from the NIH behavior change consortium. Health Psychol 2004, 23(5):443–451. References 23. Michie S, Ashford S, Sniehotta FF, Dombrowski SU, Bishop A, French DP: A refined taxonomy of behaviour change techniques to help people change their physical activity and healthy eating behaviours: The CALO-RE taxonomy. Psychol Health 2011, 26(11):1479–1498. 1. Lock K, Pomerleau J, Causer L, Altmann DR, McKee M: The global burden of disease attributable to low consumption of fruit and vegetables: implications for the global strategy on diet. Bull World Health Organ 2005, 83(2):100–108. 1. Lock K, Pomerleau J, Causer L, Altmann DR, McKee M: The global burden of disease attributable to low consumption of fruit and vegetables: implications for the global strategy on diet. Bull World Health Organ 2005, 83(2):100–108. 24. Michie S, Prestwich A: Are interventions theory-based? Development of a theory coding scheme. Health Psychol 2010, 29(1):1–8. 2. U.S. Department of Agriculture, U.S. Department of Health and Human Services: Dietary Guidelines for Americans. 7th edition. Washington, DC: U.S. Government Printing Office; 2010. 2. U.S. Department of Agriculture, U.S. Department of Health and Human Services: Dietary Guidelines for Americans. 7th edition. Washington, DC: U.S. Government Printing Office; 2010. 25. National Institue for Health and Clinical Excellence: The guidelines manual -Appendix D: Methodology checklist: randomised controlled trials. London: National Institute for Health and Clinical Excellence; 2009. 3. Streppel MT, Ocké MC, Boshuizen HC, Kok FJ, Kromhout D: Dietary fiber intake in relation to coronary heart disease and all-cause mortality over 40 y: the Zutphen Study. Am J Clin Nutr 2008, 88(4):1119–1125. 3. Streppel MT, Ocké MC, Boshuizen HC, Kok FJ, Kromhout D: Dietary fiber intake in relation to coronary heart disease and all-cause mortality over 40 y: the Zutphen Study. Am J Clin Nutr 2008, 88(4):1119–1125. 26. Higgins JPT, Green S (Eds): Cochrane Handbook for Systematic Reviews of Intervention Version 5.1.0 [updated March 2011] chapter 7.7.3. The Cochrane Collaboration. 2011. Available from www.cochrane-handbook.org. 4. Murphy N, Norat T, Ferrari P, Jenab M, Bueno-de-Mesquita B, Skeie G, Dahm CC, Overvad K, Olsen A, Tjønneland A, et al: Dietary Fibre Intake and Risks of Cancers of the Colon and Rectum in the European Prospective Investigation into Cancer and Nutrition (EPIC). PLoS ONE 2012, 7(6):e39361. 27. Takahashi Y, Sasaki S, Okubo S, Hayashi M, Tsugane S: Blood pressure change in a free-living population-based dietary modification study in 27. References doi:10.1186/1471-2458-13-1203 Cite this article as: Bhattarai et al.: Effectiveness of interventions to promote healthy diet in primary care: systematic review and meta- analysis of randomised controlled trials. 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IMPROVING COMMUNITY UNDERSTANDING THROUGH SOCIALIZATION, LITERATURE AND EDUCATION OF SHARIA BANKING IN THE CITY OF PALANGKARAYA, CENTRAL KALIMANTAN
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Hasnita State Islamic Insitute of Palangka Raya, G. Obos St., Palangka Raya, Central Kalimantan, Indonesia E-mail: hasnita@gmail.com M h d I b l Correspondence Email: isra.misra@iain-palangkaraya.ac.id Article Info Abstract: Community service focuses on the lack of public knowledge of Islamic banking in Indonesia so that socialization, literacy, and education activities are needed for Micro, Small, and Medium Enterprises (UMKM). The expected goal is none other than to increase UMKM related to Islamic banking, both systems, products, and mechanisms so that it has an impact on public interest in using Islamic banking services in Central Kalimantan. The UMKM involved in the service activities are UMKM in Kereng Bangkirai Village as many as 55 UMKM and 5 employees of Bank Syariah Indonesia Palangka Raya Branch. The method used in this service is Community Based Articipatory Research where the service is carried out by involving stakeholders with Focus Group Discussion (FGD) activities. The results of the service show that by carrying out community-based service activities and involving stakeholders, especially banking, the public's understanding of Islamic banking is increasing. These activities also have an impact on the community, especially UMKM, to use Islamic banking services. Article history: Received 10 Mei 2022 Revised 24 Mei 2022 Accepted 25 Mei 2022 Keywords: Understanding, Socialization, Literacy, Education, Sharia Banking Correspondence Email: isra.misra@iain-palangkaraya.ac.id Article Info Abstract: Community service focuses on the lack of public knowledge of Islamic banking in Indonesia so that socialization, literacy, and education activities are needed for Micro, Small, and Medium Enterprises (UMKM). The expected goal is none other than to increase UMKM related to Islamic banking, both systems, products, and mechanisms so that it has an impact on public interest in using Islamic banking services in Central Kalimantan. The UMKM involved in the service activities are UMKM in Kereng Bangkirai Village as many as 55 UMKM and 5 employees of Bank Syariah Indonesia Palangka Raya Branch. The method used in this service is Community Based Articipatory Research where the service is carried out by involving stakeholders with Focus Group Discussion (FGD) activities. The results of the service show that by carrying out community-based service activities and involving stakeholders, especially banking, the public's understanding of Islamic banking is increasing. These activities also have an impact on the community, especially UMKM, to use Islamic banking services. Article history: Received 10 Mei 2022 Revised 24 Mei 2022 Accepted 25 Mei 2022 Keywords: Understanding, Socialization, Literacy, Education, Sharia Banking AMALA Jurnal Pengabdian kepada Masyarakat Penerbit Fakultas Ekonomi dan Bisnis Islam Institut Agama Islam Negeri Palangka Raya Vol. 1, No. 1, Mei, 2022, pp. 44-55 https://doi.org/10.23971/amala.v1i1.9 https://e-journal.iain-palangkaraya.ac.id/index.php/amala AMALA Jurnal Pengabdian kepada Masyarakat Penerbit Fakultas Ekonomi dan Bisnis Islam Institut Agama Islam Negeri Palangka Raya Vol. 1, No. 1, Mei, 2022, pp. 44-55 https://doi.org/10.23971/amala.v1i1.9 https://e-journal.iain-palangkaraya.ac.id/index.php/amala AMALA Jurnal Pengabdian kepada Masyarakat Penerbit Fakultas Ekonomi dan Bisnis Islam Institut Agama Islam Negeri Palangka Raya Vol. 1, No. 1, Mei, https://doi.org/10.2397 https://e-journal.iain-palangkaraya.ac.id/in Improving Community Understanding Through Socialization, Lite Education of Sharia Banking in the City of Palangkaraya, Central Kalim Aditya Achmad State Islamic Insitute of Syekh Nurjati Cirebon, Perjuangan ByPass St., Sunyaragi Cirebon, West Java E-mail: aditya.achmad@iain-syekhnurjati.ac.id Ali Sadikin State Islamic Insitute of Palangka Raya, G. Obos St., Palangka Raya, Central Kalimantan, Indonesia E-mail: sadikin.ali@iain-palangkaraya.ac.id Enriko Tedja Sukmana State Islamic Insitute of Palangka Raya, G. Obos St., Palangka Raya, Central Kalimantan, Indonesia E-mail: enikotedjasukmana@gmail.com Isra Misra State Islamic Insitute of Palangka Raya, G. Obos St., Palangka Raya, Central Kalimantan, Indonesia E-mail: isra.misra@iain-palangkaraya.ac.id Fadiah Adlina State Islamic Insitute of Palangka Raya, G. Obos St., Palangka Raya, Central Kalimantan, Indonesia E-mail: fadiahadlina@gmail.com Hasnita State Islamic Insitute of Palangka Raya, G. Obos St., Palangka Raya, Central Kalimantan, Indonesia E-mail: hasnita@gmail.com Muhammad Iqbal State Islamic Insitute of Palangka Raya, G. Obos St., Palangka Raya, Central Kalimantan, Indonesia E-mail: miqbal@gmail.com Nadia Safitri State Islamic Insitute of Palangka Raya, G. Obos St., Palangka Raya, Central Kalimantan, Indonesia E-mail: nadiasafitri@gmail.com Asrofi Aldianto State Islamic Insitute of Palangka Raya, G. Obos St., Palangka Raya, Central Kalimantan, Indonesia E-mail: asrofi@gmail.com Correspondence Email: isra.misra@iain-palangkaraya.ac.id Article Info Abstract: Community service focuses on the lack of public knowledge of Islamic ban so that socialization, literacy, and education activities are needed for Micro, Sm Enterprises (UMKM). The expected goal is none other than to increase UMKM banking, both systems, products, and mechanisms so that it has an impact on publi Islamic banking services in Central Kalimantan. The UMKM involved in the se UMKM in Kereng Bangkirai Village as many as 55 UMKM and 5 employees Indonesia Palangka Raya Branch. The method used in this service is Community B Research where the service is carried out by involving stakeholders with Focus G (FGD) activities. The results of the service show that by carrying out community-bas and involving stakeholders, especially banking, the public's understanding of Is increasing. These activities also have an impact on the community, especially UMKM banking services. Aditya Achmad State Islamic Insitute of Palangka Raya, G. Obos St., Palangka Raya, Central Kalimantan, Indonesia E-mail: sadikin.ali@iain-palangkaraya.ac.id j State Islamic Insitute of Palangka Raya, G. Obos St., Palangka Raya, Central Kalimantan, Indonesia E-mail: enikotedjasukmana@gmail.com State Islamic Insitute of Palangka Raya, G. Obos St., Palangka Raya, Central Kalimantan, Indonesia E-mail: isra.misra@iain-palangkaraya.ac.id State Islamic Insitute of Palangka Raya, G. Obos St., Palangka Raya, Central Kalimantan, Indonesia E-mail: fadiahadlina@gmail.com Hasnita AMALA Jurnal Pengabdian kepada Masyarakat Penerbit Fakultas Ekonomi dan Bisnis Islam Institut Agama Islam Negeri Palangka Raya Vol. 1, No. 1, Mei, 2022, pp. 44-55 https://doi.org/10.23971/amala.v1i1.9 https://e-journal.iain-palangkaraya.ac.id/index.php/amala Article history: Received 10 Mei 2022 Revised 24 Mei 2022 Accepted 25 Mei 2022 Keywords: Understanding, Socialization, Literacy, Education, Sharia Banking Introduction Financial literacy which means financial literacy, according to the Indones Strategy for Financial Literacy guidebook, what is meant by financial literacy is processes or activities to increase knowledge, confidence and skills of consumers an AMALA Jurnal Pengabdian kepada Masyarakat Penerbit Fakultas Ekonomi dan Bisnis Islam Institut Agama Islam Negeri Palangka Raya Vol. 1, No. 1, Mei, 2022, pp. 44-55 https://doi.org/10.23971/amala.v1i1.9 https://e-journal.iain-palangkaraya.ac.id/index.php/amala AMALA AMALA Jurnal Pengabdian Kepada Masyarakat Vol. 1, No. 1, Mei 2022, pp. 44-55 expected not only to know and understand financial service institutions and financial products and services, but also to be able to change or improve people's behavior in financial management so as to improve their welfare.1 expected not only to know and understand financial service institutions and financial products and services, but also to be able to change or improve people's behavior in financial management so as to improve their welfare.1 The low penetration of Islamic banking in Indonesia is below 10%, in fact the introduction of Islamic banks has experienced two decades, even though Indonesia with a Muslim majority in Indonesia and an Islamic atmosphere in Indonesia has enormous potential, especially in the Palangka Raya area, Central Kalimantan, which often has difficulties looking for financial products that comply with Sharia principles, ethics and social morals. p p y p p The government has issued many regulations to support Islamic finance, as well as Law No. 21 of 2018 concerning Islamic Banking for the development of Islamic Banking, but the biggest challenge for Islamic banking industry players is the lack of public knowledge about finance and Islamic banking itself. With a literacy index that is far below the conventional banking literacy index which reaches 28%, it illustrates the inclusion of Islamic banking products that are not widely known and used by the public. This is very contrary to the beliefs of the majority of Indonesian people who are Muslim, which should be the main market for Islamic banking.2 g The financial literacy index shows a significant increase. OJK data in 2019 shows that the national financial literacy index reached 38 percent from the previous 29.7 percent in 2016. Meanwhile, the national financial inclusion index also showed a significant increase, from 67.8 percent in 2016, up to 76.2 percent in 2019. However Nevertheless, the increase in the financial literacy index in the sharia sector is still below the national level. The Islamic financial literacy index was previously 8.1 percent in 2016 to 8.93 percent in 2019.3 The challenges faced by the Islamic finance industry in Indonesia are no less great, namely the lack of Islamic financial literacy and inclusion. 1Muhammad Iqbal Isra Misra, Muhammad Ragil, Manajemen Perbankan Syariah (Yogyakarta: K-Media Publiser, 2021). 2Ibid. 3Ibid. 4Ibid. 5Pedro Abrantes, “Opening the Black Box of Socialization: Emotions, Practices and (Biographical) Identities,” International Journal of Sociology and Anthropology 5, no. 9 (2013): 391–401. 6Alexander O. Karpov, “Socialization for the Knowledge Society,” International Journal of Environmental and Science Education 11, no. 10 (2016): 3487–3496. 7Risa Umasyah and Alfiasari Alfiasari, “Effects of Socialization Methods and Peer Attachment on Character Strength of School-Aged Children,” Journal of Child Development Studies 1, no. 2 (2016): 1. Introduction Financial literacy which means financial literacy, according to the Indonesian National Strategy for Financial Literacy guidebook, what is meant by financial literacy is a series of processes or activities to increase knowledge, confidence and skills of consumers and the general public so that they are able to manage their finances better. Based on this understanding, it can be concluded that consumers of financial products and services as well as the general public are 44 7Risa Umasyah and Alfiasari Alfiasari, “Effects of Socialization Methods and Peer Attachment on Character Strength of School-Aged Children,” Journal of Child Development Studies 1, no. 2 (2016): 1. p ) J f gy p gy ( ) 6Alexander O. Karpov, “Socialization for the Knowledge Society,” International Journal of mental and Science Education 11, no. 10 (2016): 3487–3496. 5Pedro Abrantes, “Opening the Black Box of Socialization: Emotions, Practices and phical) Identities,” International Journal of Sociology and Anthropology 5, no. 9 (2013): 391–401. 8Jennifer Rowsell and Kate Pahl, “The Routledge Handbook of Literacy Studies,” The Routledge Handbook of Literacy Studies (2015): 1–679. 9Isra Misra, Muhammad Ragil, Manajemen Perbankan Syariah. 10Ziauddin Sardar and Muhammad Nafik H.R, “Kesejahteraan Dalam Perspektif Islam Pada Karyawan Bank Syariah,” Jurnal Ekonomi Syariah Teori dan Terapan 3, no. 5 (2017): 391. 11Agus Pramana Isra Misra, Sofyan Hakim, Manajemen Risiko (Yogyakarta: K-Media, 2020). AMALA AMALA Jurnal Pengabdian Kepada Masyarakat Vol. 1, No. 1, Mei 2022, pp. 44-55 learning and adjustment so that they can play a role and function in their groups in society and in their personal lives. learning and adjustment so that they can play a role and function in their groups in society and in their personal lives. p According to Mason and Wilson financial literacy is a person's ability to obtain, understand, and evaluate relevant information to make decisions by understanding the financial consequences that arise.8 Financial literacy occurs when an individual has a set of skills and abilities that enable that person to utilize existing resources to achieve pre-planned goals. The Financial Services Authority (OJK) states that by definition literacy is defined as the ability to understand, so financial literacy is the ability to manage funds owned so that they develop and so that life can be more prosperous in the future.9 An understanding of Islamic financial literacy, especially Islamic banking, is very important because:10 (1) consumers must care about managing their own hard-earned funds because no other party will do it except the consumers themselves; (2) consumers are the ones who know in detail what their financial needs are (3) consumers are currently faced with various types of financial products that vary so that they demand adequate knowledge in choosing financial products that can meet their expectations; (4) understanding Islamic financial literacy is part of the obligations of a Muslim. Muslims must be concerned about the prohibition of elements of usury, maysir, and gharar in choosing financial products. This has further implications for the realization of al-falah, both in this world and the hereafter. Public knowledge about Islamic banking can influence the attitude of the community towards the products offered so that the better the public's knowledge about Islamic banking, the more people will become customers. Conversely, if the public's knowledge of Islamic banking is limited, it will result in an unfavorable perception of the banking sector. Therefore Islamic banking must be more aggressive in marketing or introducing its products.11 Participation as an academic community in carrying out the knowledge gained for this service will also take part through a program that will be carried out in the City of Palangka Raya, Central Kalimantan. Besides that, it is the realization of the Tri dharma of higher education, namely at the point of dedication. AMALA Palangka Raya was chosen because of the location of the service because it was considered to have the potential of having an Islamic Bank Institution and an easily accessible location. The selection of a sample of the people of the city of Palangka Raya would be the right target to eradicate literacy illiteracy for Islamic banks in Palangka Raya. AMALA It is a joint task to increase Islamic financial literacy and inclusion, because based on a 2016 OJK survey, the level of literacy and inclusion in Islamic finance is only 8 percent and only 11 percent use Sharia products. Even in the 2019 data, Islamic financial literacy only rose to 8.9 percent while the inclusion rate actually dropped from 11 percent to 9 percent.4 Karel J. Veeger, defines socialization as a process of teaching and learning, through individuals learning to become members of society, where the process does not merely teach patterns of social behavior to individuals, but also the individual develops himself or carries out a process of maturing himself.5 Robert M.Z. Lawang, socialization is the process of learning norms, values, roles, and all other requirements needed to enable effective participation in social life.6 Sitorus, socialization is a process in which a person learns patterns of life in society in accordance with 12 values, norms and habits that apply to develop as members of society and as individuals (personal).7 So socialization is a process that helps individuals through the process of 45 12Margarita Echeverri, “What We Should Know about Community-Based Participatory Research (CBPR)” (2013): 1–53, https://www.lacats.org/documents/About CBPR 10-08- 13.pdf. 13Saiful Anwar, “Student Services Center (SSC): Upaya Pembinaan Prestasi, Karir, Dan Kewirausahaan Bagi Mahasiswa Melalui Community Based Participatory Research,” Penamas: Journal of Community Service 1, no. 1 (2021): 49–60. AMALA contribute their expertise and share knowledge in decision making, the thing that is assessed in participation is the level of attendance and contribution of each type of representative starting from the Focus Group Discussion to problem-solving simulations.12 The service involves agencies such as Bank Syariah Indonesia (BSI) Palangka Raya Branch, City Government, Lecturers and Students and MSME actors. The CBPR approach can be carried out with service, community, stakeholder and provider/government schemes. The steps taken start from mapping> identification> design> implementation> data collection> analysis and dissemination. The CBPR cycle can be seen in Figure 1 as follows:13 contribute their expertise and share knowledge in decision making, the thing that is assessed in participation is the level of attendance and contribution of each type of representative starting from the Focus Group Discussion to problem-solving simulations.12 The service involves agencies such as Bank Syariah Indonesia (BSI) Palangka Raya Branch, City Government, Lecturers and Students and MSME actors. The CBPR approach can be carried out with service, community, stakeholder and provider/government schemes. The steps taken start from mapping> identification> design> implementation> data collection> analysis and dissemination. The CBPR cycle can be seen in Figure 1 as follows:13 Figure 1. CBPR cycle Results Mapping Identificati on Design Implemen tation Data collection Analysis Disseminati on CB PR Disseminati on Figure 1. CBPR cycle Method This community service is an annual program organized by the Islamic banking study program IAIN Palangka Raya. The location of the service is in Kereng Bangkirai Village, Palangka Raya City. The Kereng Bangkirai sub-district was made as a location for dedication because in this area there are many business actors who have various businesses ranging from fisheries, handicrafts, tourism and swallow nest cultivators and other businesses. The subjects of the service are micro, small and medium enterprises as many as 55 people with various types of work and businesses. The method used in community service activities is the community based participatory research (CBPR) method. CBPR is carried out using a partnership approach by involving community members, representatives of organizations and researchers themselves, in this study all partners as a whole 46 AMALA Jurnal Pengabdian Kepada Masyarakat Vol. 1, No. 1, Mei 2022, pp. 44-55 AMALA Jurnal Pengabdian Kepada Masyarakat Vol. 1, No. 1, Mei 2022, pp. 44-55 Jurnal Pengabdian Kepada Masyarakat Vol. 1, No. 1, Mei 2022, pp. 44-55 In order to carry out community service activities between lecturers and students of the Islamic banking study program, planning is necessary. Planning is carried out to determine objectives and resources that support the success of community service activities related to socialization, literacy and education related to Islamic banking in Indonesia. pp g Mapping activities are the initial activities of the community service planning process. Mapping activities start from determining activity goals, objectives and also community service objects. The purpose of community service is none other than to increase public knowledge and interest regarding Islamic banking. Objectives are compiled from the preparation of proposals and action plans (terms of references). The target of the activity is MSME actors in the Kereng Bangkirai Village, Palangka Raya City. There are 55 UMKM in the Kelurahan which are registered with the Office of Cooperatives and UMKM in the City of Palangka Raya. Kereng Bangkirai UMKM is an independent UMKM that manages businesses such as Tourism, Fisheries, Handicrafts and others. Kereng Sub-District is a sub- district that has quite high tourist visits both locally and nationally. This village is also included in the Sebangau National Park, the largest peat national park in Indonesia. Figure 2. TEAM Meeting Figure 2. TEAM Meeting Results Community service related to literacy and education related to sharia banking is none other than growing public interest through outreach activities and increasing knowledge through seminars and group discussions. These activities start from the process of planning, implementation and evaluation. Planning for community service activities starts from the problems that arise then mapping and the design process are carried out. Then the implementation process begins with implementation, data collection and analysis of activity results. As for the evaluation in the form of dissemination of activity results. For this reason, community service activities can be explained as follows: Planning 47 AMALA Jurnal Pengabdian Kepada Masyarakat Vol. 1, No. 1, Mei 2022, pp. 44-55 AMALA Jurnal Pengabdian Kepada Masyarakat Vol. 1, No. 1, Mei 2022, pp. 44-55 owned bank, namely Bank Syariah Indonesia (BSI). BSI is the largest sharia banking in Indonesia. The agreed activity plan is in the form of literacy or distribution of brochures and materials related to sharia banking, both systems, products, fatwas and so on. then follow-up activities in the form of seminars and dissemination filled by lecturers and students as well as sharia banking employees. owned bank, namely Bank Syariah Indonesia (BSI). BSI is the largest sharia banking in Indonesia. The agreed activity plan is in the form of literacy or distribution of brochures and materials related to sharia banking, both systems, products, fatwas and so on. then follow-up activities in the form of seminars and dissemination filled by lecturers and students as well as sharia banking employees. g p y Figure 3. Preparation with Islamic Banking Figure 3. Preparation with Islamic Banking 2. Design 2. Design After the mapping or mapping process has been carried out, the next step is to carry out the activity design process. At the beginning of the program was the formation of a community service team for the Islamic banking study program. The legality of the activity is in the form of the Decree of the Dean of the Faculty of Economics and Islamic Business, IAIN Palangka Raya Number 245 of 2021. After being determined, the next step is consolidation with the Kereng Bangkirai Village Head. The purpose of consolidation is none other than to equalize perceptions between universities and city/kelurahan governments. After a joint agreement on community service activities, the next step is consolidation with stake holders, in this case Islamic banking. The selected sharia banking is a government- 48 AMALA Jurnal Pengabdian Kepada Masyarakat Vol. 1, No. 1, Mei 2022, pp. 44-55 Implementation Community service activities are carried out for 3 months starting from August 2021 to October 2021. Islamic banking community service activities collaborate with students and lecturers. The costs for this activity come from the work program of the Islamic banking study program. During the implementation of community service activities, several things were prepared by the team in the form of administration, such as assignment letters, confirmation letters, invitations and others. The implementation of community service activities related to sharia banking socialization, literacy and education are as follows: First, the team first provided socialization and education to the community, especially MSMEs in Kereng Bangkirai Village in the form of distributing brochures and materials. The brochure is in the form of a brief overview of the history of the existence of Islamic banks, then the laws and fatwas of the Indonesian Ulema Council and Islamic banking products. After distributing the brochures, the public was asked to understand and ask if there were things that the public could not digest regarding the brochures that had been given. Then next is the socialization activity in the form of providing material to participants by lecturers and students as well as from stakeholders, namely from Bank Syariah Indonesia Branch Office 3 Palangka Raya which is contained in figure 3 as follows: 49 49 AMALA Jurnal Pengabdian Kepada Masyarakat Vol. 1, No. 1, Mei 2022, pp. 44-55 Jurnal Pengabdian Kepada Masyarakat Vol. 1, No. 1, Mei 2022, pp. 44-55 Figure 4. Material from stakeholders Figure 4. Material from stakeholders Second, when delivering material on community service activities, the committee first distributed pretests and posttests to participants who took part in socialization and educational activities about a glimpse of knowledge of the sharia banking system. From the pretest questionnaire given, the average respondent knows about Islamic banks but does not yet know in depth the systems and mechanisms of Islamic banks because they have never used Islamic banking services. From there, this socialization and education activity is appropriate to be carried out both by Educational Institutions and by Islamic banking itself. The first material was delivered by Mr. Enriko as the vice chairman of the TIM for the Islamic banking study program. Figure 5. Submission of Material by Enriko about Islamic Banks Figure 5. 14Zakiah dan Enriko Tedja Sukmana Isra Misra, “Mekanisme Bagi Hasil Pada Sistem Affiliate Marketing Taqychan Saffron,” AL-IQTISHADIYAH 7 (2021). 15Isra Misra, Muhammad Ragil, Manajemen Perbankan Syariah. Implementation Submission of Material by Enriko about Islamic Banks He said on the first slide that he introduced Islamic banks in Indonesia, starting from a brief understanding of Islamic banks. He said that Islamic banks are in accordance with sharia 50 AMALA Jurnal Pengabdian Kepada Masyarakat Vol. 1, No. 1, Mei 2022, pp. 44-55 AMALA principles and have been regulated by the fatwa of the Indonesian Ulema Council (MUI). From the process, Islamic banks have carried out activities in accordance with the Qur'an and Hadith and are in accordance with Law Number 21 of 2008 concerning Islamic banking which regulates principles, types, distribution and collection and prioritizes honest and fair principles and avoids usury, maysir and gharar.14 On this occasion the team conveyed about the types of savings, both Islamic savings, Islamic deposits and Islamic mortgages. In addition, it also explained about financing in Islamic banks such as mudharobah financing, musyarakah and so on. in sharia banking there are also sharia demand deposits. Then the team also conveyed about the existence of Islamic banks in Indonesia. Where in the current 3 decades Islamic banks are well known by the public in accordance with the government's expectations that the economic ecosystem and Islamic banking must be increasingly supported.15 This is also the motivation for the government through BUMN to combine sharia banks under BUMN (Bank Syariah Mandiri, BRI Syariah and BNI Syariah) into one big entity, namely Bank Syariah Indonesia. By joining the 3 banks above, it will certainly have an impact on increasing assets and also the market share of Islamic banks. principles and have been regulated by the fatwa of the Indonesian Ulema Council (MUI). From the process, Islamic banks have carried out activities in accordance with the Qur'an and Hadith and are in accordance with Law Number 21 of 2008 concerning Islamic banking which regulates principles, types, distribution and collection and prioritizes honest and fair principles and avoids usury, maysir and gharar.14 On this occasion the team conveyed about the types of savings, both Islamic savings, Islamic deposits and Islamic mortgages. In addition, it also explained about financing in Islamic banks such as mudharobah financing, musyarakah and so on. in sharia banking there are also sharia demand deposits. Then the team also conveyed about the existence of Islamic banks in Indonesia. Where in the current 3 decades Islamic banks are well known by the public in accordance with the government's expectations that the economic ecosystem and Islamic banking must be increasingly supported.15 This is also the motivation for the government through BUMN to combine sharia banks under BUMN (Bank Syariah Mandiri, BRI Syariah and BNI Syariah) into one big entity, namely Bank Syariah Indonesia. 52 16Audia Junita, “Organizational Learning Culture, Consumer Satisfaction and Employee’s Attitude: Causality Analysis,” Jurnal Dinamika Manajemen 8, no. 1 (2017): 68–82. AMALA By joining the 3 banks above, it will certainly have an impact on increasing assets and also the market share of Islamic banks. Figure 6. Enthusiasm of Service Participants Listening to Material Figure 6. Enthusiasm of Service Participants Listening to Material In the next session, the explanation was provided by Islamic bank practitioners. From the BSI explained the importance of knowing and using Islamic banks. He explained that Islamic banks are currently experiencing very fundamental changes. This is because the economy and sharia business are currently the focus of the government through Bank Indonesia and also BUMN. The growth of market share for Islamic banks is the largest in the world. He added that with Indonesia's majority Muslim population, the potential for Islamic banks is enormous. However, the challenges ahead are not easy either. During the pandemic, all banks did not 51 51 Evaluation Community service activities related to increasing public understanding regarding the concepts and practices of Islamic banks in Indonesia by the Islamic banking study program IAIN Palangka Raya in collaboration with Indonesian Sharia Banks and the City Government of Palangka Raya have been carried out using community-based participatory research (CBPR) approaches and concepts that have been able to provide new knowledge and insights for SMEs in Kereng Bangkirai Village. This can be seen from the enthusiasm and participation of the public to know more about Islamic banking. In this activity it is also seen that so far the public considers Islamic banks not much different from other conventional banks. The socialization, education and literacy activities initiated by the Islamic banking study program have had an impact on increasing the interest of MSMEs to use the services offered by Islamic banking. In this activity it was also found that good partnerships and cooperation would be able to produce an activity that had a big impact on society, especially related to the introduction of Islamic banking. This outreach and education activity still needs to be improved by carrying out even more massive activities. Although currently face-to-face activities are still limited due to the Covid-19 pandemic, outreach activities can be carried out in a hybrid manner. Conclusion Community service activities related to increasing public understanding of Islamic banking can be concluded: (1) Socialization activities related to Islamic banking concepts and products in Kereng Bangkirai Village for Micro, Small and Medium Enterprises (MSMEs) contribute positively to increasing public insight and knowledge about concepts and products Islamic banks in Indonesia. (2) Educational and literacy activities are carried out involving stakeholders, namely Bank Syariah Indonesia by introducing the history of the birth of Islamic banks, the principles and functions of Islamic banks, legal basis, products and supports from Islamic banks. This activity contributes to increasing public interest, especially MSMEs, to conduct transactions using Islamic banks. (3) This community service activity needs to be continued by Educational Institutions, in this case the Faculty of Islamic Economics and Business as well as the sharia banking itself in order to continue to massively increase public knowledge and insight regarding the concepts and products of sharia banking. Evaluation A k l d t This community service activity is inseparable from the support and assistance from the Faculty of Islamic Economics and Business as the initiator of the activity, Bank Syariah Indonesia as stake holders and partners, and also the City Government of Palangka Raya which strongly supports socialization, education and literacy activities related to the concept and practice of Islamic banking in Indonesia. The team would like to thank you so that this community service activity was carried out well. AMALA AMALA Jurnal Pengabdian Kepada Masyarakat Vol. 1, No. 1, Mei 2022, pp. 44-55 increased knowledge will certainly affect the interest of MSMEs to be able to carry out transactions in Islamic banking. Evaluation increased knowledge will certainly affect the interest of MSMEs to be able to carry out transactions in Islamic banking. Anwar, Saiful. “Student Services Center (SSC): Upaya Pembinaan Prestasi, Karir, Dan Kewirausahaan Bagi Mahasiswa Melalui Community Based Participatory Research.” Penamas: Journal of Community Service 1, no. 1 (2021): 49–60. AMALA Jurnal Pengabdian Kepada Masyarakat Vol. 1, No. 1, Mei 2022, pp. 44-55 Jurnal Pengabdian Kepada Masyarakat Vol. 1, No. 1, Mei 2022, pp. 44-55 experience a decline in financial performance, including Islamic banks, which showed increased performance with positive growth. The growth and resilience of Islamic banks has actually increased where assets have grown 13.11%, credit 8.08% and third party funds 11.88%. He explained that Islamic banking is a bank that is actually experiencing growth amid minus national economic conditions. Figure 7. Graph of Growth and Resilience of Islamic Banks Figure 7. Graph of Growth and Resilience of Islamic Banks In the presentation by the BSI team, it was also explained clearly related to BSI Microfinance products. In this submission it was explained that BSI was also given the opportunity by the government to provide people's business loans which we are usually familiar with as KUR (Micro). Micro financing in Islamic banks consists of BSI Micro Enterprises that use murabaha, Ijarah and MMTQ contracts with a maximum financing limit of 200 million rupiah. As for BSIKUR, it consists of super micro, micro and small with a maximum limit of 10 million, 50 million and 500 million respectively. The provision of capital is adjusted to the needs of MSMEs where the assessment is carried out by Islamic bank employees by looking at the business criteria and business model being carried out. In the delivery by the BSI team, there are also many supporting products available at BSI or Islamic banks such as BSI Partners. Investment, Islamic funding and savings, Hajj and Umrah pilgrimage, pawnshops, BSI Gold, BSI priority. BSI also introduced BSI Mobile banking, where transactions can be made through a device without having to leave the house to make transfers, purchases, e-commers and many others. BSI also said that opening an account could be done online without having to come to the bank. In addition, BSI also partners through BSI Agents where every shop and supermarket can become a BSI agent. The process of transferring knowledge through socialization, education and literacy activities by the Islamic banking study program and stakeholders opens new horizons and knowledge for MSMEs about Islamic banking. Submissions from academics and practitioners provide very meaningful information for MSMEs. Increased understanding of Islamic banks has an impact on public interest in being able to use Islamic banking.16 Good understanding and 52 Echeverri, Margarita. “What We Should Know about Community-Based Participatory Research (CBPR)” (2013): 1–53. https://www.lacats.org/documents/About CBPR 10-08-13.pdf. Isra Misra, Muhammad Ragil, Muhammad Iqbal. Manajemen Perbankan Syariah. Yogyakarta: K- Media Publiser, 2021. Reference list Abrantes, Pedro. “Opening the Black Box of Socialization: Emotions, Practices and (Biographical) Identities.” International Journal of Sociology and Anthropology 5, no. 9 (2013): 391–401. Anwar, Saiful. “Student Services Center (SSC): Upaya Pembinaan Prestasi, Karir, Dan Kewirausahaan Bagi Mahasiswa Melalui Community Based Participatory Research.” Penamas: Journal of Community Service 1, no. 1 (2021): 49–60. Echeverri, Margarita. “What We Should Know about Community-Based Participatory Research (CBPR)” (2013): 1–53. https://www.lacats.org/documents/About CBPR 10-08-13.pdf. Isra Misra, Muhammad Ragil, Muhammad Iqbal. Manajemen Perbankan Syariah. Yogyakarta: K- Media Publiser, 2021. Reference list Abrantes, Pedro. “Opening the Black Box of Socialization: Emotions, Practices and (Biographical) Identities.” International Journal of Sociology and Anthropology 5, no. 9 (2013): 391–401. Anwar, Saiful. “Student Services Center (SSC): Upaya Pembinaan Prestasi, Karir, Dan Kewirausahaan Bagi Mahasiswa Melalui Community Based Participatory Research.” Penamas: Journal of Community Service 1, no. 1 (2021): 49–60. J f y ( ) Echeverri, Margarita. “What We Should Know about Community-Based Participatory Research (CBPR)” (2013): 1–53. https://www.lacats.org/documents/About CBPR 10-08-13.pdf. Isra Misra, Muhammad Ragil, Muhammad Iqbal. Manajemen Perbankan Syariah. Yogyakarta: K- Echeverri, Margarita. “What We Should Know about Community-Based Participatory Research (CBPR)” (2013): 1–53. https://www.lacats.org/documents/About CBPR 10-08-13.pdf. Isra Misra, Muhammad Ragil, Muhammad Iqbal. Manajemen Perbankan Syariah. Yogyakarta: K- Media Publiser, 2021. 53 AMALA Jurnal Pengabdian Kepada Masyarakat Vol. 1, No. 1, Mei 2022, pp. 44-55 Jurnal Pengabdian Kepada Masyarakat Vol. 1, No. 1, Mei 2022, pp. 44-55 Isra Misra, Sofyan Hakim, Agus Pramana. Manajemen Risiko. Yogyakarta: K-Media, 2020. Isra Misra, Zakiah dan Enriko Tedja Sukmana. “Mekanisme Bagi Hasil Pada Sistem Affiliate Marketing Taqychan Saffron.” AL-IQTISHADIYAH 7 (2021). Junita, Audia. “Organizational Learning Culture, Consumer Satisfaction and Employee’s Attitude: Causality Analysis.” Jurnal Dinamika Manajemen 8, no. 1 (2017): 68–82. y y J j ( ) Karpov, Alexander O. “Socialization for the Knowledge Society.” International Journal of Environmental and Science Education 11, no. 10 (2016): 3487–3496. Rowsell, Jennifer, and Kate Pahl. “The Routledge Handbook of Literacy Studies.” The Routledge Handbook of Literacy Studies (2015): 1–679. Sardar, Ziauddin, and Muhammad Nafik H.R. “Kesejahteraan Dalam Perspektif Islam Pada Karyawan Bank Syariah.” Jurnal Ekonomi Syariah Teori dan Terapan 3, no. 5 (2017): 391. Umasyah, Risa, and Alfiasari Alfiasari. “Effects of Socialization Methods and Peer Attachment on Character Strength of School-Aged Children.” Journal of Child Development Studies 1, no. 2 (2016): 1. 54
https://openalex.org/W3000085841
https://www.frontiersin.org/articles/10.3389/fpsyg.2019.02914/pdf
English
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Universal Restrictions in Reading: What Do French Beginning Readers (Mis)perceive?
Frontiers in psychology
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13,996
Keywords: reading, sonority, markedness, syllable segmentation, illusory conjunctions, French, phonological universals Edited by: Manuel Perea, University of Valencia, Spain Reviewed by: Shanti Ulfsbjorninn, University of Deusto, Spain Stephanie Mathey, Université de Bordeaux, France *Correspondence: Norbert Maïonchi-Pino norbert.maionchi_pino@uca.fr; mpinonor@gmail.com Reviewed by: Shanti Ulfsbjorninn, University of Deusto, Spain Stephanie Mathey, Université de Bordeaux, France *Correspondence: Norbert Maïonchi-Pino norbert.maionchi_pino@uca.fr; mpinonor@gmail.com Specialty section: This article was submitted to Language Sciences, a section of the journal Frontiers in Psychology Abbreviations: C, consonant; IC, illusory conjunction; SP, sonority profile; V, vowel. Universal Restrictions in Reading: What Do French Beginning Readers (Mis)perceive? Norbert Maïonchi-Pino1*, Audrey Carmona2, Méghane Tossonian1, Ophélie Lucas1, Virginie Loiseau1 and Ludovic Ferrand1 1 Laboratoire de Psychologie Sociale et Cognitive (LAPSCO), CNRS UMR 6024, Université Clermont Auvergne, Clermont-Ferrand, France, 2 UFR de Sciences Médicales et Pharmaceutiques – Orthophonie, Université de Franche-Comté, Besançon, France Despite the many reports that consider statistical distribution to be vitally important in visual identification tasks in children, some recent studies suggest that children do not always rely on statistical properties to help them locate syllable boundaries. Indeed, sonority – a universal phonological element – might be a reliable source for syllable segmentation. More specifically, are children sensitive to a universal phonological sonority-based markedness continuum within the syllable boundaries for segmentation (e.g., from marked, illegal intervocalic clusters, “jr,” to unmarked, legal intervocalic clusters, “rj”), and how does this sensitivity progress with reading acquisition? To answer these questions, we used the classical illusory conjunction (IC) paradigm. Forty-eight French typically developing children were tested in April (T1), October (T2) and April (T3; 20 children labeled as “good” readers, M chronological age at T1 = 81.5 ± 4.0; 20 children labeled as “poor” readers, M chronological age at T1 = 80.9 ± 3.4). In this short-term longitudinal study, not only we confirmed that syllable segmentation abilities develop with reading experience and level but the Condition × Sonority interaction revealed for the first time that syllable segmentation in reading may be modulated by phonological sonority-based markedness in the absence or quasi-absence of statistical information, in particular within syllable boundaries; this sensitivity is present at an early age and does not depend on reading level and sonority-unrelated features. INTRODUCTION Received: 28 March 2019 Accepted: 10 December 2019 Published: 14 January 2020 Cross-linguistic evidence indicates that there are regularities across languages. For instance, Consonant-Vowel structures – CV henceforth – are overrepresented across the world’s languages (e.g., Hyman, 2008). By contrast, some coda-onset clusters likewise (i.e., CC) tend to be systematically avoided or underrepresented across syllable boundaries (e.g., /bd/; e.g., Murray and Vennemann, 1983; Vennemann, 1988). But how French beginning readers perceive and segment syllable boundaries they have never read, never heard, or not learnt yet? To address this issue, we focused on sonority-based linguistic principles that rule the well-formedness and distinctiveness of Cross-linguistic evidence indicates that there are regularities across languages. For instance, Consonant-Vowel structures – CV henceforth – are overrepresented across the world’s languages (e.g., Hyman, 2008). By contrast, some coda-onset clusters likewise (i.e., CC) tend to be systematically avoided or underrepresented across syllable boundaries (e.g., /bd/; e.g., Murray and Vennemann, 1983; Vennemann, 1988). But how French beginning readers perceive and segment syllable boundaries they have never read, never heard, or not learnt yet? To address this issue, we focused on sonority-based linguistic principles that rule the well-formedness and distinctiveness of ORIGINAL RESEARCH published: 14 January 2020 doi: 10.3389/fpsyg.2019.02914 Citation: Maïonchi-Pino N, Carmona A, Tossonian M, Lucas O, Loiseau V and Ferrand L (2020) Universal Restrictions in Reading: What Do French Beginning Readers (Mis)perceive? Front Psychol 10:2914 January 2020 | Volume 10 | Article 2914 1 Frontiers in Psychology | www.frontiersin.org Universal Phonological Restrictions in French Maïonchi-Pino et al. describes a universal, hierarchically ranked set of violable phonological constraints, sonority-based linguistic principles may be interpreted as markedness constraints (e.g., “A syllable contact pair α.β is more preferred the greater the decrease in sonority from a coda segmentα to an onset segmentβ”). Thus, in the light of the sonority scale in Figure 1, the least marked (unmarked), and the most well-formed onset cluster will preferentially exhibit a steep rise in SP (e.g., /bK/, s = + 4). Onset clusters progressively become more marked, and less well-formed (marked) as the SP decreases from high-rise to low-rise (e.g., /ml/, s = + 1), then on to plateau SP (e.g., /bd/, s = 0), low- fall SP (e.g., /sp/, s = −1), and high-fall SP (e.g., /Kb/, s = −4). As markedness increases, well-formedness decreases from high- rise SP to high-fall SP. However, this point concerns the syllable structures themselves, whereas we are interested in the syllable boundaries here. phonological sequences (see de Lacy, 2006). More specifically, our question specifically raises the question of whether sonority- based markedness affects and constrains segmentation strategies in the absence or quasi-absence of statistical – distributional – information in visual letter detection. Sonority can be envisaged as a universal, formal, scalar, feature-like phonological element that categorizes all speech sounds into a hierarchical acoustic-phonetic scale. Consonants are ranked from high-sonority phonemes (i.e., from liquid to nasal – labeled sonorant –) to low-sonority phonemes (i.e., from fricative to occlusive – labeled obstruent –; see Figure 1)1. However, sonority remains a controversial linguistic concept, whose nature and origin are a matter of debate (e.g., Clements, 1990, 2006; Ladefoged, 2001; Hayes and Steriade, 2004; Parker, 2008, 2017). Beyond the question of whether sonority is a formally grounded linguistic constraint (i.e., an innate linguistic primitive) or a functionally grounded linguistic constraint derived from speakers’ linguistic experience of the acoustic- phonetic properties of sounds (e.g., Parker, 2017), sonority has different descriptions. Clements (1990, 2006), for example, emphasizes the elusive phonetic correlates in sonority, while Parker (2008) considers that phonological sonority has concrete, quantifiable physical and perceptual properties. Citation: Indeed, it has been proposed that sonority is a phonological property of sounds, with their acoustic intensity being the most reliable correlate (e.g., Parker, 2017). In any case, this basic hierarchy of individual sonorities seems to be insufficient, so we now focus on sonority-based linguistic principles to account for restrictions and constraints on the co-occurrence of sounds within and across syllables: the Sonority Sequencing Principle (Clements, 1990, 2006) and the Syllable Contact Law (e.g., Vennemann, 1988; also see Hooper, 1972; Murray and Vennemann, 1983). To cope with the restriction of the Sonority Sequencing Principle to the internal structure of syllables, we refer to the Syllable Contact Law (e.g., Murray and Vennemann, 1983; Vennemann, 1988) that predicts a gradient preference for universally optimal syllable contact as follows: “A syllable contact pairα.β is more preferred the greater the increase in consonantal strength from a coda segmentα to an onset segmentβ.” Consonantal strength has thereafter been reinterpreted in terms of sonority to describe an optimal syllable contact between two adjacent segments (e.g., Clements, 1990). In other words, the Syllable Contact Law categorically prohibits a sonority rise across syllable boundaries (e.g., “mar.teau”, hammer > “pa.tron”, boss, not “pat.ron”; the dot represents the expected location of the syllable boundary). For instance, implemented within the Optimality Theory, the Syllable Contact Law shapes a categorical, hierarchically ranked set of violable markedness constraints to describe optimal syllable contacts, due to which “sonority should not rise across a syllable boundary” (e.g., Holt, 2004). Following this, Gouskova’s (2004) proposal is of particular interest for our study. Gouskova (2004) proposed a more sophisticated and fine-grained gradient-based formalization of the syllable contact constraint implemented within Optimality Theory: a syllable contact should be envisaged as a stratified relational hierarchy that determines the well-formedness of a coda or onset not in isolation but in relation to the adjacent onset or coda, respectively (see Figure 2). Gouskova’s (2004) collapses the individual sonority in coda position and the individual sonority in onset position into a single scale (the coda tends to be the more sonorous consonant, while the onset tends to be the less sonorous consonant; see Jespersen, 1904; Prince and Smolensky, 2004; Zec, 2007). The least marked, the most well- formed, the most harmonic SP preferentially exhibits a steep fall from the coda (C1) to the onset (C2). 2These restrictions might not be expressed phonetically but rather as algebraic optimizational rules that guide the well-formedness of syllable patterns (e.g., Berent, 2013). 1The use of 1.5 and 4.5 is based on a suggestion by Parker (2002), although the relative distance between 1 and 1.5 and 4 and 4.5 may not be equivalent. Glides are not represented, in particular since this would not be useful for our study in French. Citation: The sonority contact scale in Figure 2 indicates that a syllable contact preferentially exhibits a steep fall in SP across syllable boundaries (unmarked, most harmonic; e.g., high-fall SP such as/Kb/, s = −4); syllable contacts progressively become more marked, and less well-formed (marked, less harmonic) as the SP increases across syllable boundaries from high-fall SP to low-fall SP (e.g., /df/, s = −1), then on to plateau SP (i.e., null distance; e.g., /bd/, s = 0), low-rise SP (e.g., /ml/, s = + 1), and high-rise SP (e.g., /bK/, s = + 4). The markedness pattern across syllable boundaries is therefore the complete opposite of the markedness pattern in onset clusters. For the sake of clarity and consistency, we speak of a phonological sonority-based markedness to designate the SP in either onset or intervocalic position. the last 20 years has suggested that syllables are salient perceptual and segmental units in French newborns and preliterate children (e.g., Bertoncini et al., 1988; Duncan et al., 2006; Nazzi et al., 2006; Goslin and Floccia, 2007). They are also fundamental reading units in French children who are learning to read (e.g., Maïonchi-Pino et al., 2012a,b; Doignon-Camus and Zagar, 2014). To better understand why syllables are crucial reading units, it is worth noting that learning to read follows a small- to-large developmental sequence that progresses from grapho- phonemic processing to grapho-syllabic processing (e.g., Ehri, 2005). This hypothesis supports the idea that first children have to learn the grapheme-to-phoneme correspondences to progressively consolidate and unitize them into larger units. This is particularly suitable since grapho-syllabic processing requires “fewer connections to secure the word in memory” (Ehri, 2005, p. 175), while grapho-phonemic processing requires considerable attentional resources due to the sequential left-to- right processing of each unit in turn. To explain the developmental course and the role of syllable- sized units in reading, most research tends to reduce the syllable effects to the question of statistical properties. Of the previous studies that have specifically addressed the question of the developmental course of syllable segmentation in French children, those by Colé et al. (1999) and Maïonchi-Pino et al. 3The Manulex and Manulex-infra databases provide the distributional lexical and sublexical frequencies for French elementary-school readers (from first to fifth grade). Citation: FIGURE 2 | Stratified relational hierarchy of coda and onset proposed within the SYLLABLE CONTACT constraint, where “r” stands for rhotics, “t” for voiceless occlusives, “d” for voiced occlusives, “z” for voiced fricatives, “s” for voiceless fricatives, “n” for nasals, “l” for laterals, and “w” for glides (adapted from Gouskova, 2004). FIGURE 2 | Stratified relational hierarchy of coda and onset proposed within the SYLLABLE CONTACT constraint, where “r” stands for rhotics, “t” for voiceless occlusives, “d” for voiced occlusives, “z” for voiced fricatives, “s” for voiceless fricatives, “n” for nasals, “l” for laterals, and “w” for glides (adapted from Gouskova, 2004). Sonority Distance which is not sensitive to the type of consonant (i.e., fricative, nasal, obstruent, etc.); the way a C1C2 cluster is processed is therefore predicted not by the type of consonants but only by the sonority distance. Hence, “w.z”, “r.d”, “l.s”, and “n.t” belong to the same stratum and are theoretically equivalent since these clusters share the same sonority distance (i.e., −4; see Figure 2). The sonority contact scale in Figure 2 indicates that a syllable contact preferentially exhibits a steep fall in SP across syllable boundaries (unmarked, most harmonic; e.g., high-fall SP such as/Kb/, s = −4); syllable contacts progressively become more marked, and less well-formed (marked, less harmonic) as the SP increases across syllable boundaries from high-fall SP to low-fall SP (e.g., /df/, s = −1), then on to plateau SP (i.e., null distance; e.g., /bd/, s = 0), low-rise SP (e.g., /ml/, s = + 1), and high-rise SP (e.g., /bK/, s = + 4). The markedness pattern across syllable boundaries is therefore the complete opposite of the markedness pattern in onset clusters. For the sake of clarity and consistency, we speak of a phonological sonority-based markedness to designate the SP in either onset or intervocalic position. Sonority Distance which is not sensitive to the type of consonant (i.e., fricative, nasal, obstruent, etc.); the way a C1C2 cluster is processed is therefore predicted not by the type of consonants but only by the sonority distance. Hence, “w.z”, “r.d”, “l.s”, and “n.t” belong to the same stratum and are theoretically equivalent since these clusters share the same sonority distance (i.e., −4; see Figure 2). Citation: Consequently, the SP becomes more marked, less well-formed, and less harmonic as the coda becomes less sonorous and the onset becomes more sonorous (e.g., /lb/ > /bd/ > /bl/; > stands for “preferred over”2). It is interesting to note that any syllable contact provides a p y The Sonority Sequencing Principle (Clements, 1990, 2006) is a well-known and extensively studied sonority-based linguistic principle that takes account of the fact that all languages constrain the co-occurrence of sounds within syllables. The Sonority Sequencing Principle depicts syllables’ Sonority Profile (SP henceforth), in which universally optimal syllable structures tend to conform to a Sonority Cycle that combines and orders sounds. For example, the onset tends to grow toward a maximum sonority at the vowel and fall to a minimum sonority at the coda (e.g., “partir”, to leave; although some languages, like Russian or sometimes French and English, violate the sonority generalization made by the Sonority Sequencing Principle, we do not discuss this point here; e.g., Wright, 2004). However, the sonority cycle is not a random phenomenon. According to Clements (1990); also see Selkirk, 1984), an optimal sonority cycle requires the maximum dispersion of individual sonorities between each of the sounds within a C1C2V syllable, with a minimum rise of at least x degrees from C1 to C2 in a C1C2 onset cluster (i.e., in reality, three degrees; e.g.,/pni/is better than/pfi/; e.g., Clements, 1990). For instance, within the Optimality Theory framework (e.g., Prince and Smolensky, 2004), which 1The use of 1.5 and 4.5 is based on a suggestion by Parker (2002), although the relative distance between 1 and 1.5 and 4 and 4.5 may not be equivalent. Glides are not represented, in particular since this would not be useful for our study in French. January 2020 | Volume 10 | Article 2914 Frontiers in Psychology | www.frontiersin.org 2 Universal Phonological Restrictions in French Maïonchi-Pino et al. FIGURE 1 | Sonority scale (adapted from Jespersen, 1904; p. 186; also see Gouskova, 2004). FIGURE 2 | Stratified relational hierarchy of coda and onset proposed within the SYLLABLE CONTACT constraint, where “r” stands for rhotics, “t” for voiceless occlusives, “d” for voiced occlusives, “z” for voiced fricatives, “s” for voiceless fricatives, “n” for nasals, “l” for laterals, and “w” for glides (adapted from Gouskova, 2004). FIGURE 1 | Sonority scale (adapted from Jespersen, 1904; p. 186; also see Gouskova, 2004). Citation: For instance, in French, Doignon and Zagar (2006) and Doignon-Camus and Zagar (2014) found that, from the age of six, French children’s response patterns indicated more preservation ICs than violation ICs when the orthographic and phonological boundaries matched (e.g., “BI/MIR” and “RON/TA”; the slash indicates that the orthographic boundary coincides with the phonological boundary) but that the response patterns indicated more violation ICs than preservation ICs when the orthographic and phonological boundaries mismatched (e.g., “RO/N∗ER” and “BI∗M/BU”; the ∗indicates the orthographic boundary, whereas the slash indicates the phonological boundary). Taken Consequently, a vast body of evidence provided robust arguments on the role of statistical and distributional properties with either contrasted statistical distribution effects – e.g., the bigram trough –, or effects that contradict or modulate syllable effects – e.g., inhibitory vs. facilitatory effects – (e.g., Doignon and Zagar, 2006; Chetail and Mathey, 2009a,b, 2013; Maïonchi-Pino et al., 2010; Chetail, 2015).The widely accepted and well-documented observation that children efficiently develop robust representations of the statistical orthographic and phonological distribution of their language could be confronted with the role of phonological universals. To date, the literature provides no extensive and clear-cut answers. Critically, phonological universals such as sonority have not been a clear focus of study, except in the case of research into speech production and perception, despite the fact that it might be a credible candidate that co-contributes with or even compensates for statistical properties in silent reading (e.g., in speech perception in English, Russian, and Korean, Berent et al., 2007, 2008, 2011, 2012a; in Spanish, Berent et al., 2012b; in French, Maïonchi-Pino et al., 2013; in Mandarin Chinese, Zhao and Berent, 2016). Given that sensitivity to sonority-based constraints might be available at an early age and independently of linguistic experience and that it might also contribute to language acquisition (e.g., Gómez et al., 2014), current studies should examine how and when phonological sonority-based markedness impacts reading. Our prediction is that French children might refer to non-statistical properties when implementing syllable location and segmentation strategies in silent reading by taking advantage of their sensitivity to sonority-based constraints (e.g., Fabre and Bedoin, 2003; Maïonchi-Pino et al., 2012a,b, 2015). Empirical evidence regarding sonority in reading-related French studies remains scarce (but see Sprenger-Charolles and Siegel, 1997; Marouby-Terriou and Denhière, 2002; Maïonchi- Pino et al., 2012a,b, 2015). Citation: Two IC patterns coexist. For instance, in the case of a word like ANVIL presented either as ANVil or ANvil (where upper- and lower-case letters represent two different colors) there are: (1) ICs that preserve the syllable boundary (i.e., the participant reports that the target-letter “V” is the same color as “il” in “AN.Vil”; the dot represents the syllable boundary); and (2) ICs that violate the syllable boundary (i.e., the participant reports that the target- letter “v” is the same color as “AN” in “AN.vil”). If children really perceive syllable units in pseudowords, preservation ICs would exceed violation ICs. Based on this color misperception, which is thought to be caused by automatic syllable extraction, researchers have focused on a specific statistical argument – the bigram trough hypothesis – to explain syllable segmentation strategies (e.g., Seidenberg, 1987). The bigram trough hypothesis refers to the statistical distribution of letter co-occurrences, which follows language-specific phonotactic restrictions that govern how, and how frequently, letters occur and co-occur in specific positions. The letter co-occurrences that straddle syllable boundaries (e.g., the bigram NV in ANVIL) are of lower frequency than cluster co-occurrences that constitute syllable-sized units that precede or follow the syllable boundaries (e.g., AN and VI) and act as a powerful statistical cue that underlies syllable location and segmentation. However, previous research has confirmed that orthographic statistical properties do not occur in isolation (e.g., in Spanish, Conrad et al., 2009(in English, Rapp, 1992; Muncer et al., 2014). For instance, in French, Doignon and Zagar (2006) and Doignon-Camus and Zagar (2014) found that, from the age of six, French children’s response patterns indicated more preservation ICs than violation ICs when the orthographic and phonological boundaries matched (e.g., “BI/MIR” and “RON/TA”; the slash indicates that the orthographic boundary coincides with the phonological boundary) but that the response patterns indicated more violation ICs than preservation ICs when the orthographic and phonological boundaries mismatched (e.g., “RO/N∗ER” and “BI∗M/BU”; the ∗indicates the orthographic boundary, whereas the slash indicates the phonological boundary). Taken Given that we wanted to suppress the orthographic and phonological information to potentiate the sonority-based effects, we decided to use the IC paradigm – which does not tap into the lexical processes – in combination with pseudowords, since these limit the top-down lexical processes (IC paradigm henceforth; e.g., Prinzmetal et al., 1986, 1991). Citation: Since 2003, the IC paradigm has been the subject of renewed interest for investigating whether syllables are activated quickly and automatically, and determining how the statistical (orthographic and/or phonological) distribution influences segmentation strategies in French typically developing and dyslexic children (e.g., Fabre and Bedoin, 2003; Doignon and Zagar, 2006; Maïonchi-Pino et al., 2012a,b; Doignon-Camus et al., 2013; Doignon-Camus and Zagar, 2014). An IC is a misperception of the color of a target-letter under conditions involving high attentional and perceptual demands. Two IC patterns coexist. For instance, in the case of a word like ANVIL presented either as ANVil or ANvil (where upper- and lower-case letters represent two different colors) there are: (1) ICs that preserve the syllable boundary (i.e., the participant reports that the target-letter “V” is the same color as “il” in “AN.Vil”; the dot represents the syllable boundary); and (2) ICs that violate the syllable boundary (i.e., the participant reports that the target- letter “v” is the same color as “AN” in “AN.vil”). If children really perceive syllable units in pseudowords, preservation ICs would exceed violation ICs. Based on this color misperception, which is thought to be caused by automatic syllable extraction, researchers have focused on a specific statistical argument – the bigram trough hypothesis – to explain syllable segmentation strategies (e.g., Seidenberg, 1987). The bigram trough hypothesis refers to the statistical distribution of letter co-occurrences, which follows language-specific phonotactic restrictions that govern how, and how frequently, letters occur and co-occur in specific positions. The letter co-occurrences that straddle syllable boundaries (e.g., the bigram NV in ANVIL) are of lower frequency than cluster co-occurrences that constitute syllable-sized units that precede or follow the syllable boundaries (e.g., AN and VI) and act as a powerful statistical cue that underlies syllable location and segmentation. However, previous research has confirmed that orthographic statistical properties do not occur in isolation (e.g., in Spanish, Conrad et al., 2009(in English, Rapp, 1992; Muncer et al., 2014). Frontiers in Psychology | www.frontiersin.org Citation: (2010) have suggested that the use of syllable-sized units depends on: Investigating the influence of sonority-based contact between adjacent syllables on syllable location and segmentation strategies is particularly relevant in French, which is defined as a syllable- timed language with clear-cut syllable boundaries and mostly polysyllabic words (e.g., Kaye and Lowenstamm, 1984; i.e., >90%; Manulex database3; Lété et al., 2004). French also permits both simple and complex syllable structures as well as intervocalic C1C2 clusters (i.e., 60% of CV structures vs. 17% of CVC structures vs. 14% of CCV structures; e.g., Léon, 2007; Manulex- infra database; Peereman et al., 2007). Research conducted over (1) Reading level (“good” readers use syllables earlier and better than “poor” readers); (2) Reading instruction (grapho-syllabic processing comes after grapho-phonemic processing through a process of progressive bidirectional consolidation after 6 months of reading instruction, at least in “good” readers); 3The Manulex and Manulex-infra databases provide the distributional lexical and sublexical frequencies for French elementary-school readers (from first to fifth grade). January 2020 | Volume 10 | Article 2914 Frontiers in Psychology | www.frontiersin.org 3 Universal Phonological Restrictions in French Maïonchi-Pino et al. (3) Statistical properties (after 6 months of reading instruction, children use syllables for high-frequency syllables, while phoneme-sized units are used for low-frequency syllables, before the use of syllable-sized units becomes generalized after 2.5 years of reading instruction; e.g., Maïonchi-Pino et al., 2010). (e.g., Fabre and Bedoin, 2003; Maïonchi-Pino et al., 2012a,b) – and what derives from statistical properties or from linguistic features that might account for the universal sensitivity to C1C2 clusters within syllable boundaries. clusters within syllable boundaries. Given that we wanted to suppress the orthographic and phonological information to potentiate the sonority-based effects, we decided to use the IC paradigm – which does not tap into the lexical processes – in combination with pseudowords, since these limit the top-down lexical processes (IC paradigm henceforth; e.g., Prinzmetal et al., 1986, 1991). Since 2003, the IC paradigm has been the subject of renewed interest for investigating whether syllables are activated quickly and automatically, and determining how the statistical (orthographic and/or phonological) distribution influences segmentation strategies in French typically developing and dyslexic children (e.g., Fabre and Bedoin, 2003; Doignon and Zagar, 2006; Maïonchi-Pino et al., 2012a,b; Doignon-Camus et al., 2013; Doignon-Camus and Zagar, 2014). An IC is a misperception of the color of a target-letter under conditions involving high attentional and perceptual demands. Participants Forty-eight French typically developing children from an urban elementary school were tested in April (T1), October (T2), and April (T3). All the children were from a medium socio-economic background, were monolingual native speakers of French, right- handed (+0.80 and +1 right-handedness scores were measured with the Edinburgh Handedness Inventory; Oldfield, 1971), and had normal or corrected-to-normal vision and hearing. At T1, all the children were first-graders who had been taught to read for 8 months using a mixture of analytical GPC and global procedures. We used a 20-min French standardized age-based word reading test at T1 to categorize them as “good” and “poor” readers (TIMÉ 2; Écalle, 2003). Children with a reading level of between −2 and 6 months were considered to be “good readers” (N = 22), while children with a reading level of between −3 and −9 months were considered to be “poor readers” (N = 26). TIMÉ 2 was also used at T2 and T3 and eight children who switched from good-to-poor or poor-to-good between T1–T2 (N = 3) or T2–T3 (N = 4) or who repeated a year (N = 1) were excluded from the analyses. Forty children were therefore included in the analyses (7 boys and 13 girls labeled as “good” readers; 11 boys and 9 girls labeled as “poor” readers). Descriptive and statistical data are provided in Table 1. The children neither exhibited reading or intellectual disorders nor had reading levels that differed by more than 9 months from their chronological age between T1 and T3 (M difference = 2.7 months ± 2.2, min = −9, max = 6). All the children participated after their parents had completed and signed an informed consent form. This research was approved by the Regional School Management Office and the Regional Ethics Committee. To further determine the influence of phonological sonority- based markedness across the syllable boundaries in the absence or quasi-absence of statistical information, whether orthographic or phonological, and to track developmental changes in the segmentation strategies of children who are learning to read, we used the IC paradigm to expose French “good” and “poor” readers to target-letters that had to be detected within the syllable boundaries of two-colored pseudowords. These trials were performed in April (a), October and April (b). 4All the stimuli were pre-tested with 16 French second-graders since we used pseudowords such as umzude and ymdyve, the first two first letters of which may be pronounced/ym/and/im/or/um/,/Om/, or/˜ε/. In a reading aloud task, we found that the children mostly pronounced um as/ym/(98.4%) and ym as/im/(93.8%). 5Consonants that share the same place of articulation and are considered to be more complex than heterorganic consonants at both the phonetic and articulatory levels, and that are also more likely to lead to compensation for coarticulation as well as to the delaying or mistiming of the articulatory output due to a greater gestural overlap (e.g., Jakielski, 2002). Citation: For instance, studies of reading aloud in preliterate and literate French children found that these children often reduced complex CVC or CCV syllable structures into simple CV syllables, thus indicating a preference to maximize sonority distance and eliminate sonorant consonants (e.g., “tru” →“tu”; “bar” →“ba”; e.g., Sprenger-Charolles and Siegel, 1997; Bastien-Toniazzo et al., 1999; Gnanadesikan, 2004). Crucially, these studies are limited to attested SPs, that is to say those with quantifiable orthographic and phonological statistical properties (e.g., Fabre and Bedoin, 2003; Maïonchi- Pino et al., 2012a,b). In the case of French readers, it would be particularly important to examine syllable location and segmentation strategies. Although SPs might be crucial for syllable segmentation strategies in French children, there is no clear dissociation between what is due to SPs – not considered as markedness patterns in previous studies January 2020 | Volume 10 | Article 2914 Frontiers in Psychology | www.frontiersin.org 4 Universal Phonological Restrictions in French Maïonchi-Pino et al. MATERIALS AND METHODS together, these results have led some authors to conclude that children, including those suffering from developmental dyslexia, are sensitive to – and have developed knowledge about – orthographic statistical distribution, whereas syllable boundary location and segmentation seem to depend strictly on the overlap between statistical properties. For instance, Maïonchi-Pino et al. (2012a,b) found that French children have clear-cut sonority-driven syllable segmentation strategies, in particular in the case of the optimal ‘sonorant coda – obstruent onset’ SPs. As mentioned earlier, their conclusions unfortunately stem from some attested C1C2 with quantifiable orthographic and phonological statistical properties analyzed a posteriori. Although their studies concluded that neither phonological properties nor orthographic properties provided a straightforward account for sonority-modulated syllable segmentation, there was no clear dissociation between attested vs. unattested intervocalic clusters, while children could have inferred the well-formedness of a C1C2 cluster across syllables from its existence in real French words. together, these results have led some authors to conclude that children, including those suffering from developmental dyslexia, are sensitive to – and have developed knowledge about – orthographic statistical distribution, whereas syllable boundary location and segmentation seem to depend strictly on the overlap between statistical properties. For instance, Maïonchi-Pino et al. (2012a,b) found that French children have clear-cut sonority-driven syllable segmentation strategies, in particular in the case of the optimal ‘sonorant coda – obstruent onset’ SPs. As mentioned earlier, their conclusions unfortunately stem from some attested C1C2 with quantifiable orthographic and phonological statistical properties analyzed a posteriori. Although their studies concluded that neither phonological properties nor orthographic properties provided a straightforward account for sonority-modulated syllable segmentation, there was no clear dissociation between attested vs. unattested intervocalic clusters, while children could have inferred the well-formedness of a C1C2 cluster across syllables from its existence in real French words. Participants We decided to start after 8 months of learning to read [April (a)] since previous studies showed that syllable-based effects in French are found from February in good readers (e.g., Colé et al., 1999). The orthographic and phonological statistical properties were null or quasi-null around the syllable boundary, while sonority- based markedness was manipulated along a continuum from marked, ill-formed SPs to unmarked, well-formed SPs. Our main predictions were: Frontiers in Psychology | www.frontiersin.org Material C1 and C2 could have a different manner of articulation (i.e., obstruent, fricative, nasal, or liquid; see Appendix). since segmentation occurred either before (e.g., Ul.byre) or after (e.g., UL.Byre) the syllable boundary. The target-letters to be detected were either the second or the third letter within the syllable boundary and were always at the border of the colored segments in order to prevent lateral masking (e.g., L or b in ULbyre; l in Ulbyre; B in ULByre). Each pseudoword was repeated four times (N = 140): twice in the color-syllable compatibility condition (i.e., L in ULbyre and b in ULbyre) and twice in the color-syllable incompatibility condition (i.e., l in Ulbyre and B in ULByre). In the color-syllable compatibility condition, since color and syllable boundaries matched only violation ICs occurred when the color of “L” or “b” in “ULbyre” was misperceived as being the same color as “byre” or “UL”, respectively. In the color-syllable incompatibility condition, since color and syllable boundaries mismatched only preservation ICs occurred when the color of “l” in “Ulbyre” was misperceived as being the same color as “U” or when the color of “B” in “ULByre” was misperceived as being the same color as “yre”. Regarding the unattested status of the C1C2 clusters in word- initial position, we predicted syllable segmentation between the C1 and C2 consonants (e.g., Spencer, 1996). Theoretically speaking, three segmentations were possible: V1.C1C2V2C3V3, V1C1.C2V2C3V3, or V1C1C2.V2C3V3. Whatever the syllable segmentation, and regardless of whether the segmentation respected or violated the phonotactic restrictions of French, the initial syllable structure was among the rarest in French (i.e., V, 8%; VC, 1.9%, and VCC, 0.5%; Léon, 2007). To keep the statistical properties of the orthographic and phonological components to null or quasi-null values for the V1C1C2V2C3 pattern, we referred to the Manulex-infra database (Peereman et al., 2007), which provides grade-level sublexical frequencies for French elementary-school readers, and the Surface database computed from the Lexique 2 database (New et al., 2004), which provides the distributional sublexical frequencies for French adult readers. Using the Manulex-infra database, we calculated the mean U1–U2 frequency, which designates the mean frequency computed for first and second grades, for the different sublexical structures (orthographic and phonological) of the V1C1C2V2C3V3 patterns, while the Surface database provided a confirmation of the exact mean positional frequencies. The exclusive use of the Manulex-infra database and Surface database ensured there was no clear bigram trough. Material Thirty-five six-letter disyllabic pseudowords were created. The three initial letters of these words had a VCC structure and a final schwa-like vowel (e.g., VCCVCV, ojrule). All the pseudowords had three consonants and three vowels which had regular spelling-to-sound correspondences4. All the disyllabic pseudowords had an intervocalic C1C2 cluster (i.e., V1C1C2V2C3V3). All the intervocalic C1C2 clusters were considered unattested both in word-initial position and as syllable-initial structures in French (e.g., Dell, 1995; Peereman et al., 2007). Homorganic consonants5 and voicing differences were avoided within the C1C2 clusters (regressive/progressive voicing assimilation). We used the following homorganic consonant classification: labial (i.e., /p/,/b/,/f/,/v/, and/m/), (1) If phonological representations are modeled on the basis of sonority-based markedness constraints, we predict an early and robust sensitivity to the well-formedness of the SPs (i.e., color of the target-letter assigned preferentially to high-fall SPs than to high-rise SPs) as of April (T1) in “good” and “poor” readers; (2) If segmentation strategies require sensitivity to sonority- based markedness even in the absence or quasi-absence of statistical properties, once syllable segmentation becomes available in the developmental time course, we expect that preservation ICs should increase while violation ICs should decrease, provided the well-formedness of the SPs; (3) If syllable segmentation follows a progressive developmental course coupled with the difficulties to face unattested phonological clusters, we expect that neither “good” nor “poor” readers should exhibit syllable effects in April (a), whereas these should emerge as of October and be more pronounced in “good” readers (more preservation ICs in “good” readers than in “poor” readers).” January 2020 | Volume 10 | Article 2914 Frontiers in Psychology | www.frontiersin.org 5 Universal Phonological Restrictions in French Maïonchi-Pino et al. TABLE 1 | Descriptive data for the “good” and “poor” readers at T1, T2, and T3. Time 1 Time 2 Time 3 Chronological age Reading level Chronological age Reading level Chronological age Reading level Variation T1-T3 Chronological age Reading level Good readers 81.5 (4.0) 84.2 (3.1) 87.5 (4.0) 90.4 (3.3) 93.5 (4.0) 96.3 (3.4) +12,0 +12.1 Poor readers 80.9 (3.4) 77.5 (3.4) 86.9 (3.4) 82.7 (3.3) 92.9 (3.4) 88.4 (3.6) +12,0 +10.9 Mean 81.2 80.9∗∗∗ 87.2 86.6∗∗∗ 93.2 92.4∗∗∗ ∗∗∗Indicates a significant 0.0001 p-value; ages are in months, standard deviation in brackets. TABLE 1 | Descriptive data for the “good” and “poor” readers at T1, T2, and T3. coronal (i.e., /n/,/t/,/d/,/l/,/s/,/z/,/∫/, and/Z/), and dorsal (i.e., /k/,/g/, and/K/). Frontiers in Psychology | www.frontiersin.org Material Descriptive data are reported in Table 2. Procedure The children each individually completed a single task (M duration = 16 min ± 2). The script was designed, compiled and run with E-Prime 2 Professional (Schneider et al., 2002) on Sony X-series laptop computers running under Windows 7. The children sat 57 cm from the screen in a silent room. Each trial proceeded as follows: a vertically centered green square (0.82◦of visual angle) was displayed for 250 ms. This was then replaced by a black uppercase target-letter which was displayed in the center of the screen for 1,250 ms (0.41◦of visual angle). This was followed by a 250-ms white screen which was then replaced by a two-colored pseudoword, which covered 2.46◦ of visual angle, flashed up at a visual angle of 1.14◦from the screen. A black question mark (?) then appeared in the center of the screen, where it remained until the child responded (after 5,000 ms, a warning message indicated the absence of response and this was considered to be an IC; N = 44; 0.26%. The next trial then started. The next trial followed after a 750-ms interval. Unattested intervocalic C1C2 clusters were divided into five SPs (7 pseudowords × 5): high-fall (e.g., lb; s = −4, −3 or −2), low-fall (e.g., fk; s = −1), plateau (e.g., kp; s = 0), low-rise (e.g., zm; s = + 1), and high-rise (e.g., zr; s = + 2 or + 3). Intervocalic cluster markedness fell from high-rise SPs (the most marked and most ill-formed) to high-fall SPs (the least marked and most well- formed). Each SP comprised seven different C1C2 clusters. Two colors (red and blue) were assigned to two different segments of a pseudoword. No two segments ever had the same color. This resulted in two experimental conditions. In the color-syllable compatibility condition, the color segmentation matched the expected syllable segmentation (e.g., UL.byre), whereas in the color-syllable incompatibility condition, the color segmentation mismatched the expected syllable segmentation The duration of the flashed two-colored pseudowords was adjusted for each child in order to maintain an average IC rate of 20–25% throughout the task. The duration calculation involved a two-step adjustment. First, each child was trained with a practice list on 24 trials and received corrective feedback January 2020 | Volume 10 | Article 2914 Frontiers in Psychology | www.frontiersin.org 6 Universal Phonological Restrictions in French Maïonchi-Pino et al. RESULTS (no feedback was given in the experimental lists). The initial exposure duration in the practice list was set to 350 ms (21 refresh cycles of 16.67 ms). The exposure duration was adjusted every three trials in the practice list in decrements and increments of one refresh cycle. Second, the mean exposure duration for each child at each time was used to set the initial exposure duration in the first experimental list (“good” readers, M = 309 ms ± 31; range = 300–315; “poor” readers, M = 314 ms ± 28; range = 308–325). For the experimental lists, the increment/decrement procedure was the same as for the practice list (“good” readers, M = 281 ms ± 28; range = 266–292; “poor” readers, M = 297 ms ± 30; range = 288–304). (no feedback was given in the experimental lists). The initial exposure duration in the practice list was set to 350 ms (21 refresh cycles of 16.67 ms). The exposure duration was adjusted every three trials in the practice list in decrements and increments of one refresh cycle. Second, the mean exposure duration for each child at each time was used to set the initial exposure duration in the first experimental list (“good” readers, M = 309 ms ± 31; range = 300–315; “poor” readers, M = 314 ms ± 28; range = 308–325). For the experimental lists, the increment/decrement procedure was the same as for the practice list (“good” readers, M = 281 ms ± 28; range = 266–292; “poor” readers, M = 297 ms ± 30; range = 288–304). We used Statistica 8.0 software R⃝(StatSoft R⃝, 2011) to run a 3 × 2 × 5 × 2 mixed-design repeated-measures analysis of variance (ANOVA) by subject (F1) and by item (F2) for the ICs (±16.31% of the data), with Time (T1; T2; T3), Condition (compatibility; incompatibility), Target-letter (second letter; third letter) and SP (high-fall; low-fall; plateau; low-rise; high-rise) as within-subject factors and Group (good readers; poor readers) as between-subject factor. Correct response times were trimmed (i.e., for each subject, response times that deviated by ± two standard deviations from the mean were considered as ICs, 0.96% of the data). Descriptive data are summarized in Table 3. The pseudowords in the syllable-color compatibility and syllable-color incompatibility conditions were counterbalanced across five experimental lists that were separated by pauses. RESULTS The distribution of the pseudowords was pseudo-randomized, whereas the order of their presentation was randomized. To avoid decision bias in the experimental trials, we inserted two fillers after each pause (N = 10), i.e., at the beginning of each experimental list, and the corresponding results were not included in the statistical analysis. The children were instructed to report the color of the target-letter in the flashed pseudowords as quickly and accurately as possible. They had to press on the “blue” or “red” response keys (“r” and “n” keys, respectively). The software automatically recorded ICs and response times. Procedure TABLE 2 | Mean frequencies for the different positions of the orthographic and phonological constituents in the V1C1C2V2C3V3 pseudowords calculated with the Manulex-infra database (Peereman et al., 2007) and the Surface database (New et al., 2004). Sonority Profiles Frequency Database High-Fall SPs Low-Fall SPs Plateau SPs Low-Rise SPs High-Rise SPs Mean Orthographic frequencies Frequencies for the V1C1 bigrams (inital position) Manulex-infra 11.4 0.0 3.9 2.5 10.6 5.7 Surface 73.6 0.4 16.3 19.1 9.3 23.8 Frequencies for the C1C2 bigrams (that straddle the syllable boundary; all the C1C2 bigrams have a null frequency in initial position, M = 0 ± 0) Manulex-infra 16.4 0.1 0.4 0.7 6.2 4.8 Surface 11.9 0.1 0.0 7.9 62.9 16.5 Frequencies for the C2V2 bigrams (that follow the syllable boundary; the C2V2 bigrams have equivalent frequencies in initial position) Manulex-infra 67.7 93.4 82.1 99.8 121.1 92.8 Surface 72.2 84.3 82.8 69.2 59.0 73.5 Frequencies for the V1C1C2 trigrams Manulex-infra 0.0 0.0 0.0 0.0 0.0 0.0 Surface 0.0 0.0 0.0 0.0 0.0 0.0 Frequencies for the C1C2V2 trigrams Manulex-infra 0.0 0.0 0.0 0.0 0.0 0.0 Surface 0.0 0.0 0.0 0.0 0.0 0.0 Frequencies for the C2V2C3 trigrams Manulex-infra 0.0 0.0 0.0 0.0 0.0 0.0 Surface 0.0 0.0 0.0 0.0 0.0 0.0 Frequency distance between V1C1 bigrams and C1C2 bigrams Manulex-infra 0.3 0.1 −6.1 −1.7 −4.4 −2.4 Frequency distance between C1C2 bigrams and C2V3 bigrams 99.0 137.6 58.1 84.7 132.1 102.3 Phonological frequencies Frequencies for the V1C1 syllables Manulex-infra 25.6 4.1 4.7 8.6 8.0 10.2 Frequencies for the V1C1C2 syllables 0.0 0.0 0.0 0.0 0.0 0.0 Frequencies for the C2V2 syllables 239.9 291.7 407.1 450.6 752.4 428.3 Frequencies for the C2V2C3 syllables 0.0 0.3 9.9 0.0 0.7 2.2 Frequencies are given in occurrences per million. TABLE 2 | Mean frequencies for the different positions of the orthographic and phonological constituents in the V1C1C2V2C3V3 pseudowords calculated with the Manulex-infra database (Peereman et al., 2007) and the Surface database (New et al., 2004). Sonority Profiles Sonority Profiles Frequencies are given in occurrences per million. Frontiers in Psychology | www.frontiersin.org Discrimination Sensitivity Threshold and Decision Criterion Analysis We referred to signal detection theory to assess both the d’ value (discrimination sensitivity threshold) and the β value (decision criterion), which highlights the possible bias toward the (mis)perception of the target-letters within the two-colored pseudowords (e.g., Macmillan and Creelman, 2005). The d’ value reflects the correct detection of the color of the target-letters within the two-colored pseudowords. None of the participants had a d’ = 0 ± 5% (d’ = 0 ± 5% refers to random responses embedded between 47.5 and 52.5%). We observed that the d’ January 2020 | Volume 10 | Article 2914 Frontiers in Psychology | www.frontiersin.org 7 Universal Phonological Restrictions in French Maïonchi-Pino et al. TABLE 3 | Mean illusory conjunction rate for the Group × Time × Condition × Sonority profile × Target-letter mixed-design. Time 1 Time 2 Time 3 Color-syllable Color-syllable Color-syllable Color-syllable Color-syllable Color-syllable compatibility incompatibility compatibility incompatibility compatibility incompatibility Second letter Third letter Second letter Third letter Second letter Third letter Second letter Third letter Second letter Third letter Second letter Third letter Good readers High-fall 10.0 10.7 26.4 22.9 7.1 6.4 30.7 30.0 7.1 6.4 27.1 26.4 Low-fall 12.9 12.1 25.0 22.9 10.0 8.6 25.7 24.3 7.9 7.9 25.0 23.6 Plateau 18.6 17.1 17.1 15.7 13.6 11.4 22.9 20.7 14.3 12.9 23.6 22.9 Low-rise 23.6 19.3 12.9 13.6 20.0 16.4 11.4 15.0 18.6 15.0 12.1 17.1 High-rise 22.1 19.3 12.1 14.3 23.6 17.9 10.0 14.3 19.3 13.6 10.7 16.4 Mean 17.4 15.7 18.7 17.9 14.9 12.1 20.1 20.9 13.4 11.1 19.7 21.3 Poor readers High-fall 10.0 12.9 21.4 18.6 11.4 10.7 20.0 17.9 9.3 7.9 22.1 20.7 Low-fall 11.4 13.6 21.4 20.7 10.7 10.0 20.0 18.6 8.6 10.0 20.7 20.0 Plateau 15.0 15.7 15.7 13.6 13.6 12.1 18.6 16.4 14.3 14.3 20.0 19.3 Low-rise 19.3 16.4 12.1 13.6 19.3 16.4 11.4 15.0 20.7 19.3 12.1 15.7 High-rise 22.1 18.6 10.0 12.1 21.4 18.6 10.0 14.3 20.7 18.6 11.4 14.3 Mean 15.6 15.4 16.1 15.7 15.3 13.6 16.0 16.4 14.7 14.0 17.3 18.0 Response are given in percentage (%). Errors in the color-syllable compatibility correspond to violation ICs; errors in the color-syllable incompatibility correspond to preservation ICs. TABLE 3 | Mean illusory conjunction rate for the Group × Time × Condition × Sonority profile × Target-letter mixed-design. Discrimination Sensitivity Threshold and Decision Criterion Analysis criterion fluctuated significantly as a function of Group, F(1, 114) = 4.77, p < 0.03, η2p = 0.04 (“good” readers, M = 2.183; “poor” readers, M = 2.258). The distribution for the d’ criterion was harmonic around 2 (M = 2.221 ± 0.196; min = 1.606, max = 2.757), indicating that detection was difficult, with a low to moderate sensitivity threshold. The β criterion reflects the possible bias toward syllable or non-syllable segmentation, and we observed that the β criterion varied significantly as a function of both Time, F(2,114) = 14.12, p < 0.0001, η2p = 0.20 (T1, M = 1.246; T2, M = 1.081; T3, M = 1.012) and Group, F(1,114) = 28.04, p < 0.0001, η2p = 0.20 (“good” readers, M = 1.016; “poor” readers, M = 1.211). The distribution for the β criterion started above 1 and tended to 1 and even to below 1, thus suggesting a progressive bias toward syllable segmentation (M = 1.112 ± 0.237; min = 0.515, max = 1.762). F2(4,120) = 7.53, p < 0.001, η2p = 0.17; a Fisher’s LSD post hoc test (Bonferroni’s adjusted α level for significance, p < 0.001) revealed that preservation ICs increased continuously from high-rise SPs to high-fall SPs, while violation ICs decreased monotonically from high-rise SPs to high-fall SPs. Additionally, the Condition × Sonority × Target-letter interaction was significant in the subject only (Figure 4), F1(4,152) = 3.95, p < 0.004, η2p = 0.09, F2(4,120) = 1.75, p < 0.09, η2p = 0.06; a Fisher’s LSD post hoc test (Bonferroni’s adjusted α level for significance, p < 0.0003) revealed that preservation ICs were more frequent on the third letter than the second letter in the case of the low-rise and high-rise SPs (e.g., L in IVLyde was misperceived as IVlyde more often than V in Ivlyde was misperceived as IVlyde), while violation ICs were more frequent on the second letter than the third letter in the case of the low-rise and high-rise SPs (e.g., V in IVlyde was misperceived as Ivlyde more often than l in IVlyde was misperceived as IVLyde). Discrimination Sensitivity Threshold and Decision Criterion Analysis The Condition × Time × Group interaction was also significant (Figure 5), F1(2,76) = 14.77, p < 0.0001, η2p = 0.28, F2(2,240) = 6.15, p < 0.003, η2p = 0.05; a Fisher’s LSD post hoc test (Bonferroni’s adjusted α level for significance, p < 0.0008) confirmed that preservation ICs progressively increased from T1 to T3, while violation ICs decreased progressively from T1 to T3. Both response patterns progressed faster and more steeply in “good” readers than in “poor” readers, in particular for the violation ICs. However, preservation ICs were more important than violation ICs in T2 and T3 in good readers (ps < 0.0001), and only in T3 in poor readers (p < 0.0003; marginally significant in T2, p < 0.08). In T1, differences were not significant (in good readers, p < 0.06; in poor readers, p < 0.09). The Sonority × Group interaction was not significant, Fs < 1. Illusory Conjunctions Analysis We ran an ANOVA on the proportion of ICs (16.31% of the data). All the Fisher’s LSD post hoc tests have been conducted on the F1 only. The results showed a significant main effect of Condition, F1(1,38) = 49.22, p < 0.0001, η2p = 0.56, F2(1,120) = 24.71, p < 0.0001, η2p = 0.17, with the children producing significantly more preservation ICs (18.18%) than violation ICs (14.44%). The Condition × Group interaction was significant, F1(1,38) = 12.78, p < 0.001, η2p = 0.25, F2(1,120) = 5.62, p < 0.02, η2p = 0.04; a Fisher’s LSD post hoc test (Bonferroni’s adjusted α level for significance, p < 0.008) indicated that good readers outperformed poor readers on the preservation ICs only, while both good and poor readers produced more preservation ICs than violation ICs. The Condition × Sonority interaction was significant (Figure 3), F1(4, 152) = 39.32, p < 0.0001, η2p = 0.51, January 2020 | Volume 10 | Article 2914 Frontiers in Psychology | www.frontiersin.org 8 Universal Phonological Restrictions in French Maïonchi-Pino et al. FIGURE 3 | Illusory conjunction rate (IC) in percentage (%) for the Condition × Sonority interaction. 0 5 10 15 20 25 30 IC rate (in %) SONORITY PROFILES VIOLATION IC PRESERVATION IC 0 5 10 15 20 25 30 IC rate (in %) SONORITY PROFILES VIOLATION IC PRESERVATION IC FIGURE 4 | Illusory conjunction rate (IC) in percentage (%) for the Condition × Sonority × Target-letter interaction as a function of the Target-letter (upper panel, the second letter; lower panel, the third letter). Post hoc Tests 10 15 20 25 T1 T2 T3 IC rate (in %) TIME VIOLATION IC PRESERVATION IC 10 15 20 25 T1 T2 T3 IC rate (in %) TIME VIOLATION IC PRESERVATION IC FIGURE 5 | Illusory conjunction rate (IC) in percentage (%) for the Condition × Time × Group interaction as a function of the Group (upper panel, good readers; lower panel, poor readers). FIGURE 3 | Illusory conjunction rate (IC) in percentage (%) for the Condition × Sonority interaction. 10 15 20 25 T1 T2 T3 IC rate (in %) TIME VIOLATION IC PRESERVATION IC 25 VIOLATION IC PRESERVATION IC FIGURE 3 | Illusory conjunction rate (IC) in percentage (%) for the Condition × Sonority interaction. 6It should be remembered that we a priori excluded C1C2 clusters with voicing differences in order to avoid progressive and regressive voicing assimilation. Illusory Conjunctions Analysis 0 5 10 15 20 25 30 IC rate (in %) SONORITY PROFILES VIOLATION IC PRESERVATION IC 0 5 10 15 20 25 30 IC rate (in %) SONORITY PROFILES VIOLATION IC PRESERVATION IC FIGURE 4 | Illusory conjunction rate (IC) in percentage (%) for the Condition × Sonority × Target-letter interaction as a function of the Target-letter (upper panel, the second letter; lower panel, the third letter). 10 15 20 T1 T2 T3 IC rate (in %) TIME VIOLATION IC PRESERVATION IC 0 5 10 15 20 25 30 IC rate (in %) SONORITY PROFILES VIOLATION IC PRESERVATION IC 0 SONORITY PROFILES FIGURE 5 | Illusory conjunction rate (IC) in percentage (%) for the Condition × Time × Group interaction as a function of the Group (upper panel, good readers; lower panel, poor readers). SONORITY PROFILES on ten parameters based on manner-of-articulation parameters (tests 1–6), place-of-articulation parameters (test 7), letter confusability (test 8), and statistical properties (tests 9–10). We extended our analyses in order to ensure that our sonority-based effects (i.e., global sensitivity to SPs and syllable segmentation within the C1C2 clusters; ICs in%) did not depend on isolated parameters that are known to influence the location of the syllable boundaries. We ran the post hoc tests on the IC rate. All the p-values refer to the ICs collapsed for both conditions (i.e., color- syllable compatibility and color-syllable incompatibility) since none of the parameters that we tested interacted with Condition. SONORITY PROFILES None of the distinctiveness due to any of the manner-of- articulation parameters had a significant effect: FIGURE 4 | Illusory conjunction rate (IC) in percentage (%) for the Condition × Sonority × Target-letter interaction as a function of the Target-letter (upper panel, the second letter; lower panel, the third letter). FIGURE 4 | Illusory conjunction rate (IC) in percentage (%) for the Condition × Sonority × Target-letter interaction as a function of the Target-letter (upper panel, the second letter; lower panel, the third letter). (1) Voicing (i.e., voiced-voiced C1C2 clusters; M = 16.7% vs. voiceless-voiceless C1C2 clusters; M = 15.1%; p > 0.1)6; (2) Sonorant (i.e., we performed a comparison between C1C2 clusters with two sonorant consonants; M = 16.2% and C1C2 clusters with two non-sonorant consonants; M = 16.6%; ps > 0.1); Post hoc Tests Although we controlled a priori for some statistical and linguistic parameters, we assessed a posteriori the robustness of phonological sonority-based markedness. In doing so, we attempted to determine whether or not the syllable effects resulted from low-level similarities in gestural, spectral or acoustic-phonetic contrasts (e.g., Stevens, 2002). Our aim was not to determine what constitutes sonority parameters or what determines SPs. Rather, we carried out Tukey’s HSD post hoc tests (3) Continuancy (i.e., we performed a comparison between C1C2 clusters with two non-continuant consonants; M = 15.9%, C1C2 clusters with two continuant consonants; M = 17.2%, and C1C2 clusters with a non-continuant and M = 17.2%, and C1C2 clusters with a non-continuant and 6It should be remembered that we a priori excluded C1C2 clusters with voicing differences in order to avoid progressive and regressive voicing assimilation. Frontiers in Psychology | www.frontiersin.org January 2020 | Volume 10 | Article 2914 9 Universal Phonological Restrictions in French Maïonchi-Pino et al. deriving and applying phonological sonority-based markedness constraints to unattested intervocalic C1C2 clusters. a continuant consonant, irrespective of whether they were in C1 or C2 position; M = 16.1%; ps > 0.1); (4) Stridency (i.e., we performed a comparison between C1C2 clusters with two strident consonants; M = 17.5% and C1C2 clusters with two non-strident consonants; M = 15.7%; p > 0.1); Our first results indicated non-statistical syllable segmentation strategies. Our results revealed that the absence of either orthographic or phonological statistical cues did not prevent the reliable location of the syllable boundaries. Contrary to the classical view, our results provide clear-cut evidence that syllable segmentation does not necessarily require the reading units to be defined by both orthographic and phonological information, i.e., that orthographic and syllabic boundaries coincide and that the absence of a bigram trough does not suppress or eliminate syllable segmentation effects (e.g., Doignon and Zagar, 2006; Doignon-Camus and Zagar, 2014). Combined with the fact that the IC paradigm does not tap lexical access, suppresses lexical competition and limits the top-down lexical process, the counter-intuitive response patterns, with more preservation ICs than violation ICs being found in the absence or quasi-absence of statistical information, therefore suggest that segmentation strategies do not necessarily occur as a by- product of statistical properties. Of course, this does not mean that beginning readers are unable to learn statistical distribution through repeated exposure to explicit orthographic information that is mapped to implicit phonological information. Post hoc Tests Although literature has confirmed that syllable effects appear early and are regularly reinforced through the addition of orthographic and phonological constituents that have not been previously encountered, here the use of the IC paradigm does not encourage the statistical learning of our unattested C1C2 clusters (e.g., Doignon-Camus and Zagar, 2014). (5) Nasality (i.e., we performed a comparison between C1C2 clusters with one nasal consonant in either the C1 or C2 position; M = 15.7%, and C1C2 clusters with two non-nasal consonants; M = 16.7%; p > 0.1); p (6) Manner similarity or dissimilarity (i.e., we performed a comparison between C1C2 clusters with two consonants with the same manner-of-articulation, irrespective of the similarity between manners; M = 17.0%, and C1C2 clusters with two consonants with a different manner-of- articulation, irrespective of the dissimilarity between them; M = 16.0%; p > 0.1). Similarly, (7) The distinctiveness due to the place-of-articulation parameter did not have significant effects. We compared the direction within the C1C2 clusters (i.e., forward from anterior to posterior regions; M = 16.4%, and backward from posterior to anterior regions; M = 16.2%; p > 0.1). Also, (8) letter confusability did not produce any significant difference (low; M = 16.6%, and high; M = 16.0%; p > 0.1; i.e., DRC Letter Confusability Calculator7). (7) The distinctiveness due to the place-of-articulation parameter did not have significant effects. We compared the direction within the C1C2 clusters (i.e., forward from anterior to posterior regions; M = 16.4%, and backward from posterior to anterior regions; M = 16.2%; p > 0.1). Also, (8) letter confusability did not produce any significant difference (low; M = 16.6%, and high; M = 16.0%; p > 0.1; i.e., DRC Letter Confusability Calculator7). We next performed an analysis on (9) the frequency distance between the bigram that preceded, straddled and followed the syllable boundary. Although we tried to harmonize the sublexical frequencies, some distances varied within the pseudowords. However, no difference reached the threshold of statistical significance (e.g., null or quasi-null distance; M = 16.5%, and low distance M = 16.2%; p > 0.1). Finally, (10) the phonotactic transitional probabilities were calculated for the C1C2 clusters (Crouzet, 2000) and no difference reached the threshold of statistical significance (e.g., low; M = 16.0%, and high; M = 17.0%; p > 0.1). 7www.cogsci.mq.edu.au/∼ssaunder/utils/lc.php Post hoc Tests When reading levels were examined, we confirmed well- documented data about the progressive and “reading level- dependent” nature of syllable segmentation in French (early and sustainable sensitivity to syllables to segment and access words during learning to read; e.g., Colé et al., 1999; Doignon and Zagar, 2006). The new, informative finding that we obtained relates to the time at which syllable segmentation becomes dominant and outranks other strategies. Syllables were not activated at T1 in either the good or the poor readers, even though preservation ICs exceeded violation ICs to a greater extent in good readers than in poor readers. This first observation is consistent with the fact grapho-syllabic processing is not fully specified. By contrast, preservation ICs systematically exceeded violation ICs in the good readers at T2 and T3, while this was not the case until T3 in the poor readers. This response pattern is confirmed by the β criterion, which varied over time and progressed faster and more strongly from the absence of syllable segmentation toward syllable segmentation in the good readers than it did in the poor readers. January 2020 | Volume 10 | Article 2914 DISCUSSION The true answer seems to lie in children’s early, constant, and automatic sensitivity to phonological sonority-based markedness (e.g., Berent et al., 2011, 2012b; Gómez et al., 2014). Their response patterns indicate that syllable segmentation respects the well-formedness of the C1C2 clusters within the syllable boundaries. As markedness progressively increases from high- fall SPs (e.g., lb), i.e., the unmarked and most well-formed intervocalic clusters, to marked high-rise SPs (e.g., gm), i.e., the marked and most ill-formed intervocalic clusters, there is a gradual switch from preservation ICs to violation ICs. These response patterns indicate that preservation ICs automatically and gradually increase as the C1C2 clusters tend toward an optimal syllable contact (e.g., Vennemann, 1988; Gouskova, 2004). If splitting or grouping C1and C2 depends on sonority- based markedness, then the response patterns reveal that French children do not need to strictly adhere to V1C1.C2V2C3V3 segmentation. The non-negligible, gradual inverse patterns for the violation ICs are also consistent with the sonority-based markedness of C1C2 onset clusters. Indeed, sonority-based ill- formedness (high-rise SPs) within syllable boundaries reflects the optimal sonority-based well-formedness of onset clusters (e.g., Clements, 2006). And the children optimize the segmentation to satisfy the well-formedness induced by the sonority- based markedness of onset clusters (e.g., V1.C1C2V2C3V3 segmentation).This lends support to a linguistic rule that generally applies to French segmentation with attested ill- and well-formed C1C2 clusters: the Maximum Onset Principle (e.g., Kahn, 1976). Indeed, when a C1C2 cluster violates the principle of optimal syllable contact, the adopted segmentation strategies conform to language-specific constraints which might be based in sonority, i.e., the maximization of consonants in onset clusters in cases where the native phonological regularities permit C1C2 onset clusters (e.g., Kahn, 1976; Clements, 1990; Ettlinger et al., 2012). Indeed violation ICs with the Target-letter in second position increased with low- and high-rise SPs, which are optimal onset clusters, while preservation ICs with the Target-letter in third position increased with low- and high fall SPs, which are optimal syllable contacts. In other words, this implies the use of syllable segmentation strategies in which a high-fall SP (e.g., “rj”) is considered as phonotactically illegal and disallowed in word- initial position, with segmentation therefore occurring between C1 and C2, while a high-rise SP (e.g., “jr”) is considered as phonotactically legal and allowed in word-initial position, with the result that segmentation occurs between V and C1. 8We would like to thank one of the reviewers who suggested referring to the Substance Free Phonology to discuss our results (e.g., for an in-depth presentation of the framework, see Reiss, 2017). Although the Substance Free Phonology might be relevant since it discusses the constraints implemented in the Optimality Theory (e.g., the markedness constraints), it rejects the concept of gradient for grammaticality (which was considered for the sonority scale that we used; e.g., Gouskova, 2004). Another argument lies in the fact that Substance Free Phonology does not consider underlying ill-formedness of segments; such segments just won’t be formed since they do not exist as a surface form of a language (i.e., output). DISCUSSION The present study investigated whether – and how – universal phonological sonority-based markedness – through SP across syllable boundaries – drives syllable location and segmentation in visual identification in French children when the statistical properties of the orthographic and phonological constituents are null or quasi-null. To do this, we used the classical IC paradigm with beginning readers, who were subdivided into “good” and “poor” readers. To our knowledge, this is the first time that a short-term longitudinal study has used the IC paradigm to track developmental changes in syllable-based strategies with regard to reading level, phonological universals, and statistical parameters. We thus showed that syllable location and segmentation was possible without any statistical and distributional cue by How, then, when the grapho-syllabic processing becomes available do beginning readers locate the syllable boundaries if neither orthographic nor syllable frequency explicitly modulates syllable access and the bigram trough does not cue syllable segmentation (e.g., Doignon and Zagar, 2006; Maïonchi-Pino et al., 2010)? Although we cannot rule out other unspecified, and even more complex statistical or featural properties (e.g., Hayes and Wilson, 2008; Albright, 2009), syllable segmentation might be the consequence of an early, fast and accelerated facilitatory January 2020 | Volume 10 | Article 2914 Frontiers in Psychology | www.frontiersin.org 10 Universal Phonological Restrictions in French Maïonchi-Pino et al. activation of the connection between the orthographic and phonological syllables through a limited set of syllable structures and their neighbors (e.g., Mathey et al., 2006 for arguments on syllable activation in visual word recognition) since V and VC structures represent 10% of syllable structures (e.g., Léon, 2007). Though acceptable, this proposal seems to be insufficient to account cogently for the segmentation strategies. either in onset position or within syllable boundaries. What is clear-cut is how French children process C1C2 clusters. Their knowledge is not restricted to language-specific orthographic and phonological patterns and they do not need extensive exposure to written material in order to be sensitive to sonority-based markedness. They are able to automatically derive universal sonority-based information independently of statistical information (provided through repeated exposures to oral and written material) and this helps them use the appropriate syllable segmentation strategies. This finding might therefore strengthen the hypothesis that humans “are sensitive to putatively universal restrictions on syllable structures” (p. 5839; Gómez et al., 2014). However, we did not learn as much about the time course of sonority-based markedness during the reading processes. DISCUSSION The observed response patterns also raise questions about y g g p Another interpretation might be that children derive some abstract phonological features that are shared with attested C1C2 clusters in French (see the Sonority Projection effects found in speech perception and production; e.g., Berent et al., 2007; Daland et al., 2011; see also Hayes and Wilson, 2008)8. Furthermore, our a posteriori analyses confirm that none of the IC response patterns, i.e., preservation or violation, is directly due to statistical properties or sonority-unrelated features (i.e., manner- and place-of-articulation). Although we did not probe all the phonetic transformations that do exist in speech perception (e.g., schwa deletion, consonant lenition, etc.; e.g., Hayes and Steriade, 2004; Kirchner, 2004; Davidson, 2006, 2011; Bürki et al., 2011; Davidson and Shaw, 2012), we propose that the color misperception does not stem from spectral or acoustic- phonetic failures to encode and decode the C1C2 clusters, and is more abstract, since there is no obvious evidence that such cues are required and involved in reading strategies (e.g., Berent and Lennertz, 2010; Tamási and Berent, 2014). Furthermore, the IC patterns are not due to a gestural failure to articulate the C1C2 clusters, since there is no reliable evidence that children articulate or need to articulate them (e.g., Wright, 2004; Redford, 2008). Frontiers in Psychology | www.frontiersin.org ETHICS STATEMENT This study was carried out in accordance with the recommendations of the Regional School Management Office and the Regional Ethics Committee (Auvergne-Rhône- Alpes). All the children participated after their parents had completed and signed an informed consent form. The experiment referenced in the present manuscript followed all the ethical standards of experimental and developmental psychology in children. CONCLUSION While syllable segmentation is an early, quickly activated but progressive procedure that appears earlier in good than in poor readers, the response patterns rule out a strict left- to-right effect that might stem from orthographic letter-by-letter processing or phonological grapho-phonemic processing that supports phonological grapho-syllabic processing. Indeed, the detection of the target-letter does not depend on its position, C1 or C2, but on the sonority-based markedness of the C1C2 clusters (e.g., Maïonchi-Pino et al., 2012a,b, 2013; but see Sprenger- Charolles and Siegel, 1997). Finally, we provided further evidence in support of the idea that the IC paradigm, which possesses intrinsic strong cognitive-perceptual constraints, can be used with beginning readers to assess phonological universal and language-specific abilities (e.g., Doignon-Camus et al., 2013; Doignon-Camus and Zagar, 2014). ACKNOWLEDGMENTS The authors gratefully thank the children who participated, the parents who gave their consent, and the head teachers and teachers for hosting this research. The authors also thank Tim Pownall for proofreading the manuscript, Mickaël Berthon for the management and development of the experimental applications, and the reviewers for their insightful comments. CONCLUSION Taken together, our results provide new and valuable information about both the fine-grained short-term developmental changes in the use of syllable segmentation strategies in silent reading in French children. Children are sensitive to universal phonological sonority-based markedness for the quick and efficient location of syllable boundaries in the absence or quasi-absence of statistical cues. They thus rely on sonority-based markedness to perform a bidirectional and gradual analysis that reveals the syllabification of compliant C1C2 clusters with optimal syllable contact. They The observed response patterns also raise questions about the contribution of linguistic experience and language-specific knowledge. Indeed, both distinct sonority-based markedness patterns occurred even though the children did not learn – explicitly or implicitly – any orthographic or phonological information from unattested ill- and well-formed C1C2 clusters, January 2020 | Volume 10 | Article 2914 11 Universal Phonological Restrictions in French Maïonchi-Pino et al. also perform a resyllabification that favors onset clusters over coda clusters in the case of compliant C1C2 clusters with the Sonority Sequencing Principle lying within the syllable boundaries. This also indicates that the activation of phonological sonority-based markedness, which makes it possible to locate and discriminate the syllable boundaries, is automatic and non-suppressible, and might contribute to reading acquisition. More importantly, the sensitivity to sonority-based markedness does not depend on reading abilities in contrary to the use of syllable segmentation strategies. Poor readers were sensitive to SPs in the same way as good readers, suggesting this is an unimpaired – and not delayed – phonological competence (e.g., for similar conclusions in developmental dyslexia, see Berent et al., 2012, 2013, 2016; Maïonchi-Pino et al., 2012a,b). Despite the abundant literature suggesting its importance, the phonological and/or orthographic statistical distribution does not seem to be a requirement – even in beginning readers – for learning how to use syllables and therefore for locating syllable boundaries in French (e.g., Doignon-Camus and Zagar, 2014; Mahé et al., 2014). However, the progressive crossover between the two extreme sonority-based markedness SPs (i.e., high-fall and high-rise SPs), which leads to two distinct IC patterns, corroborates the need of a maximal sonority distance between the phonemes to describe optimal clusters and to underpin syllable segmentation (e.g., Selkirk, 1984; Marouby-Terriou and Denhière, 2002). DATA AVAILABILITY STATEMENT The datasets generated for this study are available on request to the corresponding author. AUTHOR CONTRIBUTIONS NM-P, AC, and MT designed the study. AC, MT, OL, and VL collected the data. NM-P analyzed the data and wrote the first draft of the manuscript. AC, MT, OL, VL, and LF equally contributed to read, comment, and approve the submitted manuscript. Berent, I., Lennertz, T., Jun, J., Moreno, M., and Smolensky, P. (2008). Language universals in human brains. Proc. Natl. Acad. Sci. U.S.A. 105, 5321–5325. doi: 10.1073/pnas.0801469105 REFERENCES “A new method for measuring articulatory complexity,” Paper Presented at the 2002 American Speech-Language-Hearing Association Annual Convention, (Atlanta, GA). Conrad, M., Carreiras, M., Tamm, S., and Jacobs, A. (2009). Syllables and bigrams: orthographic redundancy and syllabic units affect visual word recognition at different processing levels. J. Exp. Psychol. Hum. Percept. 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This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Rapp, B. (1992). The nature of sublexical orthographic organisation: the bigram through hypothesis examined. J. Mem. Lang. 33, 33–53. doi: 10.1016/0749- 596x(92)90004-h Redford, M. (2008). Production constraints on learning novel onset phonotactics. Cognition 107, 785–816. doi: 10.1016/j.cognition.2007.11.014 Reiss, C. (2017). “Substance free phonology,” in Handbook of Phonological Theory, eds S. Hannahs, and A. Bosch, (London: Routledge). January 2020 | Volume 10 | Article 2914 Frontiers in Psychology | www.frontiersin.org 14 Universal Phonological Restrictions in French Maïonchi-Pino et al. January 2020 | Volume 10 | Article 2914 Frontiers in Psychology | www.frontiersin.org APPENDIX TABLE A1 | Stimuli used in the experiment as a function of their sonority profile. High-fall SP Low-fall SP Plateau SP Low-rise SP High-rise SP ulbyre ozbyge okpyce ydvyme ybnyje olvyde ifkute itpyfe ugjybe ymryje urzyve yftyke ydgoze ikfuce yzryve olgoze ymjuze ufsyke otfuke ivlyde ymdyve umzyde ipkute ulryge ogmuze umgove uvgoze ovzule uzmube udmube urjyde izgove itkufe ivnyze ojryve January 2020 | Volume 10 | Article 2914 Frontiers in Psychology | www.frontiersin.org 15
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Busulfan systemic exposure and its relationship with efficacy and safety in hematopoietic stem cell transplantation in children: a meta-analysis
BMC pediatrics
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Busulfan systemic exposure and its relationship with efficacy and safety in hematopoietic stem cell transplantation in children: A meta-analysis Busulfan systemic exposure and its relationship with efficacy and safety in hematopoietic stem cell transplantation in children: A meta-analysis Busulfan systemic exposure and its relationship with efficacy and safety in hematopoietic stem cell transplantation in children: A meta-analysis Xinying Feng  Beijing Children's hospital Yunjiao Wu  Beijing children's hospital Jingru Zhang  Beijing Children's hospital Jiapeng Li  Beijing Children's hospital Guanghua Zhu  Beijing Children's Hospital Duanfang FAN  China pharmaceutical university Changqing Yang  China pharmaceutical University Libo Zhao  (  libozhao2011@163.com ) Department of Pharmacy, Beijing Children's Ho Xinying Feng  Beijing Children's hospital Yunjiao Wu  Beijing children's hospital Jingru Zhang  Beijing Children's hospital Jiapeng Li  Beijing Children's hospital Guanghua Zhu  Beijing Children's Hospital Duanfang FAN  China pharmaceutical university Changqing Yang  China pharmaceutical University Libo Zhao  (  libozhao2011@163.com ) Department of Pharmacy, Beijing Children's Hospital Busulfan systemic exposure and its relationship with efficacy and safety in hematopoietic stem cell transplantation in children: A meta-analysis Xi i F Busulfan systemic exposure and its relationship with efficacy and safety in hematopoietic stem cell transplantation in children: A meta-analysis Xi i F Review Busulfaּn, Area under the concentration-time curve, Efficacy, Veno-occlusive disease, Meta-analysis is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published at BMC Pediatrics on April 20th, 2020. See the published version at https://doi.org/10.1186/s12887-020-02028-6. Page 1/15 Page 1/15 Page 1/15 Background Hematopoietic stem cell transplantation (HSCT) is widely used for the treatment of various malignancies and inherited disorders diseases. High-dose busulfan (Bu) as an alternative to total body irradiation in many pre-transplant conditioning regimens used in clinics today [1]. Although effective, Bu has a relatively narrow therapeutic index, low drug exposure is associated with increased risk of graft failure and disease relapse in transplant recipients[2-4], whereas high drug exposure is associated with increased frequency of hepatic complications, especially veno-occlusive disease (VOD)[5-7]. To improve treatment outcomes of Bu, therapeutic drug monitoring (TDM) and dose adjustment, following the first dose, has highly recommended regardless of the dosing guideline was used[8]. The area under the drug plasma concentration time curve (AUC) or its counterpart, the concentration at steady state (CSS) (the AUC divided by dose frequency) best describes the relationship between the pharmacokinetic (PK) and pharmacodynamic (PD) properties of Bu[9]. To our knowledge, there is no conclusive evidence on the relationship between optimum exposure range of Bu and its effectiveness or toxicity in children. The guidelines from the European Medicines Agency (EMA) recommended a target Bu AUC in children of 900 to 1500 µM × min [10]. The FDA labeling recommended a target intravenous (IV) Bu AUC 900 to 1350 ± 5% µM × min after 6 hour dosing [8]. The European Society for Blood and Marrow Transplantation (EBMT) guidelines recommend a total AUC after 16 doses of 90 mg × h/L (an equivalent of 1370 µM × min after every 6 hour dosage) for myeloablative exposure, without strict distinction between children and adults[11]. Numerous observational studies have recommended target Bu exposure ranges at different cut-off values, including 900[2, 12- 17], 1000[18], 1225[11], 1350[15-17], 1500[14] and 1575[11] µM × min for every 6-hour dosage. On the contrary, some observational studies found no statistically significant differences in transplant-related toxicity (TRT) or graft failure rate between different Bu AUC [19-21]. Evidence for optimum Bu exposure range described in these studies has obvious limitations. Frist, most of the observational studies that contributed to the aforementioned guidelines had too small a sample size and had no clear inclusion/ exclusion criteria. What’s more, these studies failed to identify different patient groups of adults or children. In light of these uncertainties, we conducted this systematic review and meta-analysis to evaluate the relationship between the reported Bu AUC and clinical outcomes in children undergoing HSCT. Abstract Background: Busulfan (Bu) is a key component of several conditioning regimens used before hematopoietic stem cell transplantation (HSCT). However, the optimum systemic exposure (expressed as the area under the concentration-time curve [AUC]) of Bu for clinical outcome in children is controversial. Methods: Research on pertinent literature was carried out at PubMed, EMBASE, ClinicalTrials.gov and the Cochrane Library. Observational studies were included, which compared clinical outcomes above and below the area under the concentration-time curve (AUC) cut-off value, which we set as 800, 900, 1000, 1125, 1350, and 1500 µM × min. The primary efficacy outcome was notable in the rate of graft failure. In the safety outcomes, incidents of veno-occlusive disease (VOD) were recorded, as well as other adverse events. Results: Thirteen studies involving 548 pediatric patients (aged 0.3-18 years) were included. Pooled results showed that, compared with the mean Bu AUC (i.e., the average value of AUC measured multiple times for each patient) of > 900 µM × min, the mean AUC value of <900 µM × min significantly increased the incidence of graft failure (RR=3.666, 95% CI:1.419, 9.467). The incidence of VOD was significantly decreased with the mean AUC <1350 µM × min (RR=0.370, 95% CI: 0.205-0.666) and <1500 µM × min (RR=0.409, 95% CI: 0182-0.920). Conclusions: In children, Bu mean AUC above the cut-off value of 900 µM × min (after every 6-hour dosing) was associated with decreased rates of graft failure, while the cut-off value of 1350 µM × min were associated with increased risk of VOD in children, particularly for the patients without VOD prophylaxis therapy. Further well-designed prospective and multi centric randomized controlled trials with larger sample size are necessary before putting our result into clinical practices. Data extraction and quality assessment The primary efficacy outcomes were graft failure (defined as non-engraftment or rejection). The major safety outcomes were VOD incidence and other adverse events. High-risk ratio (RR) denoted a high rate of graft failure, VOD or other adverse events. Data abstraction was conducted independently by the same two authors (X.Y.F and Y.J.W), and any discrepancy between the investigators was resolved by a third investigator (J.R.Z). The following data were collected and organized from chosen studies: the author’s name, year of publication, study design, number of patients included, methods for measuring Bu concentration, type of AUC (initial, mean or final), cut- off value of Bu AUC, and pre-specified study outcomes of efficacy and safety. Where the study already included the cut-off value, we considered patient groups treated with Bu at an AUC below the pre-defined cut-off value as the treatment group, and those above the pre- defined cut-off value as the control. Where individual patient data were available, we extracted the number of events used all our pre- defined cut-off values to divide patients into two groups in the same way. When the AUC was measured multiple times for each patient, we extracted the first dose AUC (i.e., AUC calculated from 0 h to 6 h after Bu administration) and the mean AUC (i.e., the average value of AUC measured multiple times for each patient). When neither first dose nor mean was available, we used the reported AUC for that patient in the article. When necessary, we contacted the article’s corresponding author by email for the required information. The quality of the included studies was independently assessed by two reviewers (X.Y.F and Y.J.W) according to the Newcastle–Ottawa Scale with a maximum score of 9[28]. This tool consists of three major sections concerning the methodological quality: the representative, comparability and outcome of each included study. Any disagreements that arose between the reviewers were resolved through discussion. A third reviewer (J.R.Z) was available to settle disputes. Cut-off value establishment According to the cut-off values of target Bu AUC ranges recommended by guidelines from EMA[10], EBMT[11] and the observational studies that we mentioned above[2, 14-17, 20, 23-27] The stepwise cut-off values as 800, 900, 1000, 1225, 1350, and 1500 µM × min was established . Search strategy This meta-analysis is reported in accordance with the Cochrane Handbook for Systematic Reviews and the Meta-analysis of Observational Studies in Epidemiology guidelines [22]. Studies were accessed from the PubMed, EMBASE, Web of science, the Cochrane Library and ClinicalTrials.gov. Search terms included "busulfan" in combination with "area under the curve", “AUC”, “pharmacokinetics*” and “concentration”. Reference lists of retrieved articles and related reviews were also examined, with no language or date restrictions Page 2/15 Study selection Two authors (X.Y.F and Y.J.W) independently applied the inclusion criteria to all identified and retrieved articles, if the two authors could not reach a consensus, a third reviewer (J.R.Z) was brought in to resolve the disagreement. We included studies when: (i) it was an observational study; (ii) Bu was administered 4 times daily for 4 days (16 doses), either orally or by an IV infusion route during the conditioning regimen before HSCT; (iii) TDM was performed; (iv) AUC were reported for included patients; (v) Rate of graft failure and Bu- related adverse events at both below and above the cut-off value of the AUC were reported for included patients, or sufficient data to estimate these was provided; and (vi) sample size was ≥10 patients. The exclusion criteria were as follows: (i) the object of the study was older than 18; (ii) Data came from simulated patients or pharmacokinetic models rather than real patients and; (iii) Clinical data were not presented by Bu AUC strata. Statistical Analysis Data analysis was performed using Open Meta-Analyst software (Tufts Medical Center, Boston, MA, USA). To assess variations between studies in addition to sampling error within these, the I2 statistic was used to assess for heterogeneity across the included studies. An I2 value >50% suggests substantial heterogeneity between studies. The DerSimonian-Laird was used to calculate RR and 95% confidence interval (CI) for each study. The 95% CI of outcome among distinct groups did not overlap, showing that outcomes were statistically significant. A P value <0.05 was considered statistically significant. To explore the heterogeneity among different studies, subgroup analysis was performed when more than two studies were included in the analysis of each cut-off level. For the efficacy outcome, studies were stratified by orally or an IV infusion route during the conditioning regimen before HSCT. For the safety outcome, studies were stratified by: i) studies reporting presence or absence of VOD prophylaxis therapy. ii) Orally or an IV infusion route during the conditioning regimen before HSCT. The robustness of our meta-analysis was assessed using leave-one-out approach. We isolated each study and evaluated its effect on the summary estimates and heterogeneity of the main analysis, reporting the results for sensitivity analysis when the conclusions differed. Study characteristics A summary of descriptions of included studies is reported in Table 1, the studies were published between 1996 and 2017. Nine [13-17, 23, 24, 26, 29] were prospective studies and four [2, 20, 25, 27]were retrospective studies. Six studies were conducted in Europe [14, 16, 20, 24, 25, 27], six studies were in United States [2, 13, 15, 17, 26, 29] and one [23] was in Japan. Bu concentrations were measured by high- performance liquid chromatography by means of ultraviolet detection [23, 29], while the remainder [2, 13-17, 20, 24-27] were measured by gas chromatography with mass spectrometry detection. Results Search strategy and selection criteria Page 3/15 Page 3/15 Page 3/15 A total of 4,673 articles were initially identified. Of the 3,570 articles remaining after excluding duplicate publications, 3501 were excluded after screening the title and abstract because they were not relevant. An additional 62 articles were excluded during the full-text review owing to data proceeding from simulated patients, the subjects of the study being age over 18, insufficient data on clinical outcomes, clinical data not having been presented by Bu AUC strata or Bu not having been administered 4 times daily for 4 days, among other reasons. Consequently, a total of 13 studies involving 548 patients met the inclusion criteria and, accordingly, were included for meta- analysis [2, 13-17, 20, 23-27, 29]. The literature selection process is summarized in Figure 1. Evaluation of efficacy Table 2 displays a summary of outcomes for each study. Table 3 display summaries of meta-analysis for efficacy, Forest plots are shown in Figure 2. Raw data were shown in supplementary data (Table S1 and Figures S1–S12). Our meta-analysis demonstrated that there were no significant first dose AUC cut-off values for efficacy. We found the cut-off level (AUC mean) of < 900 µM × min to be significantly associated with higher incidence of graft failure (RR=3.666, 95% CI: 1.419, 9.467). Subgroup analyses showed that the incidence of graft failure significantly decreased above a cut-off level with mean AUC 900 µM × min in the subgroup of administration by an IV infusion route alone (RR=9.718; 95% CI: 1.499- 62.989),There were no significant differences at other cut-off levels (Table 5 ). Sensitivity analysis on each study’s effect on the summary estimates for efficacy was shown in supplementary data (Table S3), which illustrated that our results were not driven by any single study, as the RRs remained stable. Evaluation of Safety A summary of primary and subgroup analysis for safety are shown in Table 4 and Table 6. Forest plots are shown in Figure 3 and Figure 4. Raw data were shown in supplementary data (Table S2 and Figures S13-20). The definitions of VOD varied across the 10 studies (Table 2), the incidence of VOD ranged from 4.8% [2, 13-17, 20, 24-27] to 40% [27]. On average, VOD occurred between 1 and 29 days after HSCT. Our meta-analysis demonstrated a significantly lower incidence of VOD with mean AUC below cut-off levels of 1350 µM × min (RR=0.370, 95% CI: 0.205-0.666) and 1500 µM × min (RR=0.409, 95% CI: 0182-0.920). In terms of the relationship between first dose AUC and clinical outcomes, our meta-analysis demonstrated there were no significant differences at all cut-off values for VOD. Subgroup analyses showed that the rate of VOD significantly decreased below a cut-off level with mean AUC 1350 µM × min in the subgroup of without VOD prophylaxis therapy (RR=0.349; 95% CI: 0.182-0.670), administration by an IV infusion route alone (RR=0.378; 95% CI: 0.158-0.906) or not (either administration by an IV infusion route or by oral) (RR=0.363; 95% CI: 0.163-0.805). There were no significant differences at other cut-off levels. For others toxic effects, the relationship of Bu AUC with graft versus-host disease (GVHD) was not found, although two studies [30, 31] reported a higher incidence of GVHD when Bu/cyclophosphamide was combined with melphalan. Regarding neurotoxicity, as benzodiazepine or phenytoin was routinely given for seizure prophylaxis, the incidence of neurotoxicity was relatively low. We could not pool the data to perform a meta-analysis. Therefore, an association between AUC and other toxic effects could not be evaluated. On each study’s effect on the summary estimates showed that exclusion of studies by Wallet al[15], Bouligand et al[25] and Tran et al[29] resulted in an insignificant difference at a cut-off level of 1500 µM × min Raw data were shown in supplementary data (Table S4). Discussion As a bifunctional alkylating agent, Bu is a key component of several conditioning regimens used before HSCT. It has been demonstrated that low plasma Bu exposure is associated with potentially fatal outcomes including graft failure, whereas high exposure is associated with toxicity, such as VOD[3, 5, 7]. Due to the high inter- and intra-patient variability in the PK profile following oral and IV infusion[10], major guidelines support and recommend TDM for Bu to improve transplant outcomes[9, 26, 32], although the exact therapeutic window in children remains inconclusive. Our meta-analysis revealed that a Bu mean AUC above the value 900 µM × min is associated with lower incidence of graft failure. This lower threshold of exposure is similar to the guideline recommendation [8]. We conducted a subgroup analysis by orally or by an IV infusion route during the conditioning regimen before HSCT, thereby demonstrating that the incidence of graft failure significantly decreased at a cut-off level of >900 µM × min in subgroup of administration by an IV infusion route. As we know, oral Bu presents a wide inter- and intrapatient variability of plasma exposures, especially in young children, which results in poor clinical outcomes [47]. That might explain why the oral Bu subgroup did not show significance at the 900 µM × min cut-off level. Our sensitivity analysis further validated the cut-off value 900 µM × min for efficacy. In addition, numerous studies [19,47] have found that the first-dose Bu AUC was significantly lower than the subsequent daily ones and AUC remained unchanged during the following days. However, we cannot identify the relationship between AUC at the first dose and efficacy as there is insufficient data from studies to support this. Thus, the correlation remain inconclusive and further investigation is needed. Our meta-analysis also demonstrated that a target value of 1350 µM × min is associated with an increased risk of VOD. This conclusion differs from the 900-1500 µM × min threshold that some publications [11, 12, 15] have suggested. This is likely due to the fact that those studies are mainly conducted on adults and their subjects of study are relatively limited. In our subgroup analyses, we stratified studies according to administration route and whether Bu treatment was combined with VOD prophylaxis therapy. Discussion In subgroup patients without VOD prophylaxis therapy, a significantly decreased incidence of VOD was detected when Bu AUC was below the cut-off value of 1350 µM × min, which could not be seen in those patients with VOD prophylaxis therapy. Plausible explanations are as follows. First, only high-risk patients (pre-existing liver damage, history of pancreatitis, genetic polymorphisms and mutations) were considered eligible for VOD prophylaxis therapy[33], which may have physiological effects on identifying the relationship between drug exposure and VOD. Secondly, as there are only two studies that include patients with VOD prophylaxis therapy, we regard these subgroup analysis results as likely to be unreliable. The optimum Bu AUC of 900-1350 µM × min is consistent with some previous research recommendations [15, 34], but differs from a recently multicenter, retrospective cohort analysis reported by Bartelink et al [11] which showed that, in children and young adults, the optimum Bu AUC is at a cumulative AUC of 78-101 mg × h/L (equivalent to 1225-1575 µM × min after every 6 hours dosing). However, there were some discrepancies that should be noted. We enforced a restriction on enrolled patients being less than 18 years of age and to be administered with Bu 4 times a day for 4 days, while in the study by Bartelink et al[11], patients older than eighteen were included and Bu was given once or four times a day. These differences in age and frequency of administration might lead to a different optimum AUC. Our study has several strengths. First and foremost, it is the first meta-analysis focusing on the relationship of Bu AUC with efficacy and safety in children, providing certain reference to individualized therapy. Secondly, our meta-analysis allowed for comparison of commonly used cut-off levels for efficacy and safety in a single analysis for individual cut-off levels. Finally, our study takes the approaches of AUC estimation (AUC for the first dose or the mean value) among transplant centers into consideration, which allowed us carry out more comprehensive comparisons of Bu AUC, despite the fact that the patients came from different institutions. We acknowledge the following limitations to our work. Quality assessment The quality assessment of the included studies is presented in Supplementary Table S5. Overall, the subjects included were representative, and ascertainment of exposure was confirmed by secure record, six studies were comparable on basis of main factors [2, 14-16, 24-25], Page 4/15 Page 4/15 Page 4/15 and seven studies were comparable on two or more factors [13, 17, 20, 23, 26, 27, 29]. Outcome assessment was based on pharmacy and medical records, the follow-up period was sufficient for outcomes to occur, and adequacy of follow-up of cohorts. According to the NOS tool, the quality assessment showed that two studies [17, 26]were scored 6 stars, four studies 7 stars[20, 25, 27, 29], three studies[13, 16, 23] 8 stars, and four studies[2, 14, 15, 24] 9 stars. No study was excluded after rating because the study quality was always above 5 stars. and seven studies were comparable on two or more factors [13, 17, 20, 23, 26, 27, 29]. Outcome assessment was based on pharmacy and medical records, the follow-up period was sufficient for outcomes to occur, and adequacy of follow-up of cohorts. According to the NOS tool, the quality assessment showed that two studies [17, 26]were scored 6 stars, four studies 7 stars[20, 25, 27, 29], three studies[13, 16, 23] 8 stars, and four studies[2, 14, 15, 24] 9 stars. No study was excluded after rating because the study quality was always above 5 stars. Discussion First, due to the shortage of available data, a detailed analysis according to different conditioning regimens and underlying disease (malignant or non-malignant disease) was not performed, which may have drug- drug interaction, and physiological effects on identifying the cut-off value of drug exposure (patients with a different disease should be treated as separate populations as they may respond to treatment differently). Moreover, we were unable to include enough data from Asian location, because we only identified one study conducted in Japan [23]. This is a timely reminder that the optimized AUC should be considered with caution when applying the results in Asian location. Finally, the use of observational studies in the meta-analysis implies Page 5/15 Page 5/15 biases and confounding factors, given that these are inherent in the original studies. As such, there is a clear requirement for further research. biases and confounding factors, given that these are inherent in the original studies. As such, there is a clear requirement for further research. Funding This research was financially supported by the Basic Clinical Research Cooperation Project of Capital University of Medical Sciences (grant number 17JL08), and the National Science and Technology Major Project of the Ministry of Science and Technology of China (grant number 2017ZX09304029). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of manuscript. Abbreviations AUC, Area under the drug plasma concentration time curve; RR, Relative risk; HSCT, Hematopoietic stem cell transplantation; TDM, Therapeutic drug monitoring; VOD, Veno-occlusive disease; NOS: Newcastle–Ottawa Scale AUC, Area under the drug plasma concentration time curve; RR, Relative risk; HSCT, Hematopo Therapeutic drug monitoring; VOD, Veno-occlusive disease; NOS: Newcastle–Ottawa Scale Authors’ contributions LBZ conceived and designed the study. XYF, YJW, JRZ and DFF conducted the literature search, quality assessment, data extraction and synthesis. XYF, YJW, CQY and JRZ interpreted the statistical analysis and drafted the manuscript. LBZ, CQY, JPL and GHZ provided critical revision on subsequent drafts and approved of the manuscript in its final form. All of the authors read and approved the final manuscript. Conclusion This meta-analysis demonstrated that Bu mean AUC above the cut-off value of 900 µM × min (after every 6-hour dosing), was associated with decreased rates of graft failure, while the cut-off value of 1350 µM × min were associated with increased risk of VOD in children, particularly for the patients without VOD prophylaxis therapy. However, our result is a synthesis of observational studies, which are the relatively low-level evidence, and should be treated carefully. Further well-designed prospective and multi centric randomized controlled trials with larger sample size are necessary before putting our result into clinical practices. Acknowledgements The authors gratefully acknowledge the support by the Basic Clinical Research Cooperation Project of Capital University of Medical Sciences and the National Science and Technology Major Project of the Ministry of Science and Technology of China. Ethics approval and consent to participate Not applicable Not applicable Availability of data and materials Raw data from this review is available in Supplementary data. The authors declare that they have no competing interests. References 1. Sisler IY, Koehler E, Koyama T, Domm JA, Ryan R, Levine JE, Pulsipher MA, Haut PR, Schultz KR, Taylor DS: Impact of Conditioning Regimen in Allogeneic Hematopoetic Stem Cell Transplantation for Children with Acute Myelogenous Leukemia beyond First Complete Remission: A Pediatric Blood and Marrow Transplant Consortium (PBMTC) Study. Biol Blood Marrow Transplant 2009; 15(12):1620-1627. Page 6/15 Page 6/15 2. McCune JS, Gooley T, Gibbs JP, Sanders JE, Petersdorf EW, Appelbaum FR, Anasetti C, Risler L, Sultan D, Slattery JT: Busulfan concentration and graft rejection in pediatric patients undergoing hematopoietic stem cell transplantation. Bone Marrow Transplant 2002; 30(3):167-173. 3. 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Vassal G, Michel G, Espérou H, Gentet JC, Valteau-Couanet D, Doz F, Mechinaud F, Galambrun C, Neven B, Zouabi H: Prospective validation of a novel IV busulfan fixed dosing for paediatric patients to improve therapeutic AUC targeting without drug monitoring. Cancer Chemotherapy & Pharmacology 2008; 61(1):113-123. 17. Bolinger AM, Zangwill AB, Slattery JT, Glidden D, Desantes K, Heyn L, Risler LJ, Bostrom B, Cowan MJ: An evaluation of engraftment, toxicity and busulfan concentration in children receiving bone marrow transplantation for leukemia or genetic disease. Bone Marrow Transpl 2000; 25(9):925-930. 18. Zwaveling J, Bredius RG, Cremers SC, Ball LM, Lankester AC, Teepe-Twiss IM, Egeler RM, Den HJ, Vossen JM: Intravenous busulfan in children prior to stem cell transplantation: study of pharmacokinetics in association with early clinical outcome and toxicity. Bone Marrow Transpl 2005; 35(1):17-23. 18. Zwaveling J, Bredius RG, Cremers SC, Ball LM, Lankester AC, Teepe-Twiss IM, Egeler RM, Den HJ, Vossen JM: Intravenous busulfan in children prior to stem cell transplantation: study of pharmacokinetics in association with early clinical outcome and toxicity. Bone Marrow Transpl 2005; 35(1):17-23. 19. References Jacobson P, Park JJ, Defor TE, Thrall M, Abel S, Krivit W, Peters C: Oral busulfan pharmacokinetics and engraftment in children with Hurler syndrome and other inherited metabolic storage diseases undergoing hematopoietic cell transplantation. Bone Marrow Transpl 2001; 27(8):855. Page 7/15 Page 7/15 20. Faraci M, Tinelli C, Lanino E, Giardino S, Leoni M, Ferretti M, Castagnola E, Broglia M, Silvestri AD, Martino DD: Monitoring of Busulphan Concentrations in Children Undergone Hematopoietic Stem Cell Transplantation: Unicentric Experience over 10 years. European Journal of Drug Metabolism & Pharmacokinetics 2017; 43(Suppl 2):1-9. 21. Blazar BR: Relationship of plasma pharmacokinetics of high-dose oral busulfan to the outcome of allogeneic bone marrow transplantation in children with thalassemia. Bone Marrow Transplant 1997; 20(11):915-920. 22. Stroup DF, Berlin JA, Morton SC, Olkin I, Williamson GD, Rennie D, Moher D, Becker BJ, Sipe TA, Thacker SB: Meta-analysis of observational studies in epidemiology: a proposal for reporting. Meta-analysis Of Observational Studies in Epidemiology (MOOSE) group. 2008. 23. Yasuhiro O, Yoshihisa N, Yoshiyuki K, Akira K, Shunichi K, Hisato K, Yasuo H, Megumi O, Makoto K, Tetsuya M: Prospective pharmacokinetic study of intravenous busulfan in hematopoietic stem cell transplantation in 25 children. Pediatr Transplant 2014; 18(3):294-301. 24. Veal GJ, Nguyen L, Paci A, Riggi M, Amiel M, Valteau-Couanet D, Brock P, Ladenstein R, Vassal G: Busulfan pharmacokinetics following intravenous and oral dosing regimens in children receiving high-dose myeloablative chemotherapy for high-risk neuroblastoma as part of the HR-NBL-1/SIOPEN trial. Eur J Cancer 2012; 48(16):3063-3072. 25. Bouligand J, Boland I, Valteau-Couanet D, Deroussent A, Kalifa C, Hartmann O, Vassal G: In children and adolescents, the pharmacodynamics of high-dose busulfan is dependent on the second alkylating agent used in the combined regimen (melphalan or thiotepa). Bone Marrow Transpl 2003; 32(10):979-986. 26. Bolinger AM, Zangwill AB, Slattery JT, Risler LJ, Sultan DH, Glidden DV, Norstad D, Cowan MJ: Target dose adjustment of busulfan in pediatric patients undergoing bone marrow transplantation. Bone Marrow Transplant 2001; 28(11):1013-1018. 27. Vassal G, Koscielny S, Challine D, Valteau-Couanet D, Boland I, Deroussent A, Lemerle J, Gouyette A, Hartmann O: Busulfan disposition and hepatic veno-occlusive disease in children undergoing bone marrow transplantation. Cancer Chemotherapy & Pharmacology 1995; 37(3):247-253. 28. Wells G, Shea B, O'Connell J: The Newcastle-Ottawa Scale (NOS) for Assessing The Quality of Nonrandomised Studies in Meta- analyses, vol. 7; 2014. 29. References Jones RJ, Lee KS, Beschorner WE, Vogel VG, Grochow LB, Braine HG, Vogelsang GB, Sensenbrenner LL, Santos GW, Saral R: Venoocclusive disease of the liver following bone marrow transplantation. Transplantation 1987; 44(6):778-783. 39. Jones RJ, Lee KS, Beschorner WE, Vogel VG, Grochow LB, Braine HG, Vogelsang GB, Sensenbrenner LL, Santos GW, Saral R: Venoocclusive disease of the liver following bone marrow transplantation. Transplantation 1987; 44(6):778-783. 39. Jones RJ, Lee KS, Beschorner WE, Vogel VG, Grochow LB, Braine HG, Vogelsang GB, Sensenbrenner LL, Santos GW, Saral R: Venoocclusive disease of the liver following bone marrow transplantation. Transplantation 1987; 44(6):778-783. References Tran HT, Madden T, Petropoulos D, Worth LL, Felix EA, Sprigg-Saenz HA, Choroszy M, Danielson M, Przepiorka D, Chan KW: Individualizing high-dose oral busulfan: prospective dose adjustment in a pediatric population undergoing allogeneic stem cell transplantation for advanced hematologic malignancies. Bone Marrow Transpl 2000; 26(5):463. 30. Mårtensson T, Priftakis P, Casswall T, Ringdén O, Mattsson J, Remberger M, Hassan M, Gustafsson B: Increased risk of gastrointestinal acute GVHD following the addition of melphalan to busulfan/cyclophosphamide conditioning. Pediatr Transplant 2013; 17(3):285-293. 31. Malär R, Sjöö F, Rentsch K, Hassan M, Güngör T: Therapeutic drug monitoring is essential for intravenous busulfan therapy in pediatric hematopoietic stem cell recipients. Pediatr Transplant 2011; 15(6):580-588. 31. Malär R, Sjöö F, Rentsch K, Hassan M, Güngör T: Therapeutic drug monitoring is essential for intravenous busulfan therapy in pediatric hematopoietic stem cell recipients. Pediatr Transplant 2011; 15(6):580-588. 32. Zao JH, Schechter T, Liu WJ, Gerges S, Gassas A, Egeler RM, Grunebaum E, Dupuis LL: Performance of Busulfan Dosing Guidelines for Pediatric Hematopoietic Stem Cell Transplant Conditioning. Biol Blood Marrow Transplant 2015; 21(8):1471-1478. 33. Corbacioglu S, Cesaro S, Faraci M, Valteaucouanet D, Gruhn B, Rovelli A, Boelens JJ, Hewitt A, Schrum J, Schulz AS: Defibrotide for prophylaxis of hepatic veno-occlusive disease in paediatric haemopoietic stem-cell transplantation: an open-label, phase 3, randomised controlled trial. Lancet 2012; 379(9823):1301-1309. 34. Javid G, Antonella I, Alessia Francesca M, Aurèlie P, Laurent N, Cristiano I, Vincenzo D, Pietro S, Marco M, Marco A: New insights into the pharmacokinetics of intravenous busulfan in children with sickle cell anemia undergoing bone marrow transplantation. Pediatr Blood Cancer 2015; 62(4):680-686. 35. Mcdonald GB, Sharma P, Matthews DE, Shulman HM, Thomas ED: Venocclusive disease of the liver after bone marrow transplantation: diagnosis, incidence, and predisposing factors. Hepatology 1984; 4(1):116-122. 36. Deleve LD, Shulman HM, Mcdonald GB: Toxic injury to hepatic sinusoids: sinusoidal obstruction syndrome (veno-occlusive disease). Semin Liver Dis 2002; 22(01):27-42. 37. Bearman SI, Appelbaum FR, Buckner CD, Petersen FB, Fisher LD, Clift RA, Thomas ED: Regimen-related toxicity in patients undergoing bone marrow transplantation. Journal of Clinical Oncology Official Journal of the American Society of Clinical Oncology 1988; 6(10):1562-1568. 38. Mcdonald GB, Hinds MS, Fisher LD, Schoch HG, Wolford JL, Banaji M, Hardin BJ, Shulman HM, Clift RA: Veno-occlusive disease of the liver and multiorgan failure after bone marrow transplantation: a cohort study of 355 patients. Ann Intern Med 1993; 118(4):255. Page 8/15 39. NB, Neuroblastoma; AML, Acute myeloid leukemia; ALL, Acute lymphocytic leukemia; MDS, myelodysplastic syndrome; NHL, non-Hodgkin’s lymphoma; SCD, sickle cell disease; SCID, Severe combined immunodeficiency syndrome; EWS, Ewing’s sarcoma; JMML, juvenile myelomonocytic leukemia; a age was represented as median (range) or mean ±SD; bFollow-up (moths) was represented as median (interquartile range); c31 patients in the autologous group (aged 0.7 to 14.9 years; median, 4 year),follow up with (49.1 to 56.9 months; median, 52.3 months)and 36 in allogeneic group, (aged 0.3 to 17.2 years old; median, 7.5 years).follow up with (45.5 to 52.8 months; median, 48.5months);d 13 patients in the ≤ 4 years group, (aged 0.5 to 3.8 years; median, 1.6year) and 11 patients in the>4 years group, (aged 4.5 to 16.7;midian 10.7 years old); d 13 patients in the ≤ 4 years group, (aged 0.5 to 3.8 years; median, 1.6year) and 11 patients in the>4 years group, (aged 4.5 to 16.7; midian 10.7 years old); e Bu with MEL group had received more prior chemotherapy courses were not considered for this article; f 31 patients were accessible for efficacy (one patient older than 18 was not included). Tables Tables Table 1 Characteristics of included studies Reference Country Study design Age (y)a Diagnosis Dosing Sampling and analysis bFollow- up (months) Faraci(2017)[20] Italy retrospective 2.9 (1.56- 9.9) NR iv 0.8-1.2mg/kg (q6h*4d);po 16 mg/kg or 480mg/m2(q6h*4d) 0 ,1, 2, 4, and 6 h after the start of infusion/HPLC- UV 48.8(0.4- 139) Okamoto(2014)[23] Japan prospective 6 (0.5- 17 ) AML(10);ALL(4);CML(2); JMML(5);Others(4) iv 0.8- 1.2mg/kg(q6h*4d) 1, 2, 2.25, 2.5, 3, and 6 h after the start of infusion/GC- MSD 1,2, 2.5, and 6 h after dose 9; 0, 2.5and 6 h after dose 13/GC-MS ≥3.33 Maheshwari(2013) [13] USA prospective 6.2(1.2– 15.5) SCD iv1.0mg/kg or 0.8mg/kg (q6h*4d) 2, 2.25,2.5,3, 4, 5 and 6 h after the start of infusion/GC- MS 36(14.4- 72) Veal(2012)[24] UK prospective mean3.6 NB po 1.45 or 1.55 mg/kg (q6h*4d) iv 0.8-1.2mg/kg (q6h*4d) 1, 2.25, 2.5, 3 and 6 h after the start of infusion for doses 1 and 9; 0, 2.5, 6h after the start of infusion for dose 13/GC- MS ≥60 cMichel (2011)[14] France prospective - AML(17);SCD(7);CML(3); NB(27); others(10) iv 0.8-1.2mg/kg (q6h*4d) NR/GC-MS - dWall(2009)[15] USA prospective - AML(8);JMML(2);MDS(2);β-thalassemia (3);Others(9) iv1.0mg/kg or 0.8mg/kg  (q6h*4d) 2,3,4,5,6 h after the start of infusion for doses 1 and 9;2 and 6h after the start of infusion for doses 13 /GC- MS 10.2(2- 23.2) Vassal(2008)[16] France prospective 5.6(0.3- 17.2 ) NB(24);AML(14);SCD(5);EWS(3);CML(3); Others(6) iv 0.8-1.2mg/kg (q6h*4d) 0, 1, 2, 2.25, 2.5, 3 and 6h after the start of infusion for doses 1 and 9;0, 2.25 and 6 h after the start of infusion for doses13/GC- MS NR eBouligand(2003)[25] France retrospective 4.4(1.1- 15.7) malignant solid tumor Page 9/15 Page 9/15 Table 1: (Continued) Reference Country Study design Age (y)a Diagnosis Dosing Sampling and analysis bFollow- up (months)   fMcCune(2003)[2] USA retrospective 6(0.25- 16) AML (19); MDS (7);SCID (5);others (22) po total dose 11-28 mg/kg (q6h*4d) 0, 1, 2, 3, 4, 6h after the start of infusion for dose 5 and dose 9/GC-MS NR   fBolinger(2001)[26] USA prospective (0.6- 17.1) AML (6); CML(5); β- thalassemia (3); AA (4);SCD(4); others(10) po total dose 10.9- 28.9 mg/kg 0.5, 1, 2, 3, 4, 5, 6 h after the start of infusion for dose 1; 0, 0.5, 1, 2, 4, 6 h after the start of infusion for dose 5, 9 and 13/GC-MS NR   Bolinger(2000) [17] USA prospective (0.6- 18) β-thalassemia (10); AML(9); others(13) po total dose 14-20 mg/kg 0, 0.5, 1, 2, 3, 4, 5 and 6 h after the start of infusion for dose 1 and dose 13/GC-MS NR   Tran(2000)[29] USA prospective 7.6 (0.8- 18) ALL (13); AML (7); MDS (3); CML(1); NHL (1) po 40 mg/m2  0, 0.5, 1, 2, 4, 6 h after the start of infusion for dose 1,dose 5 and dose 9/HPLC-UV 32 (11- 52)   VASSAL(1996)[27] France retrospective 5.9(1- 15) NB(28);Brain tumors (13);NHL(5); others (11) po 1mg/kg or 30-37.5 mg/m2 20min, 40min as well as 1, 1.5, 2, 3, 4, and 6 h after the start of infusion for dose 1,dose 5 and dose 9/GC-MS NR   NR, not reported; GC-MS, gas chromatography with mass spectrometry detection; IV, intravenous; HPLV-UV, high-performance liquid chromatography (HPLC) with the ultraviolet (UV) detection; AA, Aplastic anemia; NB, Neuroblastoma; AML, Acute myeloid leukemia; ALL, Acute lymphocytic leukemia; MDS, myelodysplastic syndrome; NHL, non-Hodgkin’s lymphoma; SCD, sickle cell disease; SCID, Severe combined immunodeficiency syndrome; EWS, Ewing’s sarcoma; JMML, juvenile myelomonocytic leukemia; a age was represented as median (range) or mean ±SD; bFollow-up (moths) was represented as median (interquartile range); c31 patients in the autologous group (aged 0.7 to 14.9 years; median, 4 year),follow up with (49.1 to 56.9 months; median, 52.3 months)and 36 in allogeneic group, (aged 0.3 to 17.2 years old; median, 7.5 years).follow up with (45.5 to 52.8 months; median, 48.5months);d 13 patients in the ≤ 4 years group, (aged 0.5 to 3.8 years; median, 1.6year) and 11 patients in the>4 years group, (aged 4.5 to 16.7;midian 10.7 years old); d 13 patients in the ≤ 4 years group, (aged 0.5 to 3.8 years; median, 1.6year) and 11 patients in the>4 years group, (aged 4.5 to 16.7; midian 10.7 years old); e Bu with MEL group had received more prior chemotherapy courses were not considered for this article; f 31 patients were accessible for efficacy (one patient older than 18 was not included). CI, confidence interval; CI, confidence interval; NA, not applicable; IV, intravenous Tables Page 10/15 Page 10/15 Table 2 Outcomes and results of included studies Reference Type of AUC Cut-off value Reported outcome Definition of graft failure or rejection Definition of VOD Faraci[20] Initial 900 Graft failure NR Mcdonald criteria[35] Okamoto[23] Initial 800; 900; 1000; 1225; 1350; 1500 Graft failure; VOD Failure to reach ANC> 0.5*109/L by day 28 after transplantation Mcdonald criteria[35] maheshwari[13] Initial and mean 1350; 1500 VOD NR McDonald criteria[36] veal[24] Mean 1350;1500 Hepatic toxicity or VOD NR Bearman criteria[37] Michel[14] Mean 900;1350;1500 VOD NR McDonald criteria[38] Wall[15] Initial, mean and Final 800; 900; 1000; 1225; 1350; 1500 Graft failure, VOD Failure to reach ANC>0.5*109/L at any time after transplantation Jones criteria[39] vassal[16] Mean 900;1350;1500 Graft failure; VOD Failure to reach ANC> 0.5 *109/L for three consecutive days by day 100 after transplantation Jones criteria[39] Bouligand[25] Final 1350;1500 VOD NR McDonald criteria[38] McCune[2] Mean 900;1350 Graft failure; TRT Failure to reach ANC> 0.5 *10 9/L Bearman criteria[37] Bolinger[26] Mean 800; 900; 1000; 1225; Graft failure No evidence of donor cells or initial evidence of donor engraftment followed by full autologous recovery Bearman criteria[37] Bolinger[17] Initial and mean 800; 900; 1000; 1225; Graft failure No evidence of donor cells or initial evidence of donor engraftment followed by full autologous recovery Bearman criteria[37] Tran[29] Mean 1350;1500 VOD NR Bearman criteria[37] VASSAL[27] Initial 1350;1500 VOD NR McDonald criteria[38] NR, not reported; VOD, veno-occlusive disease; TRT, transplant-related toxicity Table 2 Outcomes and results of included studies Page 11/15 Page 11/15 Table 3 Summary of meta-analyses for the incidence of graft failure Type of AUC Cut-off value (µM*min/L) RR (95% CI) Number of studies Number of participants in treatment group Number of participants in control group I2% AUC first dose <800 verse ≥800 2.664 (0.857, 8.282)   4 24 67 0 <900 verse ≥900 2.208 (0.686, 7.107)   5 73 100 0 <1000verse ≥1000 1.544 (0.315, 7.561)   4 48 43 0 <1225verse ≥1225 1.007 (0.222, 4.578)   4 66 25 0 AUC mean <800 verse ≥800 5.296 (1.389, 20.191)   3 22 78 0 <900 verse ≥900 3.666 (1.419, 9.467)   7 59 216 0 <1000verse ≥1000 1.245 (0.267, 5.809)   4 62 38 0 <1225verse ≥1225 0.559 (0.125, 2.505) 4 78 22 0 Table 3 Summary of meta-analyses for the incidence of graft failure Type of AUC Cut-off value (µM*min/L) RR (95% CI) Number of studies Number of participants in treatment group Number of participants in control group I2% AUC first dose <800 verse ≥800 2.664 (0.857, 8.282)   4 24 67 0 <900 verse ≥900 2.208 (0.686, 7.107)   5 73 100 0 <1000verse ≥1000 1.544 (0.315, 7.561)   4 48 43 0 <1225verse ≥1225 1.007 (0.222, 4.578)   4 66 25 0 AUC mean <800 verse ≥800 5.296 (1.389, 20.191)   3 22 78 0 <900 verse ≥900 3.666 (1.419, 9.467)   7 59 216 0 <1000verse ≥1000 1.245 (0.267, 5.809)   4 62 38 0 <1225verse ≥1225 0.559 (0.125, 2.505) 4 78 22 0 Table 3 Summary of meta-analyses for the incidence of graft failure 78 CI, confidence interval; Table 4 Summary of meta-analyses for the incidence of VOD Type of AUC Cut-off  value (µM*min/L) RR (95% CI) Number of studies Number of participants in treatment group Number of participants in control group I2% AUC first dose ≤1350 versus>1350 0.562 (0.126,2.496) 3 51 23 26.96% ≤1500 versus>1500 0.761 (0.435,1.333) 4 87 44 0 AUC mean ≤1350 versus>1350 0.370 (0.205,0.666) 7 207 61 0 ≤1500 versus>1500 0.409 (0.182,0.920) 5 163 28 0 CI, confidence interval; Table 4 Summary of meta-analyses for the incidence of VOD Page 12/15 Page 12/15 Table 5 Summary of subgroup analysis for the incidence of graft failure Subgroup     Cut-off value (µM*min/L) RR (95% CI) Number of  studies Number of participants in treatment group Number of participants in control group I2% Administration route   IV Bu ≤800 versus>800 11.282 (0.930, 136.897)   2 2 36 0 ≤900 versus>900 9.718 (1.499, 62.989)   4 10 150 0   ≤1000 versus>1000 0.418 (0.030, 5.850)   2 23 15 0 ≤1225 versus>1225 0.139 (0.011, 1.729)   2 32 6 0 Oral Bu   ≤800 versus>800 3.904(0.800,19.055)   2 20 42 0   ≤900 versus>900 2.613(0.869,7.860)   3 49 66 0   ≤1000 versus>1000 2.189 (0.328,14.587)   2 39 23 0   ≤1225 versus>1225 1.197 (0.186,7.720)   2 46 16 0 CI confidence interval NA not applicable IV intravenous Table 5 Summary of subgroup analysis for the incidence of graft failure 0 Page 13/15 Table 6 Summary of subgroup analysis for incidence of VOD Sub         group     Cut-off value (µM*min/L) RR (95% CI) Number of  studies Number of participants in treatment group Number of participants in control group I2% Administration route   IV Bu alone ≤1350 versus>1350 0.378 (0.158,0.906) 3 106 30 0   ≤1500 versus>1500 0.485 (0.171,1.377) 3 129 17 0 IV Bu+oral Bu/oral Bu     ≤1350 versus>1350 0.363 (0.163, 0.805) 4 101 31 0 ≤1500 versus>1500 0.316 (0.087,1.145) 2 34 11 0 VOD prophylaxis Yes ≤1350 versus>1350 0.476 (0.120, 1.885) 1 42 15 NA   ≤1500 versus>1500 0.491 (0.109, 2.216) 1 56 11 NA No ≤1350 versus>1350 0.349 (0.182, 0.670) 6 165 46 0   ≤1500 versus>1500 0.380 (0.145, 0.994) 4 107 17 0 CI, confidence interval; NA, not applicable; IV, intravenous Table 6 Summary of subgroup analysis for incidence of VOD Figures Page 14/15 Figures Figure 1 Flow chart of study selection process Figures Figure 1 Page 14/15 Figures Figure 1 Flow chart of study selection process Figures Figure 1 Flow chart of study selection process Page 14/15 Page 14/15 Figure 4 Meta-analysis for incidence of VOD (mean AUC of <1500 µM × min comparison with ≥1500 µM × min, RR <1 favors ≥1500 µM × min) Meta-analysis for incidence of VOD (mean AUC of <1500 µM × min comparison with ≥1500 µM × Meta-analysis for incidence of VOD (mean AUC of <1500 µM × min comparison with ≥1500 µM × min, RR <1 favors ≥1500 µM × min) Figure 2 Meta-analysis for rate of graft failure (mean AUC of <900 µM × min comparison with ≥ 900 µM × min , RR <1 favors ≥ 900 µM × min) Meta-analysis for rate of graft failure (mean AUC of <900 µM × min comparison with ≥ 900 µM × min , RR <1 favors ≥ 900 µM × min) Meta-analysis for rate of graft failure (mean AUC of <900 µM × min comparison with ≥ 900 µM × min , RR <1 favors ≥ 900 µM × min) Figure 3 Meta-analysis for incidence of VOD (mean AUC of <1350 µM × min comparison with ≥1350 µM × min , RR <1 favors ≥1350 µM × min) Figure 3 Meta-analysis for incidence of VOD (mean AUC of <1350 µM × min comparison with ≥1350 µM × min , RR <1 favors ≥1350 µM × min) Meta-analysis for incidence of VOD (mean AUC of <1350 µM × min comparison with ≥1350 µM × min , RR <1 favors ≥1350 µM × min) Figure 4 Meta-analysis for incidence of VOD (mean AUC of <1500 µM × min comparison with ≥1500 µM × min, RR <1 favors ≥1500 µM × min) Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. Supplementarydata3.doc Page 15/15 Page 15/15 Page 15/15
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Para una lectura crítica de &lt;i&gt;Hacia la crítica de la violencia&lt;/i&gt; de Walter Benjamin: Schmitt, Kafka, Agamben
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ISEGORÍA. Revista de Filosofía Moral y Política N.º 41, julio-diciembre, 2009, 267-276 ISSN: 1130-2097 Para una lectura crítica de Hacia la crítica de la violencia de Walter Benjamin: Schmitt, Kafka, Agamben Critique of Walter Benjamin’s Critique of Violence: Schmitt, Kafka, Agamben EDUARDO MAURA ZORITA * Universidad Complutense de Madrid RESUMEN. El propósito de este artículo es evaluar críticamente la lectura de Giorgio Agamben del ensayo sobre la violencia de Walter Benjamin, muy particularmente en sus relaciones con otras obras relevantes de Franz Kafka y Carl Schmitt. A partir del intento de Agamben de derivar la «nuda vida» y ciertas determinaciones de su homo sacer del texto de Benjamin, buscaré situar la crítica de la violencia en el lugar que le corresponde, así como repensar la propia obra de Agamben, tan valiosa en algunos puntos como discutible en otros. ABSTRACT. The purpose of this article is to assess in a critical fashion the way Giorgio Agamben reads Walter Benjamin’s essay on violence. I will particularly focus on the way Agamben relates this essay to other works by Carl Schmitt and Franz Kafka. Given his effort to steer Benjamin towards the concepts of «mere life» and certain qualities of the homo sacer, I will try to relocate Benjamin where his political essay belongs and, at the same time, re-read Agamben at his strongest points, but also at his weakest. Palabras clave: violencia, Benjamin, Agamben, escatología, excepción Key words: violence, Benjamin, Agamben, eschatology, exception. 1. Ante la ley: las políticas de la violencia de Franz Kafka y Carl Schmitt Benjamin se aproxime a una materia tan importante para él como la ley, en todas sus dimensiones, sin recurrir de alguna manera a categorías estéticas o literarias (véanse, en este sentido, el Trauerspielbuch o los importantes ensayos sobre Las afinidades electivas de Goethe y Franz Kafka). Sin embargo, tanto en su configuración lingüística, amparada siempre en la escritura dialéctica como expresión bifronte (derecho natural / derecho positivo, primero, y violencia mítica / violencia pura, después) como en la exposición Benjamin redactó Zur Kritik der Gewalt en diciembre de 1920 con vistas a su publicación en la revista Archiv für Sozialwissenschaft und Sozialpolitik, de gran difusión en la época y que contaba, entre otros, con colaboradores de la talla de Carl Schmitt. A primera vista, se trata de un texto singular en la producción benjaminiana. Y es que no es habitual que [Recibido: Feb. 09 / Aceptado: Jul. 09] 267 Eduardo Maura Zorita de sus motivos, la escritura de Benjamin se presenta en su máximo esplendor. La recepción del texto ha sido tan compleja como su trama: inicialmente ignorado por los editores en favor de piezas más rutilantes, no contó siquiera con la aprobación de Peter Szondi, el cual, al ser preguntado por la selección de textos que debía aparecer en la editorial Suhrkamp bajo el rótulo Schriften, lo descartó junto con el «Karl Kraus» en favor de otros como «Destino y carácter» 1. Más adelante, sin embargo, la crítica de la violencia ha adquirido un rango elevado entre los estudiosos de Benjamin, muy particularmente en lo que atañe a la cuestión del origen mítico de la ley y sus vínculos con la literatura de Kafka. En general, diversos textos de Benjamin han tratado de iluminar aspectos relacionados con la ley. Sin embargo, sólo la crítica de la violencia nos ofrece un tratamiento sistemático del tema. Benjamin, desde el comienzo, exige cautela, ya que el propio título impone una doble lectura. Gewalt, en alemán, significa tanto violencia, en sentido enfático, como poder, en el sentido de poder establecido. Así, el propósito del ensayo es doble: primero, establecer los fundamentos para una distinción entre violencia mítica y violencia divina. Segundo, elevar desde dicha dicotomía una crítica de largo alcance de las estructuras del poder establecido. El punto de partido de esta lectura de Zur Kritik der Gewalt (1921) es la idea de que Benjamin que aspira a una estrategia revolucionaria que el término mesiánico no agota en su totalidad 2. En la crítica de la violencia, las fuerzas revolucionarias miran hacia el Paraíso. El juicio divino les presta una inmediatez que Benjamin reclama y que remite al lenguaje adánico y la facultad de nombrar. A la afinidad, en definitiva, entre la palabra y la cosa. El Paraíso, lejos de ser una categoría mesiánica como tal es, en realidad, epítome de 268 una metafísica de la presencia: la violencia divina comparece en las páginas finales de la crítica de la violencia como logos en acción, como justicia suprema, a saber, como justicia sumarísima, carente de juicio y más allá de la vista de los hombres. La violencia pura, por mor de lo vivo, se presenta como «insignia y sello» y no como acto de guerra santa. Como en las páginas finales de El proceso, para ella es como si la vergüenza fuera a sobrevivirnos. La mención a Kafka no es en absoluto casual para nuestro propósito. En una importante carta a Benjamin del veinte de septiembre de 1934, en la que Scholem trata de aclarar diversos aspectos de la obra de Kafka basándose, precisamente, en las cargas histórico-naturales que los personajes de Kafka soportan, se puede leer: «¿Me preguntas qué entiendo por “la nada de la revelación”? Por ella entiendo un estado en el que la revelación aparenta no tener significado pero, aún, se afirma a sí misma, en el que tiene validez pero no significado [in dem sie gilt, aber nicht bedeutet]. Un estado en el que la riqueza de significado se ha perdido y lo que se encuentra en proceso de comparecer (pues la revelación es precisamente tal proceso) sin embargo no desaparece, pese a verse reducido a un punto cero de contenido, por así decirlo» 3. Llama la atención, a este respecto, el parecido de familia entre ese estado de validez sin significado, que para Scholem culmina en su retracción a un punto cero de contenido, con la figura política del estado de excepción, tal y como Carl Schmitt la ha configurado y, a efectos de la crítica de la violencia, la parábola kafkiana «Ante la ley». Procediendo en orden inverso, podríamos hablar del relato de Kafka como síntoma de una afinidad electiva entre Kafka, Scholem y Benjamin por la que el judío secular Kafka (o Benjamin) asume la distinción paulina entre letra y espíritu, ISEGORÍA, N.º 41, julio-diciembre, 2009, 267-276, ISSN: 1130-2097 Para una lectura crítica de Hacia la crítica de la violencia ... esto es, interioriza su exposición a una letra muerta que ha perdido valor espiritual y que, en sus cuerpos, aparece grabada violentamente en las letras de la circuncisión y no, por el contrario, en la escritura del corazón 4. La opacidad de la ley se asemeja aquí a la figura del campesino de Kafka que, perplejo ante la apertura (de la puerta que conduce a la ley) se obsesiona con su guardián y permanece sentado junto a él hasta que, ciego y agotado, percibe un resplandor que surge del interior: «Pero entonces advierte en medio de la oscuridad un resplandor que, inextinguible, sale por la puerta de la Ley. Le queda poco tiempo de vida. Antes de su muerte se le acumulan en la cabeza todas las experiencias vividas aquel tiempo hasta concretarse en una pregunta que todavía no le había hecho al guardián. Le indica por señas que se acerque, pues ya no puede incorporar su rígido cuerpo [...] “Todos aspiran a entrar en la Ley”, dice el hombre, “¿cómo es que en tantos años nadie más que yo ha solicitado entrar?”. El guardián advierte que el hombre se aproxima ya a su fin y, para llegar aún a su desfalleciente oído, le ruge: “Nadie más podía conseguir aquí el permiso, pues esta entrada sólo estaba destinada a ti. Ahora me iré y la cerraré”» 5. La incapacidad de decidir entre la potencia-de-sí y la potencia-de-no del campesino de Kafka ha podido ser interpretada como la imposibilidad crónica de discernir que inaugura el estado de excepción. La pregunta entonces, en lo que a Benjamin concierne literalmente, sería: ¿es asumible el presupuesto kantiano de la forma de la ley que, en su pretensión de universalidad, rige con total independencia de su contenido? ¿No es acaso esto lo que le ocurre al campesino de Kafka, que la ley no les exige nada? ¿Acaso significa algo para él? Si la ley, en su vínculo indisoluble con la violencia jurídica, como parece sugerir Benjamin, es también vigencia sin significado, entonces tiende a coincidir con la mera vida y es indiscernible de ella. La tesis octava «Sobre el concepto de historia», por la que había que hacer efectivo el estado de excepción (que ha sido siempre regla para los oprimidos), se puede leer ahora como pretensión de eliminar la relación entre vida y ley, resolviéndola mediante la transformación de la vida en ley. Esta interpretación, de cuño agambeniano, alcanza su máxima expresión en la siguiente fórmula: «A la impenetrabilidad de una escritura que, convertida en indescifrable, se presenta ahora como vida, corresponde la absoluta inteligibilidad de una vida enteramente resuelta en escritura» 6. Esta relación es similar a la de escritura y vida en la correspondencia con Scholem (carta del 11 de agosto de 1934) y similar a la afirmación de Benjamin por la que la puerta de la justicia no es el derecho que se practica, sino el que se estudia (el que se lee, el que se descifra, como la escritura que, sin su cifra, coincide meramente con la vida). En Kafka, la ley, como la escritura, se confunde con la vida. A su vez, el nuevo abogado de Benjamin 7 estudia un derecho impotente, un derecho que, carente ya de vínculos con la violencia y el poder, sólo puede parecerse, proféticamente, al derecho mesiánicamente cumplido. Pero este derecho impotente no es la justicia misma, sino la puerta que conduce a ella. Y en este ámbito nuevo, el derecho tiene un nuevo uso que nos es desconocido, bajo una justicia en la que el mundo aparece como un bien ya inapropiable por el estado o, incluso, indecidible jurídicamente. Un mundo diferente en el que el estudioso y el jugador marcan la pauta. La impenetrabilidad, propia de la tarea del estudioso, y la incapacidad de discernir, propia del jugador intuitivo, son categorías que, a continuación, vincularemos con la teoría de la soberanía de Carl Schmitt. En su estudio La dictadura, ISEGORÍA, N.º 41, julio-diciembre, 2009, 267-276, ISSN: 1130-2097 269 Eduardo Maura Zorita Schmitt propone una distinción entre dictadura comisarial y dictadura soberana: la primera, de índole pasajera, se caracterizaría por la autonomía de la norma de su aplicación. La Constitución, bajo este modelo, se suspende por mor de su propia conservación. Por el contrario, la dictadura soberana plantea el problema de una Constitución saliente que ya no existe efectivamente que, al mismo tiempo, no puede someterse a una nueva, ya que ésta sólo existe en forma mínima como poder constituyente. Es, por tanto, un estado en el que la ley se aplica pero no tiene vigor formal. El lugar de la soberanía, según Schmitt, es aquél en el que la oposición entre la norma y su aplicación alcanza su máxima expresión: el estado de excepción. De aquí el origen de su influyente definición de soberanía en Teología política: «Es soberano quien decide sobre el estado de excepción» 8. El soberano, de esta manera, traza los límites del derecho desde fuera: «La decisión no es aquí la expresión de la voluntad de un sujeto jerárquicamente superior a cualquier otro, sino que representa la inscripción, en el cuerpo del nomos, de la exterioridad que anima y da sentido a éste. El soberano no decide entre lo lícito y lo ilícito, sino sobre la implicación originaria de la vida en la esfera del derecho o, en las palabras mismas de Schmitt, sobre la “estructuración normal de las relaciones de vida” de que la ley tiene necesidad» 9. El derecho tiene carácter normativo porque crea su propia referencia en la vida real: la normaliza. Hay una indistinción entre lo externo y lo interno, entre el ejemplo y la excepción, entre la vida y el derecho, entre la prioridad de la culpa y la norma —culpa que no se refiere a la trasgresión sino a la vigencia de la ley, de ahí que la ignorancia no exima de su cumplimiento o, incluso, que no haya culpa sin norma, como rezan dos dichos famosos. Esta indistinción es propia de la decisión sobe270 rana sobre la excepción que es aquí entendida como forma originaria del derecho. «El orden jurídico, más que mediante la sanción del hecho transgresivo, se presenta a través de la repetición del mismo acto sin sanción alguna, es decir, como caso de excepción» 10. Esta caracterización del soberano linda sustancialmente con la tesis octava de Benjamin en la que, como hemos visto, Benjamin reclama la necesidad, para luchar contra el fascismo, de producir el estado de excepción efectivo. Estoy de acuerdo con Agamben en que este paso no es sólo una radicalización de la tesis del Trauerspielbuch de la indecisión soberana. Simplemente, Benjamin toma conciencia de que la máquina schmittiana no puede funcionar si el estado de excepción efectivo (wirklichen Ausnahmezustand) se confundiera con la regla. Es cierto que el primado de la excepción, basado en ésta como una posibilidad de constitución de la esfera normal, está en Teología Política. Pero, ¿qué ocurre cuando excepción y regla se vuelven indecidibles? Este es el desafío que, según Agamben, Benjamin plantea a Schmitt. La decisión soberana, alcanzado el paso que Benjamin reclama, se fagocitaría a sí misma, puesto que ya no podría decidir sobre el estado de excepción sin decidir sobre sí misma. Aquí es donde, una vez determinadas las diferencias entre decisión soberana (en su desenmascaramiento por Benjamin como mantenimiento de la opresión) y violencia pura, hay que volver a precisar algunos elementos de la crítica de la violencia. En una carta a Ernst Schoen, Benjamin define perfectamente lo que entiende por Reiheit (pureza), y que no será bajo ningún concepto algo sustancial: «La pureza de un ser no es nunca incondicionada ni absoluta, está siempre subordinada a una condición. Esta condición es diferente según el ser de cuya pureza se trate; ISEGORÍA, N.º 41, julio-diciembre, 2009, 267-276, ISSN: 1130-2097 Para una lectura crítica de Hacia la crítica de la violencia ... pero no reside nunca en el ser mismo» 11. Si aplicamos esta concepción a la violencia del ensayo de 1921, ésta no pertenece esencialmente a la acción violenta. Esto es, la diferencia entre la violencia pura y la violencia jurídica no reside en su propia condición de violenta, sino en algo que la determina desde fuera. En este caso, según afirma Benjamin, se trataría de su relación con el derecho y con la justicia. La violencia pura, en ese caso, no es medio para un fin, como lo era la violencia policial. Benjamin busca el quid de su crítica en la esfera de los medios, y no en la de los fines que estos persiguen. La violencia comparece como medio puro: un medio, que pese a ser considerado siempre como medio, permanece al margen de los fines. Lo que Benjamin busca es una violencia que no pudiera ser nunca medio justo o injusto de acuerdo con fines determinados. La pregunta es, ¿cómo se comporta un medio así? ¿Existe otra manera de vincular medios y fines? Igual que el lenguaje en el ensayo sobre el lenguaje de los hombres es sólo puro en tanto que comunicabilidad total y simple y esta lengua pura no es una lengua aparte, sino que reside en todas las lenguas existentes, la violencia pura sólo comparece en la reelaboración de la relación entre violencia y derecho. La violencia pura no se vincula ya con el derecho que establece o con el que mantiene, sino que deshace el vínculo: rompe la línea existente entre violencia y derecho. Es una violencia que, por tanto, no se ejecuta, sino que, simplemente, actúa. 2. La lectura de la conspiración de Giorgio Agamben Así como hemos utilizado ya algunas herramientas exegéticas de Agamben para este ensayo de lectura de la violencia y la ley en Benjamin, creo que urge en este momento reconsiderar críticamente la lectura de Benjamin que Agamben reali- za en diversos pasajes de su Homo sacer. No solamente el estatus que Agamben ha alcanzado en el mundo académico lo aconseja, sino también el hecho de que su exégesis se ha convertido, en los últimos tiempos, en una suerte de cifra canónica de lectura. En líneas generales, Agamben deriva su análisis de la sacralidad de la vida del concepto de mera vida que Benjamin alumbra en su crítica de la violencia. Si en el ensayo político de Benjamin la violencia jurídica está vinculada a la inculpación de la vida natural y la violencia pura o divina es sangrienta pero incruenta, precisamente a causa de lo vivo, en la obra de Agamben, «de la misma manera que la excepción soberana, la ley se aplica al caso excepcional desaplicándose, retirándose de él, así también el homo sacer pertenece al dios en la forma de la insacrificabilidad [...] La vida insacrificable y a la que, sin embargo, puede darse muerte, es la vida sagrada» 12. Así, el desarrollo de la figura del homo sacer, inadvertidamente para Agamben, parece avanzar en dirección opuesta al ensayo de Benjamin. Sigrid Weigel ha apuntado algo similar cuando, de forma más restringida, recuerda que el propio Benjamin rechaza, tanto en la crítica como en los ensayos sobre Goethe, Franz Kafka y Karl Kraus, el uso de conceptos secularizados procedentes del orden de lo divino o, incluso, del texto bíblico, para luego ser transferidos directamente, sin ninguna mediación teórica, al contexto de la vida humana 13. Además de esta apropiación indebida por parte de Agamben, nos concierne singularmente su lectura específica de la crítica de la violencia en la primera secuela de su Homo sacer, titulada Estado de excepción, en la que, partiendo de la dialéctica del carácter sagrado del hombre y el paradigma de la biopolítica, Agamben plantea la problemática del estado de excepción recompo- ISEGORÍA, N.º 41, julio-diciembre, 2009, 267-276, ISSN: 1130-2097 271 Eduardo Maura Zorita niendo las relaciones entre Schmitt y Benjamin en clave dialógica. El objetivo del ensayo de Benjamin, sostiene Agamben, es probar la realidad de una violencia fuera y más allá del derecho que, como tal, pudiera romper la dialéctica entre la violencia que funda derecho y la que lo conserva. Esta violencia, que se correspondería con la violencia pura o divina (o, en la esfera humana, revolucionaria) de Benjamin, se muestra incompatible con el derecho, que, obviamente, no puede tolerar tal cosa, a saber, una violencia fuera del derecho. Y no porque los fines de la violencia en su determinado sean incompatibles con el derecho, sino por el hecho de su existencia exterior al derecho (Benjamin, GS, p. 183). Garantizar a la violencia una realidad más allá del derecho implica la posibilidad de una violencia pura, cuyo carácter no será ya el de fundación o conservación, sino el de supresión del derecho. Así inauguraría el hombre una nueva época histórica. En otro lugar, Agamben sugiere que en el debate sobre el estado de excepción entre Benjamin y Schmitt, el primer paso ha de ser el análisis de la lectura de Schmitt de la crítica de la violencia. Partiendo de una aseveración de escasa entidad como los hábitos de lectura del propio Schmitt, Agamben arguye que es imposible que colaborador asiduo de la revista no reparase en el texto de Benjamin, siendo éste de tal naturaleza que el interés para él tuvo que ser enorme. De hecho, y aquí Agamben afina más en la argumentación casuística, Schmitt cita en sus escritos el número anterior y el posterior al que incluyó el texto de Benjamin. La lectura de la conspiración que presta título a este apartado consistiría en leer la teoría de la soberanía de Schmitt como una respuesta a Zur Kritik der Gewalt, y no al revés, como la historia de la relación entre Schmitt y Benjamin ha sido tradicionalmente representada. Si 272 bien no es mi intención en estas páginas negar la reciprocidad e íntima enemistad intelectuales entre ambos, tampoco me parece justo pretender perpetuar el modelo Horkheimer-Adorno, léase confrontación izquierda-derecha, de interpretación de la misma. Así, se puede considerar que la célebre carta de Benjamin a Schmitt que acompaña al ejemplar de Ursprung des deutschen Trauerspiels, que aquél le envió con motivo de su publicación en la editorial Rohwolt de Berlín en 1928, es un documento de primera magnitud, pero no lo es menos la cita de Teología política en el propio libro sobre el barroco y, por encima de todo, el recurso a la teoría de la soberanía de Schmitt como su cifra de lectura. La carta, desterrada inicialmente de la correspondencia de Benjamin, rezaba: Reconocerá usted cuánta influencia ejerce su escrito Teología política tanto metódica como objetivamente sobre mi libro; pero lo que no puede usted saber es que su libro La dictadura y otros me han conmovido profundamente, hasta el punto de que mis intuiciones en estética y sus ideas sobre filosofía política coinciden 14. Y no es menos llamativa la secuencia que el Jacob Taubes ha descrito en su pequeño exergo «La historia Jacob Taubes-Carl Schmitt». Allí se menciona una conversación con Adorno, a propósito de la exclusión de esta carta, que, siempre según Taubes, se desarrolló así: «“Han salido ya dos volúmenes de cartas de Benjamin, ¿cómo es que esta carta no se ha impreso en ellos?”. La respuesta —de Adorno— fue: “No existe tal carta”. Y yo dije: “Teddy, yo conozco la letra, conozco la máquina con la que escribía Benjamin. No me cuente cuentos. Aquí la tengo”. “Imposible”. Típica respuesta alemana. Así que saqué copia y se la mandé. Y además había allí un archivero, el señor Tiedemann. Recibo llamada telefónica de Teddy: “Sí que hay esa carta, pero ISEGORÍA, N.º 41, julio-diciembre, 2009, 267-276, ISSN: 1130-2097 Para una lectura crítica de Hacia la crítica de la violencia ... se había traspapelado”» 15. El testimonio es suficientemente explícito. Finalmente, se podría concluir que Schmitt fuera un aguzado lector de Benjamin no modifica, al menos sustancialmente, el hecho de que Benjamin adoptara algunos de sus conceptos como herramientas cognitivas, al igual que la teoría de la confrontación Schmitt-Escuela de Frankfurt no alcanza a explicar la fascinación de buena parte de sus miembros o seguidores con la figura de Schmitt. Es por tanto necesario separar lo propio de lo ajeno sin cercenar la palabra objetivamente ambigua. Esto es, resistirse a que la utilización de textos de Schmitt en el Trauerspielbuch de Benjamin determine la balanza de la relación Benjamin-Schmitt hacia la admiración de Benjamin por Schmitt, tanto como a leer a Schmitt como si la crítica de la violencia fuera referente casi único de Teología política, libro, por cierto, escrito un año después. La estrategia de lectura de Agamben consiste, en este sentido, en la inversión de la relación a través del análisis del texto de Benjamin como piedra de toque. En cuanto al texto de Schmitt, es cierto que Benjamin no utiliza el término estado de excepción, sino otro (Ernstfall), que en Schmitt sirve como sinónimo de estado de excepción (Ausnahmezustand) en Teología Política. Asimismo, Agamben señala con razón que Benjamin utiliza el término decisión (Entscheidung) en un momento importante: «El derecho reconoce la decisión local y temporalmente determinada como categoría metafísica» 16. Agamben trata de utilizar estas coincidencias como elementos que tejerían por sí mismos una trama entre Benjamin y Schmitt, lo cual es harto complicado si nos ceñimos a un texto de Benjamin particularmente singular. No creo que se pueda basar un vínculo tal en una disquisición sobre el vocabulario de ambos puesto que Benjamin, aunque más Benjamin que nunca, está sin embargo aquí fuera de quicio. El argumento de fondo, más allá de la escritura circunstancial, es que Schmitt, según Agamben, trataría de devolver la violencia pura y anómica a un contexto jurídico. Esto es, la estrategia inversa a la de Benjamin: «El estado de excepción es el espacio en que trata de aprehender la idea benjaminiana de una violencia pura y de inscribir la anomia en el cuerpo mismo del nomos. No puede haber, según Schmitt, una violencia pura, es decir absolutamente fuera del derecho, porque, en el estado de excepción, está incluida en el derecho a través de su propia exclusión» 17. Aquí es donde Agamben saca las garras de la interpretación: aparentemente, en Teología política Schmitt abandona lo que en La dictadura era la dicotomía básica: poder constituyente y poder constituido, sustituyéndola por el concepto de decisión. Agamben lee esto es términos de contraataque. La dicotomía violencia-que-funda-derecho / violencia-que-conserva-derecho sería el equivalente teórico benjaminiano a la dicotomía poder constituyente / poder constituido. Y más allá: Schmitt reelaboraría su propia teoría para desarrollar una teoría de la soberanía que neutralizara la figura benjaminiana de la violencia pura, configurando a su vez la idea de una violencia soberana, que, como la violencia pura de Benjamin, fuera un poder que no crea ni conserva derecho, sino que lo suspende allí donde, en el ensayo de Benjamin, la violencia pura lo suprimía. La soberanía pasa a ser el lugar de la decisión última, en contra de la tesis de Benjamin de la indecibilidad de todos los problemas jurídicos. Sólo así Schmitt asegura la relación en un contexto jurídico determinado entre anomia y derecho. La violencia pura, para Benjamin, no se decide. No está sujeta a una decisión. Igualmente, Schmitt aprecia el problema de la indecibilidad y, ante la dificultad de determinar los casos extre- ISEGORÍA, N.º 41, julio-diciembre, 2009, 267-276, ISSN: 1130-2097 273 Eduardo Maura Zorita mos de decisión, enfatiza la pertinencia de la decisión soberana. Agamben pretende, desde este punto de vista, reelaborar todo el debate entre Schmitt y Benjamin, incluyendo el problema del soberano en el libro sobre el Trauerspiel alemán. Pero Benjamin, en perfecta consonancia con lo anterior, modifica, incluso aquí, donde aparentemente la deuda es mayor, la propia teoría de Schmitt. Lo hace para reintroducir la exclusión necesaria del orden jurídico contra la inclusión schmittiana. Cuando Benjamin cita a Schmitt en el Trauerspiel ya desecha la posibilidad de esta inclusión: «Es el soberano quien representa a la historia, sosteniendo en la mano el acontecer histórico como un cetro [...] Esta doctrina extrema del poder del príncipe fue en sus orígenes —pese al agrupamiento en torno a ella de los partidos contrarreformistas— más ingeniosa y más profunda que su versión moderna. Si el concepto moderno de soberanía acaba por otorgar sin reservas al príncipe un supremo poder ejecutivo, el barroco se desarrolla por su parte a partir de una discusión sobre el estado de excepción, y considera que la función más importante del príncipe consiste en evitarlo. Quien manda está destinado de antemano a detentar dictatorialmente el poder durante el estado de excepción, cuando la guerra, la rebelión u otras catástrofes así lo provoquen» 18. Agamben lee excluir donde Benjamin, en mi opinión, quiere decir evitar, dando pie, sensus literalis, a toda esta polémica. La idea de la dictadura tiránica en el Barroco, sintéticamente, no sería otra que la de una utopía en la que el acontecer histórico es finalmente dominado por las leyes naturales. Esta idea es desbrozada por Benjamin mediante la teoría de la indecisión soberana que ocupa las páginas siguientes. Allí plantea la imposibilidad del soberano barroco de decidir: lo que Benjamin hace es escindir el poder sobe274 rano de su realización. Para el tirano del barroco, la decisión se vuelve casi imposible, lo cual se percibe en «ese complemento de la tiranía sanguinaria que es la indecisión» 19. Citaré ahora otro pasaje relevante de Benjamin: «En el modo de pensar teológico-jurídico tan característico del siglo se expresa la exaltación de la trascendencia que subyace a los acentos provocativos que el Barroco pone siempre en el más acá. Pues a éste la idea de catástrofe se le antoja cabalmente antitética del ideal histórico de la restauración. Y sobre esta antítesis se acuña la teoría del estado de excepción [...] El hombre religioso del Barroco tiene tanto apego al mundo dado que se siente arrastrado con él hacia una catarata. No hay una escatología barroca; y justamente por ello sí hay un mecanismo que reúne y exalta todo lo nacido sobre la tierra antes de que se entregue a su final. El más allá es vaciado de todo aquello en que se mueve hasta el más leve hálito del mundo, y el Barroco le arrebata una profusión de cosas, normalmente sustraídas a cualquier figuración, que ahora en su apogeo saca a la luz con una figura drástica, a fin de despejar un último cielo y, en cuanto vacío, ponerlo en condiciones de aniquilar algún día en sí a la tierra con catastrófica violencia» 20. Pues bien, Agamben lee aquí un desplazamiento del paradigma del milagro como estado de excepción al paradigma de la catástrofe y acusa a los editores de las Gesammelte Schriften de no haber percibido las implicaciones de este desplazamiento. Si bien, de nuevo, Agamben incurre en la secularización del milagro en catástrofe, es todavía más peculiar el argumento filológico: «Donde el texto benjaminiano rezaba: Es gibt eine barocke Eschatologie, “hay una escatología barroca”, los editores, con singular menosprecio de cualquier cautela filológica, han corregido, Es gibt keine... “no hay una escatología barroca”» 21. Por mi ISEGORÍA, N.º 41, julio-diciembre, 2009, 267-276, ISSN: 1130-2097 Para una lectura crítica de Hacia la crítica de la violencia ... parte, creo que los editores de Benjamin tienen razón: no hay una escatología barroca. De ahí la necesidad artificial, si se quiere, de un mecanismo de reunión de todas las criaturas antes de su final. Pero su final es la tesis octava, intuyo, no la acción directa divina-no-mediada. Que el Barroco no posee una escatología, además, Benjamin lo propone en otros lugares: «Mientras que tanto el misterio cristiano como la crónica cristiana presentan la totalidad del curso de la historia, la historia universal, como historia de la salvación, por su parte la acción principal y de Estado tiene que ver con una mera parte de lo que es pragmático acontecer» 22. Más adelante, leemos: «La constitución del lenguaje formal del Trauerspiel puede entenderse como el desarrollo de las necesidades contemplativas inherentes a la situación teológica de la época. Y una de ellas, tal como comporta la desaparición de toda escatología, es el intento de encontrar consuelo a la plena renuncia a un estado de gracia en la consumada regresión al estado creatural [...] Mientras que la Edad Media exhibe la precariedad de la historia universal y la caducidad de la criatura como etapas en el camino de salvación, el Trauerspiel alemán se sume por entero en el desconsuelo de la condición terrena. Si reconoce una redención, ésta se encuentra más en lo profundo de dicha fatalidad que en la idea de consumación de un plan divino de la salvación» 23. La dialéctica de la escatología barroca existe realmente en el texto de Benjamin en forma de eschaton, de final del tiempo, pero las nociones que gravitan alrededor de ambas citas, unidas a la teoría de la soberanía, no permiten afirmar la existencia palmaria de una escatología barroca cuya inmanencia tomaría la forma de una cáscara vacía. Aquí, el carácter creatural de la naturaleza de la soberanía se vuelve contra Agamben: «Casi po- dría llevar como divisa cierto pasaje de Gracián que ilustra cuán escrupulosamente en los Trauerspiele ha de ajustarse el papel del príncipe al estereotipo y el extremo: “No hay medianía en los reyes. Son conocidos, o por muy buenos, o por muy malos” [...] El tirano y el mártir son en el Barroco las cabezas de Jano del coronado, las plasmaciones necesariamente extremas de la misma esencia principesca [...] La teoría de la soberanía, para la que el caso excepcional, con su despliegue de instancias dictatoriales, resulta ejemplar, obliga a complementar virtualmente la imagen del soberano en el sentido concreto del tirano, incluso en aquel caso que no lo exige la situación; exactamente del mismo modo en que a la aparición en escena del gobernante sólo excepcionalmente le faltarán el ornato completo, la corona y el cetro» 24. Cuando Agamben busca en este soberano benjaminiano una tierra de nadie catastrófico-jurídica, olvida que la incapacidad de decidir del soberano es tal en cuanto habitante del reino de las criaturas, en su sometimiento a las leyes naturales, y no en su función política. No se justifica, ni filológica ni filosóficamente, la maniobra biopolítica de arrancar al soberano del Trauerspiel de su escenario para situarlo en la el Despacho Oval como, intuyo, Agamben aspira a hacer con su «olvido» de la dialéctica entre la función política del soberano y su realización como criatura. Para Benjamin la vida nuda, si como tal queremos considerarla, siempre queda fuera de la ley. Su exclusión no deja lugar a dudas. Es cierto, por lo tanto, que Benjamin y Schmitt escriben en la misma constelación, como es igualmente plausible la influencia sobre éste de la definición del soberano como aquél que decide sobre el estado de excepción. Pero lo que no es admisible es que Benjamin y Schmitt mantengan una suerte de correspondencia secreta. El soberano se convierte en el ISEGORÍA, N.º 41, julio-diciembre, 2009, 267-276, ISSN: 1130-2097 275 Eduardo Maura Zorita ensayo de Benjamin en el lugar donde se aprecia más claramente la fractura del poder. Por eso se puede afirmar, sin comprometer a Benjamin con el paradigma biopolítico, que «el paradigma del estado de excepción ya no es, como en la Teología Política, el milagro, sino la catástrofe» 25. Pero no se sostiene, al menos en esta formulación, que las motivaciones religiosas y la violencia mítica se hayan secularizado durante ese proceso. Agamben necesita de una escatología barroca para configurar textualmente el estado de excepción en el barroco en forma de catástrofe, y esto sólo puede ser porque necesita mantener la simetría Benja- min—Schmitt. Esta idea se aleja del Benjamin de la crítica de la violencia tanto como el campo de fuerzas que constituye la secularización de los conceptos políticos de Schmitt, a saber, la correspondencia entre las categorías Rey y Dios. En Benjamin, y es aquí precisamente donde se impone el ensayo político sobre cualquier otro, incluso el Trauerspielbuch, el soberano es una criatura más. La lectura de la conspiración de Agamben se pierde, paradójicamente en una recta paralela que escamotea los antagonismos por conocer en la dialéctica Benjamin-Schmitt, en su contradicción objetiva, en definitiva, en su núcleo de verdad. NOTAS * Eduardo Maura es licenciado en Filosofía y Sociología por la Universidad de Deusto y escribe actualmente su tesis doctoral, centrada en las implicaciones filosóficas y potencial cognitivo de la filosofía de la historia de Walter Benjamin. Ha publicado artículos y reseñas sobre estas cuestiones en revistas como La Torre del Virrey (Valencia) o Logos (Madrid). Dirección postal: Facultad de Filosofía, Departamento Filosofía IV (ed. A). Ciudad Universitaria, 28040, Madrid. Correo electrónico: emauraz81@hotmail.com. 1 Szondi, P., Briefe, Frankfurt/M, Suhrkamp, 1993, p. 25. 2 Benjamin, W., «Zur Kritik der Gewalt», en Gesammelte Schriften, II/1, Frankfurt/M, Suhrkamp, 1972-1989, pp. 179-203. En adelante, GS remite siempre a Gesammelte Schriften. Salvo mención expresa las citas pertenecen a la edición española de Jorge Navarro Pérez, «Hacia la crítica de la violencia», en Obras, II/1, Madrid, Abada, 2007, pp. 183-206. 3 Benjamin, W., Gesammelte Briefe, II. Frankfurt/M, Suhrkamp, 2000, pp. 756-764 (la cita procede de la edición española, Benjamin, W. / Scholem, G., Correspondencia, 1933-1940, Madrid, Taurus, pp. 159160). 4 Cfr. Santner, Eric L., On creaturely life. Benjamin, Rilke, Sebald. Chicago, University of Chicago Press, 2006, pp. 39-40. 5 Kafka, F., Obras completas, III. Narraciones y otros escritos, Barcelona, Galaxia Gutenberg, 2003, p. 193. 6 Agamben, G., Homo sacer. El poder soberano y la nuda vida. Valencia, Pre-textos, 2006, p. 75. 7 Cfr. Benjamin, W., «Franz Kafka», en Gesammelte Schriften, II/2. Frankfurt/M, Suhrkamp, 276 1972-1989, pp. 409-438. (traducción de R. Blatt, Para una crítica de la violencia y otros ensayos. Iluminaciones IV, Madrid, Taurus, 1999, p. 155). Otros textos relevantes de Benjamin sobre Kafka son «Franz Kafka: Beim Bau der Chinesischen Mauer», en GS, II/2, pp. 676-683 y la reseña «Max Brod. Franz Kafka», en GS, III, pp. 526-529. 8 Schmitt, C., «Teología política», en Carl Schmitt, teólogo de la política. Madrid: FCE, 2001, p. 24. 9 Agamben, G., op. cit., p. 41. 10 Agamben, G., ibid. 11 Benjamin, W., Briefe, op. cit., p. 205. 12 Agamben, G., op. cit., pp. 107-108. 13 Cfr. Weigel, «The Critique of Violence. Or, The Challenge to Political Theology of Just Wars and Terrorism with a Religious Face», en Telos, 135. Germany After the Totalitarianism (vol. 1). Summer 2006, pp. 61-76. 14 Citado en Taubes, J., La teología política de Pablo. Madrid, Trotta, 2007, p. 114. 15 Taubes, J., ibid. 16 Benjamin, W., GS, II/1, p. 189. 17 Agamben, G., Homo sacer, II/1. Estado de excepción. Valencia, Pre-textos, 2003, p. 82. 18 Benjamin, W., Obras, I/1, Madrid, Abada, 2005, p. 268. 19 Benjamin, W., op. cit., p. 275. 20 Benjamin, W., op. cit., p. 269. 21 Agamben, G., Excepción, op. cit., pp. 84-85. 22 Benjamin, W., op. cit., p. 283. 23 Benjamin, W., op. cit., p. 285. 24 Benjamin, W., op. cit., p. 273. 25 Agamben, G., Excepción, op. cit., p. 84. ISEGORÍA, N.º 41, julio-diciembre, 2009, 267-276, ISSN: 1130-2097
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Nasal Cavity and Paranasal Sinuses Cancer Clinical Distant Metastasis TNM Finding v8
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Nasal Cavity and Paranasal Sinuses Cancer Clinical Distant Metastasis TNM Finding v8 National Cancer Institute Qeios ID: D4WGHP · https://doi.org/10.32388/D4WGHP Qeios · Definition, February 2, 2020 Source National Cancer Institute. Nasal Cavity and Paranasal Sinuses Cancer Clinical Distant Metastasis TNM Finding v8. NCI Thesaurus. Code C133032. A clinical finding about one or more characteristics of nasal cavity and paranasal sinuses cancer, following the rules of the TNM AJCC v8 classification system as they pertain to distant metastases. Qeios ID: D4WGHP · https://doi.org/10.32388/D4WGHP 1/1
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Studies on Seed Vigour in Bt Cotton Hybrids
Madras Agricultural Journal
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Studies on Seed Vigour in Bt Cotton Hybrids R. Ankaiah, N. Balayenkulu, K.V.S. Meena Kumari, V. Sandeep Varma*, M. Ganesh and K. Kanaka Durga Seed Research and Technology Centre, Acharya N.G. Ranga Agricultural University Rajendranagar, Hyderabad, AP, India Studies on seed vigour tests in Bt and non Bt cotton hybrids were taken up during 2007 at Seed Research and Technology Centre, Rajendranagar, Hyderabad. Irrespective of Bt and Non Bt cotton hybrids, TP (87%), S methods (85%) recorded higher germination than BP (83%) and soil method (82%). Among the vigour tests, first count (78%), final count (86%), brick gravel test (81%) and cold test (79%) recorded higher germination in all the Bt cotton hybrids than Non Bt hybrids which recorded superior values for speed of germination (74%), field emergence (76%), paper exhaustion test (68%) and tetrazolium test for viability. Bt cotton hybrids especially TCH 9 and PRCH 31 recorded higher germination in all germination testing methods as well as vigour tests. Germination and seedling vigour gradually declined with an increase in period of accelerated ageing from 0 to 30 days. Such decline was more in Non Bt cotton hybrids when compared to Bt cotton hybrids. Among different Bt cotton hybrids, PRCH 31 and TCH 9 have shown higher values in all vigour tests as against the other hybrids. There was a rapid decline in germination and vigour in NCS 145, NCS 207, Rudra and Sandeep. Key words: Bt and Non Bt cotton hybrids, germination and vigour tests. Cotton popularly known as ‘white gold’ is an important commercial crop vis-a-vis commodity and plays an important role in agrarian and industrial activities of the nation.India stands third in world production, albeit substantially low productivity. Cotton production in India witnessed a quantum leap during last five years with the introduction of transgenic technology and inter and intra specific hybrids with high yield potential. Presently it is cultivated over an area of 11.96 m. ha with a production of 36.10 m. tonnes in 2011. Monsanto, in collaboration with Mahyco introduced Bt cotton technology in India, which carries a gene derived from soil bacterium Bacillus thuringiensis var. kurstaki that confers resistance to the bollworm complex. Bt cotton has increased yield up to 50 per cent, reduced insecticide sprays by half, with accrued environmental and health benefits and has contributed to social benefits and alleviation of poverty. Seed vigour is an important aspect of quality, which predicts the plant stand, establishment and field performance. Studies on Seed Vigour in Bt Cotton Hybrids Problems associated with establishing vigorously growing cotton seedlings are often related to poor seed quality. High quality cotton seeds have the capacity to provide vigorous seedlings over a wide range of environments. Hence, an attempt was made to study the seed vigour of six Bt and non-Bt cotton counter parts. Keeping in view the meager research in this field, the present study was undertaken to study the seed vigour of Bt and non Bt cotton hybrids. Materials and Methods The present experiment was carried out at Seed Research and Technology Centre, Rajendranagar, Hyderabad during the year 2007- 2008. Seeds of twelve varieties of cotton hybrids both Bt and Non Bt in three replications using factorial RBD were assessed for seed germination by different methods such as top of paper method, between paper method, sand method, soil method and various seed vigour assessment techniques like seedling length (cm), seedling dry weight (mg), cold test, germination percentage (first and final count), accelerated ageing, field emergence index, speed of germination, seed leacheate, brick gravel, tetrazolium and paper exhaustion tests. Madras Agric. J., 99 (10-12): 662-666, December 2012 https://doi.org/10.29321/MAJ.10.100165 Madras Agric. J., 99 (10-12): 662-666, December 2012 https://doi.org/10.29321/MAJ.10.100165 *Corresponding author email: sandeepvannam81@gmail.com Results and Discussion According to Axay Kumar (2008), highest germination was observed in sand where as lowest germination was recorded on top of paper method in jatropa. This clearly indicates that sand is the best substrata for conducting germination tests because seed is covered fully there by maintaining the required level of moisture necessary for germination and seedling development. The lowest germination was observed on top of paper method which may be attributed to unavailability of sufficient moisture for seed germination. Similar results were also reported by Uppar et al., (2006) in dicoccum wheat. Coming to soil method, the germination percentage differed significantly due to hybrids of Bt and non Bt. All the Bt cotton hybrids gave higher germination compared to their non-Bt counter parts except NCS 145. Among the cotton hybrids, PRCH 31 recorded maximum germination (93% and 88%) in both Bt and Non-Bt types, respectively. Among the yp p y Table 2. Effect of total seedling length (cm) in six Bt and Non Bt cotton hybrids Treatment First count Final count BT NBT Mean BT NBT Mean NCS 145 11.50 9.07 10.68 23.49 22.50 22.99 NCS 207 11.50 9.75 10.62 23.16 22.82 22.99 TCH 9 12.00 10.12 11.06 21.26 19.94 20.60 RUDRA 10.37 9.12 9.75 22.05 20.50 21.27 PRCH 31 11.12 10.50 10.91 22.64 24.75 23.69 SANDEEP 11.37 8.75 10.06 23.14 18.67 20.97 Mean 11.35 12.81 12.08 22.62 21.53 22.08 VBt x Non Bt I VBt x Non Bt I S.E± 5.42 3.13 7.67 1.11 0.64 1.58 C.D @ 5% 11.0 6.36 15.50 2.20 1.31 3.21 V = Variety,I = Interaction between V x Bt x Non Bt that significantly higher germination percentage in sand method and in between paper method, when compared to soil media. It might be due to the large Table 3. Results and Discussion Effect of germination methods on seedlin g g of germination methods on seedling length (cm) in six Bt and Non-Bt cotton hybrids Treatment Top of paper method Between paper method Sand method Soil method Brick gravel test BT NBT Mean BT NBT Mean BT NBT Mean BT NBT Mean BT NBT Mean NCS 145 7.30 6.75 7.02 20.11 17.87 18.99 13.33 12.70 13.02 13.36 11.31 12.24 13.27 11.90 12.59 NCS 207 7.20 6.75 6.97 19.65 17.48 18.56 13.27 12.23 12.76 13.67 13.70 12.59 13.80 13.79 13.80 TCH9 7.80 7.22 7.51 17.92 16.27 17.10 12.87 11.49 12.18 13.91 11.96 12.93 13.89 11.80 12.84 RUDRA 7.35 6.87 7.11 17.60 8.67 13.13 13.49 12.00 13.15 14.05 11.63 12.04 14.43 13.13 13.78 PRCH31 7.87 7.19 7.53 19.18 17.87 18.52 16.00 14.20 15.14 14.45 14.27 14.35 14.42 15.33 14.88 SANDEEP 7.37 6.80 7.08 14.80 7.80 11.30 13.03 12.12 12.57 13.37 11.98 12.67 13.40 11.84 12.62 MEAN 7.48 6.93 7.20 18.21 14.32 16.27 13.60 12.59 13.40 13.80 12.47 13.14 13.87 12.97 13.42 V Bt x Non Bt I V Bt x Non Bt I V Bt x Non Bt I V Bt x Non Bt I V Bt x Non Bt I S.E ± 0.24 0.139 0.34 0.51 0.29 0.72 0.18 0.10 0.26 0.21 0.12 0.30 0.35. 0.20 0.50 CP@5% 0.491 0.283 0.695 1.04 0.60 1.47 0.37 0.21 0.53 0.43 0.28 0.61 0.72 0.41 1.02 V = Variety, I= Interaction between V x Bt x Non Bt length (Table 3) revealed that the seedling length in top of the paper method, between paper method, sand method, soil method and brick gravel method has also shown similar trend like germination percentage. The cotton hybrid PRCH 31 recorded the maximum seedling length of 7.53 cm, 15.14 cm, 14.35 cm and 14.88 cm in TP method, sand method, soil method and brick gravel methods, respectively while NCS 145 recorded the maximum seedling length (18.99 cm) in BP method. length (Table 3) revealed that the seedling length in top of the paper method, between paper method, sand method, soil method and brick gravel method has also shown similar trend like germination percentage. The cotton hybrid PRCH 31 recorded the maximum seedling length of 7.53 cm, 15.14 cm, 14.35 cm and 14.88 cm in TP method, sand method, soil method and brick gravel methods, respectively while NCS 145 recorded the maximum seedling length (18.99 cm) in BP method. Results and Discussion Irrespective of cotton hybrids, Bt cotton hybrids recorded significantly higher germination percentage than non-Bt counter parts by top of paper method (Table 1). Among the cotton hybrids, PRCH 31 recorded maximum germination (91%) followed by Rudra and TCH 9 (89% and 88%, respectively) which were significantly superior to the other cotton hybrids except Rudra and Sandeep recorded the minimum germination (82%). PRCH 31 recorded maximum germination in both Bt (93%) and non Bt (89%) categories among the six cotton hybrids in top of paper method. In between paper method (Table 2), PRCH 31 recorded the maximum germination (93% and 87%) *Corresponding author email: sandeepvannam81@gmail.com *Corresponding author email: sandeepvannam81@gmail.com 663 Table 1. Effect of different germination methods on germination (%) in six Bt and Non Bt cotton hybrids Treatment Top of paper method Between paper method Sand method Soil method BT NBT Mean BT NBT Mean BT NBT Mean BT NBT Mean NCS 145 85.25 82.75 84.00 81.75 81.75 81.75 90.50 82.75 86.60 76.75 77.50 77.12 NCS 207 83.50 86.50 85.00 89.50 77.35 83.62 76.50 76.50 76.50 81.50 76.50 74.08 TCH9 93.25 82.50 87.75 91.75 83.25 87.50 91.75 86.75 89.27 90.00 83.25 86.62 RUDRA 89.75 88.25 89.00 82.35 78.25 80.50 89.25 81.50 85.37 80.75 76.75 78.75 PRCH 31 93.25 89.25 91.25 93.25 86.50 90.00 94.50 91.75 93.12 92.75 88.08 90.37 SANDEEP 87.25 77.00 82.12 77.25 76.25 77.00 77.50 76.25 76.87 78.00 76.25 77.17 MEAN 88.70 84.33 86.52 86.08 80.71 83.40 86.67 82.58 84.62 83.29 79.71 81.50 V Bt x Non Bt I V Bt x Non Bt I V Bt x Non Bt I V Bt x Non Bt I SE ± 1.31 0.25 1.84 1.33 0.77 1.88 0.994 0.57 1.41 1.82 1.05 2.58 C.D @ 5% 2.65 1.53 3.74 2.71 1.56 3.80 2.02 1.16 2.85 3.69 2.13 5.22 V = Varieties,I = Interaction between Varieties x Bt vs. non Bt able 1. Effect of different germination methods on germination (%) in six Bt and Non Bt cotton hybrids followed by TCH 9 (92 % and 83%) for Bt and Non Bt types, respectively. By sand method, PRCH 31 (95% and 92%) recorded the maximum germination, followed by TCH 9 (92% and 87%) in both Bt and non- Bt types, respectively. These results indicated seed size nature of cotton. Similar results have been reported by various workers in medicinal plants (Bahaguna et al., 1987 and Parihar et al., 2006). Results and Discussion cotton hybrids, Bt hybrids recorded maximum germination than other non-Bt counter parts. It might be due to genetic makeup. Similar results were reported by Frietas et al., 2002 in cotton, Gowda and Shobha in 2003 in soybean. With respect to evaluation of seedling vigour on seedling length basis (Table 2), TCH 9 recorded the maximum seedling length (11.06 cm) during the first count. In the final count, PRCH 31 recorded the maximum seedling length (23.69 cm) irrespective of Bt and Non Bt cotton hybrids. Seedling vigour index on seedling length basis, revealed that, the cotton hybrid PRCH 31 recorded the maximum SV I in both Bt and Non Bt categories Comparison of effect of different methods of testing of germination percentage on seedling 664 Table 4. Results and Discussion These variations observed may be due to the fact of using different testing media and hybrids and their interaction effect between them. The highest seedling vigour index I and II was due to the seedling length and seedling dry weight, respectively and the germination potentiality of the cotton hybrids. Similar results were reported by Parvathamma et al., (1991) in sorghum and Rohini (2005) and Siva Jyothi (2008) in medicinal plants. Coming to vigour tests, the germination percentage of PRCH 31 recorded maximum in both Bt (80%) and Non Bt (79%) cotton hybrids by using first count method of germination (Table 6). In final count of germination method, PRCH 31 recorded the maximum germination (92% and 90%) Table 6. Effect of different vigour tests on germination(%) in six Bt and Non-Bt cotton hybrids. Table 6. Effect of different vigour tests on germination(%) in six Bt and Non-Bt cotton hybrids. Treatment First count Final count Speed of germination Field emergence BT NBT Mean BT NBT Mean BT NBT Mean BT NBT Mean NCS 145 78 77 78 87 84 85 76 13 74 80 75 78 NCS 207 79 77 78 84 82 83 75 72 73 74 68 71 TCH 9 79 77 78 92 87 89 78 75 77 89 75 82 RUDRA 79 76 78 88 83 85 71 68 70 73 69 71 PRCH 31 80 79 80 92 90 91 78 73 76 84 77 80 SANDEEP 77 76 77 83 79 81 78 69 74 79 74 76 MEAN 79 77 78 87 84 86 76 72 74 80 73 76 V Bt x Non Bt I V Bt x Non Bt I V Bt x Non Bt I V Bt x Non Bt I SE ± 0.95 0.55 1.34 1.4 0.81 1.99 1.64 0.95 2.32 1.64 0.94 2.32 CD@5% 1.93 1.11 2.7 2.86 1.65 4.04 3.34 1.92 4.72 3.33 1.92 4.72 V = Variety, I= Interaction between V x Bt x Non Bt aspect of seed quality that concern growers and a higher percentage of germination serves as a pre requisite for seeds to be sown. Similar reports were also reported by Pieto Filho and Ellis (1991) and Siva Jyothi (2008). followed by TCH 9 (92% and 87%) in both Bt and Non Bt categories, respectively. The germination rate was higher in case of PRCH 31 (76%) while lowest was recorded for Rudra (70%). Results and Discussion Seedling vigour index-I on seedling length basis for six Bt and Non Bt cotton hybrids Treatment Top of paper method Between paper method Sand method Soil method First count Final count BT NBT Mean BT NBT Mean BT NBT Mean BT NBT Mean BT NBT Mean BT NBT Mean NCS 145 623 558 590 1689 1495 1592 1205 829 1017 1092 866 979 875 763 819 2015 1867 1941 NCS 207 642 580 611 1693 1507 1599 983 938 961 1114 1048 1081 914 745 830 1951 1890 1920 TCH9 709 594 652 1649 1355 1502 1181 997 1089 1247 993 1120 950 786 868 1946 1726 1836 RUDRA 659 607 633 1455 714 1084 1203 1041 1122 1268 947 1108 820 693 757 1935 1692 1813 PRCH31 730 640 685 1794 1596 1695 1519 1255 1387 1338 1255 1297 897 834 866 2071 2055 2063 SANDEEP 655 524 589 1148 596 872 1010 924 966 1042 908 975 880 669 774 1908 1487 1697 MEAN 670 584 627 1571 1210 1391 1183 997 1090 1184 1003 1093 889 748 819 1971 1786 1878 V Bt x Non Bt I V Bt x Non Bt I V Bt x Non Bt I V Bt x Non Bt I V Bt x Non Bt I V Bt x Non Bt I S.E± 19.83 11.45 28.04 43.47 26.83 65.72 73.20 42.20 103.50 26.61 15.36 37.63 37.88 21.87 53.57 98.99 57.15 139.99 CD@5% 40.25 23.24 56.93 94.34 54.46 133.41 148.60 85.50 210.10 54.02 31.19 76.40 76.90 44.39 108.75 200.95 116.02 284.19 V = Variety, I= Interaction between V x Bt x Non Bt in TP method (730 and 640), BP method (1794 and 1596), sand method (1519 and 1255), soil method (1338 and 1255) and even in final count (2071 and 2055) as shown in Table 4. Bt cotton hybrids recorded highest SVI II compared to the Non Bt cotton hybrids (Table 5). Highest seedling vigour index I on dry weight basis was recorded in the cotton hybrid PRCH 31 of 62, Table 4. Seedling vigour index-I on seedling length basis for six Bt and Non Bt cotton hybrids Bt cotton hybrids recorded highest SVI II compared to the Non Bt cotton hybrids (Table 5). Highest seedling vigour index I on dry weight basis was recorded in the cotton hybrid PRCH 31 of 62, Table 5. Results and Discussion Seedling vigour index-I on dry weight basis in six Bt and noin Bt cotton hybrids Table 5. Seedling vigour index-I on dry weight basis in six Bt and noin Bt cotton hybrids Treatment Top of paper method Between paper method Sand method Soil method First count Final count BT NBT Mean BT NBT Mean BT NBT Mean BT NBT Mean BT NBT Mean BT NBT Mean NCS 145 43 40 42 55 52 54 50 46 48 53 49 51 48 46 47 58 54 56 NCS 207 53 43 48 60 58 59 56 53 54 58 54 56 54 51 52 61 58 59 TCH 9 62 59 61 67 61 54 65 60 62 66 60 63 53 59 61 78 62 66 RUDRA 60 44 47 61 59 60 56 51 53 57 53 55 49 46 48 63 60 61 PRCH 31 64 59 62 71 65 68 66 61 64 69 61 65 65 58 61 75 64 70 SANDEEP 46 41 43 54 51 52 50 47 48 53 51 52 47 44 45 56 52 54 MEAN 53 48 50 61 58 59 57 53 55 59 55 57 54 51 52 64 58 61 V Bt x Non Bt I V Bt x Non Bt I V Bt x Non Bt I V Bt x Non Bt I V Bt x Non Bt I V Bt x Non Bt I S.E ± 1.74 1.0 2.48 1.38 0.79 1.95 1.86 1.07 2.65 2.07 1.20 2.94 1.27 0.73 1.80 1.20 0.69 1.70 CD@5% 3.54 2.04 5.0 2.80 1.61 3.96 3.77 2.18 5.34 4.22 .2.43 5.96 .2.59 1.49 3.66 2.44 1.41 3.46 V = Variety, I= Interaction between V x Bt x Non Bt 68, 64, 65 and 70 in TP method, BP method, sand method, soil method and final count, respectively. These variations observed may be due to the fact of using different testing media and hybrids and their interaction effect between them. The highest seedling vigour index I and II was due to the seedling length and seedling dry weight, respectively and the germination potentiality of the cotton hybrids. Similar results were reported by Parvathamma et al., (1991) 68, 64, 65 and 70 in TP method, BP method, sand method, soil method and final count, respectively. Results and Discussion First count of germination and speed of germination also serve as effective indicators for vigour tests for obtaining maximum germination and similar results were also reported by Zode et al. (1994). The field emergence was found to be higher in TCH 9 (82%) followed by PRCH 31 (80%). Field emergence ability is the major followed by TCH 9 (92% and 87%) in both Bt and Non Bt categories, respectively. The germination rate was higher in case of PRCH 31 (76%) while lowest was recorded for Rudra (70%). First count of germination and speed of germination also serve as effective indicators for vigour tests for obtaining maximum germination and similar results were also reported by Zode et al. (1994). The field emergence was found to be higher in TCH 9 (82%) followed by PRCH 31 (80%). Field emergence ability is the major Among different stress tests evaluated, PRCH 31 recorded maximum germination i.e. 87% and 85% by using brick gravel method and cold test, respectively. Martin et al., (1988) reported that the 665 Table 7. Effect of different vigour tests on germination(%) in six Bt and Non-Bt cotton hybrids. Results and Discussion Treatment Brick Gravel Test Cold test Paper exhaustion test Tetrazolium test Electrical conductivity BT NBT Mean BT NBT Mean BT NBT Mean BT NBT Mean BT NBT Mean NCS 145 79.25 77.00 78.12 77.25 75.35 76.50 75.25 78.50 71.85 84.50 84.25 84.37 542.50 522.50 533.87 NCS 207 77.75 77.50 76.62 78.25 75.50 76.87 77.50 76.00 76.75 84.25 78.50 81.37 664.50 482.50 573.50 TCH 9 88.25 81.50 84.87 87.50 82.75 85.00 76.25 61.00 68.75 100.00 100.00 100.00 612.50 610.75 611.62 RUDRA 87.25 78.50 82.87 77.75 76.50 76.87 81.25 37.50 59.37 100.00 90.00 95.00 451.50 583.50 507.50 PRCH 31 90.50 82.75 86.62 87.75 83.00 85.25 85.50 57.50 71.50 100.00 100.00 100.00 442.00 585.75 513.87 SANDEEP 76.00 75.75 75.87 71.25 75.35 76.50 55.00 71.25 63.12 74.00 85.50 79.75 612.00 568.00 590.37 MEAN 83.17 78.83 81.00 80.87 78.12 79.50 74.45 62.66 68.56 90.46 89.71 90.08 554.17 559.42 556.79 V Bt x Non Bt I V Bt x Non Bt I V Bt x Non Bt I V Bt x Non Bt I V Bt x Non Bt I S.E ± 1.22 0.70 1.72 0.97 0.56 1.37 2.83 1.63 4.01 1.45 0.84 2.05 84.24 4.75 11.65 CD@5% 2.47 1.43 3.50 1.97 1.14 2.79 5.75 3.32 8.14 2.95 1.70 4.17 16.73 9.66 23.66 V = Variety, I= Interaction between V x Bt x Non Bt Table 7. Effect of different vigour tests on germination(%) in six Bt and Non-Bt cotton hybrids. of cotton hybrids, PRCH 31 and TCH 9 were 100 percent w.r.t both Bt and non Bt cotton hybrids. The electric conductivity of seed lechates was lower for PRCH 31 (442.00) in case of Bt hybrid and NCS 207 cold test was a superior predictor of field emergence in corn. In case of paper exhaustion test, the seed germination was higher in NCS 207 (77%). The tetrazolium test revealed that the viability of the seeds Fig. 1. Effect of accelerating ageing on seedling vigour index I of six Bt and Non-Bt cotton hybrids at 30 days. Fig. 1. Effect of accelerating ageing on seedling vigour index I of six Bt and Non-Bt cotton hybrids at 30 days. (482.50) in case of Non Bt cotton hybrids while it was higher for TCH 9 (611.62) in the non Bt hybrids and NCS 207 (664.50) for Bt hybrids. Lower the electrical conductance better will be the germination and quality of seeds. Conclusion Martin, B.A., Smith, O.S. and Neil, M.O. 1988. Relationships between laboratory germination tests and field emergence of corn inbreds. Crop Sci., 28: 801–805. Bt cotton hybrids especially PRCH 31 recorded higher germination in all the germination testing methods as well as vigour tests. Germination and seedling vigour gradually declined with an increase in period of accelerated ageing from 0 to 30 days. Such decline was more in Non Bt cotton hybrids when compared to Bt cotton hybrids. However, TCH 9 and PRCH 31 have shown reduced declinein germination and seedling vigour. These varieties were shown to posses resistance to seed deterioration against adverse factors and considered as resistant varieties under stress conditions. Parvathamma, S., Prakash, H.S. and Shetty, H.S. 1991. Evaluation of seed vigour in sorghum and sunflower. Adv. Pl. Sci., 4: 35-42. Pieto Filho, C. and Ellis, R.H. 1991. The development of seed quality in spring barley in four environments. I. germination and longevity. Seed Sci. Res., 1:163- 177. Parihar, P., Leena Parihar. and Achaleshwar Bohra. 2006. Antibacterial activity of Athyrium pectinatum (Wall.) Presl. Nat. Prod. Rad., 5: 262-265. Singh, Raj Kumar. 2011. Morphological, chemical and molecular characterization and seed vigour studies in coriander (Coriandrum sativum L.). Ph.D (Seed Science and Technology) Thesis, CCSHAU, Hisar. Results and Discussion Similar results were also reported by several researchers (Rohini, 2005). Khan et al. (2005) observed that turnip seed showed a gradual reduction in seedling vigour as accelerated ageing duration increased. other hybrids. The decline in germination (%) after accelerated ageing was less in PRCH 31 (88.60% in 0 days and 82.12% after 30 days). The accelerated ageing on SVI I basis revealed that TCH 9 recorded the highest SVI (1703 initially and 1318 after 30 days) among the Bt cotton hybrids. The Non Bt hybrids, NCS 145 and PRCH 31 recorded the maximum SVI initially (1605) while PRCH 31 maintained the same trend at 30 days of accelerated ageing (1290) (Fig 1). The increased duration of accelerated ageing decreased, the percent germination and after 15 days of ageing there has been sharp decline to the extent of 27 % in rice seeds (Gangwar and Kanaujia, 2006). With respect to accelerated ageing test, the germination percentage declined with the duration of accelerated ageing test. According to Raj Kumar (2011), standard germination, tetrazolium test and accelerated ageing could be the most suitable predictors for seedling establishment in coriander. Torres et al., (2004) concluded that accelerated ageing test is the best vigour test for estimating the field emergence in soybean. PRCH 31 followed by TCH 9 had shown higher germination at all periods of accelerated ageing and significantly superior to The data on effect of AAT on SVI II (Fig 2) revealed that the Bt cotton hybrids recorded high SVI II as compared to Non Bt cotton hybrids. The seedling vigour index II was found to be higher in TCH 9 (59 initially and 38 after 30 days) among the Bt hybrids. Within the Non Bt hybrids, TCH 9 (51) followed by 666 Fig. 2. Effect of days of accelerating on seedling vigour index (SVI -II) on dry weight (g) basis of six Bt and Non-Bt cotton hybrids at 30 days Fig. 2. Effect of days of accelerating on seedling vigour index (SVI -II) on dry weight (g) basis of six Bt and Non-Bt cotton hybrids at 30 days PRCH 31 (50) recorded the maximum SVI-II initially, and finally (37 and 35, respectively). PRCH 31 (50) recorded the maximum SVI-II initially, and finally (37 and 35, respectively). Khan, M.M., Iqbal, M.J. and Abbas, M. 2005. Loss of viability correlates with membrane damage in aged turnip (Brassica rapus). Seed Sci. and Tech., 33: 517-520. Received: June 28, 2012; Accepted: July 23, 2012 References Rohini. 2005. Standardization of seed testing procedures and storability studies in some medicinal plants. M.Sc. (Ag) Thesis, ANGRAU, Rajendranagar, Hyderabad. Axay Kumar, 2008. Standardization of seed testing procedures and seed vigour studies in Jatropa. Ph.D (Seed Science and Technology) Thesis, CCSHAU, Hisar. Siva Jyothi, T. 2008. Standardization of seed testing procedures in seven medicinal plants. M.Sc (Ag) Thesis, ANGRAU, Rajendranagar, Hyderabad. Bahuguna, V.K., Rawat, M.M.S., Joshi, S.R. and Maithani, G.P. 1987. Studies on the viability, germination and longevity of Terminalia myriocarpa seeds. J. Trop. Forst., 3: 318-323. Torres, R.M., Vieira, R.D. and Panobianco, M. 2004. Accelerated ageing and seedling field emergence in soybean. Sci. Agric., 61: 476-443. Freitas, R. A., Dias, D.C.F.S., Cecon, P. R., Reis, M. S. and Dias, L.A.S. 2002. Storability of cotton seeds predicted by vigour tests. Seed Sci. and Tech., 30: 403- 410. Uppar, D.S., Kurdikeri, M.B., Chetti, M. B., Hanchinal, R. R., Nalini, A.S. and Shashidhara, S.D. 2006. Temperature and substrata requirement for germination of Dicoccum wheat. Seed Res., 34: 209-211. Gangwar, C.B., Singh. and Kanaujia, V. P. 2006. Germination and seed vigour in accelerated aged seeds of rice (Oryza sativa. L). Seed Res., 34: 70-73. Zode, N.G., Zade, V.R., Dighe, R.S. and Changade, S. T. 1994. Relationship of some seed vigour tests with field emergence in cotton. PKV Res. J., 18: 131-132. Gowda, R. and Shobha, B.N. 2003. Influence of seed coloring on seed quality and storability in soybean (Glycine max (L.) Merrill). Seed Treatment Conf., 12 – 13 June. Bangalore, India. Received: June 28, 2012; Accepted: July 23, 2012
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Cost-effectiveness analysis of use of a polypill versus usual care or best practice for primary prevention in people at high risk of cardiovascular disease
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RESEARCH ARTICLE Objective Received: November 17, 2016 Accepted: July 21, 2017 Published: September 5, 2017 To determine whether a polypill is cost-effective compared to usual care and optimal guide- line-recommended treatment for primary prevention in people already on statins and/or blood pressure lowering therapy. Copyright: © 2017 Jowett 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. Methods A Markov model was developed to perform a cost-utility analysis with a one year time cycle and a 10 year time horizon to compare the polypill with usual care and optimal implementa- tion of NICE Guidelines, using patient level data from a retrospective cross-sectional study. The model was run for ten age (40 years+) and gender-specific sub-groups on treatment for raised CVD risk with no history of CVD. Published sources were used to estimate impact of different treatment strategies on risk of CVD events. Data Availability Statement: Data from the screening study will be archived and made available for future secondary analyses and data pooling purposes, subject to agreement about the use of the data, from the authors. There are legal restrictions, as the data custodian, the University of Birmingham, does not allow release of the data without an explicit data sharing agreement to be set up with the requesting institution. Applications from interested researchers to access the data can be sent to foi@contacts.bham.ac.uk. Editor: Rudolf Kirchmair, Medical University Innsbruck, AUSTRIA Editor: Rudolf Kirchmair, Medical University Innsbruck, AUSTRIA Editor: Rudolf Kirchmair, Medical University Innsbruck, AUSTRIA Received: November 17, 2016 Accepted: July 21, 2017 Published: September 5, 2017 Copyright: © 2017 Jowett et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. OPEN ACCESS OPEN ACCESS Citation: Jowett S, Barton P, Roalfe A, Fletcher K, Hobbs FDR, McManus RJ, et al. (2017) Cost- effectiveness analysis of use of a polypill versus usual care or best practice for primary prevention in people at high risk of cardiovascular disease. PLoS ONE 12(9): e0182625. https://doi.org/ 10.1371/journal.pone.0182625 * jm677@medschl.cam.ac.uk Sue Jowett1, Pelham Barton1, Andrea Roalfe2, Kate Fletcher2, F. D. Richard Hobbs3, Richard J. McManus3, Jonathan Mant4* Sue Jowett1, Pelham Barton1, Andrea Roalfe2, Kate Fletcher2, F. D. Richard Hobbs3, Richard J. McManus3, Jonathan Mant4* a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 1 Health Economics Unit, Institute of Applied Health Research, University of Birmingham, West Midlands, United Kingdom, 2 Primary Care Clinical Sciences, Institute of Applied Health Research, University of Birmingham, West Midlands, United Kingdom, 3 Nuffield Department of Primary Care Health Sciences, University of Oxford, Oxfordshire, United Kingdom, 4 Primary Care Unit, Department of Public Health & Primary Care, Strangeways Research Laboratory, University of Cambridge, Wort’s Causeway, Cambridge, Cambridgeshire, United Kingdom * jm677@medschl.cam.ac.uk * jm677@medschl.cam.ac.uk Background Clinical trials suggest that use of fixed-dose combination therapy (‘polypills’) can improve adherence to medication and control of risk factors of people at high risk of cardiovascular disease (CVD) compared to usual care, but cost-effectiveness is unknown. Conclusions For most people already on treatment to modify CVD risk, a polypill strategy may be cost- effective compared with optimising treatment as per guidelines or their current care, as long as the polypill cost is sufficiently low. Competing interests: The authors have declared that no competing interests exist. Economic evaluation of a polypill for CVD prevention 49 and men aged 75+. Results were sensitive to polypill cost, and if the annual cost was less than £150, this approach was cost-effective compared to the other strategies. 49 and men aged 75+. Results were sensitive to polypill cost, and if the annual cost was less than £150, this approach was cost-effective compared to the other strategies. Funding: This paper presents independent research funded by the National Institute for Health Research (NIHR) under its Programme Grants for Applied Research programme (grant number: RP- PG-0606-1153). The funders did not influence the study design or the writing of this article. The views expressed in this publication are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health. Methods A Markov cohort model developed in TreeAge Pro estimated cost-effectiveness of primary prevention with a polypill strategy compared with i) current therapy and ii) optimal therapy as per guidelines. The model considered patients aged 40 and over prescribed a statin and / or blood pressure lowering therapy with no history of cardiovascular disease. The model was run over a ten year time horizon with a one year cycle. All patients started healthy and moved to other health states if they suffered stroke, myocar- dial infarction (MI), angina, heart failure or peripheral vascular disease (PVD) or died. Once a cardiovascular event occurred, they either died, or remained in this health state and incurred costs and a reduction in quality of life as assigned to that disease state until death (Fig 1). Introduction Poor uptake of pharmacotherapy for people at high risk of cardiovascular disease, and lack of adherence in people who are prescribed drugs, has generated interest in the potential for fixed dose combination pills (‘polypills’).[1,2] These can bring about important reductions in blood pressure and LDL cholesterol,[3] and are associated with improved adherence to therapy. [4,5,6,7] However, despite evidence from trials demonstrating that polypills are largely safe and effective, [8] availability still remains limited compared with other disease areas [9]. Fur- thermore, no polypill for prevention of cardiovascular disease is currently licensed for use in the United Kingdom. Previous cost-effectiveness analyses of polypills have primarily been concerned with treat- ment of secondary prevention patients [10,11], or, in primary prevention, comparing their use to no treatment, rather than to usual care or improved implementation of guidelines.[12,13] The aim of this study was to estimate the cost-effectiveness of a polypill strategy compared with current treatment or treatment as per guidelines for primary prevention for patients with known high cardiovascular risk who are already prescribed statins and/or blood pressure low- ering therapy. Results A polypill strategy was potentially cost-effective compared to other strategies for most sub- groups ranging from dominance to up to £18,811 per QALY depending on patient sub- group. Optimal implementation of guidelines was most cost-effective for women aged 40– 1 / 15 PLOS ONE | https://doi.org/10.1371/journal.pone.0182625 September 5, 2017 Study population A cross sectional retrospective study of primary care medical records in 19 West Midland gen- eral practices in England provided data on risk factor profiles and current treatment.[14] Ten year cardiovascular risk was calculated using an updated Framingham equation.[15,16] The dataset was subdivided into ten age/gender subgroups (40–49, 50–59, 60–69, 70–74, 75 and over). Within each sub-group, eight treatment/cardiovascular risk strata were identified (S1 Table) that would be treated differently according to UK National Institute for Health and Care Excellence (NICE) guidelines.[17,18] 2 / 15 PLOS ONE | https://doi.org/10.1371/journal.pone.0182625 September 5, 2017 Economic evaluation of a polypill for CVD prevention Fig 1. Model health states. https://doi.org/10.1371/journal.pone.0182625.g001 Fig 1. Model health states. Fig 1. Model health states. https://doi.org/10.1371/journal.pone.0182625.g001 https://doi.org/10.1371/journal.pone.0182625.g001 PLOS ONE | https://doi.org/10.1371/journal.pone.0182625 September 5, 2017 Economic evaluation of a polypill for CVD prevention The guideline strategy assumed optimal treatment as per UK NICE guidelines.[17, 18] Statin therapy (simvastatin 40mg) was prescribed if cardiovascular risk was 20% or higher, and antihypertensives if blood pressure was greater than 140/90mm/Hg and cardiovascular risk was 20% or greater.[17] In those patients already on antihypertensives, it was assumed that additional drugs would be added in order to reach a target systolic blood pressure of 140mmHg, up to a maximum of three drugs. We estimated the additional number of antihy- pertensive drugs that would be required using the results of a meta-analysis.[21] For each sub- group we used the starting systolic blood pressure and the degree of blood pressure lowering required to determine through linear interpolation how many additional drugs would be needed. Impact of treatment The baseline calculated 10 year cardiovascular risk was assumed to reflect benefit of current treatment (S1 Table), since the values of blood pressure and cholesterol in these patients reflected their current use of blood pressure lowering and lipid lowering drugs. For optimal guideline care, the impact of additional treatments was based on results of meta-analysis of randomised controlled trials (Table 1).[21, 22] We assumed 85% of people prescribed statins were fully compliant in taking their medication.[23] For the polypill strategy, treatment already being received was taken into account. If already on statins, then no additional effect from statins was applied. If antihypertensives were already being taken, the baseline systolic blood pressure and average number of drugs taken was used to determine the amount of BP lowering already being achieved, and what effect switching to three half dose drugs would have.[21] If switching to the polypill resulted in a lower dose of antihypertensives than current practice, risk estimates were adjusted accordingly. Outcomes Outcomes were measured in cardiovascular events and quality-adjusted life years (QALYs). A baseline utility value was applied depending upon age and gender.[33] When a cardiovascular event occurred, the health state value for that event was applied as a multiplier (Table 1). For consistency with other model-based analyses, utility values for CVD health states were obtained from previous UK NICE guidelines, where values were obtained through systematic review [18,34]. No reduction in quality of life was assumed for any drugs.[40] Gender-specific life tables were used to determine the probability of death at different ages. [41] The risk of death was adjusted to ensure there was no double counting of cardiovascular death.[42] There was an increased risk of death once in a cardiovascular event health state. PLOS ONE | https://doi.org/10.1371/journal.pone.0182625 September 5, 2017 Treatment strategies Current treatment for each stratum was characterised by whether a statin was being taken, and if antihypertensives were being taken, the average number per strata. The polypill strategy consisted of a pill a day containing a statin (40mg simvastatin) and three antihypertensives at half-dose (12.5mg hydrochlorothiazide, 5mg lisinopril, 2.5mg amlo- dipine).[19] As the patients were already taking medication, it was assumed the majority would take the polypill, with 16% discontinuing it (and therefore no longer incurring the cost of the polypill) and returning to their original treatment.[20] The polypill strategy was applied regardless of baseline cardiovascular risk or systolic blood pressure. 3 / 15 PLOS ONE | https://doi.org/10.1371/journal.pone.0182625 September 5, 2017 Costs Costs assumed a UK NHS and personal social services perspective (Table 1). Polypill costs comprised: £171 (€192) a year for the pill, an initial GP visit and blood test in the first month, and an annual practice nurse visit and blood test thereafter. Due to the absence of a UK cost for a polypill, the cost was assumed to be in line that of an existing secondary prevention poly- pill (Trinomia1), which has a different composition. The cost was calculated using the mean of available prices and converted from US$ to UK£. In the current treatment and guideline strategies, the most commonly prescribed generic antihypertensive in each class (indapamide, amlodopine, ramipril) and the statin simvastatin were assumed.[35] Patients on antihyperten- sives were allocated four consultations (mix of GP and practice nurse) per year.[43] Two addi- tional visits (one GP, one practice nurse) were included for guideline treatment in patients above target blood pressure. 4 / 15 PLOS ONE | https://doi.org/10.1371/journal.pone.0182625 September 5, 2017 Economic evaluation of a polypill for CVD prevention Table 1. Summary of model inputs. Data Sources Baseline mortality and risk of cardiovascular disease Probability of stroke (10 years) 0.7–6.2%(age and sex dependent) Calculated with Framingham [15,16] and risk factor profile based on patient level data Probability of MI (10 years) 1.1–9.4% (age and sex dependent) Probability of angina (10 years) 1.5–13.3%(age and sex dependent) Probability of heart failure (10 years) 0.4–3.9%(age and sex dependent) Probability of PVD (10 years) 0.7–6.2% %(age and sex dependent) Assumed distribution of possible CV events within 10 year CV risk Stroke 16% D’Agostino (2008) [16] Wood (2004) [24] Myocardial infarction 24% Angina 34% Heart failure 10% PVD 16% Risk reduction with statins Stroke 0.80 (95% CI 0.73–0.86) CTT (2005),[22] HPS (2002)[23] MI, HF, angina 0.72 (95% CI 0.69–0.76) CTT (2005), HPS (2002) PVD 0.85 (95% CI 0.75–0.95) HPS (2002) Probability of death from event Fatal stroke 0.19 Ward (2007)[25] Fatal MI 0.19–0.36 (Men) Ward (2007) 0.23–0.40 (Women) Fatal heart failure 0.17 (r = 68, n = 396) Mehta (2009) [26] SMR after stroke 2.72 (95% CI 2.59–2.85) Bronnum-Hansen (2001) [27] SMR after MI 2.68 (95% CI 2.48–2.91) Bronnum-Hansen (2001) [28] SMR after Heart Failure 2.17 (95% CI 1.96–2.41) de Guili (2005) [29] SMR after Angina 2.19 (95% CI 2.05–2.33) NCGC [30] SMR after PVD 2.44 (95% CI 1.59–3.74) Leng (1996) [31] Reduction in blood pressure Number of AHT drugs required to achieve target BP 0.60–1.52 Law (2009)[21] Reduction in CV risk with reduction in BP Polypill CHD risk 10–52% Law (2009) Stroke risk 14–65% Law (2009) PVD risk 13–23% Murabito (1997)[32] (Dependent on age, sex and risk group) Treat to target CHD risk 15–37% Law (2009) Stroke risk 20–47% Law (2009) PVD risk 13–32% Murabito (1997) (Dependent on age, sex and risk group) Polypill adherence 84% TIPS (2009)[20] (Continued) Table 1. Summary of model inputs. PLOS ONE | https://doi.org/10.1371/journal.pone.0182625 September 5, 2017 5 / 15 Economic evaluation of a polypill for CVD prevention Table 1. (Continued) Data Sources Utilities No cardiovascular event (age and sex dependent) General population utilities from EQ-5D (UK Tariff) (NCSR, 2006)[33] Death 0 By definition Quality of life multipliers Acute MI 0.76 (0.018) Cooper (2008)[18], NICE (2014) [34] Post MI 0.88 (0.018) As above Acute angina 0.77 (0.038) As above Post-acute angina 0.88 (0.018) As above Heart failure 0.68 (0.020) As above Stroke 0.63 (0.040) As above PVD 0.90 (0.020) As above Costs £ per year Simvastatin 40mg 15.26 BNF March 2013 [35] Amlodopine 5mg 12.13 BNF March 2013 Indapamide 2.5mg 11.87 BNF March 2013 Ramipril 5mg 18.13 BNF March 2013 Polypill 171 Assumed same price as Trinomia Unit cost £ Blood test 15 Ward (2007) GP visit 33 Curtis (2012) [36] Practice nurse visit 11.25 Curtis (2012) Acute events: One-off cost £ Stroke 11,020 Youman (2003) [37] MI 5,487 Palmer (2002) [38] Angina 3,292 Assumed 60% of MI cost PVD 1,971 NHS Reference costs 2011/12 [39] Heart failure 2,699 NHS Reference costs 2011/12 Long-term costs £ per year Stroke 2721 Youman (2003) MI 572 Cooper (2008) [18] Angina 572 Cooper (2008) PVD 302 Cooper (2008) Heart failure 572 Cooper (2008) SMR: Standardised Mortality Ratio; MI: Myocardial infarction; PVD: Peripheral Vascular Disease; CV: Cardiovascular PLOS ONE | https://doi.org/10.1371/journal.pone.0182625 September 5, 2017 Results In the base-case analysis, the polypill strategy led to fewer CV events and was cost-effective over current practice and optimal treatment as per guidelines for men aged 50–74 and women over the age of 50. Subpopulation results varied from the polypill strategy being dominant (i.e. less costly and more effective), to Incremental Cost Effectiveness Ratios (ICERs) up to £18,811 (€21,162) per QALY gained (Tables 2 and 3). Optimal guideline care was dominant over the polypill for men aged over the age of 75 (but with very small differences in costs and QALYs), and most cost-effective in women aged 40–49. The probabilistic sensitivity analysis for all three treatment options showed that the polypill had a high probability (over 90%) of being cost-effective at a £20,000 (€22,500)/QALY thresh- old for men except for the youngest and oldest sub-groups. There was much more uncertainty in the results for women, with only the 60–69 and 70–74 sub-groups having a high probability of the polypill being the most cost-effective option. with considerable uncertainty around the results for those aged 50–59 (54% probability of being the most cost-effective option at £20,000/QALY). (Tables 2 and 3). Deterministic sensitivity analyses for men aged 60–69 demonstrated that the superior cost effectiveness of a polypill over optimal guideline care over was robust to some underlying assumptions made in the model, with some key exceptions. Optimal guidelines became the most favourable strategy if take up of a polypill was low, if polypill was associated with a small reduction in quality of life, if polypill was less effective than assumed, and if the population was restricted to those with uncontrolled risk factors only (Table 4). The results were particularly sensitive to the cost of the polypill, with dominance achieved by halving the price or further reducing to the cost of the individual components. The superiority of the polypill compared with current practice in men aged 60–69 was sensitive to the cost of polypill, but robust to changes to the other assumptions (Table 5). Threshold analysis showed that the annual price of the polypill would need to be £152 (€171) or less to ensure cost-effectiveness at the £20,000 (€22,500)/QALY threshold for all sub-groups when compared with guidelines (Table 6). Analysis An incremental cost-utility analysis was undertaken with a threshold of £20,000 per QALY taken to indicate cost-effectiveness. Future costs and QALYs were discounted at 3.5% per annum.[44] Costs were in UK pounds for 2011/12. Conversion into Euros was via the Pur- chasing Power Parity (PPP) Index for 2012, using a conversion rate of £1 to €1.125.[45] A half- cycle correction was applied to costs and effectiveness. We explored the impact of changing key parameters in a deterministic sensitivity analysis in a single age-sex stratum (men aged 60–69). Analysis of impact of price involved halving and doubling the price of a ‘polypill’ and reducing the cost to £57 (€64) a year, to reflect cost of individual generic agents.[35] The threshold price at which a polypill would become cost effective for each sub-group was PLOS ONE | https://doi.org/10.1371/journal.pone.0182625 September 5, 2017 6 / 15 Economic evaluation of a polypill for CVD prevention determined. Where available, data were entered into the model as distributions so that a probabilistic sensitivity analysis could be undertaken. A log-normal distribution was used for all risk reductions and standardised mortality ratios after cardiovascular events, a beta distri- bution for cardiovascular event probabilities, risk of death from cardiovascular events and compliance with screening and a gamma distribution for acute and long-term costs. A Proba- bilistic Sensitivity Analysis (PSA) was run with 10,000 simulations and cost-effectiveness acceptability curves were produced (not shown) to provide information on the probability of interventions being cost-effective at different cost per QALY thresholds. PLOS ONE | https://doi.org/10.1371/journal.pone.0182625 September 5, 2017 Discussion Our base-case analysis suggests that using a polypill is more cost effective than usual practice for all age groups over 40 years apart from 40–49 year old women. The polypill is also cost- effective compared with optimal implementation of guidelines for most age and sex strata. Probabilistic sensitivity analysis showed that the superiority of polypill over usual care was very unlikely to be a chance finding, but there was more uncertainty over the comparison with optimal implementation of guidelines. This pattern was reflected in the deterministic sensitiv- ity analysis which found that the superiority of polypill over usual care was robust to changing our assumptions, but not in comparison to optimal implementation of guidelines. Switching to a polypill strategy may be a more cost effective way of improving cardiovascular prevention in people on treatment for raised cardiovascular risk than current practice or better PLOS ONE | https://doi.org/10.1371/journal.pone.0182625 September 5, 2017 7 / 15 PLOS ONE | https://doi.org/10.1371/journal.pone.0182625 September 5, 2017 Economic evaluation of a polypill for CVD prevention Table 2. Results of the base-case analysis and probabilistic sensitivity analysis: Men. Age group Strategy Mean cost (£) Mean QALYs Mean CV events Incremental cost Incremental QALYs ICER (£ per QALY gained) Probability of cost- effectiveness at £20,000/ QALY Polypill vs current practice ICER(£ per QALY gained) 40–49 Current practice 1,625 7.202 0.0956 0 0 - 0% Optimal guideline care 1,634 7.216 0.0822 8 0.014 604 41% Polypill 1,878 7.229 0.0683 244 0.014 18,057 59% 9,166 50–59 Current practice 2,008 6.740 0.1499 0 0 - 0% Optimal guideline care 2,013 6.765 0.1290 5 0.025 182 0% Polypill 2,136 6.784 0.1119 123 0.019 6,466 100% 2,897 60–69 Optimal guideline care 2,315 6.524 0.1714 0 0 - 0% Current practice 2,343 6.477 0.2064 28 -0.047 Dominated 0% Polypill 2,386 6.539 0.1592 71 0.015 4,791 100% 698 70–74 Optimal guideline care 2,429 5.916 0.1890 0 0 - 9% Current practice 2,457 5.853 0.2334 28 -0.063 Dominated 0% Polypill 2,459 5.922 0.1861 31 0.006 5,068 91% 33 Optimal guideline care 2,320 4.782 0.1988 0 0 - 69% Polypill 2,327 4.781 0.2005 7 -0.001 Dominated 31% Dominant 75+ Current practice 2,395 4.692 0.2564 68 -0.089 Dominated 0% https://doi.org/10.1371/journal.pone.0182625.t002 Table 2. Results of the base-case analysis and probabilistic sensitivity analysis: Men. Table 2. Results of the base-case analysis and probab https://doi.org/10.1371/journal.pone.0182625.t002 implementation of guidelines in most patient sub-groups. However, this result was highly sensi- tive to cost of a polypill, take up of this treatment and potential effectiveness in reducing CV risk. At current individual drug prices, if a polypill cost £150 (€169) per year (i.e. a cost of 41p (€0.46) per pill), a polypill would be more cost effective than achieving optimal guideline care for all peo- ple over the age of 40 who are on treatment. Given that the costs of prescribing the individual components of the polypill are only around £57 (€64) per annum, this seems a feasible price not withstanding any technical difficulties of combining components in a single pill. implementation of guidelines in most patient sub-groups. However, this result was highly sensi- tive to cost of a polypill, take up of this treatment and potential effectiveness in reducing CV risk. At current individual drug prices, if a polypill cost £150 (€169) per year (i.e. a cost of 41p (€0.46) per pill), a polypill would be more cost effective than achieving optimal guideline care for all peo- ple over the age of 40 who are on treatment. Given that the costs of prescribing the individual components of the polypill are only around £57 (€64) per annum, this seems a feasible price not withstanding any technical difficulties of combining components in a single pill. Previous cost effectiveness analyses have focussed on cost effectiveness of a polypill against no treatment, and found that this it is likely to be cost effective for primary prevention of high risk individuals in the developing world.[12, 13, 46] Trials of using a polypill compared to usual care in people at high risk of cardiovascular dis- ease have found better self-reported use of medication in the polypill arm,[5,6,7] and in one trial, this was also associated with better control of risk factors.[5] None of these trials included any intervention to enhance usual care. The results need to be interpreted in the light of certain limitations. In a number of respects, the cost effectiveness of a polypill may have been under-estimated. The analysis was restricted PLOS ONE | https://doi.org/10.1371/journal.pone.0182625 September 5, 2017 8 / 15 Economic evaluation of a polypill for CVD prevention Table 3. Results of the base-case analysis and probabilistic sensitivity analysis: Women. Age group Strategy Mean cost (£) Mean QALYs Mean CV events Incremental cost Incremental QALYs ICER (£ per QALY gained) Probability of cost- effectiveness at £20,000/ QALY Polypill vs current practice ICER (£ per QALY gained) 40–49 Current practice 1,325 7.077 0.0505 0 0 - 0% Optimal guideline care 1,343 7.083 0.0446 18 0.006 2,994 94% Polypill 1,671 7.093 0.0354 328 0.010 33,585 6% 21,798 50–59 Current practice 1,586 6.675 0.0894 0 0 - 0% Optimal guideline care 1,599 6.688 0.0770 13 0.013 950 46% Polypill 1,841 6.701 0.0644 243 0.013 18,811 54% 9,696 60–69 Current practice 1,805 6.513 0.1203 0 0 - 0% Optimal guideline care 1,829 6.530 0.1060 23 0.018 1,304 2% Polypill 1,994 6.546 0.0928 165 0.015 10,730 98% 5,667 70–74 Current practice 1,985 5.982 0.1492 0 0 - 0% Optimal guideline care 2,042 6.009 0.1281 57 0.027 2,105 0% Polypill 2,097 6.022 0.1170 55 0.013 4,245 100% 2,797 75+ Current practice 1,880 4.733 0.1644 0 0 - 0% Optimal guideline care 1,947 4.774 0.1345 66 0.041 1,606 63% Polypill 1,967 4.779 0.1303 20 0.005 4,131 37% 1,870 https://doi.org/10.1371/journal.pone.0182625.t003 Table 3. Results of the base-case analysis and probabilistic sensitivity analysis: Women. Table 3. Results of the base-case analysis and prob to higher risk people already on treatment–inclusion of people not on medication would have increased the cost-effectiveness of polypill relative to current practice. Potential benefits of improved adherence to a polypill were not included.[5] It was assumed that 100% achievement of guideline targets is possible and indeed desirable.[47] However, this has probably not had a significant impact on overall results, since blood pressure target trials tend to show that mean blood pressure for the study population is below target, even if a substantial proportion of indi- viduals have final blood pressure above target.[43,48,49] Thus, the impact of blood pressure lowering will have been over-estimated in some patients and under-estimated in others in the optimal implementation of guidelines strategy. The base-case analysis considered a 10-year time horizon as opposed to a life time horizon (which our sensitivity analysis showed tends to favour the polypill). This limited time horizon was chosen because of the complexities of esti- mating changes in risk factors (and therefore cardiovascular risk) over time. PLOS ONE | https://doi.org/10.1371/journal.pone.0182625 September 5, 2017 Economic evaluation of a polypill for CVD prevention Table 4. Deterministic sensitivity analysis results (men aged 60–69) for polypill strategy vs optimal guideline care. Cost difference vs. guidelines(£) QALY difference vs. guidelines Most CE strategy* and ICER (£/QALY) for polypill Base case 71 0.015 Polypill (£4,791) Sensitivity analysis Cost of polypill doubled 342 0.015 Guidelines (£76,849) Cost of polypill halved -462 0.015 Polypill dominates Cost of polypill reduced to £57/year -640 0.015 Polypill dominates Decreased take up of polypill (25% take polypill) 95 -0.029 Guidelines dominates Change cost of CV events increase by 30% 45 0.015 Polypill (£3,030) decrease by 30% 97 0.015 Polypill (£6,553) Quality of life reduction with polypill by 1% 71 -0.037 Guidelines dominates Reduction in polypill effectiveness Antihypertensive effect reduced (statin effect fixed): 50% 180 -0.004 Guidelines dominates 25% 126 0.006 Guidelines (£22,500) Statins effect reduced (antihypertensive effect fixed) by 25% 95 0.010 Polypill (£9,397) Antihypertensive and statin effect reduced by 25% 151 0.001 Guidelines (£228,788) Increase costs of achieving optimal guideline care‡ -582 0.015 Polypill dominates Study population restricted to people with uncontrolled risk factors at baseline† -51 - 0.013 Guidelines (£3,952)** Baseline CVD risk reduced by 30% 97 0.011 Polypill (£9,110) Alternative time horizon 20 years 49 0.048 Polypill (£1,011) 30 years 42 0.078 Polypill (£546) Lifetime 40 0.084 Polypill (£473) * CE at a £20,000/QALY gained threshold ** ICER is in the south-west quadrant and polypill is not CE as it is <£20,000/QALY † i.e. 20% ten year cardiovascular risk and not on a statin, and/or with systolic blood pressure > 140 mmHg ‡ 4 additional (2 GP and 2 practice nurse) consultations per year over usual care, rather than 2 (1 of each). https://doi org/10 1371/journal pone 0182625 t004 c sensitivity analysis results (men aged 60–69) for polypill strategy vs optimal guideline care. Table 4. Deterministic sensitivity analysis results (men aged 60–69) for polypill strategy vs optimal guidelin reported smaller combined effects.[3] The polypill was assumed to have no adverse effects on quality of life–sensitivity analysis showed that a small shift in this assumption would favour current practice. However, there is no empirical evidence of differences in quality of life between people on the polypill or usual care.[5] Optimal guideline care was based on guide- lines in force in the UK up until 2014. Recent NICE guidelines have lowered the 10 year risk threshold for statin treatment from 20% to 10%.[34] This would result in a higher proportion of the study population being treated with statins in the optimal guideline implementation. This would have little effect on older age groups (see Table 1), but would result in increased effectiveness (and cost) of optimal guideline care in younger age groups. Finally, there are sev- eral other potential formulations of a polypill, which might have different effects on cardiovas- cular risk factors.[3] Finally, the risk of further events once someone had an initial cardiovascular event was not modelled, so potential benefits of treatments of secondary prevention were ignored. to higher risk people already on treatment–inclusion of people not on medication would have increased the cost-effectiveness of polypill relative to current practice. Potential benefits of improved adherence to a polypill were not included.[5] It was assumed that 100% achievement of guideline targets is possible and indeed desirable.[47] However, this has probably not had a significant impact on overall results, since blood pressure target trials tend to show that mean blood pressure for the study population is below target, even if a substantial proportion of indi- viduals have final blood pressure above target.[43,48,49] Thus, the impact of blood pressure lowering will have been over-estimated in some patients and under-estimated in others in the optimal implementation of guidelines strategy. The base-case analysis considered a 10-year time horizon as opposed to a life time horizon (which our sensitivity analysis showed tends to favour the polypill). This limited time horizon was chosen because of the complexities of esti- mating changes in risk factors (and therefore cardiovascular risk) over time. Finally, the risk of further events once someone had an initial cardiovascular event was not modelled, so potential benefits of treatments of secondary prevention were ignored. Conversely, other assumptions favoured polypill. The separate drugs in the polypill were assumed to have additive effects. While one trial did find additive effects,[50] others have PLOS ONE | https://doi.org/10.1371/journal.pone.0182625 September 5, 2017 9 / 15 Conclusions This analysis suggests that a polypill strategy may be a cost effective means to improve primary prevention in most people aged 50 and over with high cardiovascular risk on treatment, as 10 / 15 PLOS ONE | https://doi.org/10.1371/journal.pone.0182625 September 5, 2017 Economic evaluation of a polypill for CVD prevention Table 5. Deterministic sensitivity analysis results (men aged 60–69) for polypill strategy vs current practice. Cost difference vs. current practice QALY difference vs. current practice Most CE strategy* and ICER for polypill Base case 43 0.062 Polypill (£698) Sensitivity analysis Cost of polypill doubled 1,100 0.062 Polypill (£18,045) Cost of polypill halved -490 0.062 Polypill dominates Cost of polypill reduced £57/year -668 0.062 Polypill dominates Decreased take up of polypill (25% take polypill) 67 0.018 Polypill (£3,702) Change cost of CV events. CV events increase by 30% -67 0.062 Polypill dominates CV events decrease by 30% 153 0.062 Polypill (£2.490) Quality of life reduction with polypill by 1% 43 0.001 Polypill (£4,475) Reduction in polypill effectiveness Antihypertensive effect reduced (statin effect fixed) 50% 152 0.043 Polypill (£3,517) 25% 98 0.052 Polypill (£1,865) Statins effect reduced (antihypertensive effect fixed) by 25% 66 0.057 Polypill (£1,169) Antihypertensive and statin effect reduced by 25% 122 0.047 Polypill (£2,582) Study population restricted to people with uncontrolled risk factors at baseline† -102 0.081 Polypill dominates Baseline CVD risk reduced by 30% 143 0.045 Polypill (£3,206) Alternative time horizon 20 years -5 0.190 Polypill dominates 30 years 12 0.293 Polypill (£40) Lifetime 16 0.315 Polypill (£50) * CE at a £20,000/QALY gained threshold † i.e. >20% ten year cardiovascular risk and not on a statin, and/or with systolic blood pressure > 140 mmHg https://doi.org/10.1371/journal.pone.0182625.t005 Table 6. Optimal price of polypill. Subgroup Annual cost of polypill where the polypill is CE vs optimal guideline care (£) Annual cost of polypill where the polypill is CE vs current practice (£) Male 40–49 175 215 50–59 210 285 60–69 207 361 70–74 187 408 75+ 165 542 Female 40–49 152 167 50–59 173 211 60–69 193 244 70–74 204 282 75+ 185 324 (CE = <£20,000/QALY gained),. Base case price £365.25 https://doi.org/10.1371/journal.pone.0182625.t006 Table 5. Deterministic sensitivity analysis results (men aged 60–69) for polypill strategy vs current practice. Cost difference vs. current practice QALY difference vs. current practice Most CE strategy* and ICER for polypill Base case 43 0.062 Polypill (£698) Sensitivity analysis Cost of polypill doubled 1,100 0.062 Polypill (£18,045) Cost of polypill halved -490 0.062 Polypill dominates Cost of polypill reduced £57/year -668 0.062 Polypill dominates Decreased take up of polypill (25% take polypill) 67 0.018 Polypill (£3,702) Change cost of CV events. Acknowledgments JM, RMcM & FDRH had the original idea and gained the funding. AR conducted the primary data analysis. SJ carried out the economic analysis, with support from PB. KF was responsible for the data collection. SJ and JM wrote the first draft of the paper. All authors subsequently refined the manuscript and approved the final version. JM is the study guarantor. Supporting information S1 Table. Baseline patient sub-group characteristics by age, sex and guideline category. (DOCX) S1 File. Technical appendix: Extended modelling methods. (DOCX) Author Contributions Conceptualization: Pelham Barton, F. D. Richard Hobbs, Richard J. McManus, Jonathan Mant. Conceptualization: Pelham Barton, F. D. Richard Hobbs, Richard J. McManus, Jonathan Mant. Data curation: Sue Jowett, Andrea Roalfe, Kate Fletcher, Jonathan Mant. Formal analysis: Sue Jowett, Andrea Roalfe. Formal analysis: Sue Jowett, Andrea Roalfe. Funding acquisition: Pelham Barton, F. D. Richard Hobbs, Richard J. McManus, Jonathan Mant. Investigation: Richard J. McManus, Jonathan Mant. Investigation: Richard J. McManus, Jonathan Mant. Investigation: Richard J. McManus, Jonathan Mant. Methodology: Sue Jowett, Pelham Barton, Andrea Roalfe, Richard J. McManus, Jonathan Mant. Project administration: Kate Fletcher, Jonathan Mant. Supervision: Pelham Barton, Richard J. McManus, Jonathan Mant. Validation: Sue Jowett. Validation: Sue Jowett. Visualization: Sue Jowett. Visualization: Sue Jowett. Writing – original draft: Sue Jowett, Jonathan Mant. Writing – original draft: Sue Jowett, Jonathan Mant. Writing – review & editing: Sue Jowett, Pelham Barton, Andrea Roalfe, Kate Fletcher, F. D. Richard Hobbs, Richard J. McManus, Jonathan Mant. However, despite the growing evidence base of the effectiveness of polypills,[3,5] such combinations are not yet generally available. This perhaps in part reflects reluctance of pharmaceutical companies to invest in multi-component pills and the hurdles posed by regulatory approval.[51] At the right price, a polypill strategy could be the most cost effective way of ensuring optimal cardiovascu- lar risk reduction in people who are on treatment with antihypertensives or lipid lowering agents to lower their cardiovascular risk. CV events increase by 30% -67 0.062 Polypill dominates CV events decrease by 30% 153 0.062 Polypill (£2.490) Quality of life reduction with polypill by 1% 43 0.001 Polypill (£4,475) Reduction in polypill effectiveness Antihypertensive effect reduced (statin effect fixed) 50% 152 0.043 Polypill (£3,517) 25% 98 0.052 Polypill (£1,865) Statins effect reduced (antihypertensive effect fixed) by 25% 66 0.057 Polypill (£1,169) Antihypertensive and statin effect reduced by 25% 122 0.047 Polypill (£2,582) Study population restricted to people with uncontrolled risk factors at baseline† -102 0.081 Polypill dominates Baseline CVD risk reduced by 30% 143 0.045 Polypill (£3,206) Alternative time horizon 20 years -5 0.190 Polypill dominates 30 years 12 0.293 Polypill (£40) Lifetime 16 0.315 Polypill (£50) * CE at a £20,000/QALY gained threshold nsitivity analysis results (men aged 60–69) for polypill strategy vs current practice. Table 5. Deterministic sensitivity analysis results (men aged 60–69) for polypill strategy vs current practice Table 6. Optimal price of polypill. Subgroup Annual cost of polypill where the polypill is CE vs optimal guideline care (£) Annual cost of polypill where the polypill is CE vs current practice (£) Male 40–49 175 215 50–59 210 285 60–69 207 361 70–74 187 408 75+ 165 542 Female 40–49 152 167 50–59 173 211 60–69 193 244 70–74 204 282 75+ 185 324 (CE = <£20,000/QALY gained),. Base case price £365.25 https://doi.org/10.1371/journal.pone.0182625.t006 Table 6. Optimal price of polypill. PLOS ONE | https://doi.org/10.1371/journal.pone.0182625 September 5, 2017 PLOS ONE | https://doi.org/10.1371/journal.pone.0182625 September 5, 2017 11 / 15 Economic evaluation of a polypill for CVD prevention long as the cost of a polypill is sufficiently low. If the cost of a polypill is lower than £150 (€169) per year, then this approach becomes cost effective for all sub-groups. However, despite the growing evidence base of the effectiveness of polypills,[3,5] such combinations are not yet generally available. This perhaps in part reflects reluctance of pharmaceutical companies to invest in multi-component pills and the hurdles posed by regulatory approval.[51] At the right price, a polypill strategy could be the most cost effective way of ensuring optimal cardiovascu- lar risk reduction in people who are on treatment with antihypertensives or lipid lowering agents to lower their cardiovascular risk. long as the cost of a polypill is sufficiently low. If the cost of a polypill is lower than £150 (€169) per year, then this approach becomes cost effective for all sub-groups. References 1. Sheppard JP, Fletcher K, McManus RJ, Mant J. Missed opportunities in prevention of cardiovascular disease in Primary Care: cross sectional study. 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https://europepmc.org/articles/pmc3596534?pdf=render
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The remedial effect of soluble interleukin-1 receptor type II on endometriosis in the nude mouse model
Journal of Biomedical Research/Journal of biomedical research
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Abstract Objective: Recent studies have shown that the local expression of soluble interleukin (IL) -1 receptor type Ⅱ (sIL-1 RⅡ) in endometrial tissue of women with endometriosis is decreased, and the depression of IL-1 RⅡ was more significant in infertile women than that in fertile women with endometriosis. In this research, we investigated the remedial effect of sIL-1-RⅡ administration on endometriosis in the nude mouse model. Methods: Nineteen nude model mice with endometriosis were randomly divided into three groups: group A was treated by intraperitoneal administration with only sIL-1 RⅡ for two weeks, group B was similarly treated with only IL- 1, and group C (control) was administered saline . After 2 weeks, the size of the ectopic endometrial lesions was calculated, and the expression of vascular endothelial growth factor (VEGF) and B-cell lymphoma leukemia-2 (Bcl- 2) were detected by immunohistochemistry. The IL-8 and VEGF levels in the peritoneal fluid (PF) and serum were also measured by enzyme-linked immunosorbent assay (ELISA). Results: The mean size of ectopic endometrial lesion did not differ between the three groups (P > 0.05). Compared with the control, the expression of VEGF and Bcl-2 was significantly lower in group A, and higher in group B. In the three groups, the levels of IL-8 in the PF and serum were highest in group A, and lowest in group B. Conclusion: sIL-1 RⅡ may suppresse hyperplasia of ectopic endometriosis, perhaps by reducing the expression of certain cytokines, such as VEGF, IL-8, and Bcl-2, which could provide a new clinical strategy for the treatment of endometriosis. Key words: Interleukin-1, soluble interleukin-1 receptor type Ⅱ, endometriosis, nude mouse model ☆This study was supported by funding from Innovative Research Team in Nanjing Medical University (IRT0631) and the collaborating Grants (30611120524). 荭These authors contributed equally to this work. E-mail address: jyliu_nj@126.com 荭 *Corresponding author: JBR www.jbr-pub.org www.jbr-pub.org The remedial effect of soluble interleukin-1 receptor type II on endometriosis in the nude mouse model ☆ Liying Gao a,b荭, Liang Sun a荭, Yugui Cui a, Zhen Hou a, Li Gao a, Jing Zhou a, Yundong Mao a, Suping Han a, Jiayin Liu a* Received 20 August 2009 Acquisition of human tissues Eutopic endometrial tissues at the proliferative phase of the menstrual cycle were obtained from 5 women (aged from 23 to 35 years) undergoing laparoscopy for endometriosis. Endometriosis in these 5 women was staged as Ⅱ or Ⅲ during the laparoscopy, according to the revised American Fertility Society classification system [20]. All participants had regular menstrual cycles, had not received hormonal treatment, and had not been pregnant, breast-feeding, or using an intrauterine device for the past 3 months. Endometrial biopsies were placed immediately into a culture medium and transferred to the laboratory. Written informed consent was obtained from each patient using consent forms and protocols approved by the Ethics Committee of the First Affiliated Hospital of the Nanjing Medical University. The IL-1 family consists of two distinct molecular forms, IL-1α and IL-1ß, which are encoded by different genes, but have comparable biological activities [9]. Cell activations in response to IL-1 are mediated via the IL-1 receptor type Ⅰ (IL-1 RⅠ), the functional signaling receptor [10]. However, IL-1 receptor type Ⅱ (IL-1 RⅡ) is not a signaling molecule and is reported to be a decoy target of IL-1. Moreover, IL-1 RⅡ could be shed from the cell surface as a soluble molecule (sIL-1 RⅡ) that captures IL-1 and inhibits IL-1 from binding to IL-1 RⅠ, thereby neutralize the bioactivity of IL-1 [11,12,13]. Recent studies have indicated that local expression of sIL-1 RⅡ in endometrial tissue decreased in women suffering from endometriosis, and defective IL-1 RⅡ was more significant in infertile women than in fertile women with endometriosis, which suggests its involvement in endometriosis associated infertility [14,15]. INTRODUCTION result of the dystopic location of endometrial cells in tissues other than the uterine cavity [1]. The origin of endometriotic cells remains elusive and the most widely accepted theory on the aetiology of endometriosis is retrograde menstruation [2]. However, this phenomenon has also been described in healthy women and can be viewed as a physiological process [3,4]. Thus factors other than retrograde menstruation are necessary for the development of endometriosis. Several lines of evidence indicate that the cytokines in the peritoneal fluid (PF) of women Endometriosis is a common gynecological disorder that affects 10%-15% of women of reproductive age and up to 50% of infertile patients. It is characterized by pelvic pain, dyspareunia and infertility as a L.Gao et al. / Journal of Biomedical Research, 2010, 24(1): 43-50 44 with endometriosis establishes a microenvironment for the development of the disease and undergoes a number of pathological changes, including inflammatory processes with locally and systemically altered functions of the immune system [5-7]. Increased levels of Interleukin (IL) -1ß mRNA expression have been identified in the peritoneal macrophages of patients with mild endometriosis [8]. IL-1 is a major proinflammatory cytokine which has been isolated from the PF of patients with endometriosis. It is mainly produced by monocytes and macrophages, and is believed to have an important role in endometriosis pathophysiology. shown that increased expression of the Bcl-2 protein is present in the proliferative eutopic endometrium of patients with endometriosis [19]. The purpose of this study was to evaluate the therapeutic potential of sIL-1 RⅡ in counteracting the effect of IL-1 using an in vivo nude mouse model of experimentally-induced endometriosis. Transplantation of the endometrial fragments into nude mice Nineteen 6- 8-week-old female nude mice purchased from the Shanghai Laboratory Animal Corporation were maintained in a barrier unit in a well-controlled pathogen-free environment with regulated cycles of light/dark (12 h/12 h). All equipment and food entering the barrier was autoclaved. Mice had free access to food and water. Approval from the Animal Ethics Committee of the Nanjing Medical University was obtained before the study. y The critical role of IL-1 in endometriosis has been confirmed in mice models, whereby successful implantation is blocked by administration of exogenous IL-1 receptor antagonist (IL-1ra), another natural specific inhibitor for IL-1 signaling [16]. In endometriotic stromal cells, IL-1β has been shown to stimulate angiogenic factors such as vascular endothelial growth factor (VEGF) and IL- 8, cytokines endowed with neutrophil chemotactic and angiogenic properties [17], which is not observed in normal endometrial stromal cells. Our previous study also revealed that IL-1β significantly increased the production of IL-6 and IL-8, and this effect could be counteracted by recombinant adenovirus IL-1 RⅡ (rAd-RⅡ) [18]. Furthermore, we showed that overexpression of rAd-RⅡ could suppress Bcl-2 expression in ectopic endometrium, suggesting that IL-1 RⅡ may promote apoptosis of endometriotic stromal cells through inhibition of B-cell lymphoma leukemia-2 (Bcl-2) expression. The Bcl-2 gene was defined as a new class of proto-oncogenes that block cell death in the absence of cell proliferation. Studies have Mice were weighed, and anesthetized with intraperitoneal injections of pentobarbital. The endometrial tissue fragments were washed three times with PBS and cut into 2-3 mm 2 sections that were implanted with a suture of polypropylene 3/0, in the parietal peritoneum in two sections on each side of the midline incision with a separation of 0.5-1.0 cm. The abdominal wall was then closed with Prolene 3-0 non-absorbable polypropylene sutures (Holycon, Nantong, Jiangsu, China). Each mouse received an intramuscular injection of benzestrofol (2 mg/Kg) every 4 days from the day of tissue transplantation L.Gao et al. / Journal of Biomedical Research, 2010, 24(1): 43-50 45 retrieval. Incubation with 0.01% trypsin for 30 min at 37°C was used to detect Bcl-2, and steam-heat incubation with EDTA for 30 min at 97°C pH 8 was used for VEGF. Endogenous peroxidase was removed by treatment with 0.3% hydrogen peroxide for 30 min at 37°C, after which nonspecific binding was blocked by incubation with normal goat serum. Measure of IL-8 and VEGF At the end of the experimental period, animals were sacrificed, the blood and PF were collected, and PF and serum levels of VEGF and IL-8 were measured by standard cytokine-specific enzyme-linked immunosorbent assay (ELISA) using commercial kits (Adlitteram Diagnostic Laboratories, USA). All measurements were performed in duplicate, according to the manufacturer’s instructions. The sensitivity of the kits was approximately 1 pg/ml for both VEGF and IL-8. After measuring, the lesions were immediately fixed in 4% paraformaldehyde and then paraffin embedded. The presence of viable endometiosis was confirmed by conventional hematoxylin and eosin staining in one 5 μm thickness section per implant. The histologic diagnosis of endometriosis was based on the morphologic identification of endometrial glandular tissue and stroma. An individual other than the authors evaluated three sections from each implant in a blinded fashion. Statistical analysis All data are expressed as mean ±SD. Differences among groups were analyzed by one-way analysis of variance (ANOVA), and the post hoc Student- Newman-Keuls (SNK) method was used for multiple comparison. The P-value reported was two-sided and value of P < 0.05 was considered statistically significant. All analyses were performed using the SPSS software (Version 11.0, SPSS Inc., USA). Finally, the expression of Bcl-2 and VEGF were detected in serial sections by immunohistochemistry according to the laboratory protocol. Sections were deparaffinized in xylene and rehydrated through graded alcohols to distilled water. In sections used to detect VEGF this was followed by microwaving in 0.01 mol/L sodium citrate buffer for antigen Drug administration After transplantation with endometrial tissue, nineteen nude mice were randomly divided into three groups: group A (n = 7) was treated with only sIL-1 RⅡ for two weeks, group B(n = 6) was treated with only IL-1 for the same time period and group C(n = 6) was treated with saline as control. The optimal dosage was unknown due to the lack of previous studies on sIL-1 RⅡ for experimental endometriosis. Therefore, we based the dosage on sIL-1 RⅡ treatment in the experimental orthodontic tooth movement in rats, namely 2 μg/215 g [21]. Each group of mice was treated with an intraperitoneal injection of 0.2 ml of their respective treatments every other day for 2 weeks. Treatment with human recombinant IL-1 (5 ng/kg) (R&D System, USA) and human recombinant sIL- 1 RⅡ (25 μg/kg) (R&D System) was started at day 1 after tissue transplantation. Mice in the control group received the same volume of saline. Transplantation of the endometrial fragments into nude mice Tissue sections were incubated with anti-human Bcl-2 rabbit polyclonal antibody (1:100, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or VEGF anti-human rabbit monoclonal antibody (1:200, Zeta, Sierra Madre, CA, USA) overnight at 4°C, then incubated with anti-rabbit-peroxidase conjugate (Zhongshan Ltd., Beijing, China) for 60min at 37°C. Binding was visualized by incubating sections with 3, 3'-Diaminobenzidine (DAB). After rinsing, slides were counterstained with hematoxylin, dehydrated through a graded ethanol series to xylene, and then coverslips put in place. Negative control specimens were processed by omitting the primary antibody. The intensity of Bcl-2 and VEGF staining was assessed in a blind fashion at 200 magnifications by microscopy (Leica, Germany). Images were captured and the gray value was measured using Leica Qwin, standard V2.8 software. A quantitative assessment method was used to compare the gray value of 3 randomly selected fields of each tissue specimen. for 2 weeks. All animal studies were approved and conducted in accordance with Institutional Animal Care Committee guidelines. Evaluation of ectopic uterine tissue Two weeks after the beginning of treatment, animals were sacrificed and the lesions were collected to determine the growth of each implant based on the area of lesions. The growth of lesions per mouse was measured using the average size of the four implants, which were calculated by measuring two perpendicular diameters (a×b, length and width) with a caliper and the area of each lesion was calculated by the formula of an ellipse (a×b×∏/4) [22]. The person performing the laparotomy and the evaluation of the endometriotic implants had no prior knowledge of the animal group allocation. RESULTS The behavior of the mice during the experiment was normal. No evidence of toxicity was noted in both the sIL-1 RⅡ group and IL-1 group. The body L.Gao et al. / Journal of Biomedical Research, 2010, 24(1): 43-50 46 weights of three groups (20.1±1.9 g of sIL-1 RⅡ group, 19.7±2.4 g of IL-1 group, and 20.3±2.2 g of control) were similar (P > 0.05). and eosin detected the lesions taken from all groups at day 14 after transplantation revealed typical endometriotic lesions; the morphology of endometriotic lesions in all treatment groups was diverse, with typical endometrium glands and stroma as well as tubal metaplasia with large cysts and flattened epithelium. The observed morphology of the lesions was similar to that seen in human endometriosis (Fig. 1 C and 1D). Of a total of 19 mice which received endometrial fragments, all mice developed 4 endometriotic lesions in the peritoneum at 2 weeks (Fig. 1 A). Macroscopically, the deposits appeared as reddish cystic areas and were found to be well vascularized and attached to the abdominal wall peritoneum (Fig. 1 B). Histological sections stained with hematoxylin Fig.1 The lesions on day 14 after transplantation (A), The lesion viewed by stereo microscope, 12×(B). The morphology of the lesions at day 14 after transplantation, 200×(C) and 400×(D). A B C D A Fig.1 The lesions on day 14 after transplantation (A), The lesion viewed by stereo microscope, 12×(B). The morphology of the lesions at day 14 after transplantation, 200×(C) and 400×(D). Effects of sIL1-RⅡ on the growth of endometriotic implants cells of the endometrium (Fig. 3), while the expression of VEGF was mostly limited to the epithelium (Fig. 4). The negative control sections showed an absence of specific staining. Fig. 5 showed the gray value of the lesions' glandular epithelium staining for each of the three groups. The expression of protein was the inverse of the gray value, so the expression of VEGF and Bcl-2 were highest in the IL-1 group, but lowest in the sIL-1 RⅡ group and both proteins in each group were significantly different (P < 0.05) from the control group values. After two weeks of treatment, the average area of ectopic endometrial lesions in the sIL-1 RⅡ treated nude mice decreased compared with the other two groups, but there was no significant difference among the three groups (Fig. 2). area (mm ) 35 30 25 20 15 10 5 0 IL-1 control sIL-1 RⅡ n = 6 n = 6 n = 7 area (mm ) 35 30 25 20 15 10 5 0 IL-1 control sIL-1 RⅡ n = 6 n = 6 n = 7 Fig.2 The average area of lesions per mouse in each of the three groups (sIL-1R Ⅱ treated group, IL-1 treated group and control group treated with saline) at day 14 after transplantation (P = 0.645). DISCUSSION It has been demonstrated that the expression of IL- 1ß by peritoneal macrophages was elevated during the initial stages of endometriosis but the IL-1ra was usually elevated during the late stages of the disease [15]. An up-regulation of IL-1 expression was recently observed in the eutopic endometrial tissue of endometriosis patients [23]. It has also been found that sIL-1 RⅡ binds and blocks processing of the IL- 1ß precursor, inhibits its maturation, loses affinity for The IL-8 and VEGF levels in the peritoneal fluid and serum In the PF and serum, the concentrations of IL-8 and VEGF were lowest in the sIL1-RⅡgroup and highest in the IL-1 group. The differences in IL-8 concentration between any two groups reached statistical significance (P < 0.05), but the VEGF did not (P > 0.05) (Fig. 6). Fig.2 The average area of lesions per mouse in each of the three groups (sIL-1R Ⅱ treated group, IL-1 treated group and control group treated with saline) at day 14 after transplantation (P = 0.645). Treatment of sIL1-RⅡ inhibits the expression of VEGF and Bcl-2 in endometriotic lesions The immunohistochemical staining of the slides were reviewed on three separate occasions by different combinations of investigators. The Bcl-2 and VEGF were mainly located in the plasma membrane and membranes of intracellular organelles. Expression of Bcl-2 was present in both epithelial and stromal L.Gao et al. / Journal of Biomedical Research, 2010, 24(1): 43-50 47 Fig.3 The expression of Bcl-2 on day 14 after transplantation in the 3 groups: the IL-1 treated group (A) and the negative control (D), the saline treated control group (B) and the negative control (E), and the sIL-1 RⅡ treated group (C) and the negative control (F) A B C D E F Fig.3 The expression of Bcl-2 on day 14 after transplantation in the 3 groups: the IL-1 treated group (A) and the negative control (D), the saline treated control group (B) and the negative control (E), and the sIL-1 RⅡ treated group (C) and the negative control (F) A D Fig.4 The expression of VEGF on day 14 after transplantation in the 3 groups: the IL-1 treated group (A) and the negative control (D), the saline treated control group (B) and the negative control (E), and the sIL-1 RⅡ treated group (C) and the negative control (F). A B C D E F C F B E Fig.4 The expression of VEGF on day 14 after transplantation in the 3 groups: the IL-1 treated th li t t d t l (B) d th ti t l (E) d th IL 1 RⅡt t d A B D E day 14 after transplantation in the 3 groups: the IL-1 treated group (A) and the negative c B C E F Fig.4 The expression of VEGF on day 14 after transplantation in the 3 groups: the IL-1 treated group (A) and the negative control (D), the saline treated control group (B) and the negative control (E), and the sIL-1 RⅡ treated group (C) and the negative control (F). VEGF gray 120 100 80 60 40 20 0 IL-1 control sIL-1 RⅡ * * Bcl-2 gray * * 120 100 80 60 40 20 0 IL-1 control sIL-1 RⅡ Fig.5 The gray values (inversely proportional to protein expression) of Bcl-2 (A) and VEGF (B) of the immunohistochemical slides of the 3 groups. Significantly different from the control (*P < 0.05). Treatment of sIL1-RⅡ inhibits the expression of VEGF and Bcl-2 in endometriotic lesions A B Bcl-2 gray * * 120 100 80 60 40 20 0 IL-1 control sIL-1 RⅡ A VEGF gray 120 100 80 60 40 20 0 IL-1 control sIL-1 RⅡ * * B B Fig.5 The gray values (inversely proportional to protein expression) of Bcl-2 (A) and VEGF (B) of the immunohistochemical slides of the 3 groups. Significantly different from the control (*P < 0.05). L.Gao et al. / Journal of Biomedical Research, 2010, 24(1): 43-50 48 Fig.6 The IL-8 and VEGF concentrations in the PF and serum for each group of animals. Significantly different from control (*P < 0.05). A: The IL-8 concentration in PF; B: The IL-8 concentration in serum; C:The VEGF concentration in PF; D: The VEGF concentration in serum. IL-1 control sIL1-RⅡ PF IL-8 concentration (ng/ml) 6 5 4 3 2 1 0 * * IL-1 control sIL1-RⅡ serum IL-8 concentration (ng/ml) 6 5 4 3 2 1 0 * * IL-1 control sIL1-RⅡ PF VEGF concentration (ng/ml) 7 6 5 4 3 2 1 0 IL-1 control sIL1-RⅡ serum VEGF concentration (ng/ml) 6 5 4 3 2 1 0 A C B D IL-1 control sIL1-RⅡ serum IL-8 concentration (ng/ml) 6 5 4 3 2 1 0 * * 6 B D B Fig.6 The IL-8 and VEGF concentrations in the PF and serum for each group of animals. Significantly different from control (*P < 0.05). A: The IL-8 concentration in PF; B: The IL-8 concentration in serum; C:The VEGF concentration in PF; D: The VEGF concentration in serum. the IL-1ra and does not therefore interfere with IL- 1ra mediated inhibition of IL-1 effects [24]. Finally, sIL- 1 RⅡ appears to act by sequestering IL-1ß within the extracellular compartment, thus restricting its availability and interaction with IL-1 RI. of VEGF in PF and serum showed no significant difference (P > 0.05) between the three groups after two weeks of treatment of nude mice, this may be caused by the short treatment time. In endometriosis, abnormal adhesion of endometrial fragments to ectopic sites could be due to an increase in the adhesive and proliferative properties of the menstrual endometrium, a decrease in apoptosis, and anomalities in the target tissue microenvironment. Previous studies demonstrated that endometrial cells (ECs) from women with endometriosis exhibited low apoptosis, when compared to ECs from women without endometriosis [31]. Treatment of sIL1-RⅡ inhibits the expression of VEGF and Bcl-2 in endometriotic lesions Dmowski and colleagues [32], using a cell death detection ELISA assay, demonstrated that endometrial apoptosis in the eutopic endometrium from women with endometriosis was lower than controls, and that endometrial apoptosis in the ectopic endometrium further decreased when compared to the eutopic endometrium. The evidence suggested that the normal balance between proliferative and apoptotic cyclicity of the endometrium was disturbed in women with endometriosis [33], with a reduced apoptosis which was observed primarily in the early proliferative and late secretory phases of the menstrual cycle [34]. IL-1 induces an angiogenic phenotype in endometriotic cells and stimulates the secretion of VEGF. Recently, several studies have proposed that angiogenesis represents an important step during the process of endometriosis, because similar to tumour metastases, endometriotic implants require neovascularization to establish, proliferate and invade [25-27]. The endometrium has angiogenic potential [28], and endometriotic lesions are larger in areas with a rich blood supply [29]. In a recent study about the growth of human menstrual endometrium in nude mice, a high VEGF score was observed in stromal cells as early as 5 days after transplantation, suggesting that an active vascular network is a necessary condition for the survival of the graft [30]. Our results suggest that sIL-1 RⅡ treatment throughout the development of endometriosis decreases the expression of VEGF protein, which then affects the angiogenesis of the lesions. Although, the change L.Gao et al. / Journal of Biomedical Research, 2010, 24(1): 43-50 49 The disturbed expression of IL-1 RⅡ on the eutopic endometrium resulted in disturbances in normal macrophage trafficking into the eutopic endometrium in women with endometriosis. There may also be a positive correlation between apoptosis and macrophage numbers in the eutopic endometrium [35]. These results provided further evidence for an intrinsic abnormality in apoptotic sensitivity of the endometrium of women with endometriosis. The Bcl-2 is defined as a new class of proto-oncogenes that block cell death without promoting cell proliferation [36]. Under normal conditions, epithelial cells require interactions with the extracellular matrix to survive and grow. Apoptosis is thought to prevent the dissemination and attachment of differentiated epithelial cells at inappropriate locations when they lose contact with the matrix. It is likely that in healthy women, ECs and tissue fragments expelled during menses do not survive in ectopic locations because of programmed cell death. Disruption of cell matrix interactions induces cell death through a process called anoikis [37]. References [1] Goldman MB, Cramer DW. The epidemiology of endometriosis. Prog Clin Biol Res 1990;323:15-31. [2] Brosens IA, Brosens JJ. Endometriosis. Eur J Obstet Gynecol Reprod Biol 2000;90:159-64. [3] Halme J, Hammond MG, Hulka JF, Raj SG, Talbert IM.. Retrograde menstruation in healthy women and in patients with endometriosis. Obstet Gynecol 1984;64:151-4. [4] Chung HW, Lee JY, Moon HS, Hur SE, Park MH, Wen Y, et al. Matrix metalloproteinase-2, membranous type 1 matrix metalloproteinase, and tissue inhibitor of metalloproteinase-2 expression in ectopic and eutopic endometrium. Fertil Steril 2002;78:787-95. [5] Lebovic DI, Mueller MD, Taylor RN. Immunobiology of endometriosis. Fertil Steril 2001;75:1-10. [6] Seli E, Arici A. Endometriosis: interaction of immune and endocrine system. Semin Reprod Med 2003;21:135-44. IL-8 is elevated in the PF of women with endometriosis [39-41], and the IL-8 level is also correlated with the severity of the disease [40]. It has recently been shown that IL-8 stimulates the adhesion of endometrial cells to fibronectin [42]. Thus, IL-8 may be relevant for stimulating the attachment of endometrial implants in the pathogenesis of endometriosis, and the adherence of ECs induces further IL-8 expression by an integrin-dependent mechanism [43]. [7] Wu MY, Ho HN. The role of cytokines in endometriosis. Am J Reprod Immunol 2003;49:285-96. [8] Mori H, Sawairi M, Nakagawa M, Itoh N, Wada K, Tamaya Tl. Expression of interleukin-1 (IL-1) beta messenger ribonucleic acid (mRNA) and IL-1 receptor antagonist mRNA in peritoneal macrophages from patients with endometriosis. Fertil Steril 1992;57:535-42. [9] Dinarello CA. Biologic basis for interleukin-1 in disease. Blood 1996; 87: 2095-147. Our previous results demonstrated that IL-8 secretion induced by IL-1ß was significantly decreased after infection with recombinant adenovirus expressing IL-1RⅡ [18]. Now, we have found that in an endometriosis nude mice model, the IL-8 concentration in the peritoneal fluid and serum of the sIL-1 RⅡ treated group is significantly lower than the other two groups, and is in fact increased in the IL-1 treated group. Although the cytokine network in the endometrium is complex, our results provide further insights into the mechanism of immunologic modulation of sIL-1 RⅡ during the process of [10] Sims JE, Gayle MA, Slack JL, Alderson MR, Bird TA, Giri JG, et al. Interleukin 1 signaling occurs exclusively via the type I receptor. Proc Natl Acad Sci USA 1993;90:6155-9. [11] Colotta F, Dower SK, Sims JE, Mantovani A. The type II ‘decoy’ receptor: a novel regulatory pathway for interleukin 1. endometriosis. Taken together, these findings further support the contention that the pathogenesis of endometriosis is multifactorial and involves many mediators. Presently, endometriosis is mainly treated by estrogen antagonist, which only suppresses symptoms, but will not eradicate the ectopic implants. Moreover, there are significant side effects. Alternatively, endometriosis can be treated surgically, but symptoms recur in time in a majority of women. An effective therapeutic agent for endometriosis would be a compound that not only prevents the development of endometriotic lesions but is also effective against the growth of established lesions. Our results suggest that sIL-1 RⅡ may provide a new clinical strategy for the treatment of endometriosis. Treatment of sIL1-RⅡ inhibits the expression of VEGF and Bcl-2 in endometriotic lesions This might prevent detached normal cells from implanting in a new and inappropriate location. Endometriotic lesions show an increase in Bcl-2 expression compare with the endometrium and such an elevated Bcl-2 expression could promote the survival of endometriotic tissue at ectopic sites [38]. Our results demonstrate that in the formed lesions, sIL- 1 RⅡ decreased the expression of Bcl-2 and VEGF compared with both control and IL-1 groups, and may contribute to decreasing the size of already formed lesions in the endometriosis nude mouse model. References Immunol Today 1994;15:562-6. [12] Symons JA, Young PR, Duff GW. Soluble type II interleukin 1 (IL-1) receptor binds and blocks processing of IL-1 beta precursor and loses affinity for IL-1 receptor antagonist. Proc Natl Acad Sci USA 1995;92:1714-18. [13] Dinarello CA. Interleukin-1, interleukin-1 receptor’s and L.Gao et al. / Journal of Biomedical Research, 2010, 24(1): 43-50 50 Hum Reprod 2001;16:1557-61. Hum Reprod 2001;16:1557-61. interleukin-1 receptor antagonist. Int Rev Immunol 1998; 16:457-99. [29] Nisolle M, Casanas-Roux F, Anaf V, Mine JM, Donnez J. Morphometric study of the stromal vascularization in peritoneal endometriosis. Fertil Steril 1993;59:681-4 . [14] Kharfi A, Boucher A, Akoum A. Abnormal interleukin-1 receptor type II gene expression in the endometrium of women with endometriosis. Biol Reprod 2002;66:401-6. [30] Nisolle M, Casanas-Roux F, Donnez J. Early-stage endometriosis:adhesion and growth of human menstrual endometrium in nude mice. Fertil Steril 2000;74:306-12. [15] Akoum A, Jolicoeur C, Kharfi A, Aube M. Decreased expression of the decoy interleukin-1 receptor type Ⅱ in human endometriosis. Am J Pathol 2001;158:481-9. [31] Gebel HM, Braun DP, Tambur A, Frame D, Rana N, Dmowski WP.. Spontaneous apoptosis of endometrial tissue is impaired in women with endometriosis. Fertil Steril 1998;69:1042-7. [16] Simon C, Frances A, Piquette G. The immune mediator interleukin-1 receptor antagonist (IL-1ra) prevents embryo implantation. Endocrinology 1994;134:521-8. [32] Dmowski WP, Gebel H, Braun DP. Decreased apoptosis and sensitivity to macrophage mediated cytolysis of endometrial cells in endometriosis. Hum Reprod Update 1998;4:696-701. [17] Akoum A, Lawson C, McColl S, Villeneuve Ml. Ectopic endometrial cells express high concentrations of interleukin (IL)-8 in vivo regardless of the menstrual cycle phase and respond to oestradiol by up-regulating IL-1-induced IL-8 expression in vitro. Mol Hum Reprod 2001; 7: 859-66. [33] Braun DP, Ding J, Shaheen F, Willey JC, Rana N, Dmowski WP. Quantitative expression of apoptosis- regulating genes in endometrium from women with and without endometriosis. Fertil Steril 2007;87:263-8. [18] Hou Z, Zhou J, Ma X, Fan L, Liao L, Liu JY. Role of Interleukin-1 receptor type II in the pathogenesis of endometriosis. Fertil Steril 2008;89:42-51. [34] Dmowski WP, Ding J, Shen J, Rana N, Fernandez BB, Braun DP. Apoptosis in endometrial glandular and stromal cells in women with and without endometriosis. Hum Reprod 2001;16:1802-8. [19] Meresman GF, Vighi S, Buquet RA, Contreras-Ortiz O, Tesone M, Rumi LS. Apoptosis and expression of Bcl- 2 and Bax in eutopic endometrium from women with endometriosis. Fertil Steril 2000;74:760-6. References [35] Braun DP, Ding J, Shen J, Rana N, Fernandez BB, Dmowski WP. Relationship between apoptosis and the number of macrophages in eutopic endometrium from women with and without endometriosis. Fertil Steril 2002;78:830-5. [20] American Fertility Society. Revised American Fertility Society classification of endometriosis. Fertil Steril 1985;43:351-2. [21] Jager A, Zhang D, Kawarizadeh A, Tolba R, Braumann B, Lossodrfer S, et al. Soluble cytokine receptor treatment in experimental orthodontic tooth movement in the rat. Eur J Orthod 2005;27:1-11. [36] Korsmeyer SJ. Bcl-2 initiates a new category of oncogenes: regulators of cell death. Blood 1992;80:879- 86. [37] Frisch SM, Francis H. Disruption of epithelial cell-matrix interactions induces apoptosis. J Cell Biol 1994;124:619-26. [22] Becker CM, Sampson DA, Short SM, Javaherian K, Folkman J, et al. Short synthetic endostatin peptides inhibit endothelial migration in vitro and endometriosis in a mouse model. Fertil Steril 2006;85:71-7. [38] Jones R, Searle R, Bulmer J. Apoptosis and bcl-2 expression in normal human endometrium, endometriosis and adenomyosis. Hum Reprod 1998;13:3496-3502. [23] Bergqvist A, Bruse C, Carlberg M, Carlstrom K.. Interleukin 1 beta, interleukin-6, and tumor necrosis factor-alpha in endometriotic tissue and in endometrium. Fertil Steril 2001;75:489-95. [39] Arici A, Tazuke SI, Attar E, Kliman HJ, Olive DL. Interleukin-8 concentration in peritoneal fluid of patients with endometriosis and modulation of interleukine-8 expression in human mesothelial cells. Mol Hum Reprod 1996;2:40-5. [24] Dunne A, O’Neill LA. The interleukin-1 receptor/Toll- like receptor superfamily: signal transduction during inflammation and host defense. Sci STKE 2003;171:3- 17. [40] Gazvani MR, Christmas S, Quenby S, Kirwan J, Johnson PM, Kingsland CR. Peritoneal fluid concentrations of interleukine-8 in women with endometriosis: relationship to stage of disease. Hum Reprod 1998;13:1957-61. [25] Fujishita A, Hasuo A, Khan KN, Masuzaki H, Nakashima H, Ishimaru T. Immunohistochemical study of angiogenic factors in endometrium and endometriosis. Gynecol Obstet Invest 1999;48:36-44. [41] Iwabe T, Harada T, Tsudo T, Tanikawa M, Onohara Y, Terakawa N. Pathogenic significance of increased levels of interleukin-8 in the peritoneal fluid of patients with endometriosis. Fertil Steril 1998;69:924-30. [26] Taylor RN, Lebovic DI, Mueller MD. Angiogenic factors in endometriosis. Ann NY Acad Sci 2002;955:89-100. [27] Gescher DM, Haensel A, Meyhofer-Malik A, Malik E. The importance of angiogenesis for the pathogenesis of endometriosis. Zentralbl Gynakol 2003;125:243-6. [42] Garcia-Velasco JA, Arici A. Interleukin-8 stimulates the adhesion of endometrial stromal cells to fibronectin. Fertil Steril 1999;72:336-40. [43] Garcia-Velasco JA, Arici A. References Interleukin-8 expression in endometrial stromal cells is regulated by integrin- dependent cell adhesion. Mol Hum Reprod 1999;5:1135-40. [28] Maas JW, Groothuis PG, Dunselman GA, de Goeij AF, Struyker-Boudier HA, Evers JL. Endometrial angiogenesis throughout the human menstrual cycle.
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Treatment of Trypanosoma evansi-Infected Mice With Eucalyptus camaldulensis Led to a Change in Brain Response and Spleen Immunomodulation
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Treatment of Trypanosoma evansi-Infected Mice With Eucalyptus camaldulensis Led to a Change in Brain Response and Spleen Immunomodulation Mohamed A. Dkhil1*, Esam M. Al-Shaebi2, Rewaida Abdel-Gaber2, Abdulsalam Alkhudhayri3, Felwa A. Thagfan4 and Saleh Al-Quraishy2 Mohamed A. Dkhil1*, Esam M. Al-Shaebi2, Rewaida Abdel-Gaber2, Abdulsalam Alkhudhayri3, Felwa A. Thagfan4 and Saleh Al-Quraishy2 1 Department of Zoology and Entomology, Faculty of Science, Helwan University, Cairo, Egypt, 2 Department of Zoology, College of Science, King Saud University, Riyadh, Saudi Arabia, 3 Department of Biology, College of Science, University of Hafr Al Batin, Hafr Al Batin, Saudi Arabia, 4 Department of Biology, College of Science, Princess Nourah Bint Abdulrahman University, Riyadh, Saudi Arabia ORIGINAL RESEARCH published: 21 March 2022 doi: 10.3389/fmicb.2022.833520 Keywords: Surra, Trypanosoma, brain, spleen, Eucalyptus camaldulensis Surra is a parasitic disease caused by the eukaryotic, unicellular hemoprotozoan, Trypanosoma evansi, which affects the development of animal production and is widespread among both domestic and wild animals. As such, in this research, we studied the antiparasitic activity and the ameliorative impact of Eucalyptus camaldulensis leaf extracts (ELE) against T. evansi-induced brain injury and spleen immune response in mice. As a result, we found that ELE decreased the amount of trypanosomes in the blood and improved the weight loss caused by infection. In addition, ELE reduced the parasite-induced brain and spleen histopathological damage. The parasite affected the levels of dopamine and serotonin, but after treatment with ELE, their concentrations significantly decreased to 154 ± 7 and 258 ± 11 µg/g, respectively. We clearly observed the antioxidant activity of ELE because of its ability to increase the induced change in the brain’s total antioxidant capacity and the nitric oxide level. The histopathological changes in the spleen also improved after ELE application. Based on our results, we concluded that ELE possesses antitrypanosomal antioxidant and protective effects in the brains of mice infected with T. evansi. Additional phytochemical screening and molecular studies are required to understand the mechanism underlying the effect of ELE. Edited by: Sónia Silva, University of Minho, Portugal Reviewed by: Lachhman Das Singla, Guru Angad Dev Veterinary and Animal Sciences University, India Vikrant Sudan, Guru Angad Dev Veterinary and Animal Sciences University, India *Correspondence: Mohamed A. Dkhil mohameddkhil@yahoo.com Reviewed by: Lachhman Das Singla, Guru Angad Dev Veterinary and Animal Sciences University, India Vikrant Sudan, Guru Angad Dev Veterinary and Animal Sciences University, India *C d Reviewed by: Lachhman Das Singla, Guru Angad Dev Veterinary and Animal Sciences University, India Vikrant Sudan, Guru Angad Dev Veterinary and Animal Sciences University, India *Correspondence: Mohamed A. Dkhil *Correspondence: Mohamed A. Dkhil mohameddkhil@yahoo.com Specialty section: This article was submitted to Infectious Agents and Disease, a section of the journal Frontiers in Microbiology Specialty section: This article was submitted to Infectious Agents and Disease, a section of the journal Frontiers in Microbiology Received: 11 December 2021 Accepted: 31 January 2022 Published: 21 March 2022 Citation: Dkhil MA, Al-Shaebi EM, Abdel-Gaber R, Alkhudhayri A, Thagfan FA and Al-Quraishy S (2022) Treatment of Trypanosoma evansi-Infected Mice With Eucalyptus camaldulensis Led to a Change in Brain Response and Spleen Immunomodulation. Front. Microbiol. 13:833520. doi: 10.3389/fmicb.2022.833520 Frontiers in Microbiology | www.frontiersin.org INTRODUCTION D i i l d i l active chemicals, such as alkaloids and flavonoids (Ghareeb et al., 2018). Extracts from this plant were used to treat animals suffering from malaria (Anigboro et al., 2020) and animal TABLE 2 | Effect of Eucalyptus camaldulensis on the survival and trypanosomes number per 5 fields on day 5 post-infection. Group Survival Trypanosomes/5 fields Infected Survived 645 ± 45 100 Survived 276 ± 70* FIGURE 1 | Phytochemical screening by FTIR spectroscopic analysis of Eucalyptus camaldulensis leaf extracts. FIGURE 1 | Phytochemical screening by FTIR spectroscopic analysis of Eucalyptus camaldulensis leaf extracts. TABLE 1 | FT-IR spectrum of Eucalyptus camaldulensis extract. Absorption (cm−1) Appearance Transmittance (%) Group Compound class 3,399.50 Medium 6.6 N–H stretching Aliphatic primary amine 2,930.61 Medium 19.9 C–H stretching Alkane 1,712.46 Strong 23.8 C = O stretching Carboxylic acid 1,615.19 Strong 17.5 C = C stretching α,β-unsaturated ketone 1,514.50 Strong 39.3 N–O stretching Nitro compound 1,450.66 Medium 22.7 C–H bending Alkane 1,352.18 Strong 20.5 S = O stretching Sulfonamide 1,225.05 Strong 21.8 C–O stretching Alkyl aryl ether 1,042.56 Strong, broad 12.9 CO–O–CO stretching Anhydride 871.80 Strong 64.6 C–H bending 1,3-disubstituted 819.64 Medium 64.8 C = C bending Alkene 766.97 Strong 55.1 C–H bending 1,2-disubstituted 603.51 Strong 46.1 C–Br stretching Halo compound TABLE 1 | FT-IR spectrum of Eucalyptus camaldulensis extract. active chemicals, such as alkaloids and flavonoids (Ghareeb et al., 2018). Extracts from this plant were used to treat animals suffering from malaria (Anigboro et al., 2020) and animal of the nervous system (Desquesnes et al., 2013; Dkhil et al., 2021a). Human infection with T. evansi can also occur (Joshi et al., 2005; Powar et al., 2006). Symptoms of trypanosomosis can vary with animal species (Degneh et al., 2017). The increased parasite levels in the blood of the host disturb the level of neurotransmitters. Dkhil et al. (2021a) reported increased dopamine and serotonin levels in the brains of mice infected with T. evansi. In addition, epinephrine has been reported to be increased during T. congolense and T. lewisi infections (Sanchez, 1973; Kalu and Haruna, 1985). of the nervous system (Desquesnes et al., 2013; Dkhil et al., 2021a). Human infection with T. evansi can also occur (Joshi et al., 2005; Powar et al., 2006). Symptoms of trypanosomosis can vary with animal species (Degneh et al., 2017). The increased parasite levels in the blood of the host disturb the level of neurotransmitters. INTRODUCTION Citation: Dkhil MA, Al-Shaebi EM, Abdel-Gaber R, Alkhudhayri A, Thagfan FA and Al-Quraishy S (2022) Treatment of Trypanosoma evansi-Infected Mice With Eucalyptus camaldulensis Led to a Change in Brain Response and Spleen Immunomodulation. Front. Microbiol. 13:833520. doi: 10.3389/fmicb.2022.833520 Surra is a widespread hemoprotozoan disease caused by the infection of domestic and wild animals with the single-cellular eukaryote Trypanosoma evansi (Sazmand et al., 2022). Trypanosomosis poses a barrier to the development of animal production in many tropical regions of the world. The disease pattern generated by T. evansi is similar to that of T. brucei gambiense, which causes sleep sickness in humans. The pathogenicity of T. evansi varies across strains and animal species (De Menezes et al., 2004). The tsetse fly is considered the vector transmitting trypanosomes to the host, and trypanosomosis leads to the appearance of severe weakness and anemia, and the impairment March 2022 | Volume 13 | Article 833520 Frontiers in Microbiology | www.frontiersin.org 1 Dkhil et al. Control of Surra Using Eucalyptus camaldulensis FIGURE 1 | Phytochemical screening by FTIR spectroscopic analysis of Eucalyptus camaldulensis leaf extracts. TABLE 1 | FT-IR spectrum of Eucalyptus camaldulensis extract. Absorption (cm−1) Appearance Transmittance (%) Group Compound class 3,399.50 Medium 6.6 N–H stretching Aliphatic primary amine 2,930.61 Medium 19.9 C–H stretching Alkane 1,712.46 Strong 23.8 C = O stretching Carboxylic acid 1,615.19 Strong 17.5 C = C stretching α,β-unsaturated ketone 1,514.50 Strong 39.3 N–O stretching Nitro compound 1,450.66 Medium 22.7 C–H bending Alkane 1,352.18 Strong 20.5 S = O stretching Sulfonamide 1,225.05 Strong 21.8 C–O stretching Alkyl aryl ether 1,042.56 Strong, broad 12.9 CO–O–CO stretching Anhydride 871.80 Strong 64.6 C–H bending 1,3-disubstituted 819.64 Medium 64.8 C = C bending Alkene 766.97 Strong 55.1 C–H bending 1,2-disubstituted 603.51 Strong 46.1 C–Br stretching Halo compound of the nervous system (Desquesnes et al., 2013; Dkhil et al., 2021a). Human infection with T. evansi can also occur (Joshi et al., 2005; Powar et al., 2006). Symptoms of trypanosomosis can vary with animal species (Degneh et al., 2017). The increased parasite levels in the blood of the host disturb the level of neurotransmitters. Dkhil et al. (2021a) reported increased dopamine and serotonin levels in the brains of mice infected with T. evansi. In addition, epinephrine has been reported to be increased during T. congolense and T. lewisi infections (Sanchez, 1973; Kalu and Haruna, 1985). INTRODUCTION Dkhil et al. (2021a) reported increased dopamine and serotonin levels in the brains of mice infected with T. evansi. In addition, epinephrine has been reported to be increased during T. congolense and T. lewisi infections (Sanchez, 1973; Kalu and Haruna, 1985). TABLE 2 | Effect of Eucalyptus camaldulensis on the survival and trypanosomes number per 5 fields on day 5 post-infection. Group Survival Trypanosomes/5 fields Infected Survived 645 ± 45 100 Survived 276 ± 70* 200 Dead – 300 Dead – Drug Survived 105 ± 10* *Significance against the infected group at p ≤0.01. TABLE 2 | Effect of Eucalyptus camaldulensis on the survival and trypanosomes number per 5 fields on day 5 post-infection. Due to parasite resistance to currently used antitrypanosomal medications, scientists are looking for effective natural-resource- based therapies, such as natural products, to combat the infection. Eucalyptus camaldulensis is a species of Eucalyptus that belongs to the Myrtaceae family and is known for its biologically Frontiers in Microbiology | www.frontiersin.org March 2022 | Volume 13 | Article 833520 2 Control of Surra Using Eucalyptus camaldulensis Dkhil et al. MATERIALS AND METHODS trypanosomosis (Kabiru et al., 2013), and it was reported to possess antioxidant properties (Shakibaie et al., 2021). In vitro studies carried out by Habila et al. (2010) showed that E. camaldulensis oil was effective against T. evansi. Infrared Spectroscopy FIGURE 4 | E. camaldulensis improves the brain histopathology after inf with T. evansi. Normal structure of control cerebellum (A) and ELE-treate mice (C) Infected cerebell m of mice ith inj red P rkinje cells and dilat p py For ELE analysis, we used a Nicolet 6700 Fourier-transform infrared spectroscopy (FT-IR) optical spectrometer from Thermo Scientific (Waltham, MA, United States). We mixed the powder of the extract (10 mg) with 100 mg of potassium bromide powder (1:99 wt%) to obtain a translucent sample disk that we then loaded into an FTIR spectroscope with a scan range of 400–4000 cm−1. The chemical bonds in a molecule can be determined by interpreting the infrared absorption spectra (Pakkirisamy et al., 2017). Flavonoid and Phenolic Content of Eucalyptus camaldulensis Leaf Extracts FIGURE 4 | E. camaldulensis improves the brain histopathology after infection with T. evansi. Normal structure of control cerebellum (A) and ELE-treated (B) mice. (C) Infected cerebellum of mice with injured Purkinje cells and dilated sinusoids between the molecular layers. (D) ELE-treated infected cerebellum with improved structure. (E) Drug-treated cerebellum with improved structure. Scale bar = 25 µm. Total flavonoid content was quantified using an aluminum chloride colorimetric assay, and total phenolic content was calculated using the Folin-Ciocalteu technique described by Lin and Tang (2007). The flavonoids in ELE were estimated using a calibration curve of quercetin, a standard flavonoid. A gallic acid standard curve was used to determine the phenolic concentration. Extract Preparation We collected E. camaldulensis leaves from Qassim, Saudi Arabia. The samples were authenticated by a specialist from the herbarium of Helwan University. The leaves were cleaned, dried, and powdered. We macerated the obtained powder (100 g) by mixing it at 4◦C for 24 h in 70% methanol. We filtered the obtained extract and then dried it using a vacuum evaporator (Lubbad et al., 2015). We used distilled water to dissolve the residue. To the best our knowledge, no reports have been published on the brain-protective effect and spleen-immune modulation of E. camaldulensis during T. evansi infection. As such, during trypanosomosis, we examined antioxidant activity in the brain as well as the status of neurotransmitters in mice. We also examined any morphological and histological alterations in the spleen. p FIGURE 2 | Effect of E. camaldulensis extract on body weight of mice infected with T. evansi. *p < 0.01, significance against the control group. #p < 0.01, significance against the infected group. FIGURE 3 | E. camaldulensis induced change in spleen index of mice infected with T. evansi. *p ≤0.01, significance against the control group. #p ≤0.01, significance against the infected group. FIGURE 2 | Effect of E. camaldulensis extract on body weight of mice infected with T. evansi. *p < 0.01, significance against the control group. #p < 0.01, significance against the infected group. FIGURE 3 | E. camaldulensis induced change in spleen index of mice infected with T. evansi. *p ≤0.01, significance against the control group. #p ≤0.01, significance against the infected group. FIGURE 3 | E. camaldulensis induced change in spleen index of mice infected with T. evansi. *p ≤0.01, significance against the control group. #p ≤0.01, significance against the infected group. FIGURE 3 | E. camaldulensis induced change in spleen index of mice infected with T. evansi. *p ≤0.01, significance against the control group. #p ≤0.01, significance against the infected group. March 2022 | Volume 13 | Article 833520 3 Frontiers in Microbiology | www.frontiersin.org Control of Surra Using Eucalyptus camaldulensis Dkhil et al. Animals and Infection Additionally, we estimated the NO level according to Green et al. (1982). Animals and Infection We bred 25 C57Bl/6 female mice (11 ± 2 weeks old) under specific-pathogen-free conditions. They were housed in plastic cages under standard conditions of illumination with a 12-h light/dark cycle at 25 ± 1◦C. Animals received a balanced diet and water ad libitum. We maintained T. evansi in mice by weekly passage with infected blood. The mice were infected intraperitoneally with 103 trypanosomes of T. evansi (Dkhil et al., 2019). We calculated the mean number of trypanosomes/5 fields. The animals in the first group received only water by oral gavage, whereas the second group received ELE for 4 days (100 mg/kg). Three groups of animals (third to fifth) were infected and, 1 h later, the fourth group received 100 mg/kg ELE (Anigboro et al., 2020). The fifth group was treated with 1 mg/kg Cymelarsan (Merial, Lyon, France) (Hagos et al., 2010). We sampled the brains of the mice on day 4 post-infection after killing the animals by CO2 asphyxiation. We dissected the brains of the mice, which were then washed twice in ice-cold 50 mM Tris-HCl to remove any blood. Subsequently, we carefully divided longitudinally each brain into two halves: we used the first half for histopathological analysis, and we weighed and immediately homogenized the second half in an ice-cold medium containing 50 mM Tris-HCl (pH 7.4) to yield a 10% (w/v) homogenate. We centrifuged the supernatants obtained from the homogenates at 1,000 × g for 10 min at 4◦C to determine the concentrations of nitric oxide (NO), dopamine, and serotonin. We calculated body and spleen weights on day 4 p.i. We determined spleen index as described by Dkhil (2009) by calculating the ratio of spleen weight per milligram of mouse to the mouse weight per gram. The project was approved (approval No., HU2021/Z/AD/1121-01) by the Department of Zoology, Faculty of Science, Helwan University’s Committee of Research Ethics for Laboratory Animal Care. FIGURE 4 | E. camaldulensis improves the brain histopathology after infection with T. evansi. Normal structure of control cerebellum (A) and ELE-treated (B) mice. (C) Infected cerebellum of mice with injured Purkinje cells and dilated sinusoids between the molecular layers. (D) ELE-treated infected cerebellum with improved structure. (E) Drug-treated cerebellum with improved structure. Scale bar = 25 µm. March 2022 | Volume 13 | Article 833520 Frontiers in Microbiology | www.frontiersin.org 4 Control of Surra Using Eucalyptus camaldulensis Dkhil et al. method described in Koracevic et al. (2001). Dopamine and Serotonin We weighed the brain and measured the levels of dopamine and serotonin as described by Ciarlone (1978). Histological Study method described in Koracevic et al. (2001). Additionally, we estimated the NO level according to Green et al. (1982). We fixed the brains and spleens of infected and non-infected mice in 10% formalin. After fixation, the specimens were dehydrated, embedded in wax, and sectioned into 5-µm-thick slices. Finally, we stained the sections with hematoxylin and eosin and then examined them. Spleen histology was semiquantified as described by Giamarellos-Bourboulis et al. (2006). Oxidative Status We evaluated multiple variable comparisons using one-way ANOVA, and the data are presented as the mean standard error of the mean. We compared the significance between the classes using Duncan’s test. Statistical significance was set at p ≤0.05. According to Tsakiris et al. (2004), we prepared the brain homogenate to measure the total antioxidant capacity (TAC) using commercial kits (Biodiagnostic, Egypt) by the colorimetric FIGURE 5 | Change in brain dopamine of mice infected with T. evansi and treated with E. camaldulensis. *, significance against control group at p ≤0.01. #, significance against infected group at p ≤0.01. FIGURE 6 | E. camaldulensis induced change in the level of Serotonin of mice infected with T. evansi. *, significance against control group at p ≤0.01. #, significance against infected group at p ≤0.01. FIGURE 5 | Change in brain dopamine of mice infected with T. evansi and treated with E. camaldulensis. *, significance against control group at p ≤0.01. #, significance against infected group at p ≤0.01. FIGURE 5 | Change in brain dopamine of mice infected with T. evansi and treated with E. camaldulensis. *, significance against control group at p ≤0.01. #, significance against infected group at p ≤0.01. FIGURE 6 | E. camaldulensis induced change in the level of Serotonin of mice infected with T. evansi. *, significance against control group at p ≤0.01. #, significance against infected group at p ≤0.01. FIGURE 6 | E. camaldulensis induced change in the level of Serotonin of mice infected with T. evansi. *, significance against control group at p ≤0.01. #, significance against infected group at p ≤0.01. FIGURE 6 | E. camaldulensis induced change in the level of Serotonin of mice infected with T. evansi. *, significance against control group at p ≤0.01. #, significance against infected group at p ≤0.01. March 2022 | Volume 13 | Article 833520 5 Frontiers in Microbiology | www.frontiersin.org Control of Surra Using Eucalyptus camaldulensis Dkhil et al. RESULTS peaks appeared at 3,399.50, 2,930.61, 1,450.66, and 819.64 cm−1. The expected classes of compounds were aliphatic primary amines, alkanes, carboxylic acids, α,β-unsaturated ketones, nitro compounds, sulfonamides, alkyl aryl ethers, anhydrides, and halo compounds (Table 1). The FT-IR spectra for ELE are shown in Figure 1. We extracted Table 1 from the IR spectrum table.1 Our analysis of ELE using FTIR showed strong peaks at 1,712.46, 1,615.19, 1,514.50, 1,225.05, 1,042.56, 871.80, 766.97, and 603.51 cm−1. Medium Eucalyptus camaldulensis leaf extracts has a total flavonoid and phenol content of 3.2 QE/g and 29.8 GAE/mg, respectively. 1sigmaaldrich.com flavonoid and phenol content of 3.2 QE/g and 29.8 GAE/mg, respectively. FIGURE 7 | Effect of E. camaldulensis on brain total antioxidant capacity of mice infected with T. evansi. *, significance against control group at p ≤0.01. #, significance against infected group at p ≤0.01. FIGURE 8 | Brain nitric oxide level after treatment of T. evansi-infected mice with E. camaldulensis. *, significance against control group at p ≤0.01. #, significance against infected group at p ≤0.01. Frontiers in Microbiology | www.frontiersin.org 6 March 2022 | Volume 13 | Article 833520 1sigmaaldrich.com FIGURE 7 | Effect of E. camaldulensis on brain total antioxidant capacity of mice infected with T. evansi. *, significance against control group at p ≤0.01. #, significance against infected group at p ≤0.01. FIGURE 7 | Effect of E. camaldulensis on brain total antioxidant capacity of mice infected with T. evansi. *, significance against control group at p ≤0.01. #, significance against infected group at p ≤0.01. FIGURE 7 | Effect of E. camaldulensis on brain total antioxidant capacity of mice infected with T. evansi. *, significance against control group at p ≤0.01. #, significance against infected group at p ≤0.01. FIGURE 7 | Effect of E. camaldulensis on brain total antioxidant capacity of mice infected with T. evansi. *, significance against control group at p ≤0.01. #, significance against infected group at p ≤0.01. | p y g g g p p #, significance against infected group at p ≤0.01. FIGURE 8 | Brain nitric oxide level after treatment of T. evansi-infected mice with E. camaldulensis. *, significance against control group at p ≤0.01. #, significance against infected group at p ≤0.01. FIGURE 8 | Brain nitric oxide level after treatment of T. evansi-infected mice with E. camaldulensis. *, significance against control group at p ≤0.01. 1sigmaaldrich.com DISCUSSION Trypanosoma evansi infection generated significant neurohistopathological alterations in Purkinje cells, including inflammation, bleeding, and structural abnormalities. In addition, infection reduced the number of cells in the Purkinje layer (Figure 3). Because of the parasite, dopamine and serotonin levels were much higher, but after treatment with ELE, the levels of these neurotransmitters significantly decreased (Figures 4, 5). We found that the infection decreased the TAC, but treatment with ELE ameliorated this decrease (Figure 6). Moreover, the level of NO decreased after treatment of the infected mice with ELE (Figure 7). Trypanosoma evansi infection generated significant neurohistopathological alterations in Purkinje cells, including inflammation, bleeding, and structural abnormalities. In addition, infection reduced the number of cells in the Purkinje layer (Figure 3). Because of the parasite, dopamine and serotonin levels were much higher, but after treatment with ELE, the levels of these neurotransmitters significantly decreased (Figures 4, 5). Trypanosomosis continues to be a serious health issue in both humans and animals, characterized by a complicated relationship between the host’s responses and the parasite’s actions, particularly the parasite’s generation of oxidative stress. This oxidative damage caused by parasite activity is considered to be the result of the physiological, histological, and biochemical changes that occur during infection. We found that the infection decreased the TAC, but treatment with ELE ameliorated this decrease (Figure 6). Moreover, the level of NO decreased after treatment of the infected mice with ELE (Figure 7). Eucalyptus camaldulensis leaf extracts contains phenolic and flavonoid active compounds capable of suppressing the number of trypanosomes in mouse blood. Similar compound classes present in other plants, such as Indigofera oblongifolia, were found to have antitrypanosomal effects (Dkhil et al., 2019). Due to the presence of active phenolic compounds, E. camaldulensis shows significant antibacterial and antischistosomal activity (Ghareeb et al., 2018). The histopathological effects on the brain were induced by the rapid penetration of the trypanosome and its metabolites (Biswas et al., 2010), causing inflammation (Berlin et al., 2009). Few investigations on cerebral trypanosomosis The infection caused marked changes in the spleen. Splenomegaly was pronounced when the spleen index increased approximately twofold (Figure 8). Our examination of spleen sections showed that the architecture of the spleen’s red and white pulps changed after infection (Figure 9). ELE treatment improved the induced histopathological changes. This was observed through the calculated spleen histology score, which FIGURE 9 | Spleen histology of mice. RESULTS #, significance against infected group at p ≤0.01. FIGURE 8 | Brain nitric oxide level after treatment of T. evansi-infected mice with E. camaldulensis. *, significance against control group at p ≤0.01. #, significance against infected group at p ≤0.01. March 2022 | Volume 13 | Article 833520 Frontiers in Microbiology | www.frontiersin.org 6 Control of Surra Using Eucalyptus camaldulensis Dkhil et al. indicated an ameliorative effect similar to that of the reference drug (Figure 10). Eucalyptus camaldulensis leaf extracts (100 mg/kg) reduced the number of trypanosomes to approximately 57.2% (Table 2) on day 5 post-infection, whereas animals treated with 200 and 300 mg/kg ELE died on day 4 due to infection (Table 2). The parasite decreased the weight of the mice, but ELE increased their weight after treatment (Figure 2). DISCUSSION Normal structure of control spleen (A) and ELE-treated (B) mice. (C) Infected spleen of mice with dilated and fused white and red pulps and increased apoptotic bodies. (D) ELE-treated infected spleen with improved structure. (E) Drug-treated spleen with improved structure. Scale bar = 50 µm. FIGURE 9 | Spleen histology of mice. Normal structure of control spleen (A) and ELE-treated (B) mice. (C) Infected spleen of mice with dilated and fused white and red pulps and increased apoptotic bodies. (D) ELE-treated infected spleen with improved structure. (E) Drug-treated spleen with improved structure. Scale bar = 50 µm. FIGURE 10 | Spleen histology score of mice. *, significance against control group at p ≤0.01. #, significance against infected group at p ≤0.01. FIGURE 9 | Spleen histology of mice. Normal structure of control spleen (A) and ELE-treated (B) mice. (C) Infected spleen of mice with dilated and fused white and red pulps and increased apoptotic bodies. (D) ELE-treated infected spleen with improved structure. (E) Drug-treated spleen with improved structure. Scale bar = 50 µm. bar = 50 µm. FIGURE 10 | Spleen histology score of mice. *, significance against control group at p ≤0.01. #, significance against infected group at p ≤0.01. FIGURE 10 | Spleen histology score of mice. *, significance against control group at p ≤0.01. #, significance against infected group at p ≤0.01. March 2022 | Volume 13 | Article 833520 Frontiers in Microbiology | www.frontiersin.org 7 Control of Surra Using Eucalyptus camaldulensis Dkhil et al. have been conducted in Surra; however, in our prior work, we described the behavioral and histological effects of T. evansi in mice (Dkhil et al., 2021a). In addition to the histopathological effects on the brain, T. evansi causes liver and spleen injury (Biswas et al., 2010). In this study, splenomegaly with histopathological damage was pronounced, especially in the disorganization of white and red pulps. (Whalley, 2020). Herr et al. (2017) reported that T-cell activation could be mediated by serotonin. In addition, Matt and Gaskill (2020) reported that dopamine plays an important role in immune function regulation. In this study, the neurotransmitters dopamine and serotonin increased after infection, leading to spleen cell activation. Because of the presence of phytoactive substances, ELE was able to regulate these alterations. We concluded that ELE has antitrypanosomal and immune-neuroprotective activities against T. evansi infection. DISCUSSION However, further investigation into the molecular mechanisms underlying the neuroprotective and spleen response capabilities of E. camaldulensis is required. Due to the enrichment of ELE with active compounds and the antioxidant properties of these compounds, both brain and spleen histology improved after treatment of T. evansi- infected mice. In this study, we found that brain dopamine and serotonin levels increased after infection. The disturbance in dopamine and serotonin due to infection was also investigated by Amole et al. (1989) after infection with T. brucei brucei, which was attributed to the production of reactive oxygen species, which cause cellular damage and lead to the pathogenesis of the disease (Gupta et al., 2009). FUNDING This study was supported by Princess Nourah Bint Abdulrahman University Research Supporting Project number (PNURSP2022R96), Princess Nourah Bint Abdulrahman University, Riyadh, Saudi Arabia, and was also supported by Researchers Supporting Project (RSP2021/03), King Saud University, Riyadh, Saudi Arabia. The spleen is a key site for T-cell activation and B-cell differentiation. These activities are controlled in the brain ETHICS STATEMENT The animal study was reviewed and approved (Approval No., HU2021/Z/AD/1121-01) by the Department of Zoology, Faculty of Science, Helwan University’s Committee of Research Ethics for Laboratory Animal Care. Written informed consent was obtained from the owners for the participation of their animals in this study. The spleen is the body’s largest secondary lymphoid organ, and it performs a variety of immunological processes in addition to its hematopoiesis and erythrocyte clearing functions. In this study, the increased spleen index and splenomegaly were evident during infection due to splenic cell hyperplasia and disease progression (Dkhil et al., 2019). Both spleen white and red pulps increase in size due to the response of macrophage activation, which occurs in the presence of the parasite. Furthermore, trypanosomes release toxins that disrupt organs and cause cell injury (Bal et al., 2012). This has an impact on the structure and function of the spleen. Similar improvements to those produced by ELE treatment after infection with T. evansi in the spleen in this study were reported in mice treated with I. oblongifolia extract (Dkhil et al., 2019). DATA AVAILABILITY STATEMENT The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. Nitric oxide is measured for assessing immunological responses and oxidative stress levels (Chung et al., 2022). Activated macrophages create NO, which is harmful to a host of diseases, including Trypanosoma (Gazzinelli et al., 1992). Dkhil et al. (2021b) reported brain oxidative damage due to an increase in NO levels in mice infected with T. evansi. Increased NO production was reported in the brains of T. brucei-infected mice, suggesting that NO and its derivatives’ cytotoxicity may cause brain abnormalities (Keita et al., 2000). In our study, we found that ELE acted as an antioxidant agent that reduced oxidative damage in the brain (Nasr et al., 2019). AUTHOR CONTRIBUTIONS MD, EA-S, RA-G, and SA-Q designed the study. MD, EA-S, RA-G, and FT carried out the experiments and analyzed the data. All authors wrote and revised the manuscript. Chung, C. W., Liao, B. W., Huang, S. W., Chiou, S. J., Chang, C. H., Lin, S. J., et al. (2022). Magnetic Responsive Release of Nitric Oxide from an MOF-Derived Fe3O4@PLGA Microsphere for the Treatment of Bacteria-Infected Cutaneous Wound. ACS Appl. Mater. Interfaces 5, 6343–6357. doi: 10.1021/acsami.1c20802 Ciarlone, A. E. (1978). Further modification of a fluorometric method for analyzing brain amines. Microchem. J. 23, 9-12. doi: 10.1016/0026-265X(78)90034-6 Berlin, D., Loeb, E., and Baneth, G. (2009). Disseminated central nervous system disease caused by Trypanosoma evansi in a horse. Vet. Parasitol. 161, 316–319. doi: 10.1016/j.vetpar.2009.01.030 Biswas, D., Choudhury, A., and Misra, K. K. (2010). Histopathology of Trypanosoma (Trypanozoon) evansi infection in bandicoot rat. II Brain and choroid plexus. Proc. Zool. Soc. 63, 27–37. doi: 10.1007/s12595-010-0004-6 Berlin, D., Loeb, E., and Baneth, G. (2009). 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Dis. 2009:190354. doi: 10.1155/2009/190354 Conflict of Interest: 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. Habila, N., Agbaji, A. S., Ladan, Z., Bello, I. A., Haruna, E., Dakare, M. A., et al. (2010). Evaluation of In Vitro Activity of Essential Oils against Trypanosoma brucei Astrolabe and Trypanosoma evansi. J. Parasito. Res. 2010:534601. doi: 10.1155/2010/534601 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. 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Efficacy of Cymelarsan and Diminasan against Trypanosoma equiperdum infections in mice and horses. Vet. Parasitol. 171, 200–206. doi: 10.1016/j.vetpar.2010.03.041 Herr, N., Bode, C., and Duerschmied, D. (2017). The effects of serotonin in immune cells. Front. Cardiovasc. Med. 4:48. doi: 10.3389/fcvm.2017.00048 Copyright © 2022 Dkhil, Al-Shaebi, Abdel-Gaber, Alkhudhayri, Thagfan and Al- Quraishy. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Joshi, P. P., Shegokar, V. R., Power, R. M., Herder, S., Katti, R., Salkar, H. R., et al. Joshi, P. P., Shegokar, V. R., Power, R. M., Herder, S., Katti, R., Salkar, H. R., et al. (2005). Human trypanosomiasis caused by Trypanosoma evansi in India: the first case report. Am. J Trop. Med. Hyg. 73, 491–495. doi: 10.4269/ajtmh.2005. 73.491 Kabiru, Y. A., Ogbadoyi, E. O., Okogun, J. I., Gbodi, T. A., and Makun, H. A. (2013). Anti-trypanosomal potential of Eucalyptus camaldulensis. Br. J. Pharmacol. Toxicol. 4, 25–32. doi: 10.19026/bjpt.4.5374 March 2022 | Volume 13 | Article 833520 Frontiers in Microbiology | www.frontiersin.org 9
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Digital education: cultural aspects of communication in the modern educational environment
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ERROR: type should be string, got "https://doi.org/10.1051/e3sconf/202127312104 https://doi.org/10.1051/e3sconf/202127312104 E3S Web of Conferences 273, 12104 (2021)\nINTERAGROMASH 2021 * Corresponding author: lncs@springer.com Digital \neducation: \ncultural \naspects \nof \ncommunication in the modern educational \nenvironment \nInna Zilberova*, Marina Alyaeva, Fotina Shuraeva, and Konstantin Petrov \nDon State University, 344000, Rostov-on-Don, Russia Abstract. The article presents an analysis of modern approaches to the \nconcepts of \"information and educational environment\" and\" digital \neducational environment\", in which modern students - future bachelors-\ndevelop competencies in the field of intercultural communication. Special \nattention is paid to the consideration of the features of professional \ntraining, as well as the requirements for future specialists. The problems of \nmodern digital education closely follow from the problems of verbal \ninteraction of communicants in the educational environment. The \nconsidered issues of updating the needs for an educational product cannot \nbe separated from the realization of the possibilities of didactic and \nmethodological support for digital education, which is realized in the \ncommunicative interaction of a student and a teacher. The authors \nconducted a number of experimental studies in the field of studying the \nneeds of students in educational services and the opportunities for teachers \nto effectively realize their educational potential. The authors offer \nrecommendations for improving educational technologies in the context of \ndigitalization, improving the technical and communicative literacy of \nteachers, conducting trainings and trainings aimed at increasing the stress \ntolerance of teachers, providing them with comfortable working conditions \nin remote mode, modernizing the system of technical regulation in the \nprocess of providing educational activities, conducting explanatory work \nwith students, motivating them to contact work in remote access, \nimproving the culture of group and interpersonal communication © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative\nCommons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). 2 Materials and methods When increasing the activity of the interaction between people, in terms of improving the \ntechnical means of communication formed a new type of culture - the culture of virtual \ncommunication. Characterizing this phenomenon, we can distinguish its basic properties: \nnon-linear mode of existence of the communicating subject, virtualization of public \nrelations, the increasing role of social institutions that form the space of virtual \ncommunications, etc. This study examines the problems of the correlation of contemporary culture and \ncommunication in the digital educational environment [1]. The cultural and economic basis of the civilization development has recently become \nthe subject of hard discussions. Does culture represent the same intangible value as other \nobjects that have an immaterial value, and what is the place of culture in the information \nworld? The abundance of educational programs, blogs, courses, distance education among \nuniversities and private resources raise questions “what to teach and how to teach” in a \ncompletely different plane. In the modern world, we can talk about the existence of an \nentire economy of virtual educational services, including technical profile. There is a \nnecessity for effective interaction between the student and the teacher in the context of \nremote education. In our study, we can defined necessity as the need for something, \nwithout which the natural life of a person and his formation as a member of society are\nimpossible. This definition helps us to understand the objective essence of the necessity, its \nobjective expression. However, any necessity also has a subjective expression, determined by the impact of \nneeds on the subject (personality, collective, society and its organizations), on biosocial, \nintellectual-cognitive, aesthetic-emotional, moral and other spheres of his life and which \nfinds expression in the form of a natural reaction subject - a feeling of lack or unwanted \nexcess of something [2]. Consequently, if we ignore instinctive reactions that cannot be influenced, then the \nessence of subjective expression consists in awareness, understanding, etc., of a given \nnecessity. This, as a rule, finds an active reflection of the personal position, expressed in \nspeech behavior and reactions to certain material or moral stimulus. Personal priorities may be in conflict with the public need. Public need corresponds to \nthe objective and subjective sides of the need. If a real, natural or public demand for \nsomething exists, but is not realized by the subjects, then we cannot talk about the need. 1 Introduction The increase of digitization in the economy predetermines the enlargement the volume of \nvirtual communication. In turn, this presupposes the formation of new forms of social and \nverbal human behavior, the revision of values and categories that determine interaction in \nsociety. Communication flows penetrating into all spheres of human activity make virtual \ninteraction massive and significant. On the one hand, the emerging culture requires \nreflection within the framework of culture-philosophical analysis - testing technologies for \ntheir cultural consistency and value in the aspect of analyzing the fundamental problems of \ntransforming the human being and society, because it is formed not one - common to all © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative\nCommons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). E3S Web of Conferences 273, 12104 (2021)\nINTERAGROMASH 2021 E3S Web of Conferences 273, 12104 (2021)\nINTERAGROMASH 2021 https://doi.org/10.1051/e3sconf/202127312104 reality, but multitude. On the other hand, it calls for the renewal of the culture-philosophical \nknowledge itself and the ways of its translation into the scientific and educational \nenvironment, taking into account the possibilities and requirements of new technological \nrealities. E3S Web of Conferences 273, 12104 (2021)\nINTERAGROMASH 2021 In contemporary digital education, there are signs of the formation of the follow\nneeds in the educational system “teacher-student”: y\nthe universality of the individual needs, including the factors of choice and freedom; а) the universality of the individual needs, including the factors of choice and freedom; b) the full development of all creative abilities of a person [13]; c) the all-round development of individuals ceases to be a means, but becomes a prim\ngoal, becomes a measure of the economic and social development of society; d) free self-affirmation of the person as a member of society [14-15]; e) free self-realization of personality in professional and other spheres. e) free self-realization of personality in professional and other spheres. The criterion of rationality (irrationality) of educational needs is: e) free self-realization of personality in professional and other spheres. The criterion of rationality (irrationality) of educational needs is: )\np\ny\np\np\nThe criterion of rationality (irrationality) of educational needs is: y\ny\nа) its qualitative aspect (primary definition), that is, does this need reflect the objective\nnecessary professional, cultural and social conditions of a person and trends in their\ndevelopment; а) its qualitative aspect (primary definition), that is, does this need reflect the objective \nnecessary professional, cultural and social conditions of a person and trends in their \ndevelopment; b) its quantitative aspect (secondary definition), i.e., to what extent this need corresponds \n(or does not correspond) to the objective need for education, mastering competencies, \nskills, etc. 2 Materials and methods Subjective expression of necessity, that is, a certain idea of the need for something or even \na feeling of lack (undesirable excess) of something, can exist relatively independently of \nthose primary objective conditions that contributed to the emergence of the need. If, \nhowever, the subjective expression of needs not related to its objective character and if the \nfirst does not take into account the restrictions imposed on needs by the reproduction \nprocess, then we cannot talk about social needs, although they can declare themselves as \nsuch really and sometimes very intensely [3-5]. The mismatch between the needs of the system and its environment is overcome bas\non: \u0002\nchanging the environment by increasing the production of material products and \nservices, changing the mode of production and the way of consumption in this case by \ncreating a digital educational product. At the same time, it is assumed that need is an \u0002\nchanging the environment by increasing the production of material products and \nservices, changing the mode of production and the way of consumption in this case by \ncreating a digital educational product. At the same time, it is assumed that need is an 2 E3S Web of Conferences 273, 12104 (2021)\nINTERAGROMASH 2021 E3S Web of Conferences 273, 12104 (2021)\nINTERAGROMASH 2021 https://doi.org/10.1051/e3sconf/202127312104 existing ideal image of the future, desired consumption, it is a generalized internal \ndriving force of consumption, in our case - the desire of learning to expand one's own \ncapabilities; existing ideal image of the future, desired consumption, it is a generalized internal \ndriving force of consumption, in our case - the desire of learning to expand one's own \ncapabilities; \u0002\nadjusting needs in the course of their social verification in a given system of public \nrelations, that is, a qualitative transition to the level of virtual reality, the objectification \nof spatial and personal properties. \u0002\nadjusting needs in the course of their social verification in a given system of public \nrelations, that is, a qualitative transition to the level of virtual reality, the objectification \nof spatial and personal properties. Public need as a category has a complex character; its various sides are expressed in \nobjective and subjective dimensions and find themselves in constant internal contradiction\n[6]. If the “external”, objective, expression of the need has been determined, and the \n“internal” has not yet taken shape, then it is premature or ineffective to convince the subject \nof the need for this necessity. Therefore, first, it is necessary to create conditions in the field \nof the formation of subjective needs. If the subjective, “internal”, state has already been formed, and the objective, \n“external”, has not yet matured, then this can lead to a loss of internal incentives to satisfy \nthis need, and then either there will be a reorientation to other needs, or even an \nunreasonable dissatisfaction will appear. In education, this is fixed in the form of the \nimpossibility of realizing the acquired knowledge or skills on the one hand, and on the \nother hand, in the formation of a cognitive barrier that prevents a person from learning from \nthe student's side [7-9]. What explains the subjectivity in the approach to educational needs? In our opinion, the \nreason for subjectivity lies in the confusion of the two sides of the issue. On the one hand, \nall anthropological needs must be taken into account. On the other hand, the objective \nnature of needs lies in the fact that social-production relations and the economic basis, \nwhich determine the conditions for the formation and satisfaction of needs, objectively \ndetermine them [10-12]. 3 Results and discussion The problems of modern digital education closely follow from the problems of verbal \ninteraction of communicants in the educational environment. The considered issues of \nactualizing the needs for an educational product cannot be separated from the \nimplementation of the possibilities of didactic and methodological support of digital \neducation, which is implemented in the communicative interaction of a student and a \nteacher. 3 3 E3S Web of Conferences 273, 12104 (2021)\nINTERAGROMASH 2021 https://doi.org/10.1051/e3sconf/202127312104 Today, the main attention and time of the teacher's educational work is concentrated on \nproviding students with data, familiarizing them with information, transferring knowledge \nand forming their understanding. As the study shows, there is a definite relationship \nbetween the presence of a perlocutionary effect (the presence of feedback) and the \neffectiveness of distance learning (Fig.1). Fig. 1. The mastering competencies on bachelor's programs in the direction 08.03.01 Construction in \n% from 100% competence based on the results of control and cut works during the period of distance \nlearning (Development of the authors) Fig. 1. The mastering competencies on bachelor's programs in the direction 08.03.01 Construction in \n% from 100% competence based on the results of control and cut works during the period of distance \nlearning (Development of the authors) The diagram of the development of competencies shows that the result in the studied \nperiod is quite low, and not a single competence is mastered by 50% of the possible \nmaximum. At the same time, the distribution of mastering among students shows other \ndependencies: Fig. 2. Mastering the total number of students on all competencies for bachelor`s programs in the \ndirection 08.03.01 Construction in% of 100% competence (Development of the authors)\n0\n5\n10\n15\n20\n25\n30\n35\n40\nMastered\nby 10%\nMastered\nby 30%\nMastered\nby 50%\nMastered\nby 80%\nMastered\nby 100%\n% of students\n% of students Fig. 2. Mastering the total number of students on all competencies for bachelor`s programs in the \ndirection 08.03.01 Construction in% of 100% competence (Development of the authors) Thus, the number of learners who have mastered the program is 75.2% of the total \nnumber of students who took part in the study. This shows us that the students received \nsome knowledge in the professional field, but this knowledge requires further improvement \nfor full professional implementation. E3S Web of Conferences 273, 12104 (2021)\nINTERAGROMASH 2021 6. Historical sciences\n1 priority -0%\n2 priority -7%\n3 priority -93%\n7. Philosophical sciences\n1 priority -0%\n2 priority -4%\n3 priority -96%\n8. Foreign language\n1 priority -22%\n2 priority -8%\n3 priority -70% As the analysis of the results shows, students prioritize purely practical skills in \naccordance with their personal ideas about the essence of the industry. Comparing these data with the results of teachers' feedback about the effectiveness of \nspeech communication (Fig. 4), we can draw conclusions about the patterns of the results of \nmastering the competencies of students, implemented as feedback. Fig. 4. Results of a questionnaire survey on the problems of teacher communication in a distance-\nlearning environment (Development of the authors)\n0\n5\n10\n15\n20\n25\n30\n35\n40\nTechnical\ninterference\nProblems with\nestablishing\ncommunication\nin the native\nlanguage\nAbsence of\nstudent`s\ninterest\nDifficulties with\npresenting\ninformation in a\nremote form in\nan accessible\nway, according\nto the teacher\nPsychological\ndiscomfort\nReviews of teachers Fig. 4. Results of a questionnaire survey on the problems of teacher communication in a distance-\nlearning environment (Development of the authors) Fig. 4. Results of a questionnaire survey on the problems of teacher communication in a distance-\nlearning environment (Development of the authors) The main problems, according to teachers, are difficult communication with foreign \nstudents. The lack of interest among students, difficulty in presenting information in a \ndistance mode is in second place in terms of reviews. Among the teachers` comments to the \nquestionnaire can be found the following comments: “It's hard to tell when you are here and \nthey are at home”; “Many sit imposingly lounging, some just sleep during the lecture”. These remarks indicate a low degree of teacher-student contact when the teacher is \nperforming duties and the student is serving voluntary service. In this case, the educational \nprocess does not affect in any way on the acquisition of the necessary knowledge and the \nsatisfaction of educational needs. 3 Results and discussion These data can be compared with research data on the development of competencies in \nthe study of disciplines in a full-time classroom work format. 4 E3S Web of Conferences 273, 12104 (2021)\nINTERAGROMASH 2021 https://doi.org/10.1051/e3sconf/202127312104 INTERAGROMASH 2021 Fig. 3. The mastering competencies on bachelor's programs in the direction 08.03.01 (Development \nof the authors)\n0\n10\n20\n30\n40\n50\n60\n70\n80\n1st year\n2nd year\n3rd year\n4th year\nGeneral cultural competences\nGeneral professional competencies\nProfessional competence: prospecting\nProfessional competence: production and technological\nProfessional competence: experimental and research Fig. 3. The mastering competencies on bachelor's programs in the direction 08.03.01 (Development \nof the authors) Construction in % from 100% competence based on the results of control and cut works \nduring the period of full-time classroom training As the research data show, in full-time classroom training, the percentage of mastering \ncompetencies is above average, except for the first year of study. The low percentage of \neducation shows only the development of common cultural competence. An analysis of students` necessities, carried out through a questionnaire survey, showed \nthe following differentiation of educational requests: able 1. Determining the priorities of students' needs in the development of areas of the educational \nprogram Table 1. Determining the priorities of students' needs in the development of areas of the educational\nprogram\nName of the mastered area\nPercentage specified priority\n1. Engineering surveys, design of buildings \nand constructions;\n1 priority – 74%\n2 priority - 12%\n3 priority – 14%\n2. Engineering support and equipment of \nconstruction sites and urban areas;\n1 priority - 65%\n2 priority -17%\n3 priority- 18%\n3. Application of machinery, equipment and \ntechnologies for building works;\n1 priority -54%\n2 priority -42%\n3 priority -4%\n4. Business and production management in \nthe building sector;\n1 priority -76%\n2 priority -23%\n3 priority -1%\n5. Technical and environmental safety in the \nbuilding sector\n1 priority -17%\n2 priority -36%\n3 priority -47% Table 1. Determining the priorities of students' needs in the development of areas of the ed e 1. Determining the priorities of students' needs in the development of areas of the educational \nprogram 5 https://doi.org/10.1051/e3sconf/202127312104 E3S Web of Conferences 273, 12104 (2021)\nINTERAGROMASH 2021 E3S Web of Conferences 273, 12104 (2021)\nINTERAGROMASH 2021 https://doi.org/10.1051/e3sconf/202127312104 1. Improving the technical and communication literacy of teachers. Conducting coaching \nand training courses designed to increase the resistance to stress of teachers, to provide \nthem with comfortable working conditions in a distance mode. 1. Improving the technical and communication literacy of teachers. Conducting coaching \nand training courses designed to increase the resistance to stress of teachers, to provide \nthem with comfortable working conditions in a distance mode. 2. Modernization of the technical regulation system in the process of providing educational \nactivities. 2. Modernization of the technical regulation system in the process of providing educational \nactivities. 3. Carrying out explanatory work with students, motivating them to contact work in \nconditions of remote access, increasing the culture of group and interpersonal \ncommunication. 4 Conclusion Summing up, as recommendations for improving educational technologies in the context of \ndigitalization, the authors suggest: 6 6 6 E3S Web of Conferences 273, 12104 (2021)\nINTERAGROMASH 2021 Electronic edition. Ser. \"Advances in Social Science, Education and Humanities \nResearch\" 14-18 (2018). 15. K. Gudkova, Cognitive-Pragmatic Analysis of Argumentation in the Analytical Paper \nArticle (on the British press matter), PhD Thesis, St. Petersburg (2009). 16. M.Yu. Seyranyan, Conflict discourse: sociolinguistic and pragmalinguistic aspects,\nmonograph, Moscow, 2012. References 1. E.V. Gryazeva, Social partnership as one of the conditions for quality training of \nspecialists, Modern trends in the management of industrial innovative organizations: \ncollection of scientific articles, region. Scientific and practical conf. with internat. participation (Alatyr, September 28-29, 2017), Cheboksary, Chuvash Publishing House\nun-ta, 2017. 2. V.G. Kvachev,\nI.M. Divilkovskiy,\nSocial resources of modernization,\nState \nmanagement: The Russian Federation in the modern world, Proceedings of the KHGG \nInternational Conference. (2015) 105-109. 3. S.B. Kotlyarov, E.A. Chicherov, Law and education 7, 117-122 (2017). 4. A.N. Makarevich, VestnikNovSU 74, 53-56 (2013). 5. M.A. Panfilov, S.B. Kotlyarov, E.A. Chicherov, Organizational forms of interaction \nbetween government and business at the regional level, Professional of the Year 2016: \ncollection of articles of the International Scientific and Practical Competition, Under \nthe general editorship of G.Y. Gulyaev, Penza, ICNS \"Science and Education\" (2016)\n80-84. 6. O.A. Podolsky, V.A. Pogozhina, Humanitarian studies 1, 96-103 (2016). 7. N.V. Potekhina, Yu.I. Shulinina, Theory and practice of social development 3, 2016. Information \non \nhttps://cyberieninka.ru/article/n/ \nvzaimosvyaz-tehnologicheskih-\nukladov-i-obrazovaniya-kak-chasti-chelovecheskogo-kapitala (accessed 10.09.2018). 8. N.Yu. Nevraeva, A.M. Kabanov, N.Kh. Savel'eva, N.I. Sorokina, Review of \npedagogical research 2 (3), 105-111 (2020). 9. C.M. Woodward, A History of the St. Louis Bridge (St. Louis, G. I. Jones, 1881). 10. F. van Eemeren, P. Houtlisse,\nArgumentation and Rationality, The Though: \nArgumentation 6, 7-22 (2006). 11. T. Volosovets, I, Kirillov, and A. Buyanov, Procedia - Social and Behavioral Sciences \n237, 1299-1303 (2017). doi: 10.1016/j.sbspro.2017. 02.213. 12. O.V. Kliuchnikova, O.A. Pobegaylov, Procedia Engineering 150, 2168-2172 (2016). http://www.sciencedirect.com/science/journal/18777058. doi: \n10.1016/j.proeng.2016.07.259 13. E. Shirina, G. Myasischev, The degrading value-based orientations of the linguistic \npersonality in the infomedia of the contemporary internet public communication,\nProceedings of the International Conference on the Theory and Practice of Personality \nFormation in Modern Society (ICTPPFMS 2018). Electronic edition. Ser. \"Advances \nin Social Science, Education and Humanities Research\" 4-7 (2018). 14. O. Gaibaryan G. Myasischev, Proceedings of the International Conference on the \nTheory and Practice of Personality Formation in Modern Society (ICTPPFMS 2018). 7 7 7 E3S Web of Conferences 273, 12104 (2021)\nINTERAGROMASH 2021 E3S Web of Conferences 273, 12104 (2021) https://doi.org/10.1051/e3sconf/202127312104 INTERAGROMASH 2021 15. K. Gudkova, Cognitive-Pragmatic Analysis of Argumentation in the Analytical Paper \nArticle (on the British press matter), PhD Thesis, St. Petersburg (2009). 16. M.Yu. Seyranyan, Conflict discourse: sociolinguistic and pragmalinguistic aspects,\nmonograph, Moscow, 2012. 16. M.Yu. Seyranyan, Conflict discourse: sociolinguistic and pragmalinguistic aspects,\nmonograph, Moscow, 2012. 8 8"
https://openalex.org/W2541305580
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Evaluation of Subsequent Heat Treatment Routes for Near-β Forged TA15 Ti-Alloy
Materials
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cc-by
12,754
Zhichao Sun *, Huili Wu and He Yang State Key Lab of Solidification Processing, Department of Materials Science and Engineering, Northwestern Polytechnical University, Xi’an 710072, China; 18729241566@163.com (H.W.); yanghe@nwpu.edu.cn (H.Y.) * Correspondence: zcsun@nwpu.edu.cn; Tel.: +86-29-8846-0212 (ext. 802); Fax: +86-29-8849-5632 Academic Editor: Shankar M.L. Sastry Received: 14 August 2016; Accepted: 21 October 2016; Published: 26 October 2016 Abstract: TA15 Ti-alloy is widely used to form key load-bearing components in the aerospace field, where excellent service performance is needed. Near-β forging technology provides an attractive way to form these complicated Ti-alloy components but subsequent heat treatment has a great impact on the final microstructure and mechanical properties. Therefore evaluation and determination of the heat treatment route is of particular significance. In this paper, for the near-β forged TA15 alloy, the formation and evolution of microstructures under different subsequent heat treatment routes (annealing, solution and aging, toughening and strengthening) were studied and the cooling mode after forging was also considered. Then, the type and characteristics of the obtained microstructures were discussed through quantitative metallographic analysis. The corresponding mechanical properties (tensile, impact toughness, and fracture toughness) and effects of microstructural characteristics were investigated. Finally, for a required microstructure and performance a reasonable heat treatment route was recommended. The work is of importance for the application and development of near-β forging technology. Keywords: TA15 Ti-alloy; near-β forging; heat treatment; microstructural characteristic; mechanical properties Article Evaluation of Subsequent Heat Treatment Routes for Near-β Forged TA15 Ti-Alloy Zhichao Sun *, Huili Wu and He Yang materials materials materials 1. Introduction Therefore for the near-β forged TA15 alloy, evaluating and determining the matched heat treatment route are very important for control of secondary αs and equiaxed αp, and further to meet the required performance. p For Ti-alloy, the one-step heat treatment (annealing), dual or multiple heat treatment routes (solution and aging, toughening and strengthening) after forging were applied [1,3,6,8,9,15]. According to the role and temperature of heat treatment for Ti-alloy, two types of annealing treatments, namely high temperature annealing and low temperature annealing, are defined. High temperature annealing refers to the treatment process whose heating and holding temperature is higher than the recrystallization starting temperature of the used Ti alloy, and whose purpose is to eliminate work hardening effect. While low temperature annealing, which is used to remove stress in forgings or stabilize the microstructure, denotes the annealing process with a heating and holding temperature lower than the recrystallization starting temperature. For annealing in Ti alloy, most of the existing works focused on the one-step annealing treatment. The near-β forging was not involved before it. Moreover variations in grain size and volume fraction of equiaxed αp, and thickness of lamellar αs were investigated by changing annealing temperature [15,16], holding time [17], and cooling modes [18–21]. Generally, solution and aging, and toughening and strengthening after forging were adopted to obtain a certain kind of microstructure in a near-α or α + β alloy. Meanwhile the functions of each step heat treatment can be performed. In recent years, the dual or multiple heat treatment routes for Ti-alloy were considered by Zhu et al. [22,23] and Sun et al. [24], but the forging before heat treatment was similarly not involved in these literatures. By considering the nearing-β forging before subsequent heat treatments, Zhou et al. [3] obtained a tri-modal microstructure in TC11 through near-β forging combined with subsequent high temperature toughening and low temperature strengthening. The volume fraction of equiaxed αp was controlled to about 10% and 15% through near-β forging and increased to about 20% during subsequent heat treatments. A certain number and aspect ratio of lamellar αs originating from the decomposition of the martensite formed during water quenching after forging, were obtained during toughening, and thin Widmanstätten α precipitated from β matrix during strengthening. 1. Introduction Ti-alloys, such as TA15, have been widely used to form key load-bearing components in the aerospace field, which are typical hard-to-form components due to the complex in shape and hard-to-deform Ti-alloys used [1]. Furthermore, in order to meet the extreme service requirements for these components, high macro-forming precision and particularly excellent mechanical properties are needed [2]. Near-β forging technology, in which the billet and die are heated to a relatively high temperature of 15–20 ◦C below the β transus (for TA15 alloy it is 980–985 ◦C) so as to reduce the material flow stress and improve the plasticity and cavity filling capacity, provides an attractive way to form these complicated Ti-alloy components [1,3]. The mechanical properties of Ti-alloy components are determined by the final microstructure, in general which consists of the primary equiaxed α phase (αp), secondary α phase (αs), and transformed β matrix [4]. The yield strength and ultimate tensile strength of Ti-6Al-4V alloy increase with the increase of the volume fraction of the equiaxed αp [5], and an increase of αp improves the impact toughness and plasticity within the volume fraction of 20% [3]. The volume fraction of primary equiaxed αp is related to the highest heating and holding temperature in forging or subsequent heat treatment. The secondary αs determines the fracture toughness, creep properties, and fatigue crack propagation behavior [6,7], thus it is very important for equipment reliability [8,9]. The increase of Materials 2016, 9, 872; doi:10.3390/ma9110872 www.mdpi.com/journal/materials www.mdpi.com/journal/materials Materials 2016, 9, 872 2 of 13 volume fraction and thickness of lamellar αs would improve the tensile strength, creep properties, and fracture toughness [6]. However the secondary αs forms mainly during cooling after forging or the subsequent heat treatment process. Depending on the nucleation positions, the αs can be classified as grain boundary α (αGB), grain boundary Widmanstätten α (αWGB), which nucleated on the formed αGB, and intragranular Widmanstätten α (αWI), which nucleated in the β grains [10,11]. The formation and evolution of αs are very complex due to a variety of growth behaviors [12], diverse variants and morphologies, and growth or decomposition during thermal processing [13]. The variant selection (VS) of αs is through complicated interplay between the chemical driving force (alloy chemistry, cooling rate and holding temperature) and the interfacial energy between α and β grain, etc. [14]. 1. Introduction The toughening temperature is neither too high nor too low in the two-phase field, otherwise the lamellar αs meeting the requirements in the tri-modal microstructure can not be achieved. Sun et al. [25] also got a tri-modal microstructure in TA15 alloy through near-β forging (air cooling (AC) + water quenching (WQ)) combined with solution and aging. In this method, the other two constituent phases in the tri-modal microstructure, lamellar αs and transformed β matrix, were formed during solution, and the aging was only used to stabilize the microstructure. Therefore the solution temperature must be appropriate, and the aging temperature should be much lower than the recrystallization temperature. However for the TA15 alloy components after near-β forging, it has still not been resolved as to what kind of subsequent heat treatment route should be adopted. Although the volume fraction of equiaxed αp was mainly determined by near-β forging in these methods [3,25], its content, morphology and distribution vary during cooling and subsequent heat treatment. The nucleation and growth of the secondary αs depend on the whole hot processing (forging, cooling, and heat treatment). In addition to the grain boundaries, intracrystalline places with high-energy defects (sub-grain boundaries, dislocations) caused by deformation provide the possible 3 of 13 Materials 2016, 9, 872 nucleation sites for αs, which affect not only the nucleation rate of αs, but also atomic migration [26,27]. The subsequent heat treatment provides the driving force for precipitation and growth of αs. Thus, revealing the formation and evolution of primary equiaxed αp and secondary αs under combined action of near-β forging and subsequent heat treat routes is the first problem to be solved. In this paper, for the near-β forged TA15 alloy, the microstructure evolution under different heat treatment routes (annealing, solution and aging, toughening and strengthening) was studied, then the difference in microstructure and the corresponding mechanical properties were discussed. The work is of importance for determination of a reasonable heat treatment route matched with near-β forging. 2.1. Starting Materials The TA15 Ti-alloy used in the experiments was from Western Superconducting Technologies Inc. (Xi’an, China). The chemical composition of the as-received material with a β transus temperature of 980–985 ◦C is listed in Table 1, and its microstructure consists of about 60% (volume fraction was measured by the area fraction of the equiaxed αp in microstructure) equiaxed αp, with an average diameter of 11 µm (average length of lines passing through the centroid of αp per 2 degrees), and residual transformed β matrix, as shown in Figure 1a. The blank was processed into cylindrical specimens of Φ10 mm × 15 mm with 0.2 mm-deep shallow slots on the ends to store glass lubricant. Table 1. Chemical composition of TA15 Ti-alloy. Element Al Zr Mo V Si C Fe O N H Ti Nominal composition/(wt %) 6.63–6.75 2.23–2.27 1.73–1.80 2.24–2.27 <0.04 <0.006 0.14 0.12 <0.002 0.002 Balance 2 2 Experimental Procedure Table 1. Chemical composition of TA15 Ti-alloy. Element Al Zr Mo V Si C Fe O N H Ti Nominal composition/(wt %) 6.63–6.75 2.23–2.27 1.73–1.80 2.24–2.27 <0.04 <0.006 0.14 0.12 <0.002 0.002 Balance 2.2. Experimental Procedure Table 1. Chemical composition of TA15 Ti-alloy. Table 1. Chemical composition of TA15 Ti-alloy. 2.2. Experimental Procedure 2.2. Experimental Procedure Before near-β forging, the specimens were heated to 970 ◦C at a heating rate of 5 ◦C/s and held to obtain a homogeneous microstructure with a holding time of 5 min/mm in diameter. According to the actual forging production, a strain rate of 0.1 s−1 was selected and the reduction of height was 40%, 60%, or 65%. After the forging, three kinds of cooling methods were adopted, air cooling (AC), water quenching (WQ), and a short time of AC combined with WQ (AC + WQ, air cooled to about 835 ◦C and then water quenched), which was used to consider the temperature drop during forging transfer—the forgings were transferred from press to water tank for quenching. Three kinds of subsequent heat treatment routes, annealing (low-temperature annealing, LTA, high-temperature anneal, HTA), solution and aging (SA), and toughening and strengthening (TS) were considered; a detailed experiment scheme is given in Table 2. During subsequent heat treatment the same heating rate was adopted. After the experiment for the specimens the metallographic observation was implemented by an optical microscope (Leica, Wetzlar, Germany) and quantitative metallographic analysis was carried out using an Image-Pro Plus 6.0 image analysis system (Media Cybernetics, Silver Spring, MD, USA, 2006). Meanwhile the typical mechanical properties required by aeronautic forgings, such as room and high temperature tensile properties, impact toughness, and fracture toughness, were measured according to the industry standards—“Metal test method”, HB5143-80, HB5195-81, HB5144-80, HB5142-80 [28], respectively. 4 of 13 Materials 2016, 9, 872 Table 2. Experimental scheme and microstructural characteristics. No. 3. Results and Discussion Materials 2016 9 872 3.1. Microstructural Evolution of Near β-forged TA15 Alloy under Different Heat Treatment Routes 3. Results and Discussion Figure 1 shows the optical micrographs of TA15 alloy before (Figure 1a) and after near-β forging (970 ◦C/0.1 s−1/65%) and cooled by different modes (Figure 1b–d). The water quenched microstructure in Figure 1b (water quenching after near-β forging) exhibits about 13.5% equiaxed αp and residual martensite. A high cooling rate (higher than the critical cooling rate) resulted in martensite transformation and the reservation of the distortion energy and defects caused by forging. Air cooled for a short time and then water quenched after near-β forging, the microstructure, which is shown in Figure 1c, consists of equiaxed αp, grain boundary α (αGB), grain boundary Widmanstätten α (αWGB), intragranular Widmanstätten α (αWI) and residual martensite. β→α phase transformation occurred at a low cooling rate, and the precipitated α phase preferred to nucleate at β grain boundaries, then on the formed αGB and defects in β grains [20]. Only part of β transformed into α phase during the short time of air cooling, and the residual β phase converted to martensite due to a high cooling rate. A heterogeneous precipitation of αWGB within the same or different β grains can be observed in Figure 1c, which results from both the variation selection (VS) of the αGB and αWGB [14]. When air cooled, the microstructure (Figure 1d) shows a typical bimodal microstructure, a large amount of very thin αW (αWGB and αWI) with an interlaced distribution, a small amount of equiaxed αp and residual β, and very few αGB. Since there existed lots of defects in the β grains after forging, the αWI was the main component of secondary αs in Figure 1d. 3.1. Microstructural Evolution of Near β-forged TA15 Alloy under Different Heat Treatment Routes Figure 1 shows the optical micrographs of TA15 alloy before (Figure 1a) and after near-β forging (970 °C/0.1 s−1/65%) and cooled by different modes (Figure 1b–d). The water quenched microstructure in Figure 1b (water quenching after near-β forging) exhibits about 13.5% equiaxed αp and residual martensite. A high cooling rate (higher than the critical cooling rate) resulted in martensite transformation and the reservation of the distortion energy and defects caused by forging. 3. Results and Discussion Materials 2016 9 872 Air cooled for a short time and then water quenched after near-β forging, the microstructure, which is shown in Figure 1c, consists of equiaxed αp, grain boundary α (αGB), grain boundary Widmanstätten α (αWGB), intragranular Widmanstätten α (αWI) and residual martensite. β→α phase transformation occurred at a low cooling rate, and the precipitated α phase preferred to nucleate at β grain boundaries, then on the formed αGB and defects in β grains [20]. Only part of β transformed into α phase during the short time of air cooling, and the residual β phase converted to martensite due to a high cooling rate. A heterogeneous precipitation of αWGB within the same or different β grains can be observed in Figure 1c, which results from both the variation selection (VS) of the αGB and αWGB [14]. When air cooled, the microstructure (Figure 1d) shows a typical bimodal microstructure, a large amount of very thin αW (αWGB and αWI) with an interlaced distribution, a small amount of equiaxed αp and residual β, and very few αGB. Since there existed lots of defects in the β grains after forging, the αWI was the main component of secondary αs in Figure 1d. Figure 1. Optical micrographs of TA15 alloy after near-β forging: (a) Original optical micrograph of as received TA15; (b) 970 °C/0.1 s−1/65%/(WQ); (c) 970 °C/0.1 s−1/65%/(AC + WQ); (d) 970 °C/65%/0.1 s−1/(AC). WQ: water quenching, AC: air cooling. The optical micrographs of TA15 alloy after near β forging (AC or WQ) and subsequent Figure 1. Optical micrographs of TA15 alloy after near-β forging: (a) Original optical micrograph of as received TA15; (b) 970 ◦C/0.1 s−1/65%/(WQ); (c) 970 ◦C/0.1 s−1/65%/(AC + WQ); (d) 970 ◦C/65%/0.1 s−1/(AC). WQ: water quenching, AC: air cooling. Figure 1. Optical micrographs of TA15 alloy after near-β forging: (a) Original optical micrograph of as received TA15; (b) 970 °C/0.1 s−1/65%/(WQ); (c) 970 °C/0.1 s−1/65%/(AC + WQ); (d) 970 °C/65%/0.1 s−1/(AC). WQ: water quenching, AC: air cooling. Figure 1. Optical micrographs of TA15 alloy after near-β forging: (a) Original optical micrograph of as received TA15; (b) 970 ◦C/0.1 s−1/65%/(WQ); (c) 970 ◦C/0.1 s−1/65%/(AC + WQ); (d) 970 ◦C/65%/0.1 s−1/(AC). WQ: water quenching, AC: air cooling. low-temperature annealing (LTA) (830 °C/1 h/AC) are exhibited in Figure 2. 2.2. Experimental Procedure Forging Conditions Cooling Mode Heat Treatment Route Heat Treatment Condition Microstructural Type Microstructural Characteristic 1 970 ◦C/0.1 s−1/65% WQ - - - - 2 970 ◦C/0.1 s−1/65% AC + WQ - - - - 3 970 ◦C/0.1 s−1/65% AC - - Bimodal (A1) Very thin interlaced αW, small αp 4 970 ◦C/0.1 s−1/40% AC LTA 830 ◦C/1 h/AC Bimodal (A3) Thick clustered αW, smaller αp 5 970 ◦C/0.1 s−1/40% WQ LTA 830 ◦C/1 h/AC Bimodal (A4) Thin interlaced αW, smaller αp 6 970 ◦C/0.1 s−1/60% AC + WQ HTA 930 ◦C/1 h/AC Quasi tri-modal (B1) Short and thick αs, large αp 7 970 ◦C/0.1 s−1/65% WQ SA 930 ◦C/1 h/AC + 550 ◦C/5 h/AC Tri-modal (C1) Long and very thick αs, small αp 8 970 ◦C/0.1 s−1/60% AC + WQ SA 930 ◦C/1 h/AC + 550 ◦C/5 h/AC Quasi tri-modal (B2) Short and thick αs, large αp 9 970 ◦C/0.1 s−1/65% WQ TS 950 ◦C/100 min/WQ + 800 ◦C/8 h/AC Tri-modal (C2) Short and thicker αs, smaller αp 10 970 ◦C/0.1 s−1/60% AC + WQ TS 950 ◦C/1 h/WQ + 800 ◦C/5 h/AC Tri-modal (C3) Short and thicker clustered αs, very large αp 11 965 ◦C/0.1 s−1/65% AC NTS 970 ◦C/1.5 h/WQ + 850 ◦C/1.5 h/AC Bimodal (A2) Thin and long clustered αW, moderate αp WQ—Water quenching, AC—Air cooling, LTA—Low-temperature annealing, HTA—High-temperature annealing, SA—Solution and aging, TS—toughening and strengthening, NTS—Near-β toughening and strengthening. Table 2. Experimental scheme and microstructural characteristics. 5 of 13 Materials 2016, 9, 872 3. Results and Discussion Materials 2016 9 872 In general they had the same microstructural type as that in Figure 1d (near-β forging/AC), i.e., one step subsequent low-temperature annealing did not change the microstructural type that was obtained by near-β forging/AC. Seldom very small equiaxed α (marked by red ellipse in Figure 2) appeared due to globularization of αGB or static recrystallization of αp [13]. When air cooled after forging and The optical micrographs of TA15 alloy after near-β forging (AC or WQ) and subsequent low-temperature annealing (LTA) (830 ◦C/1 h/AC) are exhibited in Figure 2. In general they had the same microstructural type as that in Figure 1d (near-β forging/AC), i.e., one step subsequent low-temperature annealing did not change the microstructural type that was obtained by near-β forging/AC. Seldom very small equiaxed α (marked by red ellipse in Figure 2) appeared due to globularization of αGB or static recrystallization of αp [13]. When air cooled after forging and 6 of 13 Materials 2016, 9, 872 annealing, the relatively long and thick clustered αW appeared, as shown in Figure 2a. Forged and air cooled (Figure 1d) clustered αW preferentially nucleated and grew on the existing αGB due to a low supercooling degree [20], meanwhile most of the distortion energy caused by forging was consumed by the precipitation and growth of the αW during air cooling after forging. When annealed, some formed αW (little ones) dissolved, while some grew both in length and width direction during heating and holding. In succedent AC, the existing αW thickened a bit, and it already had lots of α phase (αW and αp) which consumed a large amount of α-stablized elements, hence only seldom αW was formed. Short and thin disordered αW presented in Figure 2b after near-β forging (WQ) combined with LTA. The distortion energy and defects kept by water quenching provided a large driving force and enough possible sites for nucleation and growth of αW in subsequent heat treatment [26]. Moreover they promote more variants of αW [14]. As a result lots of thin αW precipitated both on formed αGB (αWGB) and inside β grains (αWI) in a disordered manner during subsequent annealing. annealing, the relatively long and thick clustered αW appeared, as shown in Figure 2a. 3. Results and Discussion Materials 2016 9 872 Forged and air cooled (Figure 1d) clustered αW preferentially nucleated and grew on the existing αGB due to a low supercooling degree [20], meanwhile most of the distortion energy caused by forging was consumed by the precipitation and growth of the αW during air cooling after forging. When annealed, some formed αW (little ones) dissolved, while some grew both in length and width direction during heating and holding. In succedent AC, the existing αW thickened a bit, and it already had lots of α phase (αW and αp) which consumed a large amount of α-stablized elements, hence only seldom αW was formed. Short and thin disordered αW presented in Figure 2b after near-β forging (WQ) combined with LTA. The distortion energy and defects kept by water quenching provided a large driving force and enough possible sites for nucleation and growth of αW in subsequent heat treatment [26]. Moreover they promote more variants of αW [14]. As a result lots of thin αW precipitated both on formed αGB (αWGB) and inside  grains (αWI) in a disordered manner during subsequent annealing. Materials 2016, 9, 872 6 of 13 annealing, the relatively long and thick clustered αW appeared, as shown in Figure 2a. Forged and air cooled (Figure 1d) clustered αW preferentially nucleated and grew on the existing αGB due to a low supercooling degree [20], meanwhile most of the distortion energy caused by forging was consumed by the precipitation and growth of the αW during air cooling after forging. When annealed, some formed αW (little ones) dissolved, while some grew both in length and width direction during heating and holding. In succedent AC, the existing αW thickened a bit, and it already had lots of α phase (αW and αp) which consumed a large amount of α-stablized elements, hence only seldom αW was formed. Short and thin disordered αW presented in Figure 2b after near-β forging (WQ) combined with LTA. The distortion energy and defects kept by water quenching provided a large driving force and enough possible sites for nucleation and growth of αW in subsequent heat treatment [26]. Moreover they promote more variants of αW [14]. As a result lots of Figure 2. Optical micrographs of TA15 alloy after near-β forging and low-temperature annealing: (a) 970 °C/0.1 s−1/40%/AC + 830 °C/1 h/AC; (b) 970 °C/0.1 s−1/40%/WQ + 830 °C/1 h/AC. Figure 2. 3. Results and Discussion Materials 2016 9 872 Optical micrographs of TA15 alloy after near-β forging and low-temperature annealing: (a) 970 ◦C/0.1 s−1/40%/AC + 830 ◦C/1 h/AC; (b) 970 ◦C/0.1 s−1/40%/WQ + 830 ◦C/1 h/AC. thin αW precipitated both on formed αGB (αWGB) and inside  grains (αWI) in a disordered manner during subsequent annealing. Figure 2. Optical micrographs of TA15 alloy after near-β forging and low-temperature annealing: in αW precipitated both on formed αGB (αWGB) uring subsequent annealing. and inside  grains (αWI) in a disordered mann Figure 2. Optical micrographs of TA15 alloy after near-β forging and low-temperature annealing: (a) 970 °C/0.1 s−1/40%/AC + 830 °C/1 h/AC; (b) 970 °C/0.1 s−1/40%/WQ + 830 °C/1 h/AC. Figure 2. Optical micrographs of TA15 alloy after near-β forging and low-temperature annealing: (a) 970 ◦C/0.1 s−1/40%/AC + 830 ◦C/1 h/AC; (b) 970 ◦C/0.1 s−1/40%/WQ + 830 ◦C/1 h/AC. Fi u e 2 O ti al i o a h of TA15 alloy afte ea β fo i a d lo te e atu e a eali mode of WQ or AC + WQ combined solution + aging (SA, 930 °C/1 h/AC + 550 °C/5 h/AC). For the near-β forged (970 °C/0.1 s−1/65%/WQ) specimen, the typical tri-modal microstructure was obtained (Figure 3a) after SA (930 °C/1 h/AC + 550 °C/5 h/AC), consisting of small equiaxed αp, thick and long secondary lamellar αs, and transformed β matrix. Its formation process was as follows. When heated at 930 °C after forging, the existing martensite decomposed to form secondary αs and β phases. A larger amount of αs nucleated at β grain boundaries (GBs) and in β grains simultaneously and grew rapidly. As holding time was prolonged, some thin αs dissolved and others thickened through merging [29]. In subsequent AC, the undissolved αs thickened further, and tertiary lamellar αs precipitated from β to form the transformed β matrix. Figure 3a,b display the optical micrographs of TA15 alloy through near-β forging with a cooling mode of WQ or AC + WQ combined solution + aging (SA, 930 ◦C/1 h/AC + 550 ◦C/5 h/AC). For the near-β forged (970 ◦C/0.1 s−1/65%/WQ) specimen, the typical tri-modal microstructure was obtained (Figure 3a) after SA (930 ◦C/1 h/AC + 550 ◦C/5 h/AC), consisting of small equiaxed αp, thick and long secondary lamellar αs, and transformed β matrix. Its formation process was as follows. 3. Results and Discussion Materials 2016 9 872 Optical micrographs of TA15 alloy after near-β forging and solution + aging treatment: (a) 970 °C/0.1 s−1/65%/WQ + 930 °C/1 h/AC + 550/5 h/AC; (b) 970 °C/0.1 s−1/60%/(AC + WQ) + 930 °C/1 h/AC + 550 °C/5 h/AC; (c) 970 °C/0.1 s−1/60%/(AC + WQ) + 930 °C/1 h/AC. Figure 3. Optical micrographs of TA15 alloy after near-β forging and solution + aging treatment: (a) 970 ◦C/0.1 s−1/65%/WQ + 930 ◦C/1 h/AC + 550/5 h/AC; (b) 970 ◦C/0.1 s−1/60%/(AC + WQ) + 930 ◦C/1 h/AC + 550 ◦C/5 h/AC; (c) 970 ◦C/0.1 s−1/60%/(AC + WQ) + 930 ◦C/1 h/AC. A quasi tri modal microstructure, including coarse equiaxed αp, scattered thick lamellar αs, and very thin tertiary αWI, was obtained after near-β forging (AC + WQ, air cooled for a short time and then water quenched) and SA treatment, as shown in Figure 3b. Figure 3c illustrates the optical micrograph without subsequent aging. When heated and held at 930 °C the driving force for nucleation of αs, which originated from the decomposition of the martensite, was insufficient because it was consumed partially during the short time of AC after forging, and the formed αs thickened and lengthened. For equiaxed αp, on one hand the growth of already-nucleated grains, meta-dynamic and static recrystallization of αp occurred and resulted in very small α, as marked by red ellipses in Figure 3b,c [25]. On the other hand the existing αp coarsened through the Ostwald ripening effect or attached-growth of α phase (as marked by the yellow ellipse in Figure 3b) [30]. It can also be seen that for near-β forged (AC + WQ) TA15 alloy, the followed low-temperature aging treatments (550 °C/5 h/AC) resulted in little variation in the microstructure only after solution treatment (930 °C/1 h/AC). However it should be noted that the aging treatment is important for stability and homogenization of the microstructure. Figure 4 presents the optical micrographs of TA15 alloy after near-β forging and toughening + strengthening (TS) After 970 °C/0 1 s−1/65%/WQ + 950 °C/100 min/WQ + 800 °C/8 h/AC the A quasi tri-modal microstructure, including coarse equiaxed αp, scattered thick lamellar αs, and very thin tertiary αWI, was obtained after near-β forging (AC + WQ, air cooled for a short time and then water quenched) and SA treatment, as shown in Figure 3b. Figure 3c illustrates the optical micrograph without subsequent aging. 3. Results and Discussion Materials 2016 9 872 When heated and held at 930 ◦C the driving force for nucleation of αs, which originated from the decomposition of the martensite, was insufficient because it was consumed partially during the short time of AC after forging, and the formed αs thickened and lengthened. For equiaxed αp, on one hand the growth of already-nucleated grains, meta-dynamic and static recrystallization of αp occurred and resulted in very small α, as marked by red ellipses in Figure 3b,c [25]. On the other hand the existing αp coarsened through the Ostwald ripening effect or attached-growth of α phase (as marked by the yellow ellipse in Figure 3b) [30]. It can also be seen that for near-β forged (AC + WQ) TA15 alloy, the followed low-temperature aging treatments (550 ◦C/5 h/AC) resulted in little variation in the microstructure only after solution treatment (930 ◦C/1 h/AC). However it should be noted that the aging treatment is important for stability and homogenization of the microstructure. strengthening (TS). After 970 C/0.1 s /65%/WQ + 950 C/100 min/WQ + 800 C/8 h/AC, the microstructural type was a typical tri-modal microstructure (Figure 4a). Compared with the obtained microstructure after solution and aging (Figure 3a), more short and disordered secondary lamellar αs appeared and thin interlaced tertiary αw on the β matrix showed a scattered distribution. This was due to two times of water quenching and a higher holding temperature of 950 °C. Heated and held at 950 °C disordered lamellar αs formed through decomposition of martensite, which resulted from WQ after forging. Meanwhile some small equiaxed αp formed through recrystallization. Water quenched again after heating at 950 °C for 100 min, the high-temperature β transformed into martensite once more. In the subsequent strengthening treatment (800 °C/8 h), the martensite decomposed to form αs and β phase. Some scattered thin interlaced tertiary αW formed through β→α phase transformation during air cooling [30]. Due to rapid WQ after the toughening temperature (950 °C), growing and coarsening of the equiaxed αp were restrained. During the subsequent strengthening process, due to a relative low temperature (800 °C) the driving force for the equiaxed αp coarsening was insufficient, which resulted in small equiaxed αp. Figure 4b shows the optical micrograph after 970 °C/0.1 s−1/60%/(AC + WQ) + 950 °C/1 h/WQ + 800 °C/5 h/AC. 3. Results and Discussion Materials 2016 9 872 When heated at 930 ◦C after forging, the existing martensite decomposed to form secondary αs and β phases. A larger amount of αs nucleated at β grain boundaries (GBs) and in β grains simultaneously and grew rapidly. As holding time was prolonged, some thin αs dissolved and others thickened through merging [29]. In subsequent AC, the undissolved αs thickened further, and tertiary lamellar αs precipitated from β to form the transformed β matrix. (a) 970 C/0.1 s /40%/AC + 830 C/1 h/AC; (b) 970 C/0.1 s /40%/WQ + 830 C/1 h/AC. Figure 3a,b display the optical micrographs of TA15 alloy through near-β forging with a cooling mode of WQ or AC + WQ combined solution + aging (SA, 930 °C/1 h/AC + 550 °C/5 h/AC). For the near-β forged (970 °C/0.1 s−1/65%/WQ) specimen, the typical tri-modal microstructure was obtained (Figure 3a) after SA (930 °C/1 h/AC + 550 °C/5 h/AC), consisting of small equiaxed αp, thick and long secondary lamellar αs, and transformed β matrix. Its formation process was as follows. When heated at 930 °C after forging, the existing martensite decomposed to form secondary αs and β phases. A larger amount of αs nucleated at β grain boundaries (GBs) and in β grains simultaneously and grew rapidly. As holding time was prolonged, some thin αs dissolved and others thickened through merging [29]. In subsequent AC, the undissolved αs thickened further, and tertiary lamellar αs precipitated from β to form the transformed β matrix. Figure 3. Cont. Figure 3. Cont. 7 of 13 Materials 2016, 9, 872 Figure 3. Optical micrographs of TA15 alloy after near-β forging and solution + aging treatment: (a) 970 °C/0.1 s−1/65%/WQ + 930 °C/1 h/AC + 550/5 h/AC; (b) 970 °C/0.1 s−1/60%/(AC + WQ) + 930 °C/1 h/AC + 550 °C/5 h/AC; (c) 970 °C/0.1 s−1/60%/(AC + WQ) + 930 °C/1 h/AC. Figure 3. Optical micrographs of TA15 alloy after near-β forging and solution + aging treatment: (a) 970 ◦C/0.1 s−1/65%/WQ + 930 ◦C/1 h/AC + 550/5 h/AC; (b) 970 ◦C/0.1 s−1/60%/(AC + WQ) + 930 ◦C/1 h/AC + 550 ◦C/5 h/AC; (c) 970 ◦C/0.1 s−1/60%/(AC + WQ) + 930 ◦C/1 h/AC. Figure 3. 3. Results and Discussion Materials 2016 9 872 Compared with that in Figure 4a, a short time of AC was involved but the microstructure showed a great difference Less clustered lamellar α were obtained but a large Figure 4 presents the optical micrographs of TA15 alloy after near-β forging and toughening + strengthening (TS). After 970 ◦C/0.1 s−1/65%/WQ + 950 ◦C/100 min/WQ + 800 ◦C/8 h/AC, the microstructural type was a typical tri-modal microstructure (Figure 4a). Compared with the obtained microstructure after solution and aging (Figure 3a), more short and disordered secondary lamellar αs appeared and thin interlaced tertiary αw on the β matrix showed a scattered distribution. This was due to two times of water quenching and a higher holding temperature of 950 ◦C. Heated and held at 950 ◦C disordered lamellar αs formed through decomposition of martensite, which resulted from WQ after forging. Meanwhile some small equiaxed αp formed through recrystallization. Water quenched again after heating at 950 ◦C for 100 min, the high-temperature β transformed into martensite once more. In the subsequent strengthening treatment (800 ◦C/8 h), the martensite decomposed to form αs and β phase. Some scattered thin interlaced tertiary αW formed through β→α phase transformation during air cooling [30]. Due to rapid WQ after the toughening temperature (950 ◦C), growing and coarsening of the equiaxed αp were restrained. During the subsequent strengthening process, due to a relative low temperature (800 ◦C) the driving force for the equiaxed αp coarsening was insufficient, which resulted in small equiaxed αp. microstructure showed a great difference. Less clustered lamellar αs were obtained but a large amount of thin tertiary Widmanstätten αW appeared. Meanwhile the equiaxed αp was coarse. The main reason was that short AC before WQ after forging changed the formation stage and source of lamellar αs. For near-β forged (AC + WQ) specimen, the final lamellar αs came from the residual Widmanstätten αW formed during AC and decomposition of residual martensite during high temperature toughening, but for near-β forged (WQ) specimen, it only came from the decomposition of martensite. Figure 4b shows the optical micrograph after 970 ◦C/0.1 s−1/60%/(AC + WQ) + 950 ◦C/1 h/WQ + 800 ◦C/5 h/AC. Compared with that in Figure 4a, a short time of AC was involved but the microstructure showed a great difference. Less clustered lamellar αs were obtained but a large amount of thin tertiary Widmanstätten αW appeared. Meanwhile the equiaxed αp was coarse. 3. Results and Discussion Materials 2016 9 872 The main reason was that short AC before WQ after forging changed the formation stage and source of lamellar αs. For near-β forged (AC + WQ) specimen, the final lamellar αs came from the residual Widmanstätten αW 8 of 13 Materials 2016, 9, 872 formed during AC and decomposition of residual martensite during high temperature toughening, but for near-β forged (WQ) specimen, it only came from the decomposition of martensite. Materials 2016, 9, 872 8 of 13 Figure 4. Optical micrographs of TA15 alloy after near-β forging and toughening + strengthening treatment: (a) 970 °C/0.1 s−1/65%/WQ + 950 °C/100 min/WQ + 800 °C/8 h/AC; (b) 970 °C/0.1 s−1/60%/(AC + WQ) + 950 °C/1 h/WQ + 800 °C/5 h/AC; (c) 965 °C/0.1 s−1/65%/AC + 970 °C/1.5 h/WQ + 850 °C/1.5 h/AC. Figure 4. Optical micrographs of TA15 alloy after near-β forging and toughening + strengthening treatment: (a) 970 ◦C/0.1 s−1/65%/WQ + 950 ◦C/100 min/WQ + 800 ◦C/8 h/AC; (b) 970 ◦C/0.1 s−1/60%/(AC + WQ) + 950 ◦C/1 h/WQ + 800 ◦C/5 h/AC; (c) 965 ◦C/0.1 s−1/65%/AC + 970 ◦C/1.5 h/WQ + 850 ◦C/1.5 h/AC. Figure 4. Optical micrographs of TA15 alloy after near-β forging and toughening + strengthening treatment: (a) 970 °C/0.1 s−1/65%/WQ + 950 °C/100 min/WQ + 800 °C/8 h/AC; (b) 970 °C/0.1 s−1/60%/(AC + WQ) + 950 °C/1 h/WQ + 800 °C/5 h/AC; (c) 965 °C/0.1 s−1/65%/AC + 970 °C/1.5 h/WQ + 850 °C/1.5 h/AC. Figure 4. Optical micrographs of TA15 alloy after near-β forging and toughening + strengthening treatment: (a) 970 ◦C/0.1 s−1/65%/WQ + 950 ◦C/100 min/WQ + 800 ◦C/8 h/AC; (b) 970 ◦C/0.1 s−1/60%/(AC + WQ) + 950 ◦C/1 h/WQ + 800 ◦C/5 h/AC; (c) 965 ◦C/0.1 s−1/65%/AC + 970 ◦C/1.5 h/WQ + 850 ◦C/1.5 h/AC. interesting phenomenon was discovered. For TA15 alloy after 965 °C/0.1 s−1/65%/AC + 970 °C/1.5 h/WQ + 850 °C/1.5 h/AC (Figure 4c), a bimodal microstructure was obtained and it was similar to that as shown in Figure 1d. However the Widmanstätten αW were thicker and longer and part of them showed a clustered distribution (Figure 4c). The residual αGB distributed along the contour of β GBs. After near-β forging (AC), about 20% equiaxed αp and a large amount of Widmanstätten αW appeared (Figure 1d). Held at 970 °C for enough time (1.5 h), the existing Widmanstätten αW dissolved into β phase and the distortion energy and defects caused by forging were consumed. 3. Results and Discussion Materials 2016 9 872 Water quenched supercooled martensite was obtained and when heated and held at 850 °C, martensite decomposed into α and β phases. Due to the low transformation temperature and inadequate driving force (cooling rate), the Widmanstätten αW nucleated on β GBs and α/β interface preferentially and grew into a clustered morphology. Meanwhile for martensite the massive transformation, which may have been caused by the composition inhomogeneity of the material, occurred as marked by the blue ellipse in Figure 4c. 3.2. Microstructural Characteristics and Mechanical Properties Based on the analysis above for the near-β forged TA15 alloy after the heat treatment routes When the toughening temperature was equal or higher than the forging temperature, an interesting phenomenon was discovered. For TA15 alloy after 965 ◦C/0.1 s−1/65%/AC + 970 ◦C/1.5 h/WQ + 850 ◦C/1.5 h/AC (Figure 4c), a bimodal microstructure was obtained and it was similar to that as shown in Figure 1d. However the Widmanstätten αW were thicker and longer and part of them showed a clustered distribution (Figure 4c). The residual αGB distributed along the contour of β GBs. After near-β forging (AC), about 20% equiaxed αp and a large amount of Widmanstätten αW appeared (Figure 1d). Held at 970 ◦C for enough time (1.5 h), the existing Widmanstätten αW dissolved into β phase and the distortion energy and defects caused by forging were consumed. Water quenched supercooled martensite was obtained and when heated and held at 850 ◦C, martensite decomposed into α and β phases. Due to the low transformation temperature and inadequate driving force (cooling rate), the Widmanstätten αW nucleated on β GBs and α/β interface preferentially and grew into a clustered morphology. Meanwhile for martensite the massive transformation, which may have been caused by the composition inhomogeneity of the material, occurred as marked by the blue ellipse in Figure 4c. involved, three types of microstructures were obtained, wh 4c) quasi-tri-modal (Figure 3b c) and tri-modal (Figures 3a 3.2. Microstructural Characteristics and Mechanical Properties ), q ( g , ) ( g ), yp , , , respectively. Comparison of the characteristics of the obtained microstructures and constituent phases and corresponding processing routes are presented in Table 2. Figure 5 shows the measured microstructural characteristic parameters and Figure 6 shows the corresponding mechanical properties. Based on the analysis above, for the near-β forged TA15 alloy after the heat treatment routes involved, three types of microstructures were obtained, which were bimodal (Figure 1d, Figure 2a,b and Figure 4c), quasi-tri-modal (Figure 3b,c) and tri-modal (Figures 3a and 4a), defined as type A, B, and C, respectively. Comparison of the characteristics of the obtained microstructures and constituent phases and corresponding processing routes are presented in Table 2. Figure 5 shows the measured microstructural characteristic parameters and Figure 6 shows the corresponding mechanical properties. Materials 2016, 9, 872 9 of 13 A1 A2 A3 A4 B1 B2 C1 C2 C3 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 (b) The grain size of equiaxed p/m The thickness of lamellar s/m, The aspect ratio of equiaxed p Microstructure type The aspect of equiaxed p The thickness of lamellar s 0 2 4 6 8 10 The grain size of equiaxed p 9 of 13 A1 A2 A3 A4 B1 B2 C1 C2 C3 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 (b) The grain size of equiaxed p/m The thickness of lamellar s/m, The aspect ratio of equiaxed p The aspect of equiaxed p The thickness of lamellar s 0 2 4 6 8 10 The grain size of equiaxed p A1 A2 A3 A4 B1 B2 C1 C2 C3 0 10 20 30 40 50 60 70 Microstructure type The volume fraction of  phase/% The lamellar s The equiaxed p (a) A1 A2 A3 A4 B1 B2 C1 C2 C3 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 (b) The grain size of equiaxed p/m The thickness of lamellar s/m, The aspect ratio of equiaxed p Microstructure type The aspect of equiaxed p The thickness of lamellar s 0 2 4 6 8 10 The grain size of equiaxed p Figure 5. Measured microstructural characteristic parameters obtained microstructures in TA15 alloy: (a) volume fraction of α phase; (b) size of α phase. Figure 5. involved, three types of microstructures were obtained, wh 4c) quasi-tri-modal (Figure 3b c) and tri-modal (Figures 3a 3.2. Microstructural Characteristics and Mechanical Properties Measured microstructural characteristic parameters obtained microstructures in TA15 alloy: (a) volume fraction of α phase; (b) size of α phase. Materials 2016, 9, 872 9 of 13 A1 A2 A3 A4 B1 B2 C1 C2 C3 0 10 20 30 40 50 60 70 Microstructure type The volume fraction of  phase/% The lamellar s The equiaxed p (a) A1 A2 A3 A4 B1 B2 C1 C2 C3 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 (b) The grain size of equiaxed p/m The thickness of lamellar s/m, The aspect ratio of equiaxed p Microstructure type The aspect of equiaxed p The thickness of lamellar s 0 2 4 6 8 10 The grain size of equiaxed p Figure 5. Measured microstructural characteristic parameters obtained microstructures in TA15 alloy: (a) volume fraction of α phase; (b) size of α phase. A1 A2 A3 A4 B1 B2 C1 C2 C3 0 10 20 30 40 50 60 70 Microstructure type The volume fraction of  phase/% The lamellar s The equiaxed p (a) Materials 2016, 9, 872 A1 A2 A3 A4 B1 B2 C1 C2 C3 0 10 20 30 40 50 60 70 The volume fraction of  phase/% The lamellar s The equiaxed p (a) Figure 5. Measured microstructural characteristic parameters obtained microstructures in TA15 alloy: (a) volume fraction of α phase; (b) size of α phase. Figure 5. Measured microstructural characteristic parameters obtained microstructures in TA15 alloy: (a) volume fraction of α phase; (b) size of α phase. c ost uctu e type Microstructure type Figure 5. Measured microstructural characteristic parameters obtained microstructures in TA15 alloy: (a) volume fraction of α phase; (b) size of α phase. A1 A2 A3 A4 B C1 C2 20 40 60 80 100 120 600 700 800 900 (b) Impact and fracture toughness Rm (MPa) K1C(MPa m 1/2) aku (J cm -2) Microstructure Type High temperature (500℃) tensile strength ? ? ? ℃ ℃ ℃ different microstructures: (a) Room-temperature A1 A2 A3 A4 B C1 C2 20 40 60 80 100 120 600 700 800 900 (b) Impact and fracture toughness Rm (MPa) K1C(MPa m 1/2) aku (J cm -2) Microstructure Type High temperature (500℃) tensile strength ? ? ? involved, three types of microstructures were obtained, wh 4c) quasi-tri-modal (Figure 3b c) and tri-modal (Figures 3a 3.2. Microstructural Characteristics and Mechanical Properties Mechanical properties of TA15 alloy with different microstructures: (a) Room-temperature tensile properties; (b) high-temperature (500 °C) tensile strength, impact, and fracture toughness. Figure 6. Mechanical properties of TA15 alloy with different microstructures: (a) Room-temperature tensile properties; (b) high-temperature (500 ◦C) tensile strength, impact, and fracture toughness. g yp β g g g yp near-β forging + NTS (near-β toughening and strengthening) (Figure 4c, type A2 in Table 2), respectively. The volume fraction of primary equiaxed αp changed between 18.2% and 21.6%, and average grain size (diameter) between 6.26 and 7.20 μm (Figure 5). The volume fraction of the Widmanstätten αW changed between 47.7% and 58.2%, but its distribution and thickness showed an obvious difference. For microstructure of type A1, there existed a great deal of very thin interlaced αW with a thickness of about 0.35 μm appearing, and spreading all over the matrix (Figure 1d, near-β forging/AC). For type A4 (Figure 2b, near-β forging/WQ + LTA) thin interlaced αW were obtained (0.45 μm). For type A3 (Figure 2a, near-β forging/AC + LTA) the αW was relatively thick, 0.75 μm, and showed a clustered distribution. For type A2 (Figure 4c, near-β forging/AC + NTS) long and thin clustered αW appeared. The quasi tri-modal microstructures were obtained through near-β forging (AC + WQ) combined with SA (Figure 3b, type B2 in Table 2) or HTA (Figure 3c, type B1 in Table 2), consisting f l i i d h t d thi k l ll d t f d β t i Th i d (Figure 1d, type A1 in Table 2), near-β forging + LTA (Figure 2, types A3 and A4 in Table 2), and near-β forging + NTS (near-β toughening and strengthening) (Figure 4c, type A2 in Table 2), respectively. The volume fraction of primary equiaxed αp changed between 18.2% and 21.6%, and average grain size (diameter) between 6.26 and 7.20 μm (Figure 5). The volume fraction of the Widmanstätten αW changed between 47.7% and 58.2%, but its distribution and thickness showed an obvious difference. For microstructure of type A1, there existed a great deal of very thin interlaced αW with a thickness of about 0.35 μm appearing, and spreading all over the matrix (Figure 1d, near-β forging/AC). For type A4 (Figure 2b, near-β forging/WQ + LTA) thin interlaced αW were obtained (0.45 μm). involved, three types of microstructures were obtained, wh 4c) quasi-tri-modal (Figure 3b c) and tri-modal (Figures 3a 3.2. Microstructural Characteristics and Mechanical Properties ℃ ℃ ℃ A1 A2 A3 A4 B C1 C2 0 20 40 60 800 900 1000 1100 1200 (a) Rm (MPa) A (Elongation %) RP0.2 (Mpa) Z (Reduction of area %) Microstructure type Room temperature tensile properties A1 A2 A3 A4 B C1 C2 20 40 60 80 100 120 600 700 800 900 (b) Impact and fracture toughness Rm (MPa) K1C(MPa m 1/2) aku (J cm -2) Microstructure Type High temperature (500℃) tensile strengt ? ? ? ℃ ℃ ℃ Figure 6. Mechanical properties of TA15 alloy with different microstructures: (a) Room-temperature tensile properties; (b) high-temperature (500 °C) tensile strength, impact, and fracture toughness. For TA15 alloy the bimodal microstructures were obtained through near-β forging and AC (Figure 1d type A1 in Table 2) near β forging + LTA (Figure 2 types A3 and A4 in Table 2) and A1 A2 A3 A4 B C1 C2 0 20 40 60 800 900 1000 1100 1200 (a) Rm (MPa) A (Elongation %) RP0.2 (Mpa) Z (Reduction of area %) Microstructure type Room temperature tensile properties A1 A2 A3 A4 B C1 C2 20 40 60 80 100 120 600 700 800 900 (b) Impact and fracture toughness Rm (MPa) K1C(MPa m 1/2) aku (J cm -2) Microstructure Type High temperature (500℃) tensile strength ? ? ? ℃ ℃ ℃ Figure 6. Mechanical properties of TA15 alloy with different microstructures: (a) Room-temperature tensile properties; (b) high-temperature (500 °C) tensile strength, impact, and fracture toughness. For TA15 alloy the bimodal microstructures were obtained through near-β forging and AC Figure 6. Mechanical properties of TA15 alloy with different microstructures: (a) Room-temperature tensile properties; (b) high-temperature (500 ◦C) tensile strength, impact, and fracture toughness. A1 A2 A3 A4 B C1 C2 0 20 40 60 800 900 1000 1100 1200 (a) Rm (MPa) A (Elongation %) RP0.2 (Mpa) Z (Reduction of area %) Microstructure type Room temperature tensile properties Figure 6 Mechanical properties of TA15 alloy with A1 A2 A3 A4 B C1 C2 0 20 40 60 800 900 1000 1100 1200 (a) Rm (MPa) A (Elongation %) RP0.2 (Mpa) Z (Reduction of area %) Microstructure type Room temperature tensile properties tensile properties; (b) high-temperature (500 °C) tensile strength, impact, and fracture toughness. For TA15 alloy the bimodal microstructures were obtained through near-β forging and AC Figure 6. involved, three types of microstructures were obtained, wh 4c) quasi-tri-modal (Figure 3b c) and tri-modal (Figures 3a 3.2. Microstructural Characteristics and Mechanical Properties For type A3 (Figure 2a, near-β forging/AC + LTA) the αW was relatively thick, 0.75 μm, and showed a clustered distribution. For type A2 (Figure 4c, near-β forging/AC + NTS) long and thin clustered αW appeared. The quasi tri-modal microstructures were obtained through near-β forging (AC + WQ) combined with SA (Figure 3b type B2 in Table 2) or HTA (Figure 3c type B1 in Table 2) consisting For TA15 alloy the bimodal microstructures were obtained through near-β forging and AC (Figure 1d, type A1 in Table 2), near-β forging + LTA (Figure 2, types A3 and A4 in Table 2), and near-β forging + NTS (near-β toughening and strengthening) (Figure 4c, type A2 in Table 2), respectively. The volume fraction of primary equiaxed αp changed between 18.2% and 21.6%, and average grain size (diameter) between 6.26 and 7.20 µm (Figure 5). The volume fraction of the Widmanstätten αW changed between 47.7% and 58.2%, but its distribution and thickness showed an obvious difference. For microstructure of type A1, there existed a great deal of very thin interlaced αW with a thickness of about 0.35 µm appearing, and spreading all over the matrix (Figure 1d, near-β forging/AC). For type A4 (Figure 2b, near-β forging/WQ + LTA) thin interlaced αW were obtained (0.45 µm). For type A3 (Figure 2a, near-β forging/AC + LTA) the αW was relatively thick, 0.75 µm, and showed a clustered distribution. For type A2 (Figure 4c, near-β forging/AC + NTS) long and thin clustered αW appeared. of large primary equiaxed αp, short and thick lamellar αs, and transformed β matrix. The equiaxed αp had a volume fraction of 19.7%–25.4%, and an average grain size of 7.78–8.26 μm. The lamellar αs had a volume fraction of 35.8%–38.6% and a thickness of 0.95–1.15 μm (Figure 5). Tri-modal microstructures were obtained after near-β forging (WQ) and SA (Figure 3a, type C1 in Table 2), and after near-β forging (WQ) and TS (Figure 4a, type C2 in Table 2), consisting of 14.5%–20.4% small equiaxed αp (6.20–6.40 μm) and 46.2%–47.8% thick lamellar αs (1.20–1.35 μm) combined with SA (Figure 3b, type B2 in Table 2) or HTA (Figure 3c, type B1 in Table 2), consisting of large primary equiaxed αp, short and thick lamellar αs, and transformed β matrix. The equiaxed αp had a volume fraction of 19.7%–25.4%, and an average grain size of 7.78–8.26 μm. involved, three types of microstructures were obtained, wh 4c) quasi-tri-modal (Figure 3b c) and tri-modal (Figures 3a 3.2. Microstructural Characteristics and Mechanical Properties The lamellar αs had a volume fraction of 35.8%–38.6% and a thickness of 0.95–1.15 μm (Figure 5). Tri-modal microstructures were obtained after near-β forging (WQ) and SA (Figure 3a, type C1 in Table 2), and after near-β forging (WQ) and TS (Figure 4a, type C2 in Table 2), consisting of yp g g g g pp The quasi tri-modal microstructures were obtained through near-β forging (AC + WQ) combined with SA (Figure 3b, type B2 in Table 2) or HTA (Figure 3c, type B1 in Table 2), consisting of large primary equiaxed αp, short and thick lamellar αs, and transformed β matrix. The equiaxed αp had a volume fraction of 19.7%–25.4%, and an average grain size of 7.78–8.26 µm. The lamellar αs had a volume fraction of 35.8%–38.6% and a thickness of 0.95–1.15 µm (Figure 5). q p ( μ ) ( μ ) (Figure 5). For microstructure of type C2 (near-β forging/WQ + TS), thinner (1.23 μm) and interlaced lamellar αs appeared but for type C1 (near-β forging/WQ + SA) αs was thicker (1.33 μm) and longer 14.5%–20.4% small equiaxed αp (6.20–6.40 μm) and 46.2%–47.8% thick lamellar αs (1.20–1.35 μm) (Figure 5). For microstructure of type C2 (near-β forging/WQ + TS), thinner (1.23 μm) and interlaced lamellar αs appeared but for type C1 (near-β forging/WQ + SA) αs was thicker (1.33 μm) and longer Tri-modal microstructures were obtained after near-β forging (WQ) and SA (Figure 3a, type C1 in Table 2), and after near-β forging (WQ) and TS (Figure 4a, type C2 in Table 2), consisting of 14.5%–20.4% small equiaxed αp (6.20–6.40 µm) and 46.2%–47.8% thick lamellar αs (1.20–1.35 µm) (Figure 5). For microstructure of type C2 (near-β forging/WQ + TS), thinner (1.23 µm) and interlaced lamellar αs appeared but for type C1 (near-β forging/WQ + SA) αs was thicker (1.33 µm) and longer (Figure 5). Materials 2016, 9, 872 10 of 13 For type C3 (Figure 4b, near-β forging/(AC + WQ) + TS) the equiaxed αp was large (8.98 µm) and there were about 15.2% moderately thick lamellar αs (Figure 5), but a large amount of very thin tertiary Widmanstätten αW appeared. Materials 2016, 9, 872 10 of 13 pp Bimodal microstructures of types A1–A4 possessed moderate room-temperature tensile strength (Rm = 1000.0–1040.0 MPa, Rp0.2 = 910.0–960.0 MPa) and plasticity (A = 17.0%–185%, Z = 49.5%–53.0%). involved, three types of microstructures were obtained, wh 4c) quasi-tri-modal (Figure 3b c) and tri-modal (Figures 3a 3.2. Microstructural Characteristics and Mechanical Properties As shown in Figure 6a, the tri-modal microstructure of type C1 (Figure 3a) exhibited the highest room-temperature tensile strength (Rm = 1098.0 MPa, Rp0.2 = 1049.0 MPa). G. Lutjering [31] studied the influence of processing on the microstructure and mechanical properties of (α + β) titanium alloys and found that α colony size was an important parameter for yield stress, high cycle fatigue (HCF) strength and low cycle fatigue (LCF) strength since it determined the effective slip length. In tri-modal microstructure C1 consisting of long and thick disordered lamellar αs, the effective slip length decrease to the width of a single lamellae plate, which is benefit for the properties aforementioned. Meanwhile ductility is improved with the increase of equiaxed αp content within 20% [30]. From Figure 5a, except microstructure of type C3, type C1 had the lowest content of equiaxed αp (14.5%), therefore, it possessed the lowest plasticity (A = 16.8%, Z = 43.8%). Research showed that equiaxed αp contents above 20% contributed little to the increase of ductility, but had detrimental effects on other performances [32]. Thus, the tri-modal microstructure of type C2 (Figure 4a) possessed low room-temperature strength (Rm = 981.3 MPa, Rp0.2 = 822.0 MPa) but the highest elongation (A = 23%). Quasi tri-modal microstructure of type B (Figure 3b,c) possessed the lowest room-temperature strength (Rm = 974.0 MPa, Rp0.2 = 866.0 MPa) but the highest percentage reduction of area (Z = 53%). On the one hand disordered lamellar αs contained in this kind of microstructure is good for the decrease of the α colony size, on the other hand a large amount of transformed β matrix increase the effective slip length. The interactions of these two effects led to the lowest room-temperature strength. (Figure 5). For type C3 (Figure 4b, near-β forging/(AC + WQ) + TS) the equiaxed αp was large (8.98 μm) and there were about 15.2% moderately thick lamellar αs (Figure 5), but a large amount of very thin tertiary Widmanstätten αW appeared. Bimodal microstructures of types A1–A4 possessed moderate room-temperature tensile strength (Rm = 1000.0–1040.0 MPa, Rp0.2 = 910.0–960.0 MPa) and plasticity (A = 17.0%–185%, Z = 49.5%–53.0%). As shown in Figure 6a, the tri-modal microstructure of type C1 (Figure 3a) exhibited the highest room-temperature tensile strength (Rm = 1098.0 MPa, Rp0.2 = 1049.0 MPa). G. involved, three types of microstructures were obtained, wh 4c) quasi-tri-modal (Figure 3b c) and tri-modal (Figures 3a 3.2. Microstructural Characteristics and Mechanical Properties Lutjering [31] studied the influence of processing on the microstructure and mechanical properties of (α + β) titanium alloys and found that α colony size was an important parameter for yield stress, high cycle fatigue (HCF) strength and low cycle fatigue (LCF) strength since it determined the effective slip length. In tri-modal microstructure C1 consisting of long and thick disordered lamellar αs, the effective slip length decrease to the width of a single lamellae plate, which is benefit for the properties aforementioned. Meanwhile ductility is improved with the increase of equiaxed αp content within 20% [30]. From Figure 5a, except microstructure of type C3, type C1 had the lowest content of equiaxed αp (14.5%), therefore, it possessed the lowest plasticity (A = 16.8%, Z = 43.8%). Research showed that equiaxed αp contents above 20% contributed little to the increase of ductility, but had detrimental effects on other performances [32]. Thus, the tri-modal microstructure of type C2 (Figure 4a) possessed low room-temperature strength (Rm = 981.3 MPa, Rp0.2 = 822.0 MPa) but the highest elongation (A = 23%). Quasi tri-modal microstructure of type B (Figure 3b,c) possessed the lowest room-temperature strength (Rm = 974.0 MPa, Rp0.2 = 866.0 MPa) but the highest percentage reduction of area (Z = 53%). On the one hand disordered lamellar αs contained in this kind of microstructure is good for the decrease of the α colony size, on the other As shown in Figure 6b, for aforementioned types of microstructures their high temperature (500 ◦C) tensile strength has a similar variation trend as that at room temperature. Microstructure of types C1 and C2 exhibited moderate fracture toughness (K1C = 88.6–91.5 MPa·m1/2) but type C2 possessed a low impact toughness (aku = 45.0 J·cm−2). Microstructures of type B possessed the highest fracture toughness (K1C = 101.3 MPa·m1/2) but low impact toughness (aku = 49.0 J·cm−2). Types A1–A4 had moderate fracture toughness (K1C = 80.8–85.5 MPa·m1/2) and impact toughness (aku = 55.4–60.0 J·cm−2). This is because the fracture toughness increased with the increase of the effective slip length [29]. g y hand a large amount of transformed β matrix increase the effective slip length. The interactions of these two effects led to the lowest room-temperature strength. As shown in Figure 6b, for aforementioned types of microstructures their high temperature (500 °C) tensile strength has a similar variation trend as that at room temperature. From the analysis above, for the near-β forged TA15 alloy a 3.3. Evaluation of Heat Treatment Routes for Near-Forged TA15 Alloy annealing (LTA) and near-β toughening + strengthening (NTS), bimodal microstructures were obtained, which possessed moderated comprehensive properties (low and high temperature tensile properties, fracture and impact toughness), types A2–A4 in Figure 6. For near-β forged (AC) specimen although subsequent NTS resulted in a higher room-temperature tensile strength than LTA, about 37.5 MPa (type A2 and A3 in Figure 6a), the NTS included a near-β heat treatment (WQ) and a LTA treatment, which was more complex. Therefore the NTS was not recommended compared with LTA. For the given subsequent LTA treatment, WQ after forging resulted in thin interlaced αW in the microstructure (Figure 2b), which increased the effective slip length. Thus the tensile strength could be enhanced to a certain degree (type A4 in Figure 6). For near-β forged (WQ) specimen the subsequent SA treatment could result in a tri-modal microstructure (Figure 3a) and excellent comprehensive performance (type C1 in Figure 6), which is d d h t i f i i d From the analysis above, for the near-β forged TA15 alloy after subsequent low-temperature annealing (LTA) and near-β toughening + strengthening (NTS), bimodal microstructures were obtained, which possessed moderated comprehensive properties (low and high temperature tensile properties, fracture and impact toughness), types A2–A4 in Figure 6. For near-β forged (AC) specimen although subsequent NTS resulted in a higher room-temperature tensile strength than LTA, about 37.5 MPa (type A2 and A3 in Figure 6a), the NTS included a near-β heat treatment (WQ) and a LTA treatment, which was more complex. Therefore the NTS was not recommended compared with LTA. For the given subsequent LTA treatment, WQ after forging resulted in thin interlaced αW in the microstructure (Figure 2b), which increased the effective slip length. Thus the tensile strength could be enhanced to a certain degree (type A4 in Figure 6). recommended when extreme service performance is required. When the near-β forged specimen was air cooled for a short time and then water quenched, the subsequent SA resulted in a quasi tri-modal microstructure (Figure 3b) and high fracture toughness (type B in Figure 6b). This was similar to the microstructure after near-β forging (AC + WQ) For near-β forged (WQ) specimen the subsequent SA treatment could result in a tri-modal microstructure (Figure 3a) and excellent comprehensive performance (type C1 in Figure 6), which is recommended when extreme service performance is required. From the analysis above, for the near-β forged TA15 alloy a 3.3. Evaluation of Heat Treatment Routes for Near-Forged TA15 Alloy ( yp g ) β g g ( Q) combined with high-temperature annealing (HTA) (Figure 3c). Under this circumstance HTA was preferred. For near-β forged (WQ) specimen subsequent TS treatment could also result in a tri-modal microstructure (Figure 4a) and a slight lower performance than that by SA. Meanwhile in the TS h i h d f h i ( 950 °C/100 i /WQ) hi When the near-β forged specimen was air cooled for a short time and then water quenched, the subsequent SA resulted in a quasi tri-modal microstructure (Figure 3b) and high fracture toughness (type B in Figure 6b). This was similar to the microstructure after near-β forging (AC + WQ) combined with high-temperature annealing (HTA) (Figure 3c). Under this circumstance HTA was preferred. process the specimen was water quenched after toughening (e.g., 950 °C/100 min/WQ), this increased the possibility of shape distortion of the component due to a large degree of supercooling in WQ, especially for large complex components [33]. 4. Conclusions (1) Bimodal microstructure possessing moderate low and high temperature tensile properties For near-β forged (WQ) specimen subsequent TS treatment could also result in a tri-modal microstructure (Figure 4a) and a slight lower performance than that by SA. Meanwhile in the TS process the specimen was water quenched after toughening (e.g., 950 ◦C/100 min/WQ), this increased the possibility of shape distortion of the component due to a large degree of supercooling in WQ, especially for large complex components [33]. involved, three types of microstructures were obtained, wh 4c) quasi-tri-modal (Figure 3b c) and tri-modal (Figures 3a 3.2. Microstructural Characteristics and Mechanical Properties Materials 2016, 9, 872 11 of 13 11 of 13 Figure 7. Scanning electron microscope (SEM) images of fracture with different magnifications for TA15 alloy after (a1) high temperature tensile test (×27); (a2) high temperature tensile test (×500); (a3) high temperature tensile test (×1000); (b1) room temperature tensile test (×27); (b2) room temperature tensile test (×500); (b3) room temperature tensile test (×1000); (c1) impact test (×27); (c2) impact test (×500); (c3) impact test (×1000). 3 3 E l ti f H t T t t R t f N F d TA15 All Figure 7. Scanning electron microscope (SEM) images of fracture with different magnifications for TA15 alloy after (a1) high temperature tensile test (×27); (a2) high temperature tensile test (×500); (a3) high temperature tensile test (×1000); (b1) room temperature tensile test (×27); (b2) room temperature tensile test (×500); (b3) room temperature tensile test (×1000); (c1) impact test (×27); (c2) impact test (×500); (c3) impact test (×1000). Figure 7. Scanning electron microscope (SEM) images of fracture with different magnifications for TA15 alloy after (a1) high temperature tensile test (×27); (a2) high temperature tensile test (×500); (a3) high temperature tensile test (×1000); (b1) room temperature tensile test (×27); (b2) room temperature tensile test (×500); (b3) room temperature tensile test (×1000); (c1) impact test (×27); (c2) impact test (×500); (c3) impact test (×1000). Figure 7. Scanning electron microscope (SEM) images of fracture with different magnifications for TA15 alloy after (a1) high temperature tensile test (×27); (a2) high temperature tensile test (×500); (a3) high temperature tensile test (×1000); (b1) room temperature tensile test (×27); (b2) room temperature tensile test (×500); (b3) room temperature tensile test (×1000); (c1) impact test (×27); (c2) impact test (×500); (c3) impact test (×1000). involved, three types of microstructures were obtained, wh 4c) quasi-tri-modal (Figure 3b c) and tri-modal (Figures 3a 3.2. Microstructural Characteristics and Mechanical Properties Microstructure of types C1 and C2 exhibited moderate fracture toughness (K1C = 88.6–91.5 MPa·m1/2) but type C2 possessed a low impact toughness (aku = 45.0 J·cm−2). Microstructures of type B possessed the highest fracture toughness (K1C = 101.3 MPa·m1/2) but low impact toughness (aku = 49.0 J·cm−2). Types A1–A4 had moderate fracture toughness (K1C = 80.8–85.5 MPa·m1/2) and impact toughness (aku = 55.4–60.0 J·cm−2). This is because the fracture toughness increased with the increase of the Figure 7 shows the scanning electron microscope (SEM) images of the fractures for TA15 alloy with a tri-modal microstructure (heat treated at a condition of 975 ◦C/2 h/WQ + 930 ◦C/3 h/AC) after high temperature tensile, room temperature tensile and impact tests. From Figure 7a1,b1, both of them display the features of ductile fracture. Figure 7c1 shows aligned fibers which is the typical characteristic of an impact fracture. When the magnification increased to 500, dimples can be observed in three figures (Figure 7a2, b2 and c2). However, the dimples are different in size, depth, and number, which can also be verified by figures of a bigger magnification. Obviously, the fracture after the high temperature tensile test possessed the deepest dimples, the next was the fracture after the room temperature tensile test, and the last was the fracture after the impact test. Thus, the material possessed a high ductility at high temperature. ( J ) g effective slip length [29]. Figure 7 shows the scanning electron microscope (SEM) images of the fractures for TA15 alloy with a tri-modal microstructure (heat treated at a condition of 975 °C/2 h/WQ + 930 °C/3 h/AC) after high temperature tensile, room temperature tensile and impact tests. From Figure 7a1,b1, both of them display the features of ductile fracture. Figure 7c1 shows aligned fibers which is the typical characteristic of an impact fracture. When the magnification increased to 500, dimples can be observed in three figures (Figure 7a2, b2 and c2). However, the dimples are different in size, depth, and number, which can also be verified by figures of a bigger magnification. Obviously, the fracture after the high temperature tensile test possessed the deepest dimples, the next was the fracture after the room temperature tensile test, and the last was the fracture after the impact test. Thus, the material possessed a high ductility at high temperature. Figure 7. Cont. Figure 7. Cont. fracture low-temp 4. Conclusions p g ( / / ) β g g g g (970 °C/1.5 h/WQ + 850 °C/1.5 h/AC). Air cooling after near-β forging resulted in thicker (0.75 μm) clustered αW, while water quenching led to thin (0.45 μm) interlaced αW. (1) Bimodal microstructure, possessing moderate low and high temperature tensile properties, fracture and impact toughness, was obtained through near-β forging combined with low-temperature annealing (830 ◦C/1 h/AC) or near-β toughening and strengthening treatment (970 ◦C/1.5 h/WQ + 850 ◦C/1.5 h/AC). Air cooling after near-β forging resulted in thicker (0.75 µm) clustered αW, while water quenching led to thin (0.45 µm) interlaced αW. p g ( / / ) β g g g g (970 °C/1.5 h/WQ + 850 °C/1.5 h/AC). Air cooling after near-β forging resulted in thicker (0.75 μm) clustered αW, while water quenching led to thin (0.45 μm) interlaced αW. (1) Bimodal microstructure, possessing moderate low and high temperature tensile properties, fracture and impact toughness, was obtained through near-β forging combined with low-temperature annealing (830 ◦C/1 h/AC) or near-β toughening and strengthening treatment (970 ◦C/1.5 h/WQ + 850 ◦C/1.5 h/AC). Air cooling after near-β forging resulted in thicker (0.75 µm) clustered αW, while water quenching led to thin (0.45 µm) interlaced αW. 12 of 13 Materials 2016, 9, 872 (2) After near-β forging (AC + WQ) combined with solution + aging (930 ◦C/1 h/AC + 550 ◦C/5 h/AC) or high-temperature annealing (930 ◦C/1 h/AC), a quasi tri-modal microstructure with low room and high temperature tensile strength and impact toughness but high fracture toughness was obtained. (2) After near-β forging (AC + WQ) combined with solution + aging (930 ◦C/1 h/AC + 550 ◦C/5 h/AC) or high-temperature annealing (930 ◦C/1 h/AC), a quasi tri-modal microstructure with low room and high temperature tensile strength and impact toughness but high fracture toughness was obtained. (2) After near-β forging (AC + WQ) combined with solution + aging (930 ◦C/1 h/AC + 550 ◦C/5 h/AC) or high-temperature annealing (930 ◦C/1 h/AC), a quasi tri-modal microstructure with low room and high temperature tensile strength and impact toughness but high fracture toughness was obtained. (2) After near-β forging (AC + WQ) combined with solution + aging (930 ◦C/1 h/AC + 550 ◦C/5 h/AC) or high-temperature annealing (930 ◦C/1 h/AC), a quasi tri-modal microstructure with low room and high temperature tensile strength and impact toughness but high fracture toughness was obtained. References 1. Shen, G.; Furrer, D. Manufacturing of Aerospace Forgings. J. Mater. Process. Technol. 2000, 98, 189–194. [CrossRef] 1. Shen, G.; Furrer, D. Manufacturing of Aerospace Forgings. J. Mater. Process. Technol. 2000, 98, 189–194. [CrossRef] 2. Sun, Z.C.; Yang, H. Microstructure and mechanical properties of TA15 titanium alloy under multi-step local loading forming. Mater. Sci. Eng. 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(4) For near-β forged TA15 alloy, when moderate mechanical properties were required, water quenching after near-β forging and subsequent low-temperature annealing were recommended; when excellent comprehensive performance was needed, solution and aging treatment was recommended; and when high fracture toughness was considered, high-temperature annealing treatment was recommended. (4) For near-β forged TA15 alloy, when moderate mechanical properties were required, water quenching after near-β forging and subsequent low-temperature annealing were recommended; when excellent comprehensive performance was needed, solution and aging treatment was recommended; and when high fracture toughness was considered, high-temperature annealing treatment was recommended. Acknowledgments: The authors would like to gratefully acknowledge the supports of the National Natural Science Foundation of China (51275560, 51675432), Research Fund of the State Key Laboratory of Solidification Processing (Northwestern Polytechnical University) (156-QP-2016), the 111 Project (B08040), the Marie Curie International Research Staff Exchange Scheme (IRSES, MatProFuture, project No.: 318968) within the 7th European Commission (EC) Framework Programme (FP7). Author Contributions: Zhichao Sun wrote the manuscript and took part in the discussion, Huili Wu completed the experiments and data measurement, He Yang participated in the discussion and determination of the experimental scheme. Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. References Yang, Y.; Xu, F.; Huang, A.J.; Li, G.P. Evolution of microstructure of full lamellar titanium alloy BT18Y solutionized at α + β phase field. 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Mechanism and kinetics of static globularization in TA15 titanium alloy with transformed structure. J. Alloys Compd. 2012, 533, 1–8. [CrossRef] 17. Xu, J.W.; Zeng, W.D.; Jia, Z.Q.; Sun, X.; Zhou, J.H. Microstructure coarsening behavior of Ti-17 alloy with equiaxed alpha during heat treatment. J. Alloys Compd. 2015, 618, 343–348. [CrossRef] 18. Li, S.K.; Xiong, B.Q.; Hui, S.X. Effects of cooling rate on the fracture properties of TA15 ELI alloy plates. Rare Met. 2007, 26, 33–38. 19. Semiatin, S.L.; Knidley, S.L.; Fagin, P.N.; Zhang, F.; Barker, D.R. Microstructure evolution during alpha-beta heat treatment of Ti-6Al-4V. Mater. Sci. Eng. A 2003, 34, 2377–2386. [CrossRef] 0. Sun, Z.C.; Guo, S.S.; Yang, H. Nucleation and growth mechanism of α-lamellae of Ti alloy TA15 cooling f an α + β phase field. Acta Mater. 2013, 61, 2057–2064. [CrossRef] 1. Zhu, S.; Yang, H.; Guo, L.G.; Fan, X.G. Effect of cooling rate on microstructure evolution during α/β treatment of TA15 titanium alloy. Mater. Charact. 2012, 70, 101–110. [CrossRef] 22. Zhu, J.C.; Wang, Y.; Lai, Z.H.; Han, L.; Liu, Y. A Heat Treatment Process for Obtaining Tri-Modal Microstructure in Two-Phase Titanium Alloy. Chinese Invention Patent 200910073419.9, 2 June 2010. 23. Zhu, J.C.; He, D.; Yang, X.W.; Liu, Y. EBSD study on dual heat treatment and microstructure evolution of TA15 titanium alloy. Rare Met. Mater. Eng. 2013, 42, 382–386. (In Chinese) 24. Sun, Z.C.; Wang, X.Q.; Zhang, J.; Yang, H. References Prediction and control of equiaxed α in near-β forging of TA15 Ti-alloy based on BP neural network: For purpose of tri-modal microstructure. Mater. Sci. Eng. A 2014, 591, 18–25. [CrossRef] 25. Sun, Z.C.; Mao, X.J.; Wu, H.L.; Yang, H.; Li, J.J. Tri-modal microstructure and performance of TA15 Ti-alloy under near-β forging and given subsequent solution and aging treatment. Mater. Sci. Eng. A 2016, 654, 113–123. [CrossRef] 26. Seshacharyulu, T.; Dutta, B. 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[CrossRef] 30. Sun, Z.C.; Li, X.S.; Wu, H.L.; Yang, H. Morphology evolution and growth mechanism of the secondary Widmanstatten α phase in the TA15 Ti-alloy. Mater. Charact. 2016, 118, 167–174. [CrossRef] 31. Lütjering, G. Influence of processing on microstructure and mechanical properties of (α + β) titanium alloys. 1. Lütjering, G. Influence of processing on microstructure and mechanical properties of (α + β) titanium al Mater. Sci. Eng. A 1998, 243, 32–45. [CrossRef] 32. Zhou, Y.G.; Zeng, W.D.; Yu, H.Q. A new high-temperature deformation strengthening and toughening process for titanium alloys. Mater. Sci. Eng. A 1996, 221, 58–62. [CrossRef] 33. Totten, G.E.; MacKenzie, D.S. Handbook of Aluminum: Vol. 1: Physical Metallurgy and Processes, 1st ed.; CRC Press: New York, NY, USA, 2003; pp. 317–323. © 2016 by the authors; licensee MDPI, Basel, Switzerland. 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Participation in and attitude towards the national immunization program in the Netherlands: data from population-based questionnaires
BMC public health
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RESEARCH ARTICLE Open Access Abstract Background: Knowledge about the determinants of participation and attitude towards the National Immunisation Program (NIP) may be helpful in tailoring information campaigns for this program. Our aim was to determine which factors were associated with nonparticipation in the NIP and which ones were associated with parents’ intention to accept remaining vaccinations. Further, we analyzed possible changes in opinion on vaccination over a 10 year period. Methods: We used questionnaire data from two independent, population-based, cross-sectional surveys performed in 1995-96 and 2006-07. For the 2006-07 survey, logistic regression modelling was used to evaluate what factors were associated with nonparticipation and with parents’ intention to accept remaining vaccinations. We used multivariate multinomial logistic regression modelling to compare the results between the two surveys. Results: Ninety-five percent of parents reported that they or their child (had) participated in the NIP. Similarly, 95% reported they intended to accept remaining vaccinations. Ethnicity, religion, income, educational level and anthroposophic beliefs were important determinants of nonparticipation in the NIP. Parental concerns that played a role in whether or not they would accept remaining vaccinations included safety of vaccinations, maximum number of injections, whether vaccinations protect the health of one’s child and whether vaccinating healthy children is necessary. Although about 90% reported their opinion towards vaccination had not changed, a larger proportion of participants reported to be less inclined to accept vaccination in 2006-07 than in 1995-96. Conclusion: Most participants had a positive attitude towards vaccination, although some had doubts. Groups with a lower income or educational level or of non-Western descent participated less in the NIP than those with a high income or educational level or indigenous Dutch and have been less well identified previously. Particular attention ought to be given to these groups as they contribute in large measure to the rate of nonparticipation in the NIP, i.e., to a greater extent than well-known vaccine refusers such as specific religious groups and anthroposophics. Our finding that the proportion of the population inclined to accept vaccinations is smaller than it was 10 years ago highlights the need to increase knowledge about attitudes and beliefs regarding the NIP. Participation in and attitude towards the national immunization program in the Netherlands: data from population-based questionnaires Liesbeth Mollema1,4*, Nancy Wijers1,2, Susan JM Hahné1, Fiona RM van der Klis1, Hendriek C Boshuizen3 and Hester E de Melker1 Mollema et al. BMC Public Health 2012, 12:57 http://www.biomedcentral.com/1471-2458/12/57 © 2012 Mollema et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. * Correspondence: Liesbeth.Mollema@rivm.nl 1Centre for Infectious Disease Control Netherlands, RIVM, Bilthoven, The Netherlands Full list of author information is available at the end of the article © 2012 Mollema et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background group C disease, measles, mumps, rubella, hepatitis B, pneumococcal disease, and cervical cancer caused by human papilloma virus). All children below the age of 13 years are eligible to receive vaccinations included in the NIP. Routine vaccination started in 1957, is nonmandatory and free of charge. Incidence rates of nearly all diseases targeted by the Dutch NIP have been reduced successfully. To maintain this success, continued high coverage and trust in vaccination by the population is of the utmost The National Immunization Program (NIP) in the Nether- lands currently offers children vaccinations against twelve infectious diseases (diphtheria, poliomyelitis, pertussis, tetanus, Haemophilus influenzae type b, meningococcal Mollema et al. BMC Public Health 2012, 12:57 http://www.biomedcentral.com/1471-2458/12/57 Page 2 of 13 importance. Vaccination coverage for infants ranged from 94.5% for diphtheria, pertussis, tetanus and polio (DTaP- IPV) to 96.0% for measles, mumps and rubella (MMR) (cohort 2005; reporting year 2008). The coverage for tod- dlers and school-aged children was over 90% for each of the vaccinations (cohort 2002 and 1997 respectively; reporting year 2008) [1]. Although vaccine coverage in infants is high [1], previous studies have identified groups who (in part) refuse vaccination. One group consists of members of Reformed Congregations who believe that vaccination is contrary to the providence of God [2]. This group is at risk for epidemics as a result of sociogeographi- cal clustering, which has been observed for polio, measles, mumps and rubella [3-6]. Another group consists of anthroposophics who believe that experiencing some childhood diseases may contribute to strengthening body and mind [2]. Anthroposophics are scattered throughout the Netherlands, but clustering in anthroposophic schools is present. The increased risk of epidemics has been observed for measles; in 2008, an outbreak occurred at several anthroposophic schools in the Netherlands [7] and also in anthroposophic communities abroad [8,9]. divided into five geographical regions of approximately equal population size. In each of the five regions a random sample of eight municipalities was drawn, proportional to the number of inhabitants. Within each municipality, an age-stratified sample (0, 1-4, 5-9, . . ., 75-79 years) of 380- 500 persons (males and females) was drawn from the municipal health registers. Background In addition to the nationwide sample (NS), an age-stratified sample (similar as for the NS) from eight municipalities with low vaccination cover- age (LVC) was drawn to assess the seroprevalence of groups of persons in sociogeographic clusters who refuse vaccination for religious reasons (known locally as the “Dutch Bible Belt”). The municipalities in the LVC sample were chosen on the basis of consistently low vaccination coverage (in the first survey for DTP-IPV for the years 1982-1993 and in the second survey for MMR and DTaP/ IPV for birth cohorts 1997-2001) and the condition of representation of several provinces. The Dutch Bible Belt stretches from the southwest of the Netherlands to just above the centre in the northeast. In the second survey (2006-07), an extra sample from non-Western migrants was drawn from 12 of the 40 municipalities in the NS. We distinguished twelve migrant groups according to country of birth (1. Morocco and Turkey, 2. Suriname, Aruba and Netherlands Antilles and 3. Other non-Western coun- tries), age (0-9 years, 10-49 years and 50-79 years) and first and second generation (only for 0-9 year-olds). A first-generation migrant was defined as somebody who was born abroad, immigrated to the Netherlands and whose parents (one or both) were born abroad. A second- generation migrant was defined as somebody who was born in the Netherlands and whose parents (one or both) were born abroad. The distribution of migrants per degree of urbanization (i.e., the number of addresses per km2) in the Netherlands was used to select the municipalities in which the oversampling of migrants took place. Figure 1 shows the number of invited persons per sample and per survey. In addition, other less well-identified groups may exist that have doubts concerning the risks and benefits of vac- cination. Paulussen et al. [10] observed that 81% of parents reported not having thought about the risks and benefits of vaccination thoroughly before making the decision. The authors hypothesized that these parents could easily be influenced by negative publicity about vaccination. In this study, we try to obtain more knowledge about the deter- minants of participation and attitude towards the NIP, which might be helpful in tailoring information campaigns for this program. In addition, over the course of a 10 year period, we were able to analyze possible changes in the opinion on vaccination during the preceding 5 years. Background In the Netherlands two nationwide seroprevalence surveys were performed, one in 1995-96, the other in 2006-07. Questionnaire data from these surveys have been used for the analyses. Approaching participants Eligible persons received an invitation package by mail two weeks prior to the prescheduled appointment time for blood donation. This included the invitation letter, a bro- chure containing information on the survey, a question- naire and an informed consent. Eligible persons were requested to donate a blood sample, to complete a ques- tionnaire and to provide immunization records. All invited persons were approached by phone by a call centre to ask if they were willing to participate, to answer their ques- tions and to remind them of the survey. When individuals refused to participate, they were asked to complete the questionnaire and if they also refused this, to answer some questions for the nonresponse survey (by telephone or mail). All participants gave signed informed consent. Study Population Sampling method Two independent population-based cross-sectional sero- surveillance surveys were carried out in the Netherlands between October 1995 and December 1996 (the first sur- vey) and between February 2006 and June 2007 (the sec- ond survey) to establish a serum bank for the general population. The serum bank was to be used to estimate age-specific antibody levels against all vaccine preventable diseases in the (future) NIP and also of other diseases. Both surveys had a similar design, which has been described previously [11,12]. Briefly, the Netherlands were Mollema et al. BMC Public Health 2012, 12:57 http://www.biomedcentral.com/1471-2458/12/57 Page 3 of 13 First survey 18217 invited persons Low vaccination coverage sample 4366 invited persons 1914 (44%) questionnaires Nationwide sample 17223 invited persons 6981 (41%) questionnaires Low vaccination coverage sample 3028 invited persons 1964 (65%) questionnaires Second survey 24147 invited persons Nationwide sample 15189 invited persons 9977 (66%) questionnaires Oversampling migrants 2558 invited persons 702 (27%) questionnaires Nationwide sample 6267 (90%) questionnaires of those <45 yrs and first- generation migrants <13 yrs 5881 questionnaires used in analyses Low vaccination coverage sample 1258 (91%) questionnaires of those <45 yrs and first- generation migrants <13 yrs 1204 questionnaires used in analyses Nationwide sample 4274 (40%) questionnaires of those <45 yrs and first- generation migrants <13 yrs 4130 and 3878 questionnaires used in first and third analyses, respectively 1739 (42%) questionnaires of those <10 yrs 1525 questionnaires used in analyses Low vaccination coverage sample 1302 (46%) questionnaires of those <45 yrs and first- generation migrants <13 yrs 1190 questionnaires used in analyses Oversampling migrants 455 (30%) questionnaires of those <45 yrs and first- generation migrants <13 yrs 376 questionnaires used in analyses 421 (30%) questionnaires of those <10 yrs 219 questionnaires used in analyses Figure 1 Overview of the number of invited persons and the number of questionnaires (response rates) per sample and per survey. Second survey 24147 invited persons First survey 18217 invited persons 6267 (90%) questionnaires of those <45 yrs and first- generation migrants <13 yrs 5881 questionnaires used in analyses 1525 questionnaires used in analyses Figure 1 Overview of the number of invited persons and the number of questionnaires (response rates) per sample and per survey. categorised as followed: 1. High NIP refusers, low vacci- nation coverage, also called “Reformed Congregations” (i.e., Reformed Congregations, Reformed Congregations in the Netherlands, Old Reformed Congregations); 2. Study Population Sampling method Moderate NIP refusers, intermediate vaccination cover- age, also called “Reformed Bonders” (i.e., Restored Reformed Church and Reformed Bond); 3. Low NIP refusers, relative high vaccination coverage, also called “other specific Protestant Church” (i.e., other specific Protestant Christian religious communities); 4. Catho- lics; 5. Muslims; 6. Other religion (i.e., religions included see text below tables); 7. No religion. The questions on the above topics were asked in the same way in both surveys. Data analyses SAS version 9.2 (SAS Institute Inc., Cary, NC, USA) was used for all analyses. Variable selection was based only on the complete cases, i.e., those without missing answers to questions. Below we consider the data ana- lyses per subject. Questionnaire h The answer cate- gories for the vaccination statements were: “strongly agree"; “agree”; “neutral”; “disagree”; and “strongly dis- agree”. Table 1 shows the questions regarding participa- tion in the NIP, intention to accept any remaining vaccinations and changes of opinion regarding vaccina- tion. Depending on the municipality, each participant was assigned a degree of urbanization and a region based on data from Statistics Netherlands available at http://www.cbs.nl. Dat SAS use on ans lyse Non Fac 1, T vey only gen part cipa whe To test deg 0.05 Table 1 Overview of question-and-answer categories regarding v Question An ca 1. Did you/does your child participate in the NIP?b Ye No Do 2. Do you intend to accept any remaining vaccinations for your child?e 1. 2. 3. ye 4. 5. no 6. 7. ap 3. Has your/parents’ opinion on vaccination changed with respect to the five years preceding the survey?b 1. 2. inc 3. inc 4. question: “How many injections are acceptable for your child?” (answer categories: 0, 1, 2, 3, 4, any number is acceptable), and to agree or disagree with the following statements regarding vaccination: a) “Childhood vaccina- tions are useful for the protection of the health of my child”; b) “There is absolutely no need to vaccinate healthy children against childhood diseases”; c) “I doubt the safety of childhood vaccinations”; d) “The immune system of my child does not develop well due to vacci- nation"; e) “Childhood vaccinations are useful for the protection of the health of others”. The answer cate- gories for the vaccination statements were: “strongly agree"; “agree”; “neutral”; “disagree”; and “strongly dis- agree”. Table 1 shows the questions regarding participa- tion in the NIP, intention to accept any remaining vaccinations and changes of opinion regarding vaccina- tion. Depending on the municipality, each participant was assigned a degree of urbanization and a region based on data from Statistics Netherlands available at http://www.cbs.nl. Questionnaire h The questionnaires were self-administered. In the first survey, three questionnaires were used for the following age groups: 0-11 years (42 questions); 12-16 years (42 questions); and 17-79 years (51 questions). The ques- tionnaires were similar in content except that the ques- tionnaire for 17 to 79-year-olds contained questions on sexual activities, alcohol and smoking. In the case of children below 12 years of age, parents mostly filled in the questionnaire. In the second survey, two questionnaires were used for the following age groups: 0-14 years (50 questions) and 15-79 years (57 questions). The questionnaires were similar in content except that the questionnaire for 0 to 14 year-olds contained statements regarding vaccination and the questionnaire for those above 15 years of age contained questions on sexual activities. In the case of children below 15 years of age, parents mostly filled in the questionnaire. The questions on net monthly household income and “whether any of the following ideas influenced the parti- cipants’/parents’ opinion on vaccination” (answer cate- gories: anthroposophy (i.e., a spiritual philosophy founded by Rudolf Steiner), homeopathy (i.e., form of alternative medicine), other alternative medicine, religion, other, or none of those ideas) were only asked in the sec- ond survey. The last answer category “none of those ideas” was left out the analyses. Both in the first and second survey, the questionnaire covered topics such as age, gender, educational level, religion, number of persons in household, health status of the participant, participation in the NIP and whether their opinion on vaccination had changed with respect to the 5 years preceding the survey. Religion was Only in the second survey, parents of children aged below 15 years were asked to answer the following Mollema et al. BMC Public Health 2012, 12:57 http://www.biomedcentral.com/1471-2458/12/57 Page 4 of 13 question: “How many injections are acceptable for your child?” (answer categories: 0, 1, 2, 3, 4, any number is acceptable), and to agree or disagree with the following statements regarding vaccination: a) “Childhood vaccina- tions are useful for the protection of the health of my child”; b) “There is absolutely no need to vaccinate healthy children against childhood diseases”; c) “I doubt the safety of childhood vaccinations”; d) “The immune system of my child does not develop well due to vacci- nation"; e) “Childhood vaccinations are useful for the protection of the health of others”. Parents’ intention to accept any remaining vaccinations for their child Factors determining reduced parental acceptance of any remaining vaccinations for their child (question 2, Table 1) were studied only in the NS of the second survey, including the oversampling of migrants. We included only partici- pants aged less than 10 years of age (i.e., eligible for vacci- nations in the NIP). For the dependent variable, we combined the answer categories “Surely yes”, “Yes”, and “Probably yes” for simplicity and, because of small num- bers, we combined the answer categories “Unsure”, “Prob- ably not”, and “Surely not”. The answer category “Not applicable” was omitted from the analyses. The analyses were similar to those conducted on determinants of non- participation in the NIP. Study population Figure 1 shows the number of questionnaires obtained (response rates) per sample and per survey. Note, the numbers of questionnaires taken into account in the ana- lyses were lower due to missing values or omitting certain answer categories. A relatively large number of the parents of migrant children less than 10 years of age did not fill in whether they agreed or disagreed with the statements regarding vaccination. Median age and interquartile range for the NS (including oversampling migrants in the second study) and the LVC sample was 31 (6-54) and 30 (6-54) in the first survey and 30 (8-55) and 25 (5-53) in the second survey, respectively. The proportion of males to females in the NS (including oversampling migrants in the second study) and the LVC sample was 0.9 and 0.9 in the first sur- vey and 0.8 and 1.0 in the second survey, respectively. Table 1 shows the numbers and percentages of partici- pants per answer category for the three questions used in the analyses in the first and second survey for the NS (including the oversampling of migrants in the second sur- vey) and LVC sample separately. Nonparticipation in the NIP There- after, a multivariate model was constructed by adding to the existing model the confounder that had the most influence on the coefficient of the variable sample. This process was repeated until no confounder remained that caused a change of more than 5% in the coefficient of the sample. The crude and adjusted ORs for the deter- minant sample and 95% CIs were calculated. analyses. A determinant remained in the model if the P value was < = 0.05. The crude and adjusted odds ratios (ORs) and 95% confidence intervals (CIs) were calculated. (O ) 9 % v (C ) w For several determinants associated with nonparticipa- tion in the NIP the population attributable fraction (PAF) was estimated using the following equation: the probability at nonparticipation in the exposed popula- tion (i.e., including the group who participates less in the NIP compared to the reference group) minus the probability at nonparticipation in the nonexposed popu- lation (i.e., not including the group who participates less in the NIP compared to the reference group), divided by the probability at nonparticipation in the exposed popu- lation, using logistic regression modelling [13]. The PAF is expressed as the percentage of reduction in the preva- lence of nonparticipation in the NIP in the population that would result from taking away the exposure to non- participation in the NIP [14]. The prevalence of the exposure to nonparticipation in the NIP used in the PAF was calculated based on the population in the NS, applying survey weights in order to make the sample representative for the Dutch population in terms of age, gender, ethnicity, degree of urbanization and region. Rao-Wu-Yue bootstrapping was used to obtain 95% confidence intervals, taking the study design into account [15]. Changes in the opinion on vaccination between the 2006-07 and 1995-96 surveys We investigated whether participants in the 2006-07 survey were more or less inclined to accept vaccination than the participants were in the 1995-96 survey. Sepa- rate analyses were performed for the NS (without the oversampling of migrants in the 2006-07 survey to be comparable with the survey in 1995-96 for which no oversampling was performed) and the LVC sample. For this purpose we analyzed the answers (“No”, “Yes, more inclined”, “Yes, less inclined”) given to the question Nonparticipation in the NIP Factors determining nonparticipation in the NIP (question 1, Table 1) were studied only in the NS of the second sur- vey, including the oversampling of migrants. We included only participants aged less than 45 years of age and first- generation migrants aged less than 13 years at the time of participation in the study, i.e., those who could have parti- cipated in the NIP. People who reported not knowing whether they had participated in the NIP were excluded. To take into account the sample design, we adjusted all test determinants for age, gender, ethnicity, region and degree of urbanization. Determinants with a P value < = 0.05 from these analyses were included in the multivariate Table 1 Overview of question-and-answer categories regarding vaccination included in the analyses Question Answer categories First survey, NS First survey, Second survey, NSa Second survey, LVC LVC N (%) N (%) N (%) N (%) 1. Did you/does your child participate in the NIP?b Yes 5951 (96) 992 (79) 4420 (94) 940 (73) No 143 (2) 242 (19) 141 (3) 330 (25) Don’t knowc 109 (2) 18 (2) 127 (3) 26 (2) 64d 6d 41d 6d 2. Do you intend to accept any remaining vaccinations for your child?e 1. Surely yes NA NA 1418 (68) 291 (46) 2. Yes 484 (23) 148 (23) 3. Probably yes 75 (4) 35 (5) 4. Unsure 28 (1) 13 (2) 5. Probably not 13 (1) 24 (4) 6. Surely not 7 (0) 82 (13) 7. Not applicablef 52 (3) 45 (7) 83d 5d 3. Has your/parents’ opinion on vaccination changed with respect to the five years preceding the survey?b 1. No 5514 (90) 1101 (89) 3866 (83) 1086 (84) 2. Yes, more inclined 517 (8) 112 (9) 311 (7) 81 (6) 3. Yes, less inclined 128 (2) 28 (2) 147 (3) 61 (5) 4. Don’t know NA NA 315 (7) 66 (5) 108d 17d 90d 8d aIncluding oversampling of non-Western migrants in the NS of the second survey bIncluding participants aged less than 45 years and first-generation migrants aged less than 13 years cCategory “Don’t know” was omitted from the analyses dNumber of missing answers at a specific question eIncluding participants aged less than 10 years fCategory “not applicable” was omitted from the analyses -and-answer categories regarding vaccination included in the analyses ble 1 Overview of question-and-answer categories regarding vaccination included in the analyses Mollema et al. Nonparticipation in the NIP BMC Public Health 2012, 12:57 http://www.biomedcentral.com/1471-2458/12/57 Page 5 of 13 “Has your opinion on vaccination changed with respect to the 5 years preceding the survey?” (See also question 3 in Table 1). Participants less than 45 years old and first-generation migrants less than 13 years old at the time of participation in the study were included in the analyses. Note, migrants who participated in the NS of both surveys were included in the analyses. For the NS the following possible confounders were tested whether they influenced the variable sample (i.e., NS in the sec- ond survey versus NS in the first survey): educational level, religion and NIP participation, adjusted for age, gender, ethnicity, degree of urbanization and region. For the LVC sample the following possible confounders were tested whether they influenced the variable sample (i.e., LVC sample in the second survey versus LVC sam- ple in the first survey): educational level, religion and NIP participation, adjusted for age and gender. There- after, a multivariate model was constructed by adding to the existing model the confounder that had the most influence on the coefficient of the variable sample. This process was repeated until no confounder remained that caused a change of more than 5% in the coefficient of the sample. The crude and adjusted ORs for the deter- minant sample and 95% CIs were calculated. “Has your opinion on vaccination changed with respect to the 5 years preceding the survey?” (See also question 3 in Table 1). Participants less than 45 years old and first-generation migrants less than 13 years old at the time of participation in the study were included in the analyses. Note, migrants who participated in the NS of both surveys were included in the analyses. For the NS the following possible confounders were tested whether they influenced the variable sample (i.e., NS in the sec- ond survey versus NS in the first survey): educational level, religion and NIP participation, adjusted for age, gender, ethnicity, degree of urbanization and region. For the LVC sample the following possible confounders were tested whether they influenced the variable sample (i.e., LVC sample in the second survey versus LVC sam- ple in the first survey): educational level, religion and NIP participation, adjusted for age and gender. Nonparticipation in the NIP The first column in Table 2 gives the numbers of parti- cipants per determinant and the second column shows the percentage of participants/parents who reported not to have participated in the NIP. Gender, degree of urba- nization, region, household size, homeopathic, other Mollema et al. BMC Public Health 2012, 12:57 http://www.biomedcentral.com/1471-2458/12/57 Page 6 of 13 Table 2 Potential determinants for reporting not to have participated in the NIPa (N = 4506) and population attributable fractions (PAF) Variable (N) Reported Crudeb Adjusted PAF in % nonparticipation in the NIP in % OR (95% CI) OR (95% CI) (95% CI) Age (yrs) 0-4 (1385) 3 1.7 (0.8-3.7) 1.4 (0.6-3.1) 5-9 (660) 7 2.7 (1.2-6.0) 2.0 (0.9-4.5) 10-29 (1470) 3 3.0 (1.4-6.2) 2.3 (1.1-4.9) 30-44 (991) 1 Reference Reference Gender Men (2051) 3 Reference Reference Women (2455) 3 1.1 (0.8-1.6) 1.1 (0.7-1.5) Ethnicity Indigenous Dutch (3640) 2 Reference Reference Other Western (226) 2 1.4 (0.5-3.5) 1.4 (0.5-3.8) Moroccan and Turkish (200) 19 11.7 (6.8-20.1) 7.1 (2.5-20.1) 21.5 (4.3-36.7) Antilles and Aruba and Surinam (223) 6 3.1 (1.5-6.1) 3.3 (1.5-7.1) 4.4 (-1.0-11.7) Other non-Western (217) 8 4.5 (2.4-8.2) 3.3 (1.6-7.1) 6.1 (-1.2-14.4) Religion Reformed Congregations (59) 17 12.5 (5.2-30.1) 15.2 (6.1-37.8) 7.4 (0.1-18.4) Reformed Bonders (129) 6 4.2 (1.7-10.4) 4.8 (1.9-12.1) 5.2 (0.1-13.1) Other specific Protestant Church (805) 2 Reference Reference Catholics (1183) 1 0.8 (0.4-1.6) 0.8 (0.4-1.6) Muslims (281) 16 1.4 (0.6-3.8) 1.2 (0.5-3.3) Other religionc (204) 7 2.2 (1.0-4.9) 1,7 (0.7-3.9) No religion (1845) 2 0.8 (0.4-1.5) 0.7 (0.4-1.4) Degree of urbanization Very high (963) 6 Reference Reference High (1998) 2 0.8 (0.5-1.3) 0.6 (0.4-1.0) Moderate (570) 2 0.9 (0.4-1.9) 0.6 (0.3-1.4) Low (452) 2 1.1 (0.4-3.3) 1.3 (0.4-4.2) Very low (523) 3 1.1 (0.6-2.2) 1.1 (0.5-2.2) Region Northeast (1023) 3 Reference Reference Central (843) 2 0.8 (0.3-1.9) 0.6 (0.2-1.6) Northwest (1017) 5 1.0 (0.6-1.8) 0.8 (0.5-1.5) Southwest (808) 4 1.2 (0.6-2.3) 1.0 (0.5-1.9) Southeast (815) 2 0.6 (0.3-1.2) 0.7 (0.3-1.5) No. Nonparticipation in the NIP of persons in household 1 (203) 2 0.8 (0.2-2.7) 2 (516) 1 0.6 (0.3-1.3) 3 (990) 3 1.2 (0.7-1.9) 4 (1718) 2 Reference > 4 (1079) 5 1.4 (0.9-2.1) Educational leveld Low (317) 13 2.1 (1.2-3.7) 2.5 (1.3-4.6) 13.7 (-1.2-29.7) Moderate (2123) 3 1.1 (0.7-1.7 1.4 (0.9-2.2) High (2066) 2 Reference Reference Net monthly household incomee ential determinants for reporting not to have participated in the NIPa (N = 4506) and population fractions (PAF) nts for reporting not to have participated in the NIPa (N = 4506) and population Mollema et al. BMC Public Health 2012, 12:57 http://www.biomedcentral.com/1471-2458/12/57 Page 7 of 13 Table 2 Potential determinants for reporting not to have participated in the NIPa (N = 4506) and population attributa- ble fractions (PAF) (Continued) Low (569) 9 2.1 (1.1-4.1) 1.9 (1.0-4.0) 13.1 (-8.1-32.1) Moderate (2051) 2 1.0 (0.5-1.8) 0.8 (0.4-1.6) High (936) 2 Reference Reference Won’t answer/missing answer (950) 2 0.97 (0.5-1.9) 0.9 (0.4-1.9) Anthroposophy Yes (46) 28 27.0 (12.8-57.0) 39.2 (17.5-87.9) 8.4 (2.3-17.6) No (4460) 3 Reference Reference Homeopathy Yes (75) 5 2.6 (0.9-7.5) No (4431) 3 Reference Other alternative medicine Yes (36) 8 2.6 (0.7-9.3) No (4470) 3 Reference Religious belief Yes (82) 17 9.5 (4.8-18.6) No (4424) 3 Reference Other ideas influencing vaccination Yes (189) 3 1.4 (0.6-3.3) No (4317) 3 Reference aThe reference category is reporting “yes” at the question whether they (had) participated in the NIP bCrude odds ratios were adjusted for age, gender, ethnicity, degree of urbanization and region cOther religion included: Judaism, Buddhism and Hinduism dLow educational level included no education and primary education; middle educational level included junior technical school, lower general secondary education and intermediate vocational education; and high educational level included senior/higher secondary education, pre-university education and university eNet monthly income was categorized as low (less than € 1.150, which is less than € 1.167 by Statistic Netherlands); middle (€ 1.151-€ 3.050, which is € 1.168-€ 2.917 by Statistic Netherlands); high (more than € 3.051, which is more than € 2.918 by Statistic Netherlands); and a won’t answer/missing answer category was included. Nonparticipation in the NIP Statistics Netherlands available at www.cbs.nl Table 2 Potential determinants for reporting not to have participated in the NIPa (N = 4506) and population attributa- ble fractions (PAF) (Continued) 3 gives the numbers of participants per determinant and the second column shows the percentages of parents who reported less likely to accept any remaining vacci- nations for their child. In the univariate analysis gender, degree of urbanization, region, religion, net monthly household income, educational level, household size, own health and proposition “vaccination protects health others” were not significantly associated with parents’ intention to accept any remaining vaccinations for their child (column 3, Table 3). Age, other beliefs, critical group (i.e., anthroposophy/homeopathy and other alter- native medicine), religious beliefs and proposition “immune system does not develop well due to vaccina- tion” were no longer significant at multivariate level. alternative medicine and other beliefs were not signifi- cantly associated with reported participation in the NIP at univariate level (column 3, Table 2) and religious beliefs were no longer significant at multivariate level. The multivariate analyses showed that participants aged 10-29 years, who had a low income, who had a low edu- cational level, who were of non-Western descent, who were members of Reformed Congregations or Reformed Bonders or who had anthroposophic beliefs regarding vaccination participated less in the NIP compared to the reference categories (column 4, Table 2). By changing the ethnicity of Moroccan or Turkish participants into indigenous Dutch a higher population attributable fraction (21.5%) was observed than by chan- ging low educational level into high educational level (13.7%) or low net monthly household income into high income (13.1%). In column 5 of Table 2 the PAFs of the various determinants are given. The multivariate analyses showed that parents of other non-Western descent, who expressed the opinion that there should be no injection at all, who disagreed (includ- ing reporting neutral) with the proposition that “child- hood vaccinations protect the health of one’s own child”, who agreed or reported neutral towards the proposition that “vaccinating healthy children is not necessary” or who agreed with the proposition “doubting the safety of childhood vaccinations” were less likely to accept any Parents’ intention to accept any remaining vaccinations for their child The proportion of parents who reported their child par- ticipated in the NIP was 96%. The first column in Table Mollema et al. BMC Public Health 2012, 12:57 http://www.biomedcentral.com/1471-2458/12/57 Page 8 of 13 Table 3 Potential determinants for parents less likely to accept any remaining vaccinations for their childa (N = 1744) Variables (N) Parents who reported less likely to accept any remaining vaccinations for their child in % Crudeb OR Adjusted OR (95% CI) (95% CI) Age (yrs) 0-4 (1237) 1 0.4 (0.2-0.9) 0.7 (0.3-1.9) 5-9 (507) 4 Reference Reference Gender Men (875) 2 Reference Reference Women (869) 2 1.2 (0.6-2.4) 0.6 (0.2-1.5) Ethnicity Indigenous Dutch (1315) 1 Reference Reference Other Western (85) 1 0.7 (0.1-5.7) 1.5 (0.1-15.7) Moroccan and Turkish (94) 7 3.7 (1.3- 11.0) 1.7 (0.4-8.1) Antilles and Aruba and Surinam (144) 2 1.0 (0.3-4.1) 1.0 (0.2-6.7) Other non-Western (106) 5 2.4 (0.8-7.1) 4.2 (1.1-16.5) Religion Reformed Congregations (22) 5 2.1 (0.2- 19.8) Reformed Bonders (53) 2 1.4 (0.2- 13.1) Other specific Protestant Church (325) 2 Reference Other religionc (679) 3 1.4 (0.5-3.8) No religion (665) 1 0.4 (0.1-1.5) Degree of urbanization Very high (391) 3 Reference Reference High (738) 1 0.7 (0.3-1.9) 0.8(0.2-3.0) Moderate (231) 1 0.7 (0.2-2.9) 1.3 (0.2-8.0) Low (181) 1 1.6 (0.1- 19.5) 1.5 (0.1-35.5) Very low (203) 4 2.3 (0.7-7.4) 4.5 (0.99-20.1) Region Northeast (379) 1 Reference Reference Central (337) 1 0.7 (0.1-6.6) 0.9 (0.1-12.0) Northwest (434) 4 2.0 (0.7-5.8) 3.1 (0.6-14.9) Southwest (302) 2 1.9 (0.5-7.6) 1.0 (0.2-6.7) Southeast (292) 2 2.0 (0.5-7.6) 5.7 (0.97-33.4) Educational leveld Low (77) 8 1.4 (0.4-5.4) Moderate (832) 2 1.0 (0.5-2.0) High (835) 2 Reference Net monthly household incomee Low (137) 7 3.0 (0.7- 11.9) Moderate (863) 2 1.5 (0.5-4.6) High (405) 1 Reference Won’t answer/missing answer (339) 2 1.3 (0.4-4.9) No. of persons in household 1-2 (42) 2 1.0 (0.1-8.7) 0.6 (0.2-1.7) 3 (451) Mollema et al. Discussion The first and second column of Table 4 shows the num- ber and percentages of participants/parents who reported to be more or less inclined to vaccinate. In both surveys about 90% of the participants or parents reported they had not changed their opinion towards vaccination in the 5 years preceding the survey. In the 2006-07 survey, fewer participants in the NS reported they were more inclined to accept vaccination and more participants reported they were less inclined to accept vaccination compared to no change in their opinion (see column 4 in Table 4). The odds ratios were adjusted for age, gender, ethnicity, degree of urbanization, region and educational level. As expected, on the basis of high vaccination coverage for infants for the vaccine-preventable diseases in the NIP [1], the attitude towards vaccination was generally positive. Not surprisingly, we observed that members of Reformed Congregations, Reformed Bonders and those with anthroposophic beliefs participated less in the NIP than those with no religion or no anthroposohic beliefs. We also observed other groups who participated less in the NIP compared to the reference categories, including those of non-Western descent, with a low income and with a low educational level. Disagree/strongly disagree (100) aReference category is the combination of answer categories “surely yes”, “yes”, and “probably yes” bCrude odds ratios were adjusted for age, gender, ethnicity, degree of urbanization and region cOther religion included: Judaism, Buddhism, Hinduism, Catholic and Muslims remaining vaccinations for their child compared to the reference categories (column 4, Table 3). vaccination and more participants reported they were less inclined to accept vaccination compared to no change in their opinion (see column 4 in Table 4). The odds ratios were adjusted for age, gender and religion. Changes in the opinion on vaccination between the 2006- 07 and 1995-96 surveys Parents’ intention to accept any remaining vaccinations for their child BMC Public Health 2012, 12:57 http://www.biomedcentral.com/1471-2458/12/57 Table 3 Potential determinants for parents less likely to accept any remaining vaccinations for their childa (N = 1744) (Continued) Disagree/strongly disagree (100) 5 2.5 (0.9-7.1) aReference category is the combination of answer categories “surely yes”, “yes”, and “probably yes” bCrude odds ratios were adjusted for age, gender, ethnicity, degree of urbanization and region cOther religion included: Judaism, Buddhism, Hinduism, Catholic and Muslims dLow educational level included no education and primary education; middle educational level included junior technical school, lower general secondary education and intermediate vocational education; and high educational level included senior/higher secondary education, pre-university education and university eNet monthly income was categorized as low (less than € 1.150, which is less than € 1.167 by Statistic Netherlands); middle (€ 1.151-€ 3.050, which is € 1.168-€ 2.917 by Statistic Netherlands); high (more than € 3.051, which is more than € 2.918 by Statistic Netherlands); and a won’t answer/missing answer category was included. Statistics Netherlands available at www.cbs.nl Table 3 Potential determinants for parents less likely to accept any remaining vaccinations for their childa (N = 1744) (Continued) Parents’ intention to accept any remaining vaccinations for their child BMC Public Health 2012, 12:57 http://www.biomedcentral.com/1471-2458/12/57 Page 9 of 13 Table 3 Potential determinants for parents less likely to accept any remaining vaccinations for their childa (N = 1744) (Continued) 4 (800) 2 Reference > 4 (451) 3 1.3 (0.6-2.7) Health status child Excellent/very well (1196) 2 Reference Well/moderate/poor (548) 3 1.6 (0.8-3.3) Anthroposophic/ homeopathic/ other alternative medicine beliefs Yes (83) 13 13.4 (5.9- 30.3) No (1661) 1 Reference Religious belief Yes (38) 11 6.8 (2.1- 22.4) No (1706) 2 Reference Other ideas influencing vaccination Yes (93) 8 5.7 (2.3- 14.1) No (1651) 2 Reference Maximum no. of injections 0 (13) 54 110.7 (9.5- > ∞) 27.7 (1.6-486.6) 1 (307) 6 3.6 (0.4- 29.1) 1.5 (0.1-16.5) 2 (1376) 1 0.4 (0.1-3.5) 0.2 (0.0-2.7) > 2 and each number is acceptable (48) 2 Reference Reference Vaccinations protect health own child Strongly agree/agree (1659) 1 Reference Reference Neutral/disagree/strongly disagree (85) 25 36.7 (16.5- 81.4) 9.2 (3.0-27.8) Vaccinating healthy children is not necessary Strongly agree/agree (57) 19 39.4 (14.4- 107.5) 6.1 (1.6-23.7) Neutral (115) 12 17.7 (7.3- 42.9) 4.0 (1.2-13.0) Disagree/strongly disagree (1572) 1 Reference Reference Doubt safety vaccinations Strongly agree/agree (181) 12 15.6 (6.9- 35.1) 3.5 (1.1-11.0) Neutral (423) 1 1.0 (0.3-3.4) 0.4 (0.1-1.8) Disagree/strongly disagree (1140) 1 Reference Reference Immune system does not develop well due to vaccination Strongly agree/agree (144) 13 15.8 (6.9- 36.1) Neutral (376) 2 1.7 (0.6-4.7) Disagree/strongly disagree (1224) 1 Reference Vaccinations protect health of others Strongly agree/agree (1275) 2 Reference Neutral (369) 2 1.2 (0.5-2.8) Page 10 of 13 Mollema et al. Table 3 Potential determinants for parents less likely to accept any remaining vaccinations for their childa (N = 1744) (Continued) 2.5 (0.9-7.1) Discussion Of interest, the analyses of population-attributable fractions showed that with regard to nonparticipation in the NIP, particular attention ought to be given to In the 2006-07 survey, fewer participants in the LVC sample reported they were more inclined to accept Table 4 Comparing opinion on vaccination in the 2006-07 surveya with the 1995-96 survey Nationwide sampleb N (%) Crude OR (95%CI) Adjusted OR (95%CI) More inclined to accept vaccination First survey 498 (8) Reference Reference Second survey 268 (7) 0.8 (0.7-0.9)c 0.8 (0.7-0.9)d Less inclined to accept vaccination First survey 122 (2) Reference Reference Second survey 131 (3) 1.4 (1.1-1.9)c 1.3 (1.0-1.7)d Low vaccination coverage samplee More inclined to accept vaccination First survey 108 (9) Reference Reference Second survey 76 (6) 0.7 (0.5-1.0)f 0.6 (0.5-0.9)g Less inclined to accept vaccination First survey 27 (2) Reference Reference Second survey 60 (5) 2.2 (1.4-3.5)f 2.0 (1.2-3.2)g aThe reference category is: “My opinion had not changed with respect to the 5 years preceding the survey” bThe number of participants in the NS in the first and second survey was 5881 and 3878, respectively cAdjusted for age, gender, ethnicity, degree of urbanization and region dAdjusted for age, gender, ethnicity, degree of urbanization, region and educational level eThe number of participants in the LVC sample in the first and second survey was 1204 and 1190, respectively fAdjusted for age and gender gAdjusted for age, gender and religion (categories are as in Table 3) Table 4 Comparing opinion on vaccination in the 2006-07 surveya with the 1995-96 survey Nationwide sampleb N (%) Crude OR (95%CI) Ad ng opinion on vaccination in the 2006-07 surveya with the 1995-96 survey 4 Comparing opinion on vaccination in the 2006-07 surveya with the 1995-96 survey id l b N (%) C d OR (9 %CI) Ad Page 11 of 13 Mollema et al. BMC Public Health 2012, 12:57 http://www.biomedcentral.com/1471-2458/12/57 Page 11 of 13 vaccinating healthy children is necessary, the effect was not significant anymore. These parental concerns have been identified previously in the literature as associated with vaccination behaviour [10,17,18,21,23-27]. The vac- cination statement “no good development of immunity” has been found in literature [10] to be associated with vaccination behaviour. Univariately, a high odds ratio was observed with those less likely to accept any remaining vaccinations, but by adding the other vaccination state- ments in the multivariate model the effect was no longer significant. Discussion Among those with low SES or of Moroccan or Turkish descent, it was not possible to study which opinions on vaccination were associated with parents’ intention to accept any remaining vaccinations for their child. More (in-depth) research would be needed to ver- ify why they are participating less in the NIP and to bet- ter understand their attitude and beliefs regarding the NIP. individuals with Moroccan or Turkish ethnicity and those with low educational and income level. They seem to contribute in large measure to the rate of nonpartici- pation in the NIP, i.e., to a greater extent than well- known vaccine refusers such as specific religious groups and anthroposophics. Lower NIP participation among those with a low edu- cational level or income compared to those with a high educational level or income is consistent with the recent observation that in areas with low socioeconomic status (SES) compared to high SES the uptake of human papil- loma virus (HPV) vaccination in the catch-up campaign in 2009 was relatively low [16]. Furthermore, in a sys- tematic review by Brown et al. [17], a lower vaccination uptake was linked to lower parental education and income compared to higher parental education and income. In contrast, several other studies showed a more negative attitude/intention or higher prevalence of non- adherents in relation to future vaccinations among per- sons with a higher educational level compared to those with a lower educational level [18-20]. Similar to our finding that NIP participation was lower among non- Western migrants compared to indigenous Dutch, the uptake of HPV vaccination was found to be lower among non-Western ethnicities than among indigenous Dutch, in particular, individuals of Moroccan and Turkish des- cent [16]. Other studies have observed a higher vaccina- tion uptake among persons of Moroccan and/or Turkish descent compared to indigenous Dutch [21,22]. In the systematic review by Brown et al. [17] both a higher and a lower vaccine uptake was observed for non-white ethni- city compared to white ethnicity. We hypothesized that the lower observed NIP participation might be caused by a potential language barrier, as the invitation letter and information on vaccines included in the NIP they receive at home are in Dutch. They have to visit the NIP website to read the information in their own language. Discussion An eva- luation might be undertaken to determine whether offer- ing non-Western migrants more written and oral information in their own language would result in a higher NIP participation in this group compared to the daily practice as was described above. Some limitations of this study must be discussed. This research was part of a larger seroepidemiological study and therefore, the number of questions related to attitude towards vaccination was limited. We also could not rule out any influence by reporting bias. A short pilot for the questionnaire was performed. The calculation of a PAF presumes that the effect of the determinant that is chan- ged on vaccination rates is a causal effect. Such causality can not be inferred from our cross-sectional study. Therefore the PAF should be interpreted as the magni- tude of the improvement of rates of reporting participa- tion in the NIP that is maximally possible by removing the exposure to nonparticipation in the NIP if the effect of this exposure would be causal. Furthermore, overall responses of those who filled in a questionnaire of 66% and 40% in the first and second survey respectively, made nonresponse bias possible. The availability of background information made it possible to weight the PAFs for the variables known to be related with nonresponse, e.g., age, gender, ethnicity, region and degree of urbanization [28]. However, nonresponse bias related to other factors could be present. It might be worrisome that both in the NS and in the LVC sample, participants in 2006-07 reported to be less inclined to accept vaccination than in 1995-96. To main- tain trust in the vaccination program, we need to monitor the acceptance of the NIP in a timely fashion and try to find reasons behind a more reluctant attitude. It is likely that doubts regarding the decision to have children vacci- nated precede changes in behaviour; use of our monitor- ing system for vaccination coverage will therefore probably be less sensitive and too slow. Future research will therefore focus on developing a system to monitor the acceptance of vaccinations among parents and (child) health care professionals and to review blogs and forums about vaccination on the internet. This system could be No associations for religion, income and educational level were found with parents’ intentions to accept any remaining vaccinations for their child in contrast to our findings for NIP participation. However, a similar trend was observed. Discussion It might be that numbers were too low to observe an association as only a subset of the population was used. Univariately, anthroposophic/homeopathic or alternative medicine beliefs were associated with parents’ intention to accept any remaining vaccinations for their child. After adding statements regarding vaccination con- cerning safety, maximum number of injections, protec- tion of the health of one’s own child and whether Mollema et al. BMC Public Health 2012, 12:57 http://www.biomedcentral.com/1471-2458/12/57 Page 12 of 13 Page 12 of 13 Mollema et al. BMC Public Health 2012, 12:57 http://www.biomedcentral.com/1471-2458/12/57 Mollema et al. BMC Public Health 2012, 12:57 http://www.biomedcentral.com/1471-2458/12/57 used to develop novel and targeted interventions to increase vaccination acceptance. 3. Conyn-van Spaendonck MA, de Melker HE, Abbink F, Elzinga-Gholizadea N, Kimman TG, van Loon T: Immunity to poliomyelitis in The Netherlands. Am J Epidemiol 2001, 153:207-214. 3. Conyn-van Spaendonck MA, de Melker HE, Abbink F, Elzinga-Gholizadea N, Kimman TG, van Loon T: Immunity to poliomyelitis in The Netherlands. Am J Epidemiol 2001, 153:207-214. used to develop novel and targeted interventions to increase vaccination acceptance. 4. Van den Hof S, Meffre CM, Conyn-van Spaendonck MA, Woonink F, de Melker HE, van Binnendijk RS: Measles outbreak in a community with very low vaccine coverage, the Netherlands. Emerg Infect Dis 2001, 7(Suppl 3):593-597. Competing interests 21. Hontelez JAC, Hahné SJM, Oomen P, de Melker H: Parental attitude towards childhood HBV vaccination in The Netherlands. Vaccine 2010, 28:1015-1020. The authors declare that they have no competing interests. The authors declare that they have no competing interests. Received: 17 August 2010 Accepted: 20 January 2012 Published: 20 January 2012 Received: 17 August 2010 Accepted: 20 January 2012 Published: 20 January 2012 Received: 17 August 2010 Accepted: 20 January 2012 Published: 20 January 2012 22. Timmermans DR, Henneman L, Hirasing RA, van der Wal G: Attitudes and risk perception of parents of different ethnic backgrounds regarding meningococcal C vaccination. Vaccine 2005, 23:3329-3335. Acknowledgements W h k S bi d G g We thank Sabine de Greeff and Alies van Lier for suggestions on this manuscript, and Sally Ebeling, Jane Whelan and Susan T. Landry for English language editing. pp p 12. Van der Klis FRM, Mollema L, Berbers GAM, de Melker HE, Coutinho RA: Second national serum bank for population-based seroprevalence studies in the Netherlands. Neth J Med 2009, 67:301-308. Author details 1 13. Deubner DC, Wilkinson WE, Helms HJ, Tyroler HA, Hames CG: Logistic model estimation of death attributable to risk factors for cardiovascular disease in Evans County, Georgia. Am J Epidemiol 1980, 112:135-143. 1Centre for Infectious Disease Control Netherlands, RIVM, Bilthoven, The Netherlands. 2Department of Earth and Life Science, VU University Amsterdam, Amsterdam, The Netherlands. 3ICT/Expertise Centre for Methodology and Information Service, RIVM, Bilthoven, The Netherlands. 4National Institute for Public Health and the Environment, Centre for Infectious Disease Control Netherlands, Epidemiology and Surveillance Unit, P.O. Box 1, 3720 BA, Bilthoven, The Netherlands. 14. In Dictionary of Epidemiology.. Fifth edition. Edited by: Porta M. New York: Oxford University Press; 2008:9-12. 15. Rao JNK, Wu CFJ, Yue K: Some recent work on re-sampling methods for complex surveys. Survey Methodology 1992, 18:209-217. 16. Rondy M, van Lier A, van de Kassteele J, Rust L, de Melker H: Determinants for HPV vaccine uptake in the Netherlands: a multilevel study. Vaccine 2010, 28:2070-2075. Authors’ contributions LM contributed greatly to the design and performance of the second population-based seroprevalence study, performed the statistical analysis of this study, and drafted the manuscript. NW assisted in performing statistical analysis of this study, and in drafting the manuscript. SH contributed to the design of the second population-based seroprevalence study, assisted in performing the statistical analysis of this study, and helped to draft the manuscript. FvdK made a large contribution to the design and performance of the second population-based seroprevalence study, and helped to draft the manuscript. HB assisted in performing the statistical analyses and helped to draft the manuscript. HdM made a large contribution to the design and performance of the first population-based seroprevalence study, contributed to the design of the second population-based seroprevalence study and revised the article critically for important intellectual content. All authors read and approved the final manuscript. 17. Brown KF, Kroll JS, Hudson MJ, Ramsay M, Green J, Long SJ, et al: Factors underlying parental decisions about combination childhood vaccinations including MMR: a systematic review. Vaccine 2010, 28:4235-4248. 18. Hak E, Schönbeck Y, de Melker H, van Essen GA, Sanders EAM: Negative attitude of highly educated parents and health care workers towards future vaccinations in the Dutch childhood vaccination program. Vaccine 2005, 23:3103-3107. 19. Paulussen ThGW, Lanting CI, Buijs GJ, Hirasing RA: Parents about the Dutch Childhood Vaccination program; satisfaction and preparedness towards vaccinations of parents of young children [In Dutch]. Leiden: TNO Prevention and Health; 2000, TNO-report PG/JGD/2000.033. 20. Opstelten W, Hak E, Verheij TJ, Van Essen GA: Introducing a pneumococcal vaccine to an existing influenza immunization program: vaccination rates and predictors of noncompliance. Am J Med 2001, 111:474-479. Conclusions Most participants had a positive attitude towards vacci- nation, although some had doubts. Groups with a lower income or educational level or of non-Western descent participated less in the NIP than those with a high income or educational level or indigenous Dutch and have been less well identified previously. Particular attention ought to be given to these groups as they con- tribute in large measure to the rate of nonparticipation in the NIP, i.e., to a greater extent than well-known vaccine refusers such as specific religious groups and anthroposophics. Our finding that the proportion of the population inclined to accept vaccinations is smaller than it was 10 years ago highlights the need to increase knowledge about attitudes and beliefs regarding the NIP. 5. Hahné SJ, Ward M, Abbink F, van Binnendijk R, Ruijs H, van Steenbergen J, Timen A, de Melker H: Large ongoing rubella outbreak in religious community in the Netherlands since September 2004. Euro Surveill 2005, 10:E050303 2. 6. Karagiannis I, van Lier A, van Binnendijk R, Ruijs H, Fanoy E, Conyn-van Spaendonck MA, de Melker H, Hahné S: Mumps in a community with low vaccination coverage in the Netherlands. Euro Surveill 2008, 13:18901. 7. De Velzen E, de Coster E, van Binnendijk R, Hahné S: Measles outbreak in an anthroposophic community in The Hague, The Netherlands, June- July 2008. Euro Surveill 2008, 13:18945. 7. De Velzen E, de Coster E, van Binnendijk R, Hahné S: Measles outbreak in an anthroposophic community in The Hague, The Netherlands, June- July 2008. Euro Surveill 2008, 13:18945. 8. Hanratty B, Holt T, Duffell E, Patterson W, Ramsay M, White JM, Jin L, Litton P: UK measles outbreak in non-immune anthroposophic communities: the implications for the elimination of measles from Europe. Epidemiol Infect 2000, 125:377-383. 9. Schmid D, Holzmann H, Schwarz K, Kasper S, Kuo H-W, Aberle SW, et al: Measles outbreak linked to a minority group in Austria, 2008. Epidemiol Infect 2010, 138:415-425. 10. Paulussen TGW, Hoekstra F, Lanting CI, Buijs GB, Hirasing RA: Determinants of Dutch parents’ decisions to vaccinate their child. Vaccine 2006, 24:644-651. 11. De Melker HE, Conyn-van Spaendonck MA: Immunosurveillance and the evaluation of national immunization programmes: a population-based approach. Epidemiol Infect 1998, 121:637-643. References Pre-publication history The pre-publication history for this paper can be accessed here: http://www.biomedcentral.com/1471-2458/12/57/prepub doi:10.1186/1471-2458-12-57 Cite this article as: Mollema et al.: Participation in and attitude towards the national immunization program in the Netherlands: data from population-based questionnaires. BMC Public Health 2012 12:57. 26. Mills E, Jadad AR, Ross C, Wilson K: Systematic review of qualitative studies exploring parental beliefs and attitudes toward childhood vaccination identifies common barriers to vaccination. J Clin Epidemiol 2005, 58:1081-1088. 27. Timmermans DR, Henneman L, Hirasing RA, van der Wal G: Parents’ perceived vulnerability and perceived control in preventing Meningococcal C infection: a large-scale interview study about vaccination. BMC Public Health 2008, 8:45. 28. De Melker HE, Nagelkerke NJD, Conyn-van Spaendonck MAE: Nonparticipation in a population-based seroprevalence study of vaccine- preventable diseases. Epidemiol Infect 2000, 124:255-262. References 23. Bonnani P, Bergamini M: Factors influencing vaccine uptake in Italy. Vaccine 2002, 20:S8-S12. 1. Van Lier EA, Oomen PJ, Oostenbrug MW, Zwakhals SL, Drijfhout IH, de Hoogh PA, de Melker HE: High vaccination coverage of the National Immunization Program in the Netherlands. Ned Tijdschr Geneesk 2009, 153:950-957, [Article in Dutch]. 1. Van Lier EA, Oomen PJ, Oostenbrug MW, Zwakhals SL, Drijfhout IH, de Hoogh PA, de Melker HE: High vaccination coverage of the National Immunization Program in the Netherlands. Ned Tijdschr Geneesk 2009, 153:950-957, [Article in Dutch]. 24. Schmitt HJ: Factors influencing vaccine uptake in Germany. Vaccine 2002, 20:S2-S4. 25. Swennen B, van Damme P, Vellinga A, Coppieters Y, Depoorter AM: Analysis of factors influencing vaccine uptake: perspectives from Belgium. Vaccine 2002, 20:S5-S7. 2. Woonink F: Objections to vaccination: from the perspective of the refuser. Infectieziekten bulletin 2009, 20:50-51, [Article in Dutch]. 2. Woonink F: Objections to vaccination: from the perspective of the refuser. Infectieziekten bulletin 2009, 20:50-51, [Article in Dutch]. Page 13 of 13 Page 13 of 13 Mollema et al. BMC Public Health 2012, 12:57 http://www.biomedcentral.com/1471-2458/12/57 26. Mills E, Jadad AR, Ross C, Wilson K: Systematic review of qualitative studies exploring parental beliefs and attitudes toward childhood vaccination identifies common barriers to vaccination. J Clin Epidemiol 2005, 58:1081-1088. 27. Timmermans DR, Henneman L, Hirasing RA, van der Wal G: Parents’ perceived vulnerability and perceived control in preventing Meningococcal C infection: a large-scale interview study about vaccination. BMC Public Health 2008, 8:45. 28. De Melker HE, Nagelkerke NJD, Conyn-van Spaendonck MAE: Nonparticipation in a population-based seroprevalence study of vaccine- preventable diseases. Epidemiol Infect 2000, 124:255-262. Pre-publication history The pre-publication history for this paper can be accessed here: http://www.biomedcentral.com/1471-2458/12/57/prepub doi:10.1186/1471-2458-12-57 Cite this article as: Mollema et al.: Participation in and attitude towards the national immunization program in the Netherlands: data from population-based questionnaires. BMC Public Health 2012 12:57. 26. Mills E, Jadad AR, Ross C, Wilson K: Systematic review of qualitative studies exploring parental beliefs and attitudes toward childhood vaccination identifies common barriers to vaccination. J Clin Epidemiol 2005, 58:1081-1088. 27. Timmermans DR, Henneman L, Hirasing RA, van der Wal G: Parents’ perceived vulnerability and perceived control in preventing Meningococcal C infection: a large-scale interview study about vaccination. BMC Public Health 2008, 8:45. 28. De Melker HE, Nagelkerke NJD, Conyn-van Spaendonck MAE: Nonparticipation in a population-based seroprevalence study of vaccine- preventable diseases. Epidemiol Infect 2000, 124:255-262. Pre-publication history The pre-publication history for this paper can be accessed here: http://www.biomedcentral.com/1471-2458/12/57/prepub The pre-publication history for this paper can be accessed here: http://www.biomedcentral.com/1471-2458/12/57/prepub doi:10.1186/1471-2458-12-57 Cite this article as: Mollema et al.: Participation in and attitude towards the national immunization program in the Netherlands: data from population-based questionnaires. BMC Public Health 2012 12:57. 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A 36-Year-Old Renal Transplant Recipient Female with Leg Ulcer: A Case Report and Brief Review
Case reports in infectious diseases
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Hindawi Case Reports in Infectious Diseases Volume 2018, Article ID 5086501, 6 pages https://doi.org/10.1155/2018/5086501 Hindawi Case Reports in Infectious Diseases Volume 2018, Article ID 5086501, 6 pages https://doi.org/10.1155/2018/5086501 Hindawi Ali Monfared,1 Eftekhari Hojat,2 Seyed Alireza Mesbah,3 Abbas Darjani,2 and Seyyede Zeinab Azimi 2 1Department of Nephrology, Urology Research Center, Razi Hospital, School of Medicine, Guilan University of Medical Sciences, Rasht, Iran 2Department of Dermatology, Skin Research Center, Razi Hospital, School of Medicine, Guilan University of Medical Sciences, Rasht, Iran 3Razi Pathobiology Laboratory Rasht Iran Department of Nephrology, Urology Research Center, Razi Hospital, School of Medicine, Guilan University of Medi Rasht, Iran Department of Dermatology, Skin Research Center, Razi Hospital, School of Medicine, Guilan University of Medi Rasht, Iran Correspondence should be addressed to Seyyede Zeinab Azimi; sz.azimi@yahoo.com Received 16 October 2017; Revised 1 December 2017; Accepted 7 December 2017; Published 11 January 2018 Academic Editor: Sin´esio Talhari Academic Editor: Sin´esio Talhari Copyright © 2018 Ali Monfared et al. Tis 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. Background. Opportunistic infections are common in organ transplant recipients. After 6 months of transplantation, patients have the highest risk of opportunistic infections such as cryptococcosis. Case Presentation. Te report presents the case of a 36-year-old female renal transplant recipient, with complaints of few subcutaneous painful and warm nodules and large, warm, erythematous, nontender plaques on the mildly edematous right leg and ankle. Incisional biopsy of the subcutaneous nodule over the leg showed panniculitis with small- to medium-sized vasculitis associated with round yeast forms, and culture of the fragments revealed C. neoformans var. grubii. Conclusions. Tis article also reviews in brief the treatment of this rare complication. Reviewing the literature showed that since the cryptococcal cutaneous lesions are often nonspecific, the clinical picture solely is not enough to construct a definite diagnosis and there must be a high clinical suspicion. painful and warm, and the skin over the nodules consisted of focal superficial ulcerations (Figure 1). She was admitted for further evaluations. 1. Introduction Cutaneous disorders in kidney transplant recipients are common. Organ transplant recipients’ survival enhanced because of advancements in immunosuppressive therapies. Due to prolonged survival among immunosuppressed patients, it is very important to pay more attention to increased risk of skin lesions and early detection, as well as appropriate management of them [1]. On admission, she was conscious, alert, and oriented. Her vital signs were stable. Tere was no history of fever, cough, hemoptysis, dysuria, oliguria, headache, or vomiting. Also there was no lymphadenopathy or hep- atosplenomegaly. Physical examination by a consultant dermatologist showed multiple erythematous plaques and nodules on the anterolateral portion of right leg and dorsum of calcaneus. Tere was a central ulceration on one plaque on the leg as well. Te margins were discrete. San- guineous and purulent exudative materials were observed from both margins and center of the lesion (Figure 1). Te pulse of the dorsalis pedis artery was well palpable. 2. Case Presentation A 36-year-old female, a resident of north of Iran, presented with a complaint of few subcutaneous nodules during a visit to renal transplant clinic. She also had history of large, warm, erythematous, nontender plaques on the mildly edematous right leg and ankle since one month ago. She had been in her usual health status until one month ago. Te nodules were She had undergone renal transplantation 15 years earlier for end-stage renal disease due probably to Alport syndrome. Her immunosuppression regimen consisted of prednisolone Case Reports in Infectious Diseases 2 (a) (b) (c) (d) (e) (f) Figure 1: (a to d) Large, warm, erythematous, nontender plaques and subcutaneous nodules on the edematous right leg and ankle in a renal transplant recipient on admission. (e and f) Hematoxylin and eosin shows fat necrosis and coagulative necrosis and rare foci of vasculitis as well as foci of RBC extravasation. (a) (b) (c) (b) (c) (c) (a) (b) (a) (a) (b) (b) (a) (c) (d) (d) (e) (f) Figure 1: (a to d) Large, warm, erythematous, nontender plaques and subcutaneous nodules on the edematous right leg and ankle in a transplant recipient on admission. (e and f) Hematoxylin and eosin shows fat necrosis and coagulative necrosis and rare foci of vascul well as foci of RBC extravasation. (d) (e) (f) (d) (e) (f) Figure 1: (a to d) Large, warm, erythematous, nontender plaques and subcutaneous nodules on the edematous right leg and ankle in a renal transplant recipient on admission. (e and f) Hematoxylin and eosin shows fat necrosis and coagulative necrosis and rare foci of vasculitis as well as foci of RBC extravasation. 5mg/day, cyclosporine 50mg/BD, and mycophenolate mofetil 500mg/BD. Laboratory investigations are shown in Table 1. tibial crest and the dorsum of calcaneus, with sharply de- marcated red border. Te lesions were almost circumfer- ential, with no hyperpigmentation or purpuric-type rash on the skin. Tere was no peripheral neuropathy. Over a period of a few days, her condition deteriorated, and the ulcerative area rapidly increased in size (Figure 2). Te leg ulcer was noted to have signs of infection, and swab cultures were reported as positive for Escherichia coli. She was treated with a course of ciprofloxacin and clinda- mycin, but no improvement was achieved after 96 hr of antimicrobial therapy. She developed erythema, tenderness, and edema of the right calf; the ulcers extended progressively over the tibial crest. 2. Case Presentation Cyclosporine was changed to rapamune 1 mg/day. Also vancomycine and ceftazidim were prescribed for bacterial super infection. Her renal functions remained stable during therapy. C3 and C4 levels were 93 and 36mg/dL, respectively; ANCA was negative (3.6) and angiotensin-converting enzyme (ACE) level was 65µL. Urine analyses and urine culture showed normal results. She was negative for hepatitis B surface antigen, hepatitis B core antibody, hepatitis C virus, human immuno- deficiency virus (HIV), and cytomegalovirus (CMV). Te pu- rified protein derivative (PPD) test was negative as well. Te chest X-ray showed no abnormal lesion. Te culture of tissue biopsy revealed the yeast colonies. Ten, she was treated with conventional amphotericin B 50 mg/day and oral fluconazole 200 mg/BD (adjusted due to her renal function) subsequently. But, the patient developed edema and tenderness over the right leg, with extensive ulcerations of her right leg and foot. Te result of a test received. Results of direct India ink examination and culture of an aspirate from the leg were positive for cryptococcus. One day later, an incisional biopsy taken from the subcutaneous nodule over the leg showed panniculitis with small- to medium-sized vasculitis (Figure 1). Reevaluation of skin biopsies showed the presence of chronic panniculitis associated with round yeast forms. Unless specifically searched for, the organisms were During this period, lower limb swelling and pain in- creased. Ulcers developed on her mid to lower leg over the Case Reports in Infectious Diseases 3 3 Table 1: Laboratory data on admission and one month later. 2. Case Presentation Laboratory data Normal range adults On admission One month after admission White cell count (per mm3) 4–10 9.5 7.2 Differential count (%) Neutrophils 40–60 85 83 Lymphocytes 20–40 13 10 Monocytes 4–8 2 2 Eosinophils 1–3 — 1 Basophils 0–1 — 1 Band forms 0–5 — — Red cell count (per µL) 4.1–5.1 4.7 3.3 Mean corpuscular volume (fl) 80–100 80.6 81.3 Mean corpuscular hemoglobin (pg/red/cell) 27–32 24.6 26.1 Mean corpuscular hemoglobin concentration (g/dL) 31–37 29 31.3 Red-cell distribution width (%) 10.6–15.7 13.8 14.5 Hemoglobin (g/dL) 14–18 9.7 9 Hematocrit (%) 42–52 40.03 30 Platelet count (per mm3) 150–450 313 189 Sodium (mmol/liter) 135–145 139 135 Potassium (mmol/liter) 3.5–5.3 3.8 3.7 Calcium (mg/dL) 8.5–10.5 8.1 7.5 Phosphorus (mg/dL) 2–4 3.5 3.2 Blood urea nitrogen (mg/dL) 7–21 41 37 Creatinine (mg/dL) 0.6–1.1 2.5 1.5 Estimated glomerular filtration rate (ml/min/1.73 m2) 90 27.01 45.01 Glucose (mg/dL) 140 145 125 Total protein (g/dL) 6.6–8.8 6.1 5.9 Albumin (g/dL) 3.5–5 3.6 3.8 Alanine aminotransferase (U/liter) <37 45 40 Aspartate aminotransferase (U/liter) <41 36 37 Alkaline phosphatase (U/liter) 80–306 93 100 Total bilirubin (mg/dL) 0–1.1 0.7 0.6 Direct bilirubin (mg/dL) 0–0.2 0.2 0.2 Iron (µg/dL) 30–150 55 7 Iron-binding capacity (µg/dL) 230–400 216 230 Ferritin (ng/ml) 10–100 54 55 Table 1: Laboratory data on admission and one month later. Table 1: Laboratory data on admission and one month later. Te serum cryptococcal antibody titer was positive (1:16), and it was confirmed by polymerase chain reaction (PCR). Cerebrospinal fluid was negative for cryptococcus. No evidence of disseminated disease was found. Two weeks later, she de- veloped right knee arthritis which was negative for cryptococcus in synovial assay. Blood cultures were also negative. Ampho- tericin B was continued (1 mg/kg/day). Chest radiography was normal. Subsequent questioning revealed that she had no contact with pigeons. difficult to find on hematoxylin and eosin preparations. Periodic acid-Schiffreagent, alcian blue, and mucicarmine showed single, small yeast forms surrounded by a clear halo. Tese organisms were present in moderate numbers in the fat and occasionally in the reticular dermis. Giemsa and Prussian blue stains were negative (Figure 3). Foci of fat necrosis, coagulative necrosis, and rare foci of vas- culitis involving small- to medium-sized vessels resulting luminal occlusion were also evident, as well as foci of RBC extravasation. Te above findings were suggestive of cryptococcal panniculitis associated with secondary vasculitis. Te skin lesions regressed gradually, and ulcers started healing, however leaving eschars. 3. Discussion Cutaneous disorders in kidney transplant recipients are common. Organ transplant recipients’ survival enhanced because of advancements in immunosuppressive therapies. Due to prolonged survival among immunosuppressed patients, it is very important to pay more attention to in- creased risk of skin lesions and early detection, as well as appropriate management of them. Tese skin conditions include aesthetic distortion, iatrogenic lesions due to im- munosuppressive drugs, infections, precancerous lesions, and malignancies [1]. In immunocompetent hosts, the infection is usually limited to the lungs, with self-limited flu-like symptoms. Although cryptococcosis has been commonly encountered among the HIV-infected population, there are reports of cryptococcosis in non-HIV-infected patients such as human stem cell transplant (HSCT) recipients, solid organ trans- plant recipients, patients with hematologic malignancies, and patients with other malignancies [10]. Cutaneous cryptococcosis is a manifestation of dis- seminated infection, and therefore, it should prompt a comprehensive investigation into cerebrospinal fluid, blood, the lungs, and serum cryptococcal antigen. Usually, cryptococcosis starts as a respiratory tract infection, followed by hematogenous dissemination to other organs. Primary cutaneous cryptococcosis is defined as lesions confined to a restricted body region with positive skin cultures for C. neoformans and without signs of concurrent dissemination [12]. Opportunistic infections are common among organ transplant recipients [1]. Cutaneous manifestations of in- fection in immunosuppressive recipients may be an im- portant clue to their existence [2, 3]. Skin infections may be primary or secondary. Sec- ondary cutaneous infections are because of an underlying systemic or disseminated infection. In the first post- operative month, most infections are due to nosocomial infections and surgical complications [4]. Staphylococcus and Streptococcus superficial pyodermas, cellulitis, and wound infections are the most common infections during this period [1]. Moreover, atypical pathogens contribute to soft tissue and wound infections in this group. Reactivation of herpes simplex virus (HSV) often occurs within the first 3 postoperative weeks. Te most common presentation of HSV is anogenital and orolabial lesions [5]. A few cases of cutaneous lesions with no evidence of systemic involvement have been reported, but this makes controversial with current knowledge that cutaneous in- volvement points to disseminated disease [13]. About 15% of patients with cryptococcosis have skin lesions. Te most common cutaneous presentations of cryptococcosis are papules, nodules on the skin resem- bling molluscum contagiosum, vesicles, nodules, mass, pustule, abscess, ulcers, ecchymosis, and rarely cellulitis. 2. Case Presentation Black eschars remained persistent despite administration of fibrinolysin ointment. Surgical debridements were carried out to remove the ne- crotic tissue, and then graft was performed for her. Te plan is to continue fluconazole for at least 1 year. Te culture of biopsy from the leg’s necrotic ulcer grew C. neoformans var. grubii. Te overall colonies appearance was creamy, smooth, and mucoid. Case Reports in Infectious Diseases 4 Figure 2: Rapid deterioration of ulcers and increase in the lesion size after 2 weeks. cryptococcosis, and nocardiosis as well as reactivation of varicella-zoster virus [7]. Figure 2: Rapid deterioration of ulcers and increase in the lesion size after 2 weeks. Te skill to connect particular pathogens and in- fections with compromised immunity can help to set up a differential diagnosis and direct a logical management plan. Te convenience of the skin to rapid diagnostic sampling procedures can save lives and prevent the most invasive kinds. Fungal infection following solid-organ transplantation is a major cause of morbidity and mortality. After candidiasis and aspergillosis, cryptococcosis is the third most common invasive fungal infection in organ transplant recipients. Te immunosuppressive regimens and antirejection therapy with pulse-dose methylprednisolone used in solid organ transplantation might have been considered as the predis- posing factor [8, 9]. Cryptococcus neoformans is present in soils enriched by bird excreta; fruits may also harbor the yeast. It is usually acquired through inhalation of airborne spores [9, 10]. However, our patient had no history of cutaneous injury, outdoor activities, and exposure to bird droppings. In the past few years, it became evident that direct inoculation of the pathogen through a minor skin wound can cause pri- mary infection of the skin [11]. Figure 2: Rapid deterioration of ulcers and increase in the lesion size after 2 weeks. 3. Discussion Necrotizing fasciitis, eschar, ecchymosis, panniculitis, vegetating plaques, palpable purpura, and pyoderma gangrenosum-like lesion and cellulitis with necrotizing vasculitis have also been reported in the organ transplant recipients [12, 13]. In 2 to 6 months after renal transplantation, opportunistic infections with organisms such as atypical mycobacterial spe- cies, mycobacterium tuberculosis, CMV, Epstein-Barr virus, nocardia, and bartonella are common [4, 6]. After 6 months of transplantation, patients with in- adequate graft function need higher than usual immuno- suppressive dosage. So these patients have the highest risk of opportunistic infections such as Pneumocystis carinii, In transplant recipients, drug-induced immune deficiency results in the extrapulmonary hematogenous spreading of cryptococcosis, with the involvement of other organs, such as Case Reports in Infectious Diseases Case Reports in Infectious Diseases 5 (a) (b) (c) (d) (e) (f) Figure 3: (a) Periodic acid-Schiff(PAS), (b) alcian blue, and (c) colloidal iron stains were positive, but (d) Giemsa and (e) Prussian blue stains were negative for cryptococcus. (f) Mucicarmine stain was positive for cryptococcosis. (b) (c) (a) (a) (b) (c) (d) (e) (f) (e) (d) Figure 3: (a) Periodic acid-Schiff(PAS), (b) alcian blue, and (c) colloidal iron stains were positive, but (d) stains were negative for cryptococcus. (f) Mucicarmine stain was positive for cryptococcosis. Figure 3: (a) Periodic acid-Schiff(PAS), (b) alcian blue, and (c) colloidal iron stains were positive, but (d) Giemsa and (e) Prussian blue stains were negative for cryptococcus. (f) Mucicarmine stain was positive for cryptococcosis. d-Schiff(PAS), (b) alcian blue, and (c) colloidal iron stains were positive, but (d) Giemsa and (e) Prussian blue ryptococcus. (f) Mucicarmine stain was positive for cryptococcosis. the central nervous system (CNS), as well as the cutaneous tissue, urinary system, bones, and joints. Cutaneous lesions may be the only sign of severe systemic disease in transplant patients. Not only do cutaneous lesions largely characterize hematogenous dissemination, but the skin also has been recognized as a portal of access to cryptococcus and a po- tential source of following disseminated disease in transplant recipients [10]. from 33 to 61 years, and the mean age of onset of panniculitis was 49.64 years [10]. Disclosure [11] J. V. Zorman, T. L. Zupanc, Z. Parac, and I. Cucek, “Primary cutaneous cryptococcosis in a renal transplant recipient: case report,” Mycoses, vol. 53, no. 6, pp. 535–537, 2010. Te abstract of this manuscript was presented before in the 18th Congress of the European Society for Organ Trans- plantation, 24–27 September 2017, Barcelona, Spain, Posters. Transpl. Int., 30: 390–576. doi:10.1111/tri.13053. [12] H. Y. Sun, B. D. Alexander, O. Lortholary et al., “Cutaneous cryptococcosis in solid organ transplant recipients,” Medical Mycology, vol. 48, no. 6, pp. 785–791, 2010. [13] M. Kimura, E. Kadota, T. Satou, E. Yoneda, and T. Furuta, “Case report. Cryptococcal cellulitis showing necrotizing vasculitis,” Mycoses, vol. 44, no. 3-4, pp. 115–118, 2001. Conflicts of Interest Te authors declare that they have no conflicts of interest. [14] N. Singh, F. Dromer, J. R. Perfect, and O. Lortholary, “Cryptococcosis in solid organ transplant recipients: current state of the science,” Clinical Infectious Diseases, vol. 47, no.10, pp. 1321–1327, 2008. Authors’ Contributions Drs. Ali Monfared and Seyyede Zeinab Azimi had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. All authors involved in acquisition and interpretation of data. Dr. Seyyede Zeinab Azimi drafted the manuscript. Drs. Ali Monfared, Seyyede Zeinab Azimi, and Seyed Alireza Mesbah involved in critical revision of the manuscript for important intellectual content. All authors involved in obtaining funding. Drs. Ali Monfared, Seyyede Zeinab Azimi, and Seyed Alireza Mesbah provided administrative and technical support. All authors involved in study supervision. [15] J. R. Perfect, W. E. Dismukes, F. Dromer, D. L. Goldman, R. John, and J. R. Graybill, “Clinical practice guidelines for the management of cryptococcal disease: 2010 update by the Infectious Diseases Society of America,” Clinical Infectious Diseases, vol. 50, no. 3, pp. 291–322, 2010. Case Reports in Infectious Diseases 6 by a lower dose of oral fluconazole (200 to 400 mg/day) for six to 12 months. [5] S. R. Smith, D. W. Butterly, B. D. Alexander, and A. Greenberg, “Viral infections after renal transplantation,” American Journal of Kidney Diseases, vol. 37, no. 4, pp. 659–676, 2001. If flucytosine was not prescribed from the beginning, four to six weeks of therapy with liposomal formulations of amphotericin B should be considered. Patients with a high- fungal burden or those on relapse benefit from liposomal amphotericin B (6 mg/kg/day). [6] S. Yodmalai, S. Chiewchanvit, and P. Mahanupab, “Cutaneous miliary tuberculosis in a renal transplant patient: a case report and literature review,” Southeast Asian Journal of Tropical Medicine and Public Health, vol. 42, no. 3, pp. 674–678, 2011. [7] S. Gourishankar, J. C. McDermid, G. S. Jhangri, and J. K. Preiksaitis, “Herpes zoster infection following solid organ transplantation: incidence, risk factors and outcomes in the current immunosuppressive era,” American Journal of Transplantation, vol. 4, no. 1, pp. 108–115, 2004. In immunocompetent patients, disseminated, non-CNS cryptococcus infection is treated with oral fluconazole for 3–6 months or with itraconazole for 6–12 months. In immunosuppressed patients, the initial treatments are the same, but lifelong maintenance treatment with fluco- nazole may be required. Te primary cutaneous disease can be treated with oral fluconazole or itraconazole [15]. [8] R. Chaya, S. Padmanabhan, V. Anandaswamy, and A. Moin, “Disseminated Cryptococcosis presenting as cellulitis in a renal transplant recipient,” Journal of Infection in Developing Countries, vol. 7, no. 1, pp. 060–063, 2013. Te present case was also started with conventional amphotericin B 50 mg/day without flucytosine in the in- duction phase of therapy and was followed by fluconazole 200 mg/twice a day. Our patient showed significant clinical improvement after systemic antifungal therapy. [9] D. Bhowmik, A. K. Dinda, I. Xess et al., “Fungal panniculitis in renal transplant recipients,” Transplant Infectious Disease, vol. 10, no. 4, pp. 286–289, 2008. [10] S. K. Kothiwala, M. Prajapat, C. M. Kuldeep, and A. Jindal, “Cryptococcal panniculitis in a renal transplant recipient: case report and review of literature,” Journal of Dermatological Case Reports, vol. 9, no. 3, pp. 76–80, 2015. 4. Management In immunocompromised patients, we need to evaluate sys- temic involvement in a complete assessment including a col- lection of large volume CSF (≥1ml or 20 drops) and blood and urine analysis (10). About 88–91% of the organ transplant recipients with cryptococcal meningitis show positive serum cryptococcal antigen; notably in non-HIV-infected recipients, the serum and CSF antigen titers are usually lower. Localized cutaneous lesion in HIV-infected patients that did not develop AIDS is often antigen negative [14]. Since the cryptococcal cutaneous lesions are often nonspecific, the clinical picture solely is not enough to construct a definite diagnosis and there must be a high clinical suspicion. We need deep skin biopsy consisting of soft tissue to make the diagnosis of panniculitis. With only scraping the skin or aspiration material, one may identify fungal infection, but panniculitis would be missed. Tis may elucidate why only occasional case reports have revealed fungal panniculitis in posttransplant patients [9]. Te Infectious Diseases Society of America (IDSA) updated guidelines on the management of cryptococcal disease consist of categories based on the organ involvement. Patients with central nervous system (CNS) disease should be treated with liposomal amphotericin B (AmB) (3-4mg/kg/day intravenously) or amphotericin B lipid complex (5mg/kg/day intravenously) plus flucytosine (100mg/kg/day in four divided doses) for at least two weeks, followed by oral fluconazole (400 to 800 mg (6 to 12 mg/kg) per day) for eight weeks and then followed Only 14 cases of cryptococcal panniculitis in solid organ transplant recipient have been reported so far. After reviewing all these cases of cryptococcal panniculitis, it has been estimated that duration between organ transplant and cryptococcal in- fection ranges from 3 months to 31 years with a mean period of 9.64 years. Age of onset of cryptococcal infection varies Case Reports in Infectious Diseases References [1] B. L. Kasiske and M. G. Zeier, “KDIGO clinical practice guideline for the care of kidney transplant recipients,” 2009, http://kdigo.org/wp-content/uploads/2017/02/KDIGO- 2009-Transplant-Recipient-Guideline-English.pdf. [2] M. Castello, M. Gregorini, T. Rampino et al., “A retrospective analysis of dermatological lesions in kidney transplant pa- tients,” Indian Journal of Medical Research, vol. 137, no. 6, pp. 1188–1192, 2013. [3] A. A. Hogewoning, W. Goettsch, H. van Loveren, J. W. de Fijter, B. J. Vermeer, and J. N. B. Bavinck, “Skin infections in renal transplant recipients,” Clinical Transplantation, vol. 15, no. 1, pp. 32–38, 2001. [4] K. Skorecki, G. Chertow, P. Marsden, M. Taal, and A. Yu, Brener and Rectors the Kidney, Elsevier, 10th edition, 2016.
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Optofluidics Research Article • DOI: 10.2478/optof-2013-0001 • Optofluidics • 2013 • 1-10 Modular Platforms for Optofluidic Systems Abstract Marko Brammer1,2∗ , Timo Mappes1,3† Optofluidics is increasingly gaining impact in a number of different fields of research, namely biology and medicine, environmental monitoring and green energy. However, the market for optofluidic products is still in the early development phase. In this manuscript, we discuss modular platforms as a potential concept to facilitate the transfer of optofluidic sensing systems to an industrial implementation. We present microfluidic and optical networks as a basis for the interconnection of optofluidic sensor modules. Finally, we show the potential for entire optofluidic networks. 1 This work has been carried out at the Karlsruhe Institute of Technology (KIT), Institute of Microstructure Technology, 76128 Karlsruhe, Germany 2 Now with Festo AG, 73734 Esslingen, Germany 3 Now with Carl Zeiss AG, Corpoate Research & Technology, 07745 Jena, Germany Received 2012-12-21 Accepted 2013-01-21 Keywords optofluidics • microfluidics • micro total analysis systems • backplane • modular platform PACS: © Versita sp. z o.o. 1. Introduction idic demonstrators in the research society. Optofluidics emerged from microfluidics and thus uses mainly the same fabrication technologies. Optofluidic systems are based on the interaction between light and fluid. Controlling the properties of the fluid allows for manipulating the properties of this interaction. Due to this significant advantage, considerable research progress has been made in the recent past towards complex system design for several applications [1–6]. One major research focus lies on analysis applications in areas such as pharmaceutical, biotechnology, and life-sciences. However, soft-lithography has not been successfully established for large-scale industrial implementation yet, due to several drawbacks, such as swelling with several solvents and of permeability of dyes [15, 16]. Until now industrial fabrication of microfluidic and miniature fluidic devices is mostly based on thermoplastic replication technologies [17, 18], such as injection molding, hot embossing or injection embossing [15, 19–21]. Examples for widely used thermoplastic materials are Polymethylmethacrylate (PMMA) and Cyclic-Olefin Copolymers (COC). Potential low-cost alternatives are cellulose-based materials [22, 23]. The technologies of choice are still to be defined. This applies for structuring as well as for assembly and packaging of the fluidic channel plates. Candidates for assembly technologies are thermally, chemically, and radiation induced bonding, adhesive bonding [24], and laser beam welding [25]. This progress in optofluidics is comparable with the development of microfluidics in the 80s and 90s [7–9]. First microfluidic devices have been fabricated by well-proven microelectromechanical systems (MEMS) technologies in silicon and glass [10–12]. In contrast to most MEMS devices, microfluidic devices are larger and often used as disposables. Thus, new cost-efficient fabrication technologies and materials were crucial for commercial success. Henceforth, most devices were made of polymers and structured by replication technologies. The polydimethylsiloxane (PDMS) based soft-lithography process offers a method for rapid prototyping with low investment costs needed for implementtation [13, 14]. Moreover, PDMSbased devices inherit several advantages, such as tunability due to its viscoelastic behavior. This caused the widespread use of this technology platform for microflu∗ † Microfluidics and optofluidics have further aspects in common besides the fabrication technologies. To date the commercially most important industrial implementation of a MEMS-based microfluidic device is the ink-jet printer head [26]. Similarly to microfluidics, the commercially most relevant industrial implementation of an optofluidic principle, the liquid crystal display [27, 28], remains isolated without strong impact for other applications. Thus, both technologies, which were driven by large consumer E-mail: marko.brammer@googlemail.com E-mail: timo.mappes@kit.edu 1 - 10.2478/optof-2013-0001 Downloaded from De Gruyter Online at 09/12/2016 10:20:52AM via free access M. Brammer, T. Mappes markets, are not fully considered to be part of the corresponding research fields. nent needs to be developed. (2) By exchanging or adding modules in an operational system it may be used for a variety of applications and with different working parameters. Moreover, (3) even if for each individual variant only a low number of pieces is available, they add up with the number of different variants. Thus, components with standardized design and dimensions may be fabricated by large-scale technologies. Establishing interconnection platforms as a basis for a product family of optofluidic analysis systems requires: (1) Switching concepts for selectively guiding fluid and light between a custom-made combination and configuration of optofluidic modules, (2) compact and defined interfaces assuring stable mechanical, leak-proof fluidic and low-loss optical coupling between the modules, (3) components are cost-efficiently mass-producible. An interconnection platform may be either designed (1) modularly in itself (Fig. 1(b)), or (2) with a predefined number of slots (Fig. 1(c)), such that each sensor module is mounted on top of a backplane module that is individually interconnected with other backplane modules. The first concept needs not only coupling interfaces to the sensor modules but also to the neighboring backplane modules. The advantage is, however, that the arrangement and complexity of the system may be user-defined and variably adapted to the target application. In this manuscript, we discuss modular concepts and implementations of microfluidic (section 2), optical (section 3), and optofluidic platforms (section 4). In particular, we compare our recently introduced platform, consisting of backplane modules with dimensions of 40 × 40 × 50 mm3 [35] with other technological solutions. We equipped the module interfaces with optical and fluidic connectors. Mechanical interconnection has been realized by reversibly detachable magnetostatic connectors. Each backplane module offers an interface with optical and fluidic connectors for a functional module, such as an optofluidic sensor. The backplane modules are built of three virtual layers: (1) a microfluidic backplane layer for guiding fluids selectively through fluidic channels and microvalves, and through the sensor modules, (2) an optical backplane layer for guiding light through optical fibers and switches to the sensor modules, and (3) an electronic backplane layer for controlling the active elements in the system, namely the microvalves and optical switches. Several stand-alone sensor systems have been commercialized, based on fluorescence (Agilent, Affymetrix, Gyros, Biosite), absorption (Nanostream), colorimetry (Eppendorf Array Technologies), surface plasmon resonance (General Electrics Healthcare, Sensata) or interferometry (Fairfield). However, in 2001, a market volume in the field of microfluidics of 3 billion USD has been predicted for 2004 [29], of which however only 10 % have been achieved. In 2011 the market volume of 3 billion USD was expected to be reached in 2014 [30]. A number of reasons may be considered, in order to achieve a progress in commercial growth, such as (1) missing well-defined coupling interfaces in between components and with the outer world [31], (2) use of different fabrication technologies and materials in research and industry [15], and (3) missing break-through applications [32]. In scientific projects there is the tendency to develop optofluidic on-chip screening platforms covering the fluidic channel size from 100 nm for single DNA molecule sequence mapping [33] to compact whole-animal on-chip imaging [34]. These approaches are expected to result in a number of commercial applications within the present decade. One of the predominant success factors for industrial implementation is cost-efficiency. The system functionality and its application will define the approach to follow. For operating analysis systems, the sensor components need to be combined with switching and manipulating elements. In the development of these systems, two major design principles are applicable. Either all elements and functions are integrated into one individual device, or a set of modules is implemented that may be individually interconnected as required (Fig. 1). If high unit numbers are produced, a fixed configuration may be more cost-efficient. However, the modular design concept may be the better choice when developing a product family of similar system configurations. Modular system design has its drawbacks in terms of functionality, since it is not optimized for each individual application. Examples are, (1) longer channel length, leading to slower dynamic response, and higher analyte and energy consumption. Furthermore, (2) modular systems necessitate more components, such as mechanical, optical, fluidic, and potentially electrical coupling interfaces, resulting in lower coupling efficiencies. 2. However, a modular approach may reduce development time and costs when implementing new variants of a product family. (1) New variants may be created by choosing different combinations, such that custom-made functions may be configured. Thus, only the new functional compo- Microfluidic Platforms Microfluidic networks in analysis systems are used to guide the fluid to be analyzed through channels to the interaction zones of the sensors. The fluid may be guided passively by capillary forces or diffusion. Higher flow rates and pressure differences may be achieved by ex2 - 10.2478/optof-2013-0001 Downloaded from De Gruyter Online at 09/12/2016 10:20:52AM via free access Modular Platforms for Optofluidic Systems Fig 1. Generalized schematic of different system designs with microfluidic (blue), optical (yellow), and electronic (green) networking layers, and sensor units (white). (a) System with all components integrated in one device. (b) System with sensor modules interconnected by backplane modules. (c) System with sensor modules mounted on one backplane. ternal pumping or by miniature pumps (e.g. Bartels Mikrotechnik mpx, bi.flow Systems, Bürkert 7604, Debiotech Nanopump) integrated into a compact system. Most miniature pumps are membrane-based, thus generating a pulsated fluid flow. Pulsation-free pumping for low flow rates is achievable by electroosmotic pumps (Osmotex, CSEM). Since the electrolysis produces gas, an intermediate element or fluid is necessary, limiting the range of possible applications. Available alternatives for higher flow rates are syringe pumps or pressure reservoirs with dosing valves, both being rather large and thus not compatible with compact portable devices. Fig 2. Schematics of microfluidic circuits consisting of fluidic channels and microvalves to be integrated into backplane modules. Fluidic connectors to the neighboring modules and a mounted device are marked with grey and blue circles, respectively. A second layer of fluidic channels and microvalves is illustrated in red, connecting channels in green. (a) Circuit with four connectors to neighboring modules in plane. (b) Circuit with eight connectors to four modules in plane. (c) Circuit with four connectors in plane and two layers (d) Circuit with connectors to six modules oriented in three dimensions and two independent layers. In order to guide the fluid selectively through the channels without applying pressure differences, external or local fields may be used. By applying external electric or magnetic fields [36], or upon acceleration of the device, e.g., coriolis acceleration [37], the fluid may be guided along the chosen path. Alternatively, electrical fields locally applied by an electrode array are used, forming the basis of digital microfluidics devices [38]. Advantages of this principle include leveraging well-developed microelectronics fabrication technology, and the possibility to manipulate and analyze several individual fluid drops in parallel [39]. 10 × 10 × 10 mm3 , leaving potential for further development. For high-density integration of microvalves, alternative concepts based on indirect actuation have been developed [43–45]. Two layers of microfluidic channels are built on top of each other and coupled either by an elastomeric intermediate membrane or two non-miscible fluids. The fluidic pressure in the first layer controls the switching states in the second layer, in which the analyte is guided. Whereas the microfluidic chip is very compact, a large external pneumatic control setup is needed. Thus, a modular system based on this principle would necessitate a large number of fluidic connectors. Modular platforms have been presented [46–48] and are already available commercially from different companies (Microfluidic Chipshop, thinXXS Microtechnology), however without integrated fluid control devices. We recently introduced the concept for fluidic circuits integrated into backplane modules and with coupling interfaces to devices to be mounted on top [49]. Depending on the circuit different flow paths are possible (Fig. 2). Microvalves mounted on top of the channel layer and controlled individually by an electronic circuit layer define the flow path between the coupling interfaces. Each mod- Higher pressures, however, require matching fluid flow control mechanisms. To fulfill this goal, integrated microvalves have been developed with several different designs and actuators [40]. They may be based on electrostatic, electromagnetic, piezoelectric or thermal actuation principles. Due to high energy density, shape memory alloy actuators are particularly interesting for compact design and have been developed in different variants [41, 42]. Drawbacks of these actuators are relatively high energy consumption and the limitation to operation temperatures below the transition temperature. For most materials the range is restricted to temperatures below 55◦ C, which is not compatible with several process automation applications. Currently commercially available valves are mostly based on solenoids (Bürkert 6650, Kendrion Tri-Tech, LEE LFV, Staiger VA 204-7, Takasago VODA) and larger than 3 - 10.2478/optof-2013-0001 Downloaded from De Gruyter Online at 09/12/2016 10:20:52AM via free access M. Brammer, T. Mappes ized the mechanical interconnection between the modules by magnetic connectors. The magnetostatic force between a permanent-magnetic plug and a soft-magnetic socket is used to compress elastomeric sealing membranes at the coupling interfaces of the microfluidic backplane layers. Taking into consideration the influence of the magnetic field on the fluid flow, the fluid has to be insulated magnetically or cleaned from magnetic particles. We integrated the permanent-magnetic plug into a soft-magnetic holder, thus enclosing the magnetic field. This approach may have the potential to overcome the difficulties in coupling microfluidic circuits or modules with the system or lab environment. Fig 3. Schematics of fluidic coupling elements for module interconnection in cross-sectional views. Fluid flow is illustrated by blue arrows, compression force by red arrows. (a) O-ring. (b) Sealing membrane in a uniform notch. (c) Sealing membrane in a notch with inclined surface. (d) Needle inserted into a sealing ring [50]. (e) Needle pierced into a sealing membrane [51]. 3. ule inherits interfaces to the neighboring modules and to the interconnected sensor module. The interface to the sensor module provides two channels to guide the fluid through the interaction zone of the sensor and back into the backplane layer. Depending on the circuit, the interfaces to the neighboring modules consist of one or two channels. Building two channel layers on top of each other enables intersection of two fluid flows without mixing. The fluidic network of choice for the dedicated application allows defined fluid control. Optical Platforms Optical networks guide light from light sources to the interaction zones of the sensors, where the electromagnetic wave and the fluid overlap and the interaction properties are analyzed. Similarly to microfluidic platforms, one may differentiate between passive and active concepts. Passive concepts consist of optical waveguides or optical fibers, whereas active principles have additional controllable components, such as optical switches, multiplexers, and amplifiers. Optical waveguides have been widely integrated into polymer chips and used as optical backplanes to guide light within sensor systems [55, 56]. E.g., PMMA offers the possibility to integrate waveguides directly into the substrate by deep-ultraviolet radiation induced modification of the refractive index [57, 58]. This process requires fewer fabrication steps than two-material waveguide fabrication. Whereas for optical communication applications signal dispersion is crucial, optimization of waveguides for sensor systems is focused on efficiency. Low losses are possible with high contrast in refractive index [59]. In order to couple light into the waveguide, different approaches have been proposed. In particular, direct integration of light sources and waveguide into the same substrate allow for the most efficient coupling [60]. Optical fibers are an alternative to integrated waveguides. Since they are not integrated directly into the substrate, assembly costs are significantly higher. However, lower guiding and coupling losses are possible. Active switching between different fibers inside the optical network is mostly achieved by opto-mechanical switches. Several systems have been published [61, 62] and also integrated into commercial solutions (Lucent, DiCon, Fujitsu, Sercalo). Recently, we presented optical backplane modules with integrated optical switches [35, 63]. Five polymer optical fibers are inserted in each module, four of which are di- Another major issue to be solved is the development of coupling interfaces between small chips and the environment [31]. For modular systems connectors have to be integrated into the module interfaces, which is often done by O-rings or sealing membranes placed around the inlet of the fluidic channels (Fig. 3(a),(b)). A promising alternative concept for fluidic coupling is realized by hollow needles pressed into a tight fit elastomeric sealing ring [50], as illustrated in Fig. 3(d). Alternatively a non-structured sealing membrane is pierced by a needle (Fig. 3(e)), and sealed again due to internal stress of the elastomeric membrane when the needle is removed [51]. This design assures sealing in the disconnected state, it is, however, significantly more complex than O-rings or sealing membranes. Furthermore, low death volume and designs that may be easily flushed are important for clean and defined fluid flow. This requires coupling elements with well-defined channel boundaries as illustrated in Fig. 3(c). In this design the membrane is compressed at the borders of the fluidic channel by the inclined surface of the notch. Alternative solutions enable leak-proof coupling, but are often not designed symmetrically, which would limit the variability of interconnection of modules [52, 53]. Mechanical connectors compress the sealing elements and provide mechanical stability between the modules. However, the reliability of compact mechanical connectors is difficult to assure, particularly when they are designed for reversibly detachable interconnection [35, 54]. We real4 - 10.2478/optof-2013-0001 Downloaded from De Gruyter Online at 09/12/2016 10:20:52AM via free access Modular Platforms for Optofluidic Systems Fig 5. Optical fiber connector [65]. (a) CAD model of the yoke-shaped fiber holder. (b) Photograph of the fiber connecter as a standalone version with magnets providing mechanical connection. Fig 4. Schematics of optical switches designed for five perpendicular orientated optical fibers. (a) Optical switch with three linearly actuated mirror elements (two double-sided plane mirrors, one pyramid mirror). (b) Linearly actuated optical switch as implemented in [63]. (c) Rotatory actuated optical switch with prism element, deflecting light by total internal reflection, mirror reflection or transmitting light. (d) Rotatory actuated optical switch with differently bent fibers. (e) MEMS mirror based optical switch with fixed pyramid mirror as implemented in [35]. (f) MEMS mirror based optical switch with bent fibers. rected horizontally to the neighboring modules and one vertically to the sensor module. The optical switch is positioned in the center of the module. Lenses are placed in front of each fiber. Light coupling out of one fiber is focused in the lens and coupled by the optical switch and through another lens into the chosen other fibers. Different opto-mechanical switching principles are illustrated in Fig. 4. Fig 6. Scheme of an exemplary optofluidic analysis system based on the system design illustrated in Fig. 1(b) with microfluidic (blue), optical (yellow), and electronic (green) networking layers, and functional modules (white). The system consists of three backplane modules interconnecting one mixer/reactor (A), one sensor (B), and one light source module (C). Light path is illustrated with orange arrows, analyte flow with blue, reagent flow with red, and mixed fluid with violet arrows. We developed two optical switches, each one optimized with respect to different parameters, (1) one with low coupling losses (2.5 - 3.6 dB) but long switching time (1 3 s), based on a linearly actuated assembly of optical elements [63] (Fig. 4(b)), and (2) another one with higher losses (7.3 - 11.9 dB) but faster switching (< 30 ms), based on an electrostatically actuated MEMS mirror [35] (Fig. 4(e)). Especially the performance of the MEMS mirror switch may be optimized, increasing the maximum angles of the quasi-static mirror deflection. This would lead to a shorter free-space distance between the fibers and thus to lower coupling losses. We used quasi-static deflection (Sercalo Microtechnology), since it offers the advantage of stable switching positions in comparison to resonant operation. The connectors are designed to be fabricated by micromachined tool fabrication and injection molding [65]. An O-ring provides the possibility to insert and seal an indexmatching gel. 4. Optofluidic Platforms Based on modular microfluidic and optical platforms, optofluidic devices may be interconnected and operated. Fig. 6 shows a possible configuration of how to interconnect an optofluidic sensor module with backplane modules. We assembled microfluidic and optical backplane layers in one module, thus supplying different optical sensor modules with light and the fluid to be analyzed (Fig. 7) [35]. When coupling two modules, alignment is particularly important between the optical backplane layers, whereas the fluidic layers allow larger tolerances. In order to achieve mechanical alignment between fibers or waveguides, precisely shaped structures are used. Fabrication within such low tolerances is possible with lithographic-based techniques and subsequent replication technologies [64]. We assured precise alignment by yoke-shaped fiber holders that are mechanically self-aligned upon assembly (Fig. 5). The proposed design is particularly advantageous for continuous monitoring of fluid flow parameters, crucial for several applications in life sciences, medicine, environmental, and process analysis [66, 67], as listed in the following. Optical and non-optical sensors operated in parallel would allow for measuring parameters, such as turbidity, color, the content of certain particles, the pH value or the oxygen content. The design holds the advantage that 5 - 10.2478/optof-2013-0001 Downloaded from De Gruyter Online at 09/12/2016 10:20:52AM via free access M. Brammer, T. Mappes For implementation of an operational optofluidic analysis systems further optimization is required. In the beginning of the optofluidic era Psaltis et al. introduced a general platform concept, built of three functional layers, (1) an optical layer, (2) a microfluidic channel layer, and (3) a fluid control layer. In order to realize such a design and thus to pave the way for optofluidic products on the market, the individual components are to be combined to an entire system. Fig 7. Photographs of the modular backplane [35]. (a) Prototype of the assembled optical and microfluidic backplane module (40 × 40 × 50 mm3 ). Fluidic connectors are marked with blue arrows, optical connectors with yellow arrows. The crossing of the tube and the fiber on top of the module illustrates the interface for an optofluidic device. (b) Measurement setup with three modules interconnecting a combined conductivity/temperature sensor module (Bürkert) and a miniature spectrometer (Hamamatsu C10988MA). The sensor modules are operated with a supply module with an integrated laser diode, two light emitting diodes, and two micropumps. 5. Conclusion In this manuscript, we discussed modular platforms for the interconnection of optofluidic devices to a total analysis system. We believe that modular concepts and welldefined coupling interfaces would promote industrial implementation of optofluidic systems. In particular, the development process of product families would benefit from this approach. microfluidic and optical backplane layers are compatible but independently implementable. The functionality of the system may be chosen depending on the application, thus potentially reducing fabrication costs. Acknowledgement The authors thank C. Megnin from the Institute of Microstructure Technology (IMT) of the Karlsruhe Institute of Technology (KIT) for the development and supply of microvalves, D. G. Rabus from Festo AG & Co. KG, for the inspiring discussions, as well as A. Voigt, S. Sobich, and C. Essig from the IMT of the KIT, C. Schmuck from Bürkert Fluid Control Systems, H. Besser from the Institute of Applied Material Research of the KIT, and A. Hofmann from the Institute for Applied Computer Science of the KIT for technical support. This work has been supported by the Bürkert Technology Center, Karlsruhe. For some applications, a direct combination of optics and fluidics would be advantageous, when not only the sensor modules but also the platform itself is based on optofluidics. Different components have been presented that have the potential to be integrated into optofluidic analysis systems, with tunability being one of the most promising advantages. This would enable the control of the system parameters during operation, e.g., the measuring range of a sensor system may be adjusted to the environmental conditions. One of the first optofluidic components were tunable lenses, of which the focal distance is controlled by changing the geometry or the refractive index [68, 69]. The geometry of a liquid surface may be tuned by electrowetting [70–72] or an applied fluid pressure difference [73, 74]. Lenses made from elastomeric polymers can also be modified by applying a pressure difference [75–77]. The refractive index can be controlled simply by changing or mixing the fluid. Alternative methods for manipulating geometry or refractive index are electrical, thermal or optical effects [68, 69]. 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A geometrical model for testing bilateral symmetry of bamboo leaf with a simplified Gielis equation
Ecology and evolution
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K E Y W O R D S K E Y W O R D S bamboo, bilateral symmetry, goodness of fit, leaf-shape parameter, polar coordinate K E Y W O R D S bamboo, bilateral symmetry, goodness of fit, leaf-shape parameter, polar coordinate O R I G I N A L R E S E A R C H O R I G I N A L R E S E A R C H Shuyan Lin1 | Li Zhang1 | Gadi V. P. Reddy2 | Cang Hui3,4 | Johan Gielis5 |  Yulong Ding1 | Peijian Shi1 1Collaborative Innovation Center of Sustainable Forestry in Southern China of Jiangsu Province, Bamboo Research Institute, College of Biology and the Environment, Nanjing Forestry University, Xuanwu District, Nanjing, China 2Western Triangle Ag Research Center, Montana State University, Conrad, MT, USA 1Collaborative Innovation Center of Sustainable Forestry in Southern China of Jiangsu Province, Bamboo Research Institute, College of Biology and the Environment, Nanjing Forestry University, Xuanwu District, Nanjing, China 2Western Triangle Ag Research Center, Montana State University, Conrad, MT, USA 3Centre for Invasion Biology, Department of Mathematical Sciences, Stellenbosch University, Matieland, South Africa 4Mathematical and Physical Biosciences, African Institute for Mathematical Sciences, Cape Town, South Africa 5Department of Biosciences Engineering, University of Antwerp, Antwerp, Belgium Correspondence Peijian Shi, Bamboo Research Institute, Nanjing Forestry University, Xuanwu District, Nanjing, China. Email: shi_peijian@163.com and Johan Gielis, Department of Bioscience Engineering, University of Antwerp, Antwerp, Belgium. Email: johan.gielis@uantwerpen.be 1Collaborative Innovation Center of Sustainable Forestry in Southern China of Jiangsu Province, Bamboo Research Institute, College of Biology and the Environment, Nanjing Forestry University, Xuanwu District, Nanjing, China 2Western Triangle Ag Research Center, Montana State University, Conrad, MT, USA 3Centre for Invasion Biology, Department of Mathematical Sciences, Stellenbosch University, Matieland, South Africa 4Mathematical and Physical Biosciences, African Institute for Mathematical Sciences, Cape Town, South Africa 5Department of Biosciences Engineering, University of Antwerp, Antwerp, Belgium Correspondence Peijian Shi, Bamboo Research Institute, Nanjing Forestry University, Xuanwu District, Nanjing, China. Email: shi_peijian@163.com and Johan Gielis, Department of Bioscience Engineering, University of Antwerp, Antwerp, Belgium. Email: johan.gielis@uantwerpen.be A geometrical model for testing bilateral symmetry of bamboo leaf with a simplified Gielis equation Shuyan Lin1 | Li Zhang1 | Gadi V. P. Reddy2 | Cang Hui3,4 | Johan Gielis5 |  Yulong Ding1 | Peijian Shi1 Revised: 16 July 2016  |  Accepted: 4 August 2016 Revised: 16 July 2016  |  Accepted: 4 August 2016 Received: 21 May 2016  |  Revised: 16 July 2016  |  Accepted: 4 August 2016 DOI: 10.1002/ece3.2407 Abstract Abstract The size and shape of plant leaves change with growth, and an accurate description of leaf shape is crucial for describing plant morphogenesis and development. Bilateral symmetry, which has been widely observed but poorly examined, occurs in both dicot and monocot leaves, including all nominated bamboo species (approximately 1,300 species), of which at least 500 are found in China. Although there are apparent differ- ences in leaf size among bamboo species due to genetic and environmental profiles, bamboo leaves have bilateral symmetry with parallel venation and appear similar across species. Here, we investigate whether the shape of bamboo leaves can be ac- curately described by a simplified Gielis equation, which consists of only two param- eters (leaf length and shape) and produces a perfect bilateral shape. To test the applicability of this equation and the occurrence of bilateral symmetry, we first meas- ured the leaf length of 42 bamboo species, examining >500 leaves per species. We then scanned 30 leaves per species that had approximately the same length as the median leaf length for that species. The leaf-­shape data from scanned profiles were fitted to the simplified Gielis equation. Results confirmed that the equation fits the leaf-­shape data extremely well, with the coefficients of determination being 0.995 on average. We further demonstrated the bilateral symmetry of bamboo leaves, with a clearly defined leaf-­shape parameter of all 42 bamboo species investigated ranging from 0.02 to 0.1. This results in a simple and reliable tool for precise determination of bamboo species, with applications in forestry, ecology, and taxonomy. Bamboo leaves consist of two major parts, a sheath and a blade, with a characteristic ligule with oral setae and auricles as ap- pendages (Stapleton, 2012). Cauline leaves have a protective func- tion in the development of young shoots and culms. The sheath in these types of leaves is very stiff, and the blade is greatly reduced. In contrast, foliage leaves have fully developed leaf blades and the blades are connected to the sheath via a pseudo-­petiole. This is a structure unique to all bamboo species and some large leaved grasses. These foliage leaf blades are usually linear, lanceolate or oblong-­lanceolate, with the tip long and acuminate, often sca- brous and the side glabrous or softly hairy. The leaf blade is gen- erally thinner than the culm sheath blade and often shows more marked dorsiventrality. The morphology and structure of leaves vary among species and can be used for species identification (Stapleton, 2012). A leaf-­shape description based on Lamé curves has been used to model foliage leaves of the bamboo genus Indocalamus (Shi, Xu, et al., 2015). To test the applicability of this work to other bamboos, with different size and shape of leaves, in this study, we extend this modeling to a wide variety of genera and species. We propose a sim- ple and adequate mathematical model for describing the leaf shape of bamboos, which allows for testing the bilateral symmetry of leaf shape in more than 40 different bamboo species we can have ac- cess to in the Nanjing Forestry University campus. In addition, we will show that this model also can be used to assist in identifying bamboo species. Such locally organized structures develop into organs, new phytomers or leaves, either vegetative leaves or modified leaves into floral structures. Vegetative leaves are the most important plant organs for photosynthesis, and their size and shape have an important influence on the morphogenesis and development of plants. As many plant organs vary over the growing season, it is important to identify a simple but adequate mathematical model for capturing morphological change in plants. This is not a simple task. Besides the classic phenotypic differences related to eco- logical habitat, there are the phenomena of heteroblasty and het- erochrony to include in any accurate model. Heteroblasty is the phenomenon whereby several types of leaves occur, depending on position or age (Bell & Bryan, 2008). Various studies have focused on integrating the development of plants and plant organs and on simulating the dynamics of crop morphological development based on L-­systems (Fournier & Andrieu, 1999; Lindenmayer, 1968a, 1968b; Prusinkiewicz, 1999). For several important crops such as maize (Fournier & Andrieu, 1999), barley (Buck-­Sorlin & Bachmann, 2000), sor- ghum (Kaitaniemi, Hanan, & Room, 2000), and rice (Ding, Zhang, Zhang, Zhu, & Chen, 2011), architectural models have been linked to physiology and to measured development and growth (Chen, Jiang, Zhu, Cao, & Chen, 2004; Liu, Tang, & Qi, 2002; Watanabe et al., 2005; Zhan, Wang, Reffye, & Hu, 2001). Alternatively, spe- cific models have been developed to address plant growth under optimal conditions that seek to reproduce the complex structure of a crop’s shape. Plant growth and development is the result of the timing and spatial organization of the development of organs from a meristematic zone. In physiology, various studies have focused on timing and spatial aspects of gene expression, in relation to the formation of organs. One example is the development of lateral outgrowths of organs from apical meristems and the direct relation of this to the spatial distribution of auxin via auxin transporters (Reinhardt, Mandel, & Kuhlemeier, 2000). However, gene action or gene regulatory networks do not give a complete picture of this process. The development of such organs is dictated by mathe- matical and physical laws, making it no surprise that Fibonacci and Lucas series are found so frequently in phyllotaxy, both in the po- sitioning of leaves along the stem and in the arrangement of parts in single and composite flowers (Jean, 2009). Phyllotactic patterns have also been linked to physical phenomena optimizing space ­utilization (Douady & Couder, 1992). Vegetative leaves of bamboo, on the other hand, are relatively simple, and their structure is stable throughout the whole sub- family. The bamboos (Poaceae: Bambusoideae) consist of approxi- mately 1,300 species of temperate, tropical woody bamboos and a tribe of herbaceous bamboos (Kelchner, 2013; Liese & Köhl, 2015; Wysocki, Clark, Attigala, Ruiz-­Sanchez, & Duvall, 2015). Unlike other grasses, bamboos are the only major lineage within the fam- ily that have adapted to and diversified within the forest habitat (Judziewicz & Clark, 2007; Judziewicz, Clark, Londono, & Stern, 1999; Liese & Köhl, 2015). The dry biomass of bamboo leaves contributes 5%–10% of the whole plant biomass for most bamboo species. Heterochrony, on the other hand, describes leaf-­shape variation over time. Differences in tim- ing can lead, for example, to simple versus compound leaves, and heterochrony has been identified as the basis for natural variation in Cardamine hirsuta, a close relative of Arabidopsis, in which differ- ences in leaf shape were correlated to flowering time (Cartolano et al., 2015). A final difficulty to mathematical modeling of leaf shape is that it can be hard to define precise landmarks in leaves for defining morphometry. Leaves and leaflets in compound leaves in general have relatively simple shapes, but leaves can be highly variable as in Begonia (McLellan, 1993), Papaya and Vasconcellea (Scheldeman et al., 2011). 1 | INTRODUCTION symmetry, and all plants are in some sense repetitions of basic phy- tomers (Clark & Fisher, 1987). Most plants, in particular monocots, have a simple structure, a repetition of phytomers consisting of an internode and a nodal zone. At each nodal zone, a bud and protective leaf struc- tures occur. This basic structure led to a great diversity of growth forms and to a variety of models from plant diversity (Bell & Bryan, 2008; Dabadie, Reffye, & Dinouard, 1991; Doust, 2007; Halle, 1986). 1 In botany, forestry, and agriculture, understanding the development and morphogenesis of plants is of primary importance. To this end, many studies have focused on exploring potential rules of plant growth, reconstructing plant morphology, and capturing their morphogenetic dynamics. Fundamental features of plants are self-­similarity and Ecology and Evolution 2016; 6: 6798–6806 6799 Lin et al. 2.3 | Data analysis where m, a, b, n1, n2, and n3 are constants, we find symmetry parame- ter m = 1 and exponents on cosine and sine terms both equal to 1 (in the general Gielis equation, the parameter m and the exponents can be other values; see Gielis (2003a, 2003b) for details). The parameter l is a constant value, namely the distance from the polar origin to the leaf tip. We have r = l when φ = 0. The number n is a parameter that can determine the overall ratio of the leaf width to length and is inde- pendent from the absolute leaf length; hereafter, we refer to n as the leaf-­shape parameter. The leaf length (L) is the sum of r when φ = 0 and when φ = π (see Fig. 1): For every species, we randomly chose more than 500 matured foli- age leaves and measured their lengths, as the distance from the leaf bottom to the tip. We need to choose the leaves whose lengths can reflect the generality of a population. If leaf length follows a normal distribution (namely a symmetric density distribution curve), the mean or median both can represent the generality of a population; if its den- sity distribution curve is skewed (that has a long left or right tail) like the Weibull distribution, the median is better than the mean in reflect- ing the generality of a population. The Shapiro–Wilk test (Quinn & Keough, 2002) was used to check the normality of leaf lengths, and the Kolmogorov–Smirnov test (Quinn & Keough, 2002) for examin- ing whether the leaf length follows the Weibull distribution. We then chose 30 leaves per species of approximately equal to the median of leaf lengths and scanned them to extract Cartesian coordinates on the leaf boundary (Shi, Huang, et al., 2015). The bilateral symmetry of bamboo leaves was tested by the goodness of fit of the simplified Gielis equation. We also tested whether the bamboo species that are more closely related in taxon have smaller difference in the leaf-­shape parameter. Tukey’s HSD (honestly significant difference) test (Quinn & Keough, 2002) was used to examine the pairwise difference of leaf-­ shape parameters between different bamboo species. (4) L= ( 1+2−1 2n ) ⋅l. (4) It is apparent that this simplified Gielis equation (i.e., eq. 2) could produce the bilateral symmetry as r(φ)=r(−φ). 2.2 | The Gielis equation Let us assume (x, y) be the Cartesian coordinates of a point on the boundary of a leaf. We can transform this point into the polar coordi- nates as the following: (1) { x=r⋅cos φ y=r⋅sin φ , (1) where r is the polar radius (i.e., the distance between the polar origin and the point; Fig. 1), and φ the angle of the radial vector. FIGURE 1 Leaf shape produced by the simplified Gielis equation FIGURE 1 Leaf shape produced by the simplified Gielis equation For testing the bilateral symmetry of plant leaves, we put forward a simplified version of the Gielis equation (Gielis, 2003a, 2003b; Gielis & Gerats, 2004). Xu, et al. (2015) by estimating three parameters: x0, y0, and θ. The first two parameters reflect the shift of coordinates from (0, 0) to (x0, y0), and the last parameter reflects the angle change in the horizon- tal axis from 0° to θ. We used the optimization algorithm proposed by Nelder and Mead (1965) to fit the parameters of the simplified Gielis equation using the “optim” function in R software (R Core Team 2015). (2) r= l ( || cos φ 4 ||+|| sin φ 4 || )1∕n . (2) Comparing this with the general Gielis equation (Gielis, 2003a) Comparing this with the general Gielis equation (Gielis, 2003a) (3) r= 1 {( || 1 a cos (φ m 4 )|| )n2 + ( || 1 b sin (φ m 4 )|| )n3}1∕n1 , (3) FIGURE 1 Leaf shape produced by the simplified Gielis equation Table S1 lists the official names of the sampled species ac- cording to the Flora of China (Wu, Raven, & Hong, 2006). If no entry was found in the Flora of China, the name was selected from Bamboos of the World (Ohrnberger, 1999). For every species, we randomly chose more than 500 leaves from different individuals and measured their lengths to check the normality of leaf length distribution and to calculate the median of leaf lengths. Then, 30 leaves with length ≈ the median were collected, scanned, and analyzed. 2.1 | Experimental design To test the generality of bilateral symmetry in bamboo leaves, we collected 42 bamboo species from the Nanjing Forestry University campus (32°04′34.53′′N, 118°48′42.06′′E; Table S1). In the Nanjing Forestry University campus, the environmental factors can be re- garded as constants because of low spatial heterogeneity. Nanjing belongs to the subtropical monsoon climate, with mean annual pre- cipitation of 1,058 mm (±237.5 mm) and a mean annual temperature of 15.6°C (±0.7°C). Mean minimum annual temperature was −8.6°C, and the mean maximum annual temperature was 37.4°C (based on the climate data of 1951–2012 downloaded from China Climate Data Online, www.data.cma.cn). 6800 Lin et al. 3.1 | Leaf length The length of the leaves of these 42 bamboo species follows the Weibull distribution rather than the normal distribution (Table S2, Fig. 2). We found leaf length to differ significantly among genera and among species within the same genus. Sampled leaves could be very small, <5 cm length in most species, while the maximum length ranged up to 35 cm for Pseudosasa amabilis var. convexa, but for most spe- cies was below 25 cm. While the variation in leaf length of the genus Phyllostachys was less than the variation of the genus Pleioblastus, Phyllostachys is a narrowly defined genus with restricted natural geo- graphic distribution while that of Pleioblastus is very broad, encom- passing bamboos from different temperate zones of the world. 3.2 The leaf shape of all bamboo species (see Fig. 3 for examples) was described by the simplified Gielis equation well (see Table S3), with the results for six bamboo species illustrated in Fig. 4. The predicted leaf shape matched the observed leaf shape well for these six bamboo species. The goodness of fit also provides convincing evidence of bi- lateral symmetry in bamboo leaves, with all coefficients of determina- tion higher than 0.980 (Fig. 5). The real leaf area fits extremely well with the predicted leaf area (Fig. 6), with the regression straight line deviated only trivially from the straight line of y = x. FIGURE 3 Scanned leaf images of six bamboo species. S36: Pleioblastus yixingensis; S1: Bambusa emeiensis var. viridiflavus; S10: Indosasa shibataeoides; S17: Phyllostachys bissetii: S19: Phyllostachys edulis; and S22: Phyllostachys edulis “Gracilis” The variation of leaf-­shape parameters for these 42 species is ­illustrated in Fig. 7. Although there were significant differences in the leaf-­shape parameters among species from different genera or from the same genus (Table S4), the calculated leaf-­shape parameters for these 42 species range only from 0.02 to 0.1. The lower values are for shapes of a more linear-­lanceolate type, such as leaves of Pleioblastus chino, Pleioblastus simonii f. heterophyllus, Pleioblastus gramineus f. monstrispiralis, Chimonobambusa tumidissinoda, and Phyllostachys edulis. Leaves described by the higher values of the shape parameter n were slightly broader in shape, such as those of Shibataea chinensis, Indosasa shibaeatoides (Fig. 3), and Bambusa multiplex var. riviereorum. The remaining leaves fell broadly within the range of shape parameters 0.03–0.08. 2.3 | Data analysis In practice, there are two issues for estimating the two parame- ters in the above simplified Gielis equation when using the leaf-­shape data extracted from a scanned image of a real leaf: (1) the polar origin usually deviates from the coordinate point of (0, 0); and (2) the angle between the line passing from the polar origin to the leaf tip and the actual horizontal axis usually also deviates from 0°. To solve these two issues, we took the method of Shi, Huang, et al. (2015) and Shi, |  6801 FIGURE 3 Scanned leaf images of six bamboo species. S36: Pleioblastus yixingensis; S1: Bambusa emeiensis var. viridiflavus; S10: Indosasa shibataeoides; S17: Phyllostachys bissetii: S19: Phyllostachys edulis; and S22: Phyllostachys edulis “Gracilis” 6801 Lin et al. 4.1 | Capturing the diversity of bamboo leaves FIGURE 2 Comparison of leaf lengths for 42 bamboo species Our results corroborate findings for some dwarf bamboos (Shi, Xu, et al., 2015) and suggest that modeling with the modified Gielis equa- tion that has a reduced number of parameters is clearly applicable to a wide variety of temperate bamboo genera and species with lanceolate leaves. A range of shapes and sizes from the small leaves of Bambusa multiplex var. riviereorum to the large leaves of Indocalamus victorialis and from the linear-­lanceolate leaves of P. linear to the broader leaves of Shibataea can all be efficiently modeled with one equation with only two parameters. The 42 bamboo species examined in this study were randomly chosen from the bamboo garden of Nanjing Forestry University. Although all are Asian bamboos, both temperate and tropi- cal, the mix was chosen to be representative of the whole subfamily. While the only tropical woody bamboos in this study are the orna- mental Bambusa species, our findings can certainly be extended to most, if not all, other tropical bamboos. FIGURE 2 Comparison of leaf lengths for 42 bamboo species Lin et al. 6802 802  |     Lin et al. Leaf length of these bamboo species shows significant differ- differentiation and hybridization of some closely related species and FIGURE 4 Comparison between scanned leaf profile (gray line) and predicted leaf profile (red line) from the simplified Gielis equation. (A) Pleioblastus yixingensis; (B) Bambusa emeiensis var. viridiflavus; (C) Indosasa shibataeoides; (D) Phyllostachys bissetii; (E) Phyllostachys edulis; (F) Phyllostachys edulis “Gracilis” FIGURE 4 Comparison between scanned leaf profile (gray line) and predicted leaf profile (red line) from the simplified Gielis equation. (A) Pleioblastus yixingensis; (B) Bambusa emeiensis var. viridiflavus; (C) Indosasa shibataeoides; (D) Phyllostachys bissetii; (E) Phyllostachys edulis; (F) Phyllostachys edulis “Gracilis” Leaf length of these bamboo species shows significant differ- ences between species, but in general, the shape parameter varies little across species, from 0.02 to 0.1. Also, three Indocalamus species had shape parameter values between 0.04 and 0.07 (Shi, Xu, et al., 2015). The only exception is Indosasa victorialis, where the value is above 0.1. Although the leaf sizes (length and width) of the 42 bam- boo species examined in this study and the four species studied in the paper (Shi, Xu, et al., 2015) differ considerably, their leaf shapes vary little and show general lanceolate characteristics. 4.1 | Capturing the diversity of bamboo leaves Our results showed that the simplified Gielis equation delineates leaf shapes of different bamboo species extremely well. Whereas in the past bamboo leaf blades were characterized by length and width with additional quali- tative characteristic such as linear-­lanceolate or oblong-­lanceolate, we now have a clear quantitative number for a qualitative characteristic. Molecular analysis and genetic assignments usually are used to study differentiation and hybridization of some closely related species and secondary for identification of established species. Our results also demonstrated the bilateral symmetry of the leaf shape of all these bamboos. The occurrence of bilateral symmetry might be helpful for the transportation of nutrients and water from branches to leaves. The greater the goodness of fit is when using the Gielis equation, the stronger the bilateral symmetry of the leaves, thereby confirming our original assumption that r(φ)=r(−φ). The in- terpretation of this symmetry is global, concerning the whole leaf, rather than local. Carefully examining the base of the leaf and the vas- cular bundles emanating from the pseudo-­petiole (Fig. 3), the bases are rarely perfectly symmetric, but on average for the population the leaves are bilaterally symmetric (Fig. 3), and this symmetry is a guiding basis of development. The fact that bilateral symmetry and leaf blades connected to the sheath part by a pseudo-­petiole are typical of all |  6803 Lin et al. FIGURE 5 Coefficients of determination for fitting the leaf shapes of 42 bamboo species to the simplified Gielis equation FIGURE 7 Comparison of the estimated leaf-­shape parameters for 42 bamboo species |  6803 FIGURE 7 Comparison of the estimated leaf-­shape parameters for 42 bamboo species |  680 FIGURE 7 Comparison of the estimated leaf-­shape parameters for 42 bamboo species 6803 Lin et al. FIGURE 5 Coefficients of determination for fitting the leaf shapes of 42 bamboo species to the simplified Gielis equation FIGURE 7 Comparison of the estimated leaf-­shape parameters for 42 bamboo species FIGURE 6 Comparison between the real and predicted leaf areas estimated from the simplified Gielis equation. The red straight line represents y = x, the small open circles are made up of the actual leaf areas and the predicted leaf areas The model established in this article can be extended to many more bilaterally symmetric foliage leaves. Bamboo leaves (and grass leaves in particular) show parallel venation characteristics of monocots. 4.1 | Capturing the diversity of bamboo leaves The ve- nation of bamboo leaves is parallel with three orders, namely the mid- rib, secondary veins, and tertiary veins. In bamboos, adjacent veins are connected by transverse distinct veinlets, visible in temperate bamboos but hidden in tropical species (Brandis, 1907). In particular, bamboo leaves show multicostate parallel convergent venation whereby the main vascular systems diverge from the pseudo-­petiole and converge at the apex of the leaves. This leads to a clear bilateral symmetry, and the model can also be used for many other monocots with similar ve- nation. However, the venation of a unicostate type in monocot leaves, with one main vein and branching along the vein as in banana leaves, is also clearly symmetric and can be modeled in the same way. The model can be extended to all bilaterally symmetric leaves, both in simple and compound leaves, and including petals in composite or simple flowers. FIGURE 6 Comparison between the real and predicted leaf areas estimated from the simplified Gielis equation. The red straight line represents y = x, the small open circles are made up of the actual leaf areas and the predicted leaf areas Compared to the general equation with six parameters, the sym- metry parameter m = 1, the exponents n2 and n3 are both equal to one, and n1 = n, the number of is reduced here to 1, namely the expo- nent n. With the general Gielis equation and more parameters, other leaf shapes such as orbicular, hastate, elliptical, cordate, and others (Gielis, 2003a; Wang, 2007) can also be described efficiently, either directly as transformation of the circle or indirectly as modifications of cardioid or similar functions (Gielis, 2003a; Wang, 2007). For plants with nonsymmetric leaves, more complicated mathematical models are needed, such as elliptic Fourier analysis or summations of eq. 2 into k-­type functions (Gielis et al., 2012). For asymmetrical leaf bases, adjusting the condition r(φ)=r(−φ) could be considered. The original Gielis equation could then be a first choice for describing nonsymmet- ric leaf shapes with smooth margins. The shape of petioles, typically concave for large leaves, has also been modeled efficiently with the same approach (Faisal, Abad, Hristozov, & Pasini, 2010). We therefore recommend simplifying the original Gielis equation contextually for different purposes. bamboos foliage leaf blades potentially reflects the evolutionary sta- bility of this solution. 4.2 | Leaf development models and the future trends Leaf-­shape models for species of the grass family have been proposed to address development (Dornbusch, Watt, Baccar, Fournier, & Andrieu, 2009; Dornbusch, Watt, Baccar, Fournier, & Andrieu, 2011; Zhu et al., 2009). In particular, Dornbusch et al. (2009) analyzed the leaf shapes of wheat, barley, and maize using an empirical shape model proposed in wheat (Dornbusch et al., 2011) and based on three-­dimensional param- eters and three shape parameters. They found that varying conditions during growth will affect leaf dimensions but not leaf shapes. The quan- tification of leaf expansion in Miscanthus species and Brachypodium, in particular duration and timing of leaf growth in relation to environmen- tal parameters, allows for distinguishing between closely related geno- types (Shi, Chen, Hui, & Grissino-­Mayer, 2016; Voorend et al., 2014). Along with leaf expansion, the model proposed by Dornbusch et al. (2011) could also be used to quantify cell elongation in leaves. Here, a simplified version of the Gielis equation was shown to be an excellent model for describing the foliage leaf blades of bamboo with lanceolate characteristics and bilateral symmetry, corroborating earlier findings on four species of Indocalamus (Shi, Xu, et al., 2015). The present study demonstrated this bilateral symmetry in leaves of 42 bamboo species, including four sympodial (clustered) species, 16 monopodial (scattered) species, and 22 mixed species (that have the characteristics of both clustering and scattering). In this study, this was performed by measuring thousands of leaves to determine lengths and a subset of over one thousand to determine shape parameters. There are very few geometrical models for modeling leaves. Many models of planar plant leaves use ellipses as a starting point. A mul- tiparametric model was developed for the description of the axial-­ symmetric convex pentagon of grass leaf shape (Dornbusch et al., 2011). In contrast, for modeling bamboo leaves with the Gielis equation, we now need only two model parameters, with extremely high good- ness of fit for all species (>0.98), and confirming the bilateral symmetry of bamboo leaves. In this study, quantitative measurements were per- formed for 500 leaves, and scans and comparison with models for 30 leaves per species. As the scanned leaves per species were of similar size, this range should also reflect differences in leaf age and position- ing, confirming the Weibull distribution also observed by Shi, Xu, et al. (2015). 4.1 | Capturing the diversity of bamboo leaves Indeed, bamboos are one of very few groups in the grass family that have evolved in forests, not in open areas, and can reach quite large sizes (up to 20 m for temperate bamboos and up to 30 m for tropical bamboos). Hollow and flexible culms and branches, protected during development with very stiff culms or branch sheaths on the one hand, and leaf blades that are connected through pseudo-­ petioles allowing for torsion on the other hand contribute to the evo- lutionary success of woody and herbaceous bamboos. The anatomy of bamboo leaves is very stable throughout the subfamily (Brandis, 1907). It is remarkable that the pseudo-­petiole, a specialized structure in all bamboos (and only some grasses with larger leaves), has hardly been studied. The course of the vascular bundle and the possible pres- ence of pulvini should be investigated, as pseudo-­petioles allow bam- boos to adjust the orientation of the blades toward the sun or to ease torsion caused by wind or snow loads. 6804 Lin et al. Lin et al. or other botanical shapes such as tree rings (Lei & Koike, 1998; Shi, Huang, et al. 2015; Shi, Xu, et al. 2015) and petioles (Faisal et al. 2010). It may become a uniform approach for studying leaves in plants or other shapes and could be better than existing models, as shown by using the Akaike information criterion (Shi, Huang, et al., 2015). From a geometric and mathematical point of view, it is a generalization of the Pythagorean theorem, retaining its structure with separated vari- ables, useful for studying development from an applied mathematics point of view (Caratelli et al., 2009; Gielis et al., 2012). FUNDING INFORMATION This work was financially supported by the Key Project of National Science & Technology Ministry (2016YFD0600901), the National Postdoctoral Fund of China (2014M560427), the National Natural Science Foundation for Young Scholars of China (31400348 and 31000294), and the Priority Academic Program Development of ACKNOWLEDGMENTS We thank Rong Zhao, Huarong Li, Xiaofei Cheng, Xiaobo Dong, Qiang Xu, Junjie Cao, Yongqi Zhi, Lin Guo, Mingyun Jiang, Ru Wang, Xiao Zheng, and Caiyun Zhang for participating in the investigation of bam- boo leaves. We also thank the editor and two anonymous reviewers for their valuable comments. 4.2 | Leaf development models and the future trends This distribution originates from the study of different particle sizes in crushed particles, sand, or volcanic ash, following power laws. This distribution implies that leaf length in bamboo follows a power law. Only two parameters are involved, one for length and the other for shape, accounting for full variations in shape and dimensions (com- pared to three-­dimensional and three shape parameters in wheat, bar- ley, and maize (Dornbusch et al., 2011)). Keeping the shape parameter constant and modify the length parameter according to the species’ minimum and maximum leaf lengths and the specific Weibull distribu- tions should suffice to faithfully regenerate leaf shapes and images en- compassing the variation on a plant within a given species. As the leaf shape of plants can be affected by many factors such as genetics, cul- tivar, and growing conditions, future research could focus on explain- ing the variation of leaf shape in bamboos at different ages and under different controlled environments such as sites with different levels of nutrients. Our method will allow for the building of reliable databases of genotypes for bamboo studies worldwide, supplementing molecular markers (Gielis, Everaert, Goetghebeur, & Deloose, 1995; Hodkinson, Renvoize, Chonghaile, Stapleton, & Chase, 2000; Lin, Ruan, Lou, Guo, & Fang, 2009; Schiessl, Kausika, Southam, Bush, & Sablowski, 2012; Suyama, Obayashi, & Hayashi, 2000) for precise identification. One advantage of our model is that the associated characteristics (perimeter, area, polar moment of inertia) can be computed directly from the analytical expressions of these characteristics for leaf-­shape variation, by observing cross sections along the petiole (Faisal et al., 2010) or stems (Dornbusch et al., 2009). While little quantitative information is available on the develop- ment of bamboo leaf blades, the development of these blades is qual- itatively quite simple. After elongation of the culm or branching, the leaves develop in an acropetal way (Banik, 2015). They develop along the axis of the midrib fully parallel to the main axis of the branch, in- side the preceding sheath, and emerge through the upper opening of that preceding sheath. When they emerge they unroll until they are planar. This process of convolute vernation (Brandis, 1907) involves the turgor state of the bulliform cells, the same process that occurs when leaf blades roll up in dry conditions. Certainly, this precise pro- cess requires further research over the whole bamboo subfamily. REFERENCES Banik, R. L. 2015. Morphology and growth. In W. Liese & M. Köhl (Eds.), Bamboo-the plant and its uses. Tropical Forestry series (pp. 43–89). Heidelberg, Germany: Springer. Gielis, J., & Gerats, T. (2004). A botanical perspective on modeling plants and plant shapes in computer graphics. In International Conference on Computer, Communication and Control Technologies. Austin, Texas. Bell, A. D., & Bryan, A. (2008). Plant form: An illustrated guide to flowering plant morphology. 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This increases the potential for modeling leaves 6805 Lin et al. Lin et al. CONFLICT OF INTEREST Gielis, J., Caratelli, D., Fougerolle, Y., Ricci, P. E., Tavkelidze, I., & Gerats, T. (2012). Universal natural shapes: From unifying shape description to simple methods for shape analysis and boundary value problems. PLoS ONE, 7, e29324. doi:10.1371/journal.pone.0029324 None declared. Wang, S. (2007). The semantics of natural language descriptions of con- tinuous quantities. PhD Dissertation, University of Manchester, Manchester. R Core Team (2015). R: A language and environment for statistical comput- ing. Vienna, Austria: R Foundation for Statistical Computing. Retrieved from http://www.R-project.org/ Watanabe, T., Hanan, J. S., Room, P. M., Hasegawa, T., Nakagawa, H., & Takahashi, W. (2005). Rice morphogenesis and plant architecture: Measurement, specification and the reconstruction of structural devel- opment by 3D architectural modelling. Annals of Botany, 95, 1131–1143. 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Shi, P., Xu, Q., Sandhu, H. S., Gielis, J., Ding, Y., Li, H., & Dong, X. (2015). Comparison of dwarf bamboos (Indocalamus sp.) leaf parameters to determine relationship between spatial density of plants and total leaf area per plant. Ecology and Evolution, 5, 4578–4589. REFERENCES American Journal of Botany, 80, 796–804. Doust, A. (2007). Architectural evolution and its implications for domesti- cation in grasses. Annals of Botany, 100, 941–950. Nelder, J. A., & Mead, R. (1965). A simplex algorithm for function minimiza- tion. The Computer Journal, 7, 308–313. Faisal, T. R., Abad, E. M. K., Hristozov, N., & Pasini, D. (2010). The impact of tissue morphology, cross-­section and turgor pressure on the mechani- cal properties of the leaf petiole in plants. Journal of Bionic Engineering, 7, S11–S23. Ohrnberger, D. (1999). The bamboos of the world: Annotated nomenclature and literature of the species and the higher and lower taxa. Amsterdam, The Netherlands: Elsevier. 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Clonal structure in a dwarf bamboo (Sasa senanensis) population inferred from amplified frag- ment length polymorphism (AFLP) fingerprints. Molecular Ecology, 9, 901–906. How to cite this article: Lin, S., Zhang, L., Reddy, G. V. P., Hui, C., Gielis, J., Ding, Y. and Shi, P. (2016), A geometrical model for testing bilateral symmetry of bamboo leaf with a simplified Gielis equation. Ecology and Evolution, 6: 6798–6806. doi: 10.1002/ece3.2407 How to cite this article: Lin, S., Zhang, L., Reddy, G. V. P., Hui, C., Gielis, J., Ding, Y. and Shi, P. (2016), A geometrical model for testing bilateral symmetry of bamboo leaf with a simplified Gielis equation. Ecology and Evolution, 6: 6798–6806. doi: 10.1002/ece3.2407 Voorend, W., Lootens, P., Nelissen, H., Roldan-Ruiz, I., Inzé, D., & Muylle, H. (2014). LEAF-­E: A tool to analyze grass leaf growth using function fitting. Plant Methods, 10, 37.
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Impact of COVID-19 pandemic on the residency programs of the country: A multicentre study
Pakistan journal of medical sciences
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Original Article Original Article ABSTRACT Objective: To provide an insight on the disruption of multiple facets of residency programs in a multi- centre study. Methods: This cross-sectional survey was carried out by enrolling the available residents from three teaching hospitals of the country by sending a questionnaire through email. The questionnaire comprised of three parts; 1) basic demographics, 2) effect on multiple facets of training and 3) the use of smart learning with the support provided by the hospitals. Data collection was started during the first week of June 2020 after acquiring ethical approval from the concerned department and the total duration of the study was one month. Data was analysed using SPSS v. 19.0. Results: A hundred-and-five completed responses were obtained with a response rate of 42%. Fifty- nine percent of the participants were female residents. Majority of the residents (69%) belonged to the age group 25-30 years. Fourth year residents (38%) showed maximum participation and the mean number of work days per month were 22±5.4. All of the aspects of training suffered complete or severe reduction except for the multi-disciplinary team (MDT) meetings, elective rotations and e-log book entries. Sixty seven to sixty-nine percent of the residents felt complete clinical, educational and psychological desertion in their departments, 59% used telemedicine and 90% reported non-availability of smart learning facilities. g Conclusion: Overall, our study confirmed that the COVID-19 pandemic has substantially affected the clinical skills, teaching and personal growth of many trainees. There is a decrease in exposure to almost all of the aspects of training with no alternative in the form of smart learning provided to many. Clinical, educational and psychological support, although an extremely important part of healthcare staffing and management, has been largely neglected as well. KEYWORDS: COVID-19, Pandemic, Residents, Survey, Training. KEYWORDS: COVID-19, Pandemic, Residents, Survey, Training. doi: https://doi.org/10.12669/pjms.37.2.3496 zmi SKH, Kazmi SAH. Impact of COVID-19 pandemic on the residency programs of the country: A multicentr i. 2021;37(2):367-372. doi: https://doi.org/10.12669/pjms.37.2.3496 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecomm which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Impact of COVID-19 pandemic on the residency programs of the country: A multicentre study Laima Alam1, Mafaza Alam2, Syed Kumail Hasan Kazmi3, Syed Ashoor Hasan Kazmi4 INTRODUCTION The second part comprised of three main aspects of residency programs affected by the current situation; clinical (active and passive learning), teaching (receiving and giving) and per- sonal growth and development, all of which were evaluated using 8 questions each, by taking help from the training module of Royal College of Phy- sicians and Australian Medical Council Limited. The percentage decrease of the residents’ involve- ment in all three aspects of training was classified as slight for <40% reduction, severe for 40-80% re- duction and complete suppression for >80% reduc- tion10 (the already defined percentages were used as surrogate metrics to represent the suppressed train- ing activities as the referenced study) than the pre- covid days. The referenced study is a pioneer study and no such study has been carried out keeping in view the multiple facets of post-graduate training. The third part had Yes/No questions regarding the use of telemedicine, use of online assessments and classes, and the support provided from the hospital psychologically and educationally. The re-percussions of the unprecedented challenges are being felt almost by all specialties and fellowship programs of the medical field.4 Many of the residents belonging to diverse specialties have been redeployed to cater COVID-19 patients and wards, causing cancellation of elective procedures (normally done by residents) and elimination of Out Patient visits which were the main sources of learning for the residents.5 The balance between clinical work, research, educational aspect of training and personal growth and development has been disrupted severely.6 Many of the residents are and will be side-lined by self-isolation, hospital admissions and possible deaths while performing their front-line duties during these chaotic times.4 This pandemic will change the educational and clinical experience of many fellows across the globe.7 The constant pressure to acquire clinical skills, provide care, stay healthy and to prevent the community spread of the virus by not becoming a vector has been emotionally and psychologically crippling for many doctors and residents.8 We present a cross-sectional survey, one of its own kind, locally and internationally, involving residents from many different specialties of three large teaching hospitals of the country to gauge the possible impact of the pandemic on their training routines. The sample size was calculated with margin of error set at 6%, confidence level at 95% and an anticipated frequency (response distribution) of 65% using OpenEpi sample size calculator. INTRODUCTION The number of active cases and deaths second- ary to COVID-19 are rising rapidly following a steep ascending curve.1 Social distancing and implementation of safe practices have impacted many post-graduate medical training programs across the country, resulting in severe disruption of clinical and educational skill procurement.2 It has become an international necessity to balance the mandatory social distancing required for slow- ing the disease spread while catering to the edu- Pak J Med Sci March - April 2021 Vol. 37 No. 2 www.pjms.org.pk 367 The number of active cases and deaths second- ary to COVID-19 are rising rapidly following a steep ascending curve.1 Social distancing and implementation of safe practices have impacted many post-graduate medical training programs across the country, resulting in severe disruption of clinical and educational skill procurement.2 It has become an international necessity to balance the mandatory social distancing required for slow- ing the disease spread while catering to the edu- Correspondence: Laima Alam, FCPS. Junior Consultant Gastroenterology, Bahria Town International Hospital, Phase VIII, Rawalpindi, Pakistan. E-mail: Laima_alam@yahoo.com * Received for Publication: August 17, 2020 * Revision Received: December 3, 2020 * Revision Accepted: December 12, 2020 Pak J Med Sci March - April 2021 Vol. 37 No. 2 www.pjms.org.pk 367 Pak J Med Sci March - April 2021 Vol. 37 No. 2 www.pjms.org.pk 367 367 Laima Alam et al. reminder was given to the residents after one week of no response and the candidates were dropped who failed to respond after another three days. cational and clinical needs of the trainee doctors who would be running the wards and clinics on their own in near future after accreditation. Also, maintaining a steady supply of workforce in these unprecedented conditions for providing clinical care to the masses, flexibility in the current health care guidelines, adjustments in trainee schedul- ing and flow, re-evaluation of resident duties and prospects, and the need for frequent assessments in a safe environment are needed.3 p y The questionnaire was developed by LA and MA after relevant literature review. It was reviewed by two medical education experts for content validity. The survey was piloted among 15 post-graduate residents before putting it to test. The questionnaire was divided in to three parts. The first encompassed the demographic data including age, gender, spe- cialty of residency, year of residency program and the number of days spent in the hospital per month during the pandemic. INTRODUCTION Thus a total of 250 surveys were sent out through email to residents of all medical, surgical and allied specialties. Data was statistically described in terms of mean ± SD for continuous data, frequencies and percentages when appropriate. Chi square test and Fisher exact test were used to compare qualitative data. All statistical analyses were performed using SPSS v 19.0. All p values ≤0.05 were considered statistically significant. METHODS This cross-sectional survey was carried out enrolling the available residents from three teaching hospitals namely, Pak Emirates Military Hospital Rawalpindi, Fauji Foundation Hospital Rawalpindi and Armed Forces Institute of Dentistry Rawalpindi by sending a questionnaire through email, using convenience sampling. Data collection was started during the first week of June 2020 after acquiring ethical approval from the concerned department (A/28/07/EC/121 dated 01-06-2020) and the total duration of the study was one month. The survey was created by an online survey generator.9 A RESULTS Hundred and nine residents filled the survey within the time frame, four responses were incomplete and thus discarded. The response rate was 42%. Female residents were in abundance Pak J Med Sci March - April 2021 Vol. 37 No. 2 www.pjms.org.pk 368 COVID-19 and residency programs of the country 59%). Majority of the residents (69%) belonged to he age group 25 to 30 years. Fourth year residents 38%) showed preponderance which happens to be the last year of residency for many specialties Fig.1). The mean number of work days per month were 22 ± 5.4. Majority of the residents reported severe to complete reduction in all of the facets of teaching except for the Multi-disciplinary Team meetings of the residents (67-69%) felt complete clinical, educational and psychological abandonment in their departments (Table-II). Table-I: Impact of the pandemic on different aspects of residency. <40% reduction (n%) 40-80% reduction (n%) >80% reduction (n%) Teaching aspect of residency MDT meetings Clinical presentations Morning meetings Case based discussions Grand rounds Clinico-pathological conferences Teaching sessions by the consultants Teaching sessions by trainees to juniors 40 (38.1) 31 (29.5) 30 (28.6) 11 (10.5) 26 (24.8) 21 (20) 16 (15.2) 13 (12.4) 26 (24.8) 26 (24.8) 27 (25.7) 25 (23.8) 22 (21) 19 (18.1) 28 (26.7) 21 (20) 39 (37.1) 48 (45.7) 48 (45.7) 69 (65.7) 57 (54.3) 65 (61.9) 61 (58.1) 71 (67.6) Clinical skills Hands-on Elective rotations OPD patient inflow OT/Procedure room inflow Elective procedure cancellation Level of direct/indirect supervision Delay in acquiring clinical skills Delay in mandatory workshops 7 (6.7) 38 (36.2) 19 (18.1) 28 (26.7) 17 (16.2) 15 (14.3) 18 (17.1) 10 (9.5) 27 (25.7) 31 (29.5) 57 (54.3) 56 (53.3) 38 (36.2) 53 (50.5) 42 (40) 32 (30.5) 71 (67.6) 36 (34.3) 29 (27.6) 21 (20) 50 (47.6) 37 (35.2) 45 (42.9) 63 (60) Personal growth and development Delay in acquiring recommendation/experience certificates Delay in post graduate exams and assessments Cancellation of in-person medical conferences Effect on clinical research Evaluations and appraisals Acquiring CMEs Maintaining e-log book Annual leaves 15 (14.3) 11 (10.5) 8 (7.6) 17 (16.2) 21 (20) 10 (9.5) 31 (29.5) 21 (20) 47 (44.8) 60 (57.1) 57 (54.3) 50 (47.6) 38 (36.2) 37 (35.2) 59 (56.2) 40 (38.1) 43 (41) 34 (32.4) 40 (38.1) 38 (36.2) 46 (43.8) 58 (55.2) 15 (14.3) 44 (41.9) Table-II: Miscellaneous questions regarding impact of the pandemic on residency. Pak J Med Sci March - April 2021 Vol. 37 No. 2 www.pjms.org.pk 369 RESULTS Yes (n%) No (n%) Alternative to the lack of educational aspect of training 29 (27.6) 76 (72.4) Table-I: Impact of the pandemic on different aspects of residency. of the residents (67-69%) felt complete clinical, educational and psychological abandonment in their departments (Table-II). (59%). Majority of the residents (69%) belonged to the age group 25 to 30 years. Fourth year residents (38%) showed preponderance which happens to be the last year of residency for many specialties (Fig.1). The mean number of work days per month were 22 ± 5.4. of the residents (67-69%) felt complete clinical, educational and psychological abandonment in their departments (Table-II). (59%). Majority of the residents (69%) belonged to the age group 25 to 30 years. Fourth year residents (38%) showed preponderance which happens to be the last year of residency for many specialties (Fig.1). The mean number of work days per month were 22 ± 5.4. Table-II: Miscellaneous questions regarding impact of the pandemic on residency. Table-II: Miscellaneous questions regarding Table-II: Miscellaneous questions regarding impact of the pandemic on residency. Table-II: Miscellaneous questions regarding impact of the pandemic on residency. Yes (n%) No (n%) Alternative to the lack of educational aspect of training 29 (27.6) 76 (72.4) Online classes 16 (15.2) 89 (84.8) Online assessments 15 (14.3) 90 (85.7) Use of telemedicine 62 (59) 43 (41) Deployment to other wards/ centers? 41 (39) 64 (61) Increase in duty hours? 37 (35.2) 68 (64.8) Do you feel clinically supported? 38 (36.2) 67 (63.8) Do you feel educationally supported? 37 (35.2) 68 (64.8) Do you feel psychologically supported? 36 (34.3) 69 (65.7) Majority of the residents reported severe to complete reduction in all of the facets of teaching except for the Multi-disciplinary Team meetings (Table-I). Clinical skills acquirement was also reduced severely or completely except for the elective rotations since the trainees could rotate in to different medical and surgical specialties according to their laid-down curriculum. E-log book entries were also largely unaffected since residents were keeping a record of the patients (COVID-19) through online portal. Majority of the residents reported non-availability of online learning and assessment (Table-II) and only 59% used telemedicine to decrease the patient load without unnecessary exposure. Increase in duty hours was relayed by 35.2% of the residents. Many Pak J Med Sci March - April 2021 Vol. 37 No. 2 www.pjms.org.pk 369 Laima Alam et al. RESULTS facility of smart learning helped the trainees feel more supported. Fig.1: Demographics of the residents enrolled in the study. DISCUSSION Healthcare (including medicines, buildings, beds, equipment, vehicles and healthcare staff) being a finite resource is severely affected by the pandemic and the resulting lockdowns. Patients with chronic illnesses, those requiring screening and surveillance and the non-emergency Out Patient follow ups are also among the badly affected population.10,11 These so called “cold cases” used to be a great learning opportunity for the residents. The patients themselves have also limited their health seeking behaviour out of fear as well as lack of transport and resources.12 Late diagnosis, misdiagnosis and considerable emotional trauma have been seen in previous pandemics as well.13 Due to the unexpected emergency condition world-wide, community-centred rather than a patient-centred approach has been advocated including social segregation, performing only emergency procedures, telemedicine etc., which might not be regarded ideal by the patients individually but are paramount in view of community safety.14 Guidelines have already been laid down to limit unnecessary exposure for the doctors’ and the patients’ safety including deferral of elective procedures and prioritizing life/limb/ sight saving procedures.15 Fig.1: Demographics of the residents enrolled in the study. Gender, specialty, number of work days per month and year of residency were all statistically related to substantial decrease in multiple aspects of the residency programs (Table-III). All these variables (except number of work days) were also statistically related to lack of support from the respective departments. On the contrary, the Many of the post graduate and entrance exams have been rescheduled causing frustration among residents planning for accreditation and higher/foreign qualifications (GMC registration through PLAB or MRCP/MRCS routes, Specialty Certificate Exams (SCE) and local post-graduate exams like FCPS, MCPS and MD/MS).16 Many of Pak J Med Sci March - April 2021 Vol. 37 No. 2 www.pjms.org.pk 370 Table-III: Relation of demographics and variables of smart learning with the impact of the pandemic on several aspects of residency. DISCUSSION Variables Clinical aspect of residency Teaching aspect Personal growth and development Increase in duty hours Lack of psychological support Lack of clinical support Lack of educational support Gender P=0.04 P=0.008 P=0.008 P=0.369 P <0.001 P<0.001 P <0.001 Specialty P<0.001 P=0.001 P=0.023 P=0.004 P <0.001 P=0.001 P=0.002 Year of residency P=0.184 P=0.017 P=0.06 P=0.127 P=0.02 P=0.001 P=0.019 Days/month in hospital P=0.004 P=0.01 P=0.006 P=0.402 P=0.57 P=0.51 P=0.12 On-line classes P=0.429 P=0.034 P=0.021 P=0.07 P=0.04 P=0.07 P=0.001 On-line assessments P=0.512 P=0.191 P=0.546 P=0.62 P=0.001 P=0.001 P=0.001 Table-III: Relation of demographics and variables of smart learning with the impact of the pandemic on several aspects of residency. COVID-19 and residency programs of the country our residents fell victim to delay in accreditation (90% reporting severe to complete delay) and final assessments (94% reporting severe to complete delay). Unlike the international training programs where relaxation to the overall requirement to get competencies signed-off in lieu of the current circumstances has been formulated already,17 no such facilitation has been provided for our residents. and psychological stress due to a decreased confidence level that may come with a reduced specialty-specific case exposure and possibly re- deployment. According to a survey, it will soon be almost impossible to evaluate the residents for their core competencies and the normal laid-out curriculum.22 This would lead to further frustration and fear among the residents as their future seems bleak at the expense of these catastrophic conditions. The rational for sequential rotation of the residents is to conserve resources including PPE and to safeguard the heath of the residents, and as a consequence, the health of the patients and the community as a whole.2,18 Despite clear guidelines, majority of the residents were working more than the designated days with no “cool off” period in our study. Interestingly, many of the residents did not report increase in duty hours in our study as most of them had already been performing 60 plus hours per week with no legislation for work hour limitation.19 It is noteworthy that female residents in our study were more prone to clinical, educational and psychological abandonment causing their training programs to be affected tremendously. Similarly, the interventional specialties (Gastroenterology, Cardiology, Dentistry and Surgery) were facing greater disruption. To our best knowledge this is one of the first multi-centre studies involving multiple specialities to provide insight in to the disruption of the many facets of residency programs. DISCUSSION The data was collected from a single province of the country which might not be a true representation of all the residents country-wide owing to logistics and differences in current pandemic situation region wise. Also the socio-economic struggles of the residents were not discussed which could massively affect the psychological stability and as a consequence performance of the trainees. Source of Funding: None. Source of Funding: None. CONCLUSION The current unprecedented condition has disrupted many aspects of life along with the training and assessment of healthcare front- liners. Overall, our study confirmed that the COVID-19 pandemic has substantially affected the clinical skills, teaching and personal growth of many trainees. There is a decrease in exposure to almost all of the aspects of training with no alternative in the form of smart learning provided to many. Clinical, educational and psychological support, although an extremely important part of healthcare staffing and management, has been largely neglected as well. g y) g g p The Multi-disciplinary team meetings (MDT) were largely unaffected in our survey which might be because of the reason that many departments lack these kind of meetings to begin with. All in-person conferences have been called off around the globe with introduction of online lectures and didactic educational resources being introduced.20,21 Smart learning has yet to be introduced in many of the teaching hospitals in our country, the importance of which is reflected from the direct relationship of online-classes/ assessments and the low level of psychological stress and the feeling of clinical and educational abandonment as shown in Table-III. It was interesting to note that although smart-learning helped in the teaching and personal development (acquiring CME for example) aspects, it didn’t correlate positively with ticking off the clinical skills (such as hands-on) check-list. REFERENCES 1. [Internet]. Covid.gov.pk. 2020 [cited 12 June 2020]. Available from: http://covid.gov.pk/ 2. Warhadpande S, Khaja M, Sabri S. The Impact of COVID-19 on Interventional Radiology Training Programs: What You Need to Know. Acad Radiol. 2020;27(6):868-871. doi: 10.1016/j.acra.2020.04.024 Residents from highly specialized fellowship programs have been redeployed to stations like screening facilities, emergency departments and acute medical intakes to supplement the available medical force.4 Many of our senior trainees reported greater disruption of training activities /j 3. ACGME Response to the Coronavirus (COVID-19) [Internet]. ACGME. 2020 [cited 12 June 2020]. Available from: https://acgme.org/Newsroom/Newsroom-Details/ ArticleID/10111/ACGME-Response-to-the-Coronavirus- COVID-19 j 3. ACGME Response to the Coronavirus (COVID-19) [Internet]. ACGME. 2020 [cited 12 June 2020]. Available from: https://acgme.org/Newsroom/Newsroom-Details/ ArticleID/10111/ACGME-Response-to-the-Coronavirus- COVID-19 Pak J Med Sci March - April 2021 Vol. 37 No. 2 www.pjms.org.pk 371 Laima Alam et al. 4. Potts J. Residency and Fellowship Program Accreditation: Effects of the Novel Coronavirus (COVID-19) Pandemic. J Am Coll Surg. 2020;230(6):1094-1097. doi:  10.1016/j. jamcollsurg.2020.03.026 17. Special Communication to Diagnostic Radiology Residents, Interventional Radiology Residents, Subspecialty Radiology Fellows, and Program Directors). [Internet]. Accreditation Council of Graduate Medical Education (ACGME), [Cited 12 June 2020]. Available from: https://www.acgme.org/ Portals/0/Documents/ COVID-19/RadCOVID19LTC.pdf. j g 5. COVID-19: Recommendations for Management of Elective Surgical Procedures [Internet]. American College of Surgeons. 2020 [cited 12 June 2020]. Available from: https:// www.facs.org/covid-19/clinical-guidance/elective- surgery p 18. Backer JA, Klinkenberg D, Wallinga J. Incubation period of 2019 novel coronavirus (2019-nCoV) infections among travellers from Wuhan, China, 20-28 January 2020. Euro Surveill. 2020;25(5). g y 6. Coronavirus Disease 2019 (COVID-19) – Prevention & Treatment [Internet]. Centers for Disease Control and Prevention. 2020 [cited 12 June 2020]. Available from: https://www.cdc.gov/coronavirus/2019-ncov/prevent- getting-sick/prevention.html. ( ) 19. Working time and breaks | Advice guides | Royal College of Nursing [Internet]. The Royal College of Nursing. 2020 [cited 12 June 2020]. Available from: https://www.rcn.org. uk/get-help/rcn-advice/working-time-rest-breaks-on-call- and-night-work g g p 7. Coe T, Jogerst K, Sell N, Cassidy D, Eurboonyanun C, Gee D, et al. Practical Techniques to Adapt Surgical Resident Education to the COVID-19 Era. Ann Surg. 2020;272(2):e139-e141. doi: 10.1097/SLA.0000000000003993 20. Critical Care Introductory Course, SIR resident fellow student (RFS) section, [Internet]. 2020 [cited 12 June 2020]. Available from: http://rfs.sirweb.org/clinical-resources/ critical-care-course/ ( ) / 8. Koh D. Occupational risks for COVID-19 infection. Occup Med. 2020;70(1):3-5. 21. American Board of Surgery COVID-19 Updates [Internet]. Absurgery.org. 2020 [cited 12 June 2020]. Available from: https://www.absurgery.org/default.jsp?news_ covid19updates 9. Create Free Online Surveys, Quizzes and Forms - FreeOnlineSurveys [Internet]. FreeOnlineSurveys. REFERENCES 2020 [cited 12 June 2020]. Available from: https:// freeonlinesurveys.com/ p 22. UPDATED: Coronavirus (COVID-19) and ACGME Site Visits, Educational Activities, and Other Meetings [Internet]. ACGME. 2020 [cited 12 June 2020]. Available from: https://acgme.org/Newsroom/Newsroom-Details/ ArticleID/10072/Coronavirus-COVID-19-and-ACGME- Site-Visits-Educational-Activities-and-Other-Meetings 10. Daniele A, Francesco C, Giovanni EC, Francesco E, Cristian F, Giovanni L, Sergio S, et al. The impact of COVID-19 pandemic on Urology residency in Italy. Minerva Urol Nefrol. 2020;72(4):505-509. doi: 10.23736/S0393- 2249.20.03868-0 11. Letters to the Editor: Non-COVID patients are skipping ER visits. That can be dangerous [Internet]. Los Angeles Times. 2020 [cited 2020 Apr 20]. Available from: https:// www.latimes.com/opinion/story/202004-10/non-covid- patients-skipping-er-visits 5 Authors’ Contribution: LA: Contributed to the idea, design and drafting of the manuscript, data collection, statistical analysis and literature review. g 12. Masroor S. Collateral damage of COVID-19 pandemic: Delayed medical care. J Card Surg. 2020;35(6):1345-1347. doi: 10.1111/jocs.14638 MA: Contributed to data collection of literature review. 13. Moody W, Loudon M, Watkin R, Steeds R, Prendergast B. Infective endocarditis: diagnosis delayed during swine flu pandemic. Postgrad Med J. 2011;87:240-240. SKHK: Contributed to data collection. SAHK: Contributed to critical review. 14. Naspro R, Da Pozzo LF. Urology in the time of corona. Nat Rev Urol. 2020;17:251-253. doi: 10.1038/s41585-020-0312-1 All the authors take equal responsibility for the accuracy and integrity of the work. All the authors take equal responsibility for the accuracy and integrity of the work. 15. Adhi M. Novel Coronavirus Disease Pandemic and Ophthalmologists Perspectives. J Pak Med Assoc. 2020;70(Suppl 3)(5):S95-S97. doi: 10.5455/JPMA.18 16. Application guidelines for early specialization in interventional radiology (ESIR). [Internet]. Accreditation Council of Graduate Medical Education (ACGME). [Cited 12 June 2020]. Available from: http://www.acgme.org/ Portals/0/PFAssets/ProgramResources/415_Application_ Guidelines_for_ESIR.pdf. Pak J Med Sci March - April 2021 Vol. 37 No. 2 www.pjms.org.pk 372 Authors: Pak J Med Sci March - April 2021 Vol. 37 No. 2 www.pjms.org.pk 372 Authors: 1. Laima Alam, FCPS. Junior Consultant Gastroenterology, Bahria Town International Hospital, Phase VIII, Rawalpindi, Pakistan. 2. Mafaza Alam Registrar Operative Dentistry, Armed Forces Institute of Dentistry, Rawalpindi, Pakistan. 3. Syed Kumail Hasan Kazmi, FCPS, Fellow Gastroenterology, Pak Emirates Military Hospital, Rawalpindi, Pakistan. 4. Syed Ashoor Hasan Kazmi Registrar Trauma and Orthopaedics, University Hospital Birmingham, United Kingdom. p // g g/ sets/ProgramResources/415_Application_ ESIR.pdf. 1. Laima Alam, FCPS. Junior Consultant Gastroenterology, Bahria Town International Hospital, Phase VIII, Rawalpindi, Pakistan. 2. Mafaza Alam Registrar Operative Dentistry, Armed Forces Institute of Dentistry, Rawalpindi, Pakistan. 3. Syed Kumail Hasan Kazmi, FCPS, Fellow Gastroenterology, Pak Emirates Military Hospital, Rawalpindi, Pakistan. 4. Syed Ashoor Hasan Kazmi Registrar Trauma and Orthopaedics, University Hospital Birmingham, United Kingdom. 1. Laima Alam, FCPS. Junior Consultant Gastroenterology, Bahria Town International Hospital, Phase VIII, Rawalpindi, Pakistan. 2. Mafaza Alam Registrar Operative Dentistry, Armed Forces Institute of Dentistry, Rawalpindi, Pakistan. 3. Syed Kumail Hasan Kazmi, FCPS, F ll G t t l Pak J Med Sci March - April 2021 Vol. 37 No. 2 www.pjms.org.pk 372
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https://aacr.figshare.com/articles/journal_contribution/Supplementary_Figure_5_from_Thrombospondin-1_Triggers_Cell_Migration_and_Development_of_Advanced_Prostate_Tumors/22390746/1/files/39836280.pdf
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Supplementary Figure 5 from Thrombospondin-1 Triggers Cell Migration and Development of Advanced Prostate Tumors
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Firlej et al, supplementary figure 5 Firlej et al, supplementary figure 5 A TRPV3 TRPV3 [Ca++], nM siRNA cont cont normoxia hypoxia *** *** *** *** 0 50 100 150 200 250 300 350 400 B 0 0.5 1 1.5 2 2.5 3 3.5 4 2h 6h 24h 48h TSP1 mRNA hypoxia/normoxia B TSP1 Vehicle CoCl2 Thg C C
https://openalex.org/W1975892498
https://link.springer.com/content/pdf/10.1007/s12471-013-0455-2.pdf
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Impact factor 2012 for cardiovascular journals: true impact?
Netherlands heart journal
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E. E. van der Wall (*) Interuniversity Cardiology Institute of the Netherlands (ICIN) - Netherlands Heart Institute (NHI), PO Box 19258, 3501 DG Utrecht, the Netherlands e-mail: Ernst.van.der.wall@icin.knaw.nl Impact factor 2012 for cardiovascular journals: true impact? 2) For the cardiovascular journals directed by Dutch Editors-in-Chief, the following remarkable results for the 2012 impact factors were observed: a) The European Journal of Heart Failure (EJHF), fitting in the family of journals belonging to the European Society of Cardiology (ESC), received a 2012 impact factor of 5.247 versus a 2011 impact factor of 4.896, a rewarding increase. The Editorial Board of the EHJF is chaired by Dirk Jan van Veldhuisen (Groningen, the Netherlands) with Wiek van Gilst and Adriaan Voors as Deputy Editors. b) For the journal Eurointervention, also part of the ESC journal family, with Patrick Serruys (Rotterdam, the Netherlands) as Editor-in-Chief, the 2012 impact fac- tor was rather similar to the 2011 impact factor (3.285 in 2011 versus 3.173 in 2012) b) For the journal Eurointervention, also part of the ESC journal family, with Patrick Serruys (Rotterdam, the Netherlands) as Editor-in-Chief, the 2012 impact fac- tor was rather similar to the 2011 impact factor (3.285 in 2011 versus 3.173 in 2012) From a European perspective, the impact factors of 2012 show the following interesting facts: c) The impact factor of the European Journal of Preven- tive Cardiology (another member of the ESC journal family), with Diederick Grobbee (Utrecht, the Neth- erlands) as Editor-in-Chief, moved from 2.634 in 2011 to 3.903 in 2012, a considerable increase! 1) Out of a total number of 122 cardiovascular journals (sub- ject category Cardiac & Cardiovascular Systems), the European Heart Journal (EHJ) moved to the second place with a 2012 impact factor of 14.097, directly behind Circu- lation (2012 impact factor of 15.202) and ahead of the Journal of the American College of Cardiology (JACC) with a 2012 impact factor of 14.086. Last year, the EHJ already ranked in the third place behind JACC (second) and Circulation (first). As a result, the EHJ Editor-in-Chief Thomas Lüscher (Zürich, Switzerland) and the EHJ d) The impact factor of the European Journal of Cardio- vascular Nursing (also part of the ESC journal family), chaired by Tiny Jaarsma (currently residing in Norrköping, Sweden) as Editor-in-Chief, increased significantly from 1.711 in 2011 to 2.042 in 2012 e) Finally, the impact factor of the International Journal of Cardiovascular Imaging (CAIM), supervised by Hans Reiber (Leiden, the Netherlands) as Editor-in- Chief, moved from 2.285 in 2011 to 2.648 in 2012, also an obvious increase The above-mentioned Editors-in-Chief should be con- gratulated on attaining a fine 2012 impact factor. Impact factor 2012 for cardiovascular journals: true impact? E. E. van der Wall Published online: 13 August 2013 # The Author(s) 2013. This article is published with open access at Springerlink.com In June 2013, the impact factors for the year 2012 were released (Thomson Reuters Journal Citation Reports® 2012, ISI Web of Knowledge). The impact factor is defined as the average number of citations received per paper divid- ed by the published articles in a specific journal during the preceding 2 years [1–3]. The 2012 impact factor of the Netherlands Heart Journal (NHJ) was 1.411. The 2012 NHJ impact factor was calculated as follows: in 2012, the total number of citations to articles published in the years 2010 and 2011 was 207. The number of articles published in 2010 and 2011 was 146. As a result, the 2012 impact factor was 206 citations divided by 144 articles: 206/146=1.411. The NHJ impact factor has therefore remained rather con- stant over the past 4 years: 1.392 in 2009, 1.447 in 2010, 1.438 in 2011, and 1.411 in 2012. Of course, we are striving to increase our impact factor over the forthcoming years (see the article by Tobias Opthof in the July/August edition of NHJ this year) [4]. Editorial Board should be complimented. First place is within reach! In June 2013, the impact factors for the year 2012 were released (Thomson Reuters Journal Citation Reports® 2012, ISI Web of Knowledge). The impact factor is defined as the average number of citations received per paper divid- ed by the published articles in a specific journal during the preceding 2 years [1–3]. The 2012 impact factor of the Netherlands Heart Journal (NHJ) was 1.411. The 2012 NHJ impact factor was calculated as follows: in 2012, the total number of citations to articles published in the years 2010 and 2011 was 207. The number of articles published in 2010 and 2011 was 146. As a result, the 2012 impact factor was 206 citations divided by 144 articles: 206/146=1.411. The NHJ impact factor has therefore remained rather con- stant over the past 4 years: 1.392 in 2009, 1.447 in 2010, 1.438 in 2011, and 1.411 in 2012. Of course, we are striving to increase our impact factor over the forthcoming years (see the article by Tobias Opthof in the July/August edition of NHJ this year) [4]. Neth Heart J (2013) 21:377–378 DOI 10.1007/s12471-013-0455-2 Neth Heart J (2013) 21:377–378 DOI 10.1007/s12471-013-0455-2 EDITOR'S COMMENT Published online: 13 August 2013 # The Author(s) 2013. This article is published with open access at Springerlink.com References 1. Van der Wall. Increasing recognition of NHJ: a first-time impact factor of 1.4! Neth Heart J. 2010;18:399. 2. van der Wall EE, de Boer MJ, Doevendans PA, et al. Journal metrics for the Netherlands Heart Journal. Neth Heart J. 2011;19:159–61. 3. Opthof T, Wilde AA. Bibliometric data in clinical cardiology revisited. The case of 37 Dutch professors. Neth Heart J. 2011;19:246–55. Nallomothu and Lüscher recently suggested that medical journals should move from the concept of ‘impact’ to ‘influ- ence’ [12]. To that purpose, article-level metrics will prob- ably play a more prominent role in the near future. As opposed to the impact factor, article-level metrics provide a major step forward in evaluating the performance of individ- ual articles published in (cardiovascular) journals [13]. The Public Library of Science (PLoS) has been instrumental in developing several article-level metrics that integrate tradi- tional approaches such as the impact factor which measures scientific impact with overall interest and readership. Perti- nent questions are: 1) How often do others comment on articles and how do peers ‘rank’ these articles? 2) How many of the articles are being downloaded, and for how long? 3) How often are text, tables, and figures from the article ‘cut- and-pasted’? 4) How often are articles being selected by blogs, twitter, and lay media outlets? [12–15]. The concept of using article-level metrics, as a more objective and reliable way of measuring journal influence, may provide the true impact of a scientific journal. 4. Opthof T. The impact factor of the Netherlands Heart Journal in 2013. Neth Heart J. 2013;21:319–21. doi :10.1007/s12471-013- 0443-6. 5. van der Wall EE. Journal impact factor: holy grail? Neth Heart J. 2012;20:385–6. doi:10.1007/s12471-012-0317-3. 6. Alfonso F, Timmis A, Pinto FJ, et al. Conflict of interest policies and disclosure requirements among European Society of Cardiology national cardiovascular journals. Neth Heart J. 2012;20:279–87. 7. van der Wall EE. Conflicts of interest: call for new editorial policies in European national journals. Neth Heart J. 2012;20:249–50. doi:10.1007/s12471-012-0287-5. 8. van der Wall EE. The NHJ 2012 in retrospect: which articles are cited most? Neth Heart J. 2012;20:481–2. doi:10.1007/s12471- 012-0336-0. 9. McAlister FA, Lawson FME, Good AH, et al. Evaluating research in cardiovascular medicine: citation counts are not sufficient. Circulation. 2011;123:1038–43. 10. Lüscher TF. Good publishing practice. Eur Heart J. 2012;33:557– 61. doi:10.1093/eurheartj/ehr506. 11. Opthof T. Inflation of impact factors by journal self-citation in cardiovascular science. Neth Heart J. 2013;21:163–5. doi:10. Impact factor 2012 for cardiovascular journals: true impact? This can be viewed as a major accomplishment, especially when 378 Neth Heart J (2013) 21:377–378 objective bibliometric index, we remain dependent on the impact factor for a journal quality index. recognising that there are currently more than 40,000 scien- tific journals worldwide. Over time, impact factors have become the holy grail in the scientific journal domain [5]. Many authors wish to publish in the journals with the highest impact factors be- cause this will increase their scientific image, their profes- sional profile, and their academic career perspectives. As a result, every journal editor works hard to improve the journal’s impact factor because it is viewed by publishers as an index of journal quality and success, determining the extent to which the journal is resourced by its sponsoring organisation or publisher [6–8]. However, there are many confounders that may influence the impact factor, at least challenging the scientific significance of an impact factor [9–12]. 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. y 15. Priem J, Piwowar HA, Hemminger BM. Altmetrics in the wild: using social media to explore scholarly impact. 2012. arXiv:1203.4745. References 1007/s12471-013-0384-0. 12. Nallamothu BK, Lüscher TF. Moving from impact to influence: measurement and the changing role of medical journals. Eur Heart J. 2012;33:2892–96. In summary, the impact factor is still considered to be the nec plus ultra for authors, editors, publishers, and academic institutions. Although it is not very likely that a valid substi- tute for the impact factor will soon be implemented, the use of article-level metrics sounds noteworthy and hopeful. Until this approach has been accepted as a solid, reliable, and 13. Neylon C, Wu S. Article-level metrics and the evolution of scien- tific impact. PLoS Biol. 2009;7:e1000242. 14. Yarkoni T. Designing next-generation platforms for evaluating scientific output: what scientists can learn from the social web. SSRN eLibrary 2011. y 15. Priem J, Piwowar HA, Hemminger BM. Altmetrics in the wild: using social media to explore scholarly impact. 2012. arXiv:1203.4745.
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LC-MS identification and preparative HPLC isolation of<i>Frankenia pulverulenta</i>phenolics with antioxidant and neuroprotective capacities in PC12 cell line
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ABSTRACT Context: Frankenia pulverulenta L. (Frankeniaceae) is a medicinal species with carminative, analgesic and antiviral properties. However, phytochemical investigations, antioxidant and neuroprotective capacities of this plant remain unclear. p bjective: This work assesses the phenolic composition of F. pulverulenta shoot and root and evaluates heir antioxidant and neuroprotective capacities. KEYWORDS b-Amyloid peptide; LC-DAD- ESI-MS; fractionation; MTT test; ORAC p p Materials and methods: Successive fractionation of F. pulverulenta shoot and root using 6 solvents were used. Antioxidant capacity of these fractions was assessed through four in vitro tests (DPPH, ABTS, Fe-che- lating activity and ORAC). Phenolic identification, purification as well as neuroprotective activity of ethyl acetate (EtOAc) fraction and purified molecules were assessed. Results: Among the tested fractions, EtOAc shoot and root fractions possessed considerable phenolic con- tents (383 and 374 mg GAE/g E, respectively) because of their important ORAC (821 and 1054 mg of TE/g E), DPPH (586 and 750 mg of TE/g) and ABTS (1453 and 1319 mg of TE/g) results. Moreover, gallic acid, quercetin, quercetin galloyl glucoside, trigalloyl hexoside, procyanidin dimers and sulfated flavonoids were identified by LC-DAD-ESI-MS for the first time in this species. The relevant cytoprotective capacity (at 300 lg/mL) against b-amyloid peptide induced toxicity in PC12 cells of EtOAc fractions were corroborated with the chemical composition. In addition, purified molecules were tested for their ORAC and neuropro- tective activity. Quercetin showed the best ORAC value (33.55 mmol TE/g polyphenols); nevertheless, pro- cyanidin dimer exhibited an exceptionally efficient neuroprotective activity (100% of viability at 50 lg/mL). Discussion and conclusions: These findings suggest that this halophyte is a promising source of antioxi- dant and neuroprotective molecules for pharmaceutical purposes. CONTACT Rim Ben Mansour rim_ben_mansour@hotmail.fr Laboratory of Aromatic and Medicinal Plants, Center of Biotechnology, Technopark of Borj-Cedria (CBBC), BP 901, Hammam-Lif 2050, Tunisia  2017 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group. 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, distri- bution, and reproduction in any medium, provided the original work is properly cited. KEYWORDS b-Amyloid peptide; LC-DAD- ESI-MS; fractionation; MTT test; ORAC Laboratory of Aromatic and Medicinal Plants, Center of Biotechnology, Technopark of Borj-Cedria LC-MS identification and preparative HPLC isolation of Frankenia pulverulenta phenolics with antioxidant and neuroprotective capacities in PC12 cell line Rim Ben Mansoura, Megdiche Ksouri Wideda, Stephanie Cluzetb, Stephanie Krisab, Tristan Richardb and Riadh Ksouria aLaboratory of Aromatic and Medicinal Plants, Center of Biotechnology, Technopark of Borj-Cedria (CBBC), Ha EA3675, University of Bordeaux, Villenave d‘Ornon, France edicinal Plants, Center of Biotechnology, Technopark of Borj-Cedria (CBBC), Hammam-Lif, Tunisia; bISVV, GESVAB , Villenave d‘Ornon, France aLaboratory of Aromatic and Medicinal Plants, Center of Biotechnology, Technopark of Borj-Cedria (CBBC), Hammam-Lif, Tunisia; bISVV, GESVAB EA3675, University of Bordeaux, Villenave d‘Ornon, France ARTICLE HISTORY Received 15 March 2016 Revised 23 December 2016 Accepted 29 December 2016 ARTICLE HISTORY Received 15 March 2016 Revised 23 December 2016 Accepted 29 December 2016 PHARMACEUTICAL BIOLOGY, 2017 VOL. 55, NO. 1, 880–887 http://dx.doi.org/10.1080/13880209.2016.1278452 PHARMACEUTICAL BIOLOGY, 2017 VOL. 55, NO. 1, 880–887 http://dx.doi.org/10.1080/13880209.2016.1278452 PHARMACEUTICAL BIOLOGY, 2017 VOL. 55, NO. 1, 880–887 http://dx.doi.org/10.1080/13880209.2016.1278452 ORAC assay The procedure was modified from the method described by Ou et al. (2001), using Trolox as a control standard. The ORAC assay was carried out in black round-bottom 96-well microplates (Costar) and absorbance was measured with an automated plate reader (Fluostar Optima; BMG Labtech). All the samples (extracts, pure polyphenols, fluorescein and AAPH) were diluted in 75 mM phosphate buffer (pH 7.4). Thirty microliters of extracts (1 mg/mL) or phosphate buffer (blank) were mixed with 180 lL of fluorescein solution (117 nM final concentration) and incubated for 5 min at 37 C. A volume of 90 lL of AAPH solu- tion (40 mM final concentration) were added and fluorescence was immediately monitored using 485 nm excitation and 520 nm emission wavelengths at 1 min intervals for 70 min. The antioxi- dant capacities of extracts or purified molecules were expressed as mg of Trolox equivalent per g of extract (mg TE/g) or mmol equivalent Trolox per g of polyphenols (mmol TE/g P), respect- ively. All samples were analyzed in quadruplicate and at least in three different experiments. Plant material and extraction Frankenia pulverulenta was collected during the vegetative stage in March 2014 from Borj-Cedria (latitude: 36460N, longitude: 10390E 16 m elevation) at 30 km to Tunis. This halophyte was identified at the Biotechnology Center by Pr Abderrazak Smaoui (CBBC, Technopark of Borj-Cedria), and a voucher specimen [PLM51] was deposited at the Herbarium of the Laboratory of Medicinal and Aromatic Plants at the CBBC. Shoot and roots were air dried. Sample extracts were obtained by magnetic stir- ring of 150 g of dry matter powder with 1500 mL methanol 80% for 2 h, then the filtrate is evaporated using a rotary evaporator. The obtained filtrate is first extracted with hexane followed by dichloromethane, EtOAc and finally with butanol. The different phases are separated by a separator funnel. Introduction research interest focus on the study of antioxidant molecules that are able to attenuate the damaging effects of Reactive Oxygen Species (ROS). Excess of ROS in the body can lead to cumulative damage in proteins, lipids and DNA, resulting in the so-called oxidative stress (Yoshikazu & Yuji 2002). Among cellular struc- ture, neurons are particularly vulnerable to ROS, and oxidative stress is one of the main causative factors in the etiology of a number of late onset disorders (Esposito et al. 2006; Tellone et al. 2015). Oxidative stress, which is one of the specific hall- marks of Alzheimer’s disease (AD), promotes neurofibrillary degeneration and death of neurons (Abramov et al. 2004; Lin & Beal 2006). Yatin et al. (1999) reported that the involvement of free radicals in AD includes the presence of elevated levels of protein oxidation, lipid peroxidation products and oxidative damage to mitochondria in AD brains. Furthermore, neurotox- icity of amyloid peptides occurs in conjunction with the presence of oxidative stress associated with the peptide, and excessive accumulation of Ab peptide in neurons triggers progressive neur- onal degeneration (Richard et al. 2011). Implication of Ab25–35 induceing apoptosis in multiple cell types in vitro and in vivo in human AD brains was suggested by many other reports (Lin & Nowadays, herbal constituents, especially phenolics, have been received increasing interest owing to their reported beneficial effects on longevity and disease prevention (Lin et al. 2015). The identification of new sources of natural antioxidants is a promis- ing alternative for their use and value in the food industry and in preventive medicine to replace synthetic antioxidants (Tadhani et al. 2007). This is mainly due to their strong biological activity, exceeding those of many synthetic antioxidants that have possible activity as promoters of carcinogenesis (Ksouri et al. 2009). Comprehensive identification of phenolic compounds in food matrices is a crucial starting point for assessing their biological, nutritional and technological properties (Gasperotti et al. 2010). This kind of compounds acts as antioxidants, flavor precursors and as ingredient of our diet, and they have been associated with several health-promoting activities such as decreasing blood sugar levels, reducing body weight, anticarcinogenic, anti-inflammatory, antiaging and antithrombotic capacities (Senevirathne et al. 2006). These activities depend on their structure, in particular the number and positions of the hydroxyl groups and the nature of substitutions on the aromatic rings. Introduction Much of the current PHARMACEUTICAL BIOLOGY 881 Beal 2006; Bolea et al. 2013; El Bitar et al. 2014; Clarke et al. 2015). Ab25–35 caused a significant decrease in viability with a concomitant increase in apoptosis and morphological abnormal- ities of nuclei in PC12 cells (Lin & Beal 2006). 96-well plate. Briefly, 100 lL of Folin-Ciocalteu’s reagent was added to 20 lL of extract (1 mg/mL). After 5 min, 80 lL of sodium carbonate (75 g/L) solution were added. After 1 h, the absorbance was measured at 765 nm. The TPC was expressed as mg gallic acid equivalent per g of extract (mg GAE/g E). Experiments were analyzed at least three times and with triplicate samples. There is an international effort to understand the biology of AD to develop primary and secondary prevention strategies, and to develop effective therapeutic interventions for individuals who are already symptomatic (Boada et al. 2014). Hence, the balance between antioxidation and oxidation is believed to be a critical concept for maintaining a healthy biological system (Finkel & Holbrook 2000). Therefore, the search of antioxidants from natural sources has received much attention, and efforts have been made to identify new natural resources for active antioxidant com- pounds. In Tunisia, a considerable diversity of medicinal halo- phytes species with multiple interests including therapeutic practices occurs, and many have not been subject to chemical investigations. For example, Frankenia pulverulenta L. (Frankeniaceae) is an endemic species from North Africa (Ozenda 1991), growing in the high plateaus and in salt and arid regions of Tunisia. The principal use of this species in local medicine is based on the oral administration of a decoction and gargle prepared from whole plant for its analgesic and carminative properties (Youssef 2013). Phytochemical studies on the genus Frankenia and the information on the chemical composition of F. pulverulenta are still scarce. Moreover, it was reported that F. pulverulenta extract is active against Herpes simplex virus type 1 (Sassi et al. 2008). Previous studies focused on the phytochemical identification of flavonoids and phenolic sodium sulfates from F. thymifolia Desf. (Harkat et al. 2007), F. pulverulenta L. (Harborne 1975), F. laevis L. (Hussein 2004) and F. thymifolia (Megdiche-Ksouri et al. 2011). Radical scavenging assays Radical scavenging ability against DPPH radical was measured as described by Blois (1958). A volume of 50 lL of each samples (1 mg/mL) were mixed with 150 lL of 200 lM methanolic solu- tion of DPPH in a 96-well plate. The plate was allowed to stand at room temperature in dark for 20 min. The absorbance was measured at 520 nm. Results were expressed as mg of Trolox equivalent per g of extract (mg TE/g). The scavenging activity of the extracts on ABTS radical cation was estimated according to the method of Re et al. (1999). Briefly, 250 lL of the diluted ABTSþsolution were added to 10 lL of extracts at the concentration of 1 mg/mL (or Trolox). Six min after initial mixing, the absorbance was measured at 734 nm at 30 C. Results were expressed as mg of Trolox equiva- lent per g of extract (mg TE/g). All samples were analyzed in triplicate in at least three different experiments. Metal chelating activity (MCA) The chelating activity of the extracts for ferrous ions Feþ2 was measured according to the method of Dinis et al. (1994). A vol- ume of 80 lL of deionized water and 40 lL of FeSO4 (0.2 mM) were added to the extract (40 lL, 1 mg/mL) and mixed o a 96- well microplate. The reaction was initiated by the addition of 40 lL of ferrozine (2 mM). After 10 min, at room temperature, the absorbance of the Fe2þ-ferrozine complex was measured at 562 nm. EDTA was used as standard and results were expressed as mg EDTA per gram of extract (mg EDTA/g). All samples were analyzed in triplicate in at least three different experiments. This study describes the optimization of shoot and root frac- tionation of F. pulverulenta using solvents with increasing polar- ity. This investigation was assessed through different antioxidant activities, such as DPPH, ABTS, metal chelating activity and ORAC tests. In addition, separation and identification of phyto- chemical composition of EtOAc fractions by LC-DAD-ESI-MS and preparative HPLC as well as the neuroprotective activity on PC12 cell line of acetate fraction and purified molecules were assessed. Total phenolic contents Cell culture and MTT assay of EtOAc fractions Cell culture and MTT assay of EtOAc fractions The total phenolic content (TPC) in aerial parts and roots of extract and fractions were determined by the Folin–Ciocalteu colorimetric method (Singleton & Rossi 1965) adapted to a Pheochromocytoma-derived PC12 cells (ATCC, Manassas, VA) were maintained routinely in DMEM-Glutamax supplemented R. BEN MANSOUR ET AL. 882 The solvents used are mixture of acetonitrile (ACN) and acidified water, the elution of phenolic compounds occurs more polar to less polar, driven by a mobile phase increasingly non polar. A stationary phase C18 reverse phase column and mobile phase solvent mixture in gradient fashion were used: solvent A (H2O/ 0.1% TFA) and solvent B (ACN/0.1% TFA). Throughout the chromatography, the concentration of trifluoroacetic acid (TFA) remains constant and provides good resolution of peaks. The gradient is according to the following order: 99–1% B (0–4 min), 99–1% B (4–12.8 min), 90% B (12.8–13 min), 10–100% B (13–55 min), 100% B (55–59 min). Injections of 3 mL/min to sol- utions of about 100 mg dry extract were dissolved in 1 mL of 50% MeOH. The solvents used are mixture of acetonitrile (ACN) and acidified water, the elution of phenolic compounds occurs more polar to less polar, driven by a mobile phase increasingly non polar. A stationary phase C18 reverse phase column and mobile phase solvent mixture in gradient fashion were used: solvent A (H2O/ 0.1% TFA) and solvent B (ACN/0.1% TFA). Throughout the chromatography, the concentration of trifluoroacetic acid (TFA) remains constant and provides good resolution of peaks. The gradient is according to the following order: 99–1% B (0–4 min), 99–1% B (4–12.8 min), 90% B (12.8–13 min), 10–100% B (13–55 min), 100% B (55–59 min). Injections of 3 mL/min to sol- utions of about 100 mg dry extract were dissolved in 1 mL of 50% MeOH. with 15% horse serum, 2.5% fetal bovine serum and 1% penicil- lin/streptomycin antibiotics at 37 C in a humidified atmosphere of 5% CO2/50% air. Cells were plated at a density of 30,000 cells per well on 96-well plates and incubated at 37 C for 24 h. Then, the cells were treated with 5 lM of Ab(25  35), with or without extracts (25, 50, 100, 200 and 300 lM) or pure molecules (10, 25, and 50 lM) in a serum-free culture medium. After 24 h of incu- bation, cell viability was determined by the conventional MTT reduction assay. Cell culture and MTT assay of EtOAc fractions Cells were treated with MTT solution (0.5 mg/ mL) for 3 h at 37 C. The dark blue formazan crystals formed in viable cells were solubilized with DMSO for 0.5 h. The absorb- ance was measured at 595 nm with a microplate reader (Dynex, Carnegie, PA). Results were expressed as the percentage of MTT reduction in relation to the absorbance of control cells at 100%. All data represent the average of four tests. Statistical analysis All samples were analyzed at least in triplicates. Data are expressed as means ± standard error mean (SEM). Differences were evaluated by one-way analysis of variance (ANOVA) com- pleted by Tukey’s test. Differences were considered statistically significant at p < 0.05. Alternatively, the results were analyzed by GraphPad Prism 5.03 for Windows (GraphPad Software, San Diego, CA). Phenolic content of F. pulverulenta The obtained fractions (crude extract, hexane, dichloromethane, ethyl acetate, butanol and water) from F. pulverulenta shoot and root were analyzed for their total phenolic contents (TPC). Results depicted in Table 1 show that the highest recovery of TPC was observed for extraction with EtOAc reaching up to 383 and 374 mg GAE/g in aerial part and roots, respectively. In both plant parts, the TPC was found to be in the order EtOAc > BuOH > methanol > water > dichloromethane > hexane, suggesting that polar solvents were more efficient in extracting phenolics from F. pulverulenta. However, given the low polarity of EtOAc (polarity index 4.4) when compared with butanol, methanol (5.1) or water (polarity index 9), it seems logical to suppose that the highest recovery of TPC by using EtOAc was presumably due to its high molecular weight (88 g/mol) which enables it to easily extract about the same molecular weight fol- lowing the concept ‘like dissolve like’. At this point, it can be expected that most of the phenolic compounds were of catechin- or epicatechin-type compounds. Support to this assumption is given by Row and Jin (2006) who reported that catechin-type phenolics are better extracted with EtOAc. Liquid chromatography-mass spectrometry Lyophilized EtOAc fractions were dissolved in 50% methanol and chromatographed using an HPLC-MS system. The chroma- tography apparatus was an Agilent 1200 from Agilent Technologies (Santa Clara, CA). The EtOAc fractions were ana- lyzed at 25 C with a 250  4 mm i.d., 5 lm, Prontosil 120-5-C18- AQ reverse phase column, Bischoff (Leonberg, Germany). Water, 0.1% HCOOH (solvent A) and acetonitrile 0.1% HCOOH (solv- ent B) were used as mobile phases. The gradient elution program was as follows (v/v): 0 min 1% B, 0.4 min 1% B, 2 min 10% B, 6 min 35% B, 7 min 50% B, 8.8 min 70% B, 10.8 min 92% B, 11 min 100% B and 12 min 100% B, followed by 10 min for re- equilibration. The optimum values of the ESI-MS parameters were: capillary voltage – 4.7 kv; drying gaz temperature 350 C; drying gas flow 10 L/min; nebulizing gas pressure 35 psi. LC/MS accurate mass spectra were recorded across the range 150–2000 m/z. The detection wavelengths were set at 280 and 360 nm. LC- ESI-MS analyses were carried out in the negative ion mode. This HPLC was coupled to an Esquire 3000 þ ion trap mass spectrom- eter using an ESI source from Bruker Daltonics (Billerica, MA). LC-DAD-ESI-MS screening Compound 6 was identified as trigalloyl hexoside (Rt 4.5 min) with precursor ions at m/z 483, corresponding to the loss of galloyl at m/z 465 (loss of gallic acid at m/z 170) (Abu-Reidah et al. 2015). The metal chelating activities of F. pulverulenta fractions were monitored in order to evaluate the ability to inhibit interactions between metals and lipids. Water fraction exhibited the strongest chelating capacity as compared with the other fractions of aerial parts (62 mg EDTA/g) (Table 1). In roots, EtOAc and butanol fractions had the most important chelating activity (23 mg EDTA/g). In addition, shoot iron chelating activities were signifi- cantly higher than the root fraction. The results were in agree- ment with Dhingra et al. (2014) on the Prunus persica fruits, these authors showed that EtOAc and n-butanol fractions possess the higher Fe2þ-chelating activity. Compound 7 (Rt 4.8 mn; kmax ¼ 275) is proposed as quercetin galloyl glucoside, yielded a prominent fragment ion at m/z 301, [M–290–H], assigned to interflavanic bond cleavage following the quinone-methide mechanism (quercetin), m/z 255 [M  H2O- pentose moiety], m/z 439 [M–152–H] corresponding to the loss of the galloyl fragment (C8H8O3) from retro-Diels-Alder (RDA) cleavage; m/z 465 [M–126–H], to the loss of the fragment (C6H4O2) from RDA (Sannomiya et al. 2005). Compound 10 at m/z 301 corresponds to quercetin and com- pound 11 had the [M  H] ion at m/z 477, which yielded the fragment ions at m/z 301 ([M  H-162] corresponding to the loss of one galactose or glucose unit), 179 and 151, identified as quercetin hexoside (Kabran et al. 2014). g g y Due to the high TPC and the important antioxidant capacities of EtOAc fraction, the identification of the main bioactive Table 2. Phenolic compounds detected in ethyl acetate fraction by LC-DAD-ESI-MS from aerial parts and roots of F. pulverulenta in negative mode. LC-DAD-ESI-MS screening Phenolic composition analysis of F. pulverulenta EtOAc shoot and root fractions has been carried out using LC-DAD-ESI-MS at a negative mode (Table 2). For the first time, the method- ology used in this work, allowed us to identify nine new phen- olic compounds (1, 2, 3, 4, 6, 7, 8, 10 and 11) which have not been reported so far in this species (Figure 1). The com- pounds detected were characterized by means of MS data, together with the interpretation of the observed MS-MS spectra in comparison with those found in the literature public databases. Measuring the ability of oxygen radical absorbance (ORAC) was widely used in the field of antioxidants and oxidative stress. The antioxidative potential of different fractions evaluated by ORAC indicated that EtOAc fraction is the most active (821 and 1054 mg TE/g for aerial parts and root, respectively). But, hexane fractions showed very low peroxyl radical scavenging activity, which was around 20 times lesser than EtOAc fraction (Table 1). In this context, Surget et al. (2015) observed that EtOAc fraction of the halophyte Salicornia amosissima showed high ORAC activ- ity. It appears that the ORAC values in the present study were comparable to or even higher than those reported by Silva et al. (2007), where found ORAC values ranged from 6.7 to 1396.4 lmol of Trolox equiv/g in 15 medicinal Amazonian plants. These results support previous studies on antioxidant tests, since EtOAc fractions usually present the highest TPC and antioxidant capacities (Dhingra et al. 2014; Saada et al. 2014; Surget et al. 2015). Compound 1 at m/z 169 corresponds to gallic acid. It was identified with fragmentation ion at m/z 125 ([M  H-44] cor- responding to the loss of carboxylate). Analysis revealed the pres- ence of three isomers of procyanidin dimers (compounds 2, 4 and 8) in the ESI- mode with ions at m/z 577. It was identified with the same fragmentation ions at m/z 451, 425 (corresponding to the neutral losses of galloyl m/z 152), 407 (loss of gallic acid m/z 170) and 289 (Kabran et al. 2014). Compound 3 (Rt 4 min) with the precursor ion at m/z 289 was assigned as catechin. MS/MS spectral data showed product ions at m/z 245, 179 and 205 (Kabran et al. 2014). Antioxidant activity of shoot and root fractions compounds by LC-DAD-ESI-MS and the purification by prepara- tive HPLC of major compounds of EtOAc shoot fraction were performed. The protective effect of these fractions and purified compounds against Ab-induced toxicity in the PC12 cell line was further investigated. Results depicted that EtOAc and BuOH fractions of F. pulveru- lenta exhibited the higher scavenging effects (Table 1). Moreover, root fraction exhibits better performance than the shoot fraction against DPPH and ABTS radicals. These results suggest that anti- oxidative phytochemicals are abundantly present in F. pulveru- lenta, particularly in EtOAc and BuOH fractions. These data are in agreement with the previous study of Saada et al. (2014), which showed that EtOAc fraction of the halophyte Retama rae- tam (Fabaceae) exhibits the highest total phenolic compounds and antioxidant activity. Desire et al. (2010) also found that the EtOAc fraction of Mascarenhasia arborescens had better antiradi- cal capacity than the other fractions like hexane, water and methanol. Accordingly, the superiority of the EtOAc fraction is probably associated to its polarity which allows the accumulation of a variety of antioxidant compounds. LC-DAD-ESI-MS screening Preparative HPLC Preparative HPLC In order to increase our production capacity of pure compounds, we used preparative HPLC Varian chain consisting of two pumps (model 218 with heads of 50 mL min1 maximum flow rate) and a dual wavelength detector UV-visible wave (model 325). The flow rate was 18 mL min1 with a C18 reverse phase column Bischoff Ultrasep Eurobond (5 lm, 20 mm diameter 250 mm) protected by a C18 guard column (20 mm diameter 50 mm). Table 1. Total phenolic content (TPC) and antioxidant capacities of F. pulverulenta aerial part and roots fractions. Extract/fraction TPC (mg of GAE/g) DPPH (mg of TE/g) ABTS (mg of TE/g) ORAC (mg of TE/g) MCA (mg of EDTA/g) Aerial part Crude extract 286 ± 10c 378 ± 42c 216 ± 33d 495 ± 43c 44 ± 3b Hexane fraction 71 ± 5f 88 ± 17e 165 ± 27d 196 ± 30e 25 ± 2d Dichloromethane 115 ± 7e 77 ± 10e 220 ± 22d 617 ± 58b 21 ± 2d Ethyl acetate 383 ± 10a 586 ± 31b 1453 ± 63a 821 ± 34a 37 ± 6c Butanol 319 ± 6b 698 ± 73a 1110 ± 67b 496 ± 38c 44 ± 4b Water 210 ± 9d 193 ± 51d 626 ± 35c 374 ± 51d 62 ± 3a Root Crude extract 202 ± 13c 540 ± 50b 230 ± 45d 398 ± 33d 15 ± 1b Hexane fraction 25 ± 3f 30 ± 10d 156 ± 10d 52 ± 17f 12 ± 1c Dichloromethane 105 ± 8e 45 ± 10d 230 ± 25d 501 ± 50c 12 ± 1c Ethyl acetate 374 ± 15a 750 ± 11a 1319 ± 76a 1054 ± 54a 23 ± 2a Butanol 289 ± 7b 529 ± 116b 1242 ± 54b 646 ± 32b 23 ± 2a Water 182 ± 6d 204 ± 66c 754 ± 41c 311 ± 23e 15 ± 1b a–eSignificant difference at p < 0.05 by Tukey’s test. 883 PHARMACEUTICAL BIOLOGY LC-DAD-ESI-MS screening Compound No Rt (min) kmax(nm) [M  H] Fragments Organ Compound 1 2.3 270-275 169 125 AP, R Gallic acid 2 3.6 260 577 407-425-451-289 R Procyanidin dimer 1 3 4 280 289 245-179-205 AP Catechin 4 4.3 275 577 407-425-451-289 AP Procyanidin dimer 2 5 4.3 275 551 291-352-415-465-529 R ND 6 4.5 275 635 483-465 AP, R Tri-galloyl hexoside 7 4.8 275 591 301-255-359-407-439-465-487-529 AP Quercetin galloyl glucoside 8 5.1 270 577 407-425-451-289 AP Procyanidin dimer 3 9 5.1 280 619 245-289-407-425 R ND 10 5.3 280/370 301 301 R Quercetin 11 5.3 285/360 477 301-151-179 AP Quercetin hexoside 12 5.5 290/320 435 168-315-345 R ND 13 6.3 280/365 551 343-491 R ND 14 6.7 290/320 685 365-458-488-514-541-617-649-667 R ND 15 7.1 290/360 423 343-80 AP, R Flavonoid-sulfated isomer 16 7.2 290/310 423 343-80 AP Flavonoid-sulfated isomer cted in ethyl acetate fraction by LC-DAD-ESI-MS from aerial parts and roots of F. pulverulenta in negative mode. Table 2. Phenolic compounds detected in ethyl acetate fraction by LC-DAD-ESI-MS from aerial parts and roots of F. pulver 884 R. BEN MANSOUR ET AL. Figure 1. LC-DAD-ESI-MS base peak chromatograms in negative ion mode and UV at 280nm for the ethyl acetate fraction of aerial parts and roots of F. pulverulenta. Figure 1. LC-DAD-ESI-MS base peak chromatograms in negative ion mode and UV at 280nm for the ethyl acetate fraction of aerial parts and roots of F. pulverulenta. gure 1. LC-DAD-ESI-MS base peak chromatograms in negative ion mode and UV at 280nm for the ethyl acetate fraction of ae Seven compounds (5, 9, 12, 13, 14, 15 and 16) eluting respectively at 4.3, 5.1, 5.5, 6.3, 6.7, 7.1 and 7.2 remain unidenti- fied. No data corresponding to their mass spectra in the literature and databases are reported. Among them, compounds 15 and 16 matched exactly with the accurate mass at m/z 423 and same fragment ions m/z 343 and m/z 80 corresponding to a loss of one sulfate and UV-Vis spectrum shape with a kmax at 290 and 360 nm attributed to two isomer sulfated flavonoids. Isolation and NMR identification might be required to identify these compounds. 0 20 40 60 80 100 F. LC-DAD-ESI-MS screening pulverulenta Cell viability (%) Control Aerial parts Roots Aβ (µM) 0 5 5 5 5 5 5 5 5 5 5 5 Extracts (µM) 0 0 25 50 100 200 300 25 50 100 200 300 Extracts (µM) 0 25 50 100 200 300 25 50 100 200 300 Aβ(µM) 0 0 0 0 0 0 0 0 0 0 0 Cell viability (%) 0 20 40 60 80 100 120 (a) (b) Figure 2. Cytotoxic activity (a) and neuroprotective activity on Ab-induced tox- icity in PC12 cell line (b) of aerial part and root EtOAc fraction. The experiment was repeated three times. 0 20 40 60 80 100 F. pulverulenta Cell viability (%) Control Aerial parts Roots Aβ (µM) 0 5 5 5 5 5 5 5 5 5 5 5 Extracts (µM) 0 0 25 50 100 200 300 25 50 100 200 300 (b) Neuroprotective activity of EtOAc fractions The cytotoxic potential of each phenolic fraction on PC12 cells was measured with the MTT assay. EtOAc fractions (F. pulveru- lenta shoot and root) were not cytotoxic to concentration up to 300 lg/mL (Figure 2(a)). As shown in Figure 2(b), treatment of PC12 cells with 5 lM Ab25–35 reduced cell viability about 40% of control. Induction of cytotoxicity by Ab25–35 at 5 lM was then used for all subsequent experiments to evaluate the protective effect of the species. Both EtOAc fractions from shoot and root exhibited cytoprotective activities against Ab-induced toxicity. The extracts increased cell viability in a dose-dependent manner. Incubation of PC12 cells with 100 lg/mL of fractions significantly prevented the cytotoxic effect of Ab25–35 at levels close to 57%. Ab-induced cytotoxicity was prevented at levels close to 80% at 200 lg/mL. The root fraction at the highest concentration tested (300 lg/mL) completely reversed the toxic effect of Ab25–35. This cytoprotective activity is mainly correlated to the nature of phen- olic compounds in these fractions. In this context, several reports have demonstrated that Lotus seedpod proanthocyanidins act as anti-aging agents for protection against memory deficits (Xiao et al. 2015). Figure 2. Cytotoxic activity (a) and neuroprotective activity on Ab-induced tox- icity in PC12 cell line (b) of aerial part and root EtOAc fraction. The experiment was repeated three times. ORAC assay and neuroprotective effect of purified compounds Pure molecule ORAC (mmol TE/g polyphenols) Gallic acid 14.176 Catechin 21.917 Procyanidin dimer 4.71 Trigalloyl hexoside 2.99 Quercetin galloyl glucoside 1.2 Flavonoid sulphate ND Quercetin 33.558 The results are expressed in mmol Trolox equivalent/g polyphenols. Table 3. Antioxidant capacity (ORAC) of purified molecules of the ethyl acetate fraction. Pure molecule ORAC (mmol TE/g polyphenols) Gallic acid 14.176 Catechin 21.917 Procyanidin dimer 4.71 Trigalloyl hexoside 2.99 Quercetin galloyl glucoside 1.2 Flavonoid sulphate ND Quercetin 33.558 The results are expressed in mmol Trolox equivalent/g polyphenols. Table 3. Antioxidant capacity (ORAC) of purified molecules of the ethyl acetate fraction. (a) (b) 0 10 20 30 40 50 60 70 80 90 100 Cell viability (%) Control Quercetin AG Catechin P. dimer TGH QGG FS Molecule (µM) 0 0 10 25 50 10 25 50 10 25 50 10 25 50 10 25 50 10 25 50 10 25 50 βA (µM) 0 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 Molecule (µM) 0 10 25 50 10 25 50 10 25 50 10 25 50 10 25 50 10 25 50 10 25 50 βA (µM) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 20 40 60 80 100 120 140 160 Cell viability (%) Control Quercetin AG Catechin P. dim er TGH QGG FS 3. Cytotoxic activity (a) and neuroprotective activity on Ab-induced toxicity in PC12 cell line (b) of purified molecules of the ethyl acetate fraction. GA: dimer: procyanidin dimer; TGH: trigalloyl hexoside; QGG: quercetin galloyl glucoside; FS: flavonoid sulfated. (a) Molecule (µM) 0 10 25 50 10 25 50 10 25 50 10 25 50 10 25 50 10 25 50 10 25 50 βA (µM) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 20 40 60 80 100 120 140 160 Cell viability (%) Control Quercetin AG Catechin P. dim er TGH QGG FS (b) 0 10 20 30 40 50 60 70 80 90 100 Cell viability (%) Control Quercetin AG Catechin P. ORAC assay and neuroprotective effect of purified compounds With regard to the discussion on phytochemicals responsible for antioxidant and cytoprotective capacities, it could be assumed that these properties were attributed to high content of phenolics such as gallic acid, quercetin, quercetin galloyl glucoside, PHARMACEUTICAL BIOLOGY 885 maximum concentration of 50 lM (Figure 3(a)). Only quercitin (at 50 lM) and trigalloyl hexoside (at 25 and 50 lM) were cyto- toxic on PC12 cells. Major purified phenolics prevent the aggre- gation of Ab (Figure 3(b)). Among them, procyanidin dimer exhibited an extremely efficient inhibition of Ab-induced cell death in PC12 cells, followed by flavonoid sulfate, in a dose- dependent manner. Ab-induced cytotoxicity was prevented at levels close to 100% and 85%, respectively, at 50 lM. Trigalloyl hexoside prevented also significantly the cytotoxic effect of Ab25–35 to 86% at the lowest concentration (10 lM) and decreased at higher concentrations to 60% (50 lM). This decrease is due to the cytotoxic effect of trigalloyl hexoside up to 10 lM on PC12 cells. Also, quercetin, quercetin galloyl glucoside and catechin are not efficient neuroprotective agents. In fact, one of the major properties of polyphenols is the important interaction with peptides and proteins, particularly in the AD (Henry-Vitrac et al. 2010). Several studies indicate that polyphenols present in high amounts in natural products could play a preventive role in the incidence of age-related neurological disorders (Basli et al. 2004). These findings have been supported by epidemiological studies and confirmed by in vitro and in vivo studies (Ono et al. 2003; Bastianetto et al. 2006). The ability of EtOAc fractionation in phenolics can explain their capacity to reduce the cell death caused by Ab. catechin, trigalloyl hexoside and procyanidine dimers. Thus, the newly isolated phenolics from F. pulverulenta EtOAc fractions were tested for their ORAC and neuroprotective activities. Results presented in Table 3 show that quercetin had the highest ORAC values (33.558 mmol TE/g polyphenols), followed by cat- echin (21.917 mmol TE/g polyphenols) and gallic acid (14.176 mmol TE/g polyphenols). Trigalloyl hexoside and quer- cetin galloyl glucoside showed very low peroxyl radical scaveng- ing activity, which was around 11- and 28-fold weaker than quercetin. Moreover, this study displayed quercetin ORAC values comparable withthat reported by Davalos et al. (2004). p p y Concerning MTT test, like EtOAc fractions, main purified molecules did not significantly affect cell viability, with the Table 3. Antioxidant capacity (ORAC) of purified molecules of the ethyl acetate fraction. References Lin MT, Beal MF. 2006. Review article mitochondrial dysfunction and oxida- tive stress in neurodegenerative diseases. Nature. 443:787–795. Abramov AY, Canevari L, Duchen MR. 2004. Beta-amyloid peptides induce mitochondrial dysfunction and oxidative stress in astrocytes and death of neurons through activation of NADPH oxidase. J Neurosci. 24:565–575. Lin Z, Zheng LC, Zhang H-J, Wai Tsang S, Bian Z-X. 2015. Anti-fibrotic effects of phenolic compounds on pancreatic stellate cells. BMC Complement Altern Med. 15:259. Abu-Reidah I, Ali-Shtayeh MS, Jamous RM, Arraez-Roman D, Segura- Carretero A. 2015. HPLC-DAD-ESI-MS/MS screening of bioactive compo- nents from Rhus coriaria L. (Sumac) fruits. Food Chem. 166:179–191. 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Identification de composes phenoliques extraits de deux plantes de la pharmacopee ivoirienne. J Soc Ouest-Afr Chim. 38:57–63. This work was supported by the Tunisian Ministry of Higher Education, Scientific Research and Information and Communication Technologies (LR15CBBC06). Ksouri R, Falleh H, Megdiche W, Trabelsi N, Mhamdi B, Chaieb K, Bakrouf A, Magne C, Abdelly C. 2009. Antioxidant and antimicrobial activities of the edible medicinal halophyte Tamarix gallica L. and related polyphenolic constituents. Food Chem Toxicol. 47:2083–2091. Disclosure statement Henry-Vitrac C, Berbille H, Merillon JM, Vitrac X. 2010. Soy isoflavones as potential inhibitors of Alzheimer-amyloid fibril aggregation in vitro. Food Res Int. 43:2176–2178. The authors declare that they have no conflict of interest. Hussein SAM. 2004. Phenolic sodium sulphates of Frankenia laevis L. Pharmazie. 59:304. ORAC assay and neuroprotective effect of purified compounds dimer TGH QGG FS Molecule (µM) 0 0 10 25 50 10 25 50 10 25 50 10 25 50 10 25 50 10 25 50 10 25 50 βA (µM) 0 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 ( ) d b d d ll l (b) f f d l l f h h l f Figure 3. Cytotoxic activity (a) and neuroprotective activity on Ab-induced toxicity in PC12 cell line (b) of purified molecules of the ethyl acetate fraction. GA: gallic acid; P dimer: procyanidin dimer; TGH: trigalloyl hexoside; QGG: quercetin galloyl glucoside; FS: flavonoid sulfated. References Re R, Pellegrini N, Proteggente A, Pannala A, Yang M, Rice-Evans C. 1999. Antioxidant activity applying an improved ABTS radical cation decoloriza- tion assay. Free Radic Biol Med. 26:1231–1237. Richard T, Poupard P, Nassra M, Papastamoulis Y, Iglesias ML, Krisa S, Waffo-Teguo P, Merillon JM, Monti JP. 2011. Protective effect of e-vini- ferin on b-amyloid peptide aggregation investigated by electrospray ioniza- tion mass spectrometry. 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Structural heterogeneity of cellular K5/K14 filaments as revealed by cryo-electron microscopy
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Zurich Open Repository and Archive Zurich Open Repository and Archive University of Zurich University Library Strickhofstrasse 39 CH-8057 Zurich www.zora.uzh.ch Year: 2021 uctural heterogeneity of cellular K5/K14 filaments as revealed by cryo-electron microscopy Weber, Miriam S ; Eibauer, Matthias ; Sivagurunathan, Suganya ; Magin, Thomas M ; Goldman, Robert D Medalia, Ohad DOI: https://doi.org/10.7554/eLife.70307 DOI: https://doi.org/10.7554/eLife.70307 Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-210310 Journal Article Published Version The following work is licensed under a Creative Commons: Attribution 4.0 International (CC BY 4.0) License. Originally published at: Weber, Miriam S; Eibauer, Matthias; Sivagurunathan, Suganya; Magin, Thomas M; Goldman, Robert D; Medalia, Ohad (2021). Structural heterogeneity of cellular K5/K14 filaments as revealed by cryo-electron microscopy. eLife, 10:e70307. DOI: https://doi.org/10.7554/eLife.70307 Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-210310 Journal Article Published Version The following work is licensed under a Creative Commons: Attribution 4.0 Int Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-210310 Journal Article Published Version ollowing work is licensed under a Creative Commons: Attribution 4.0 International (CC BY 4.0) License. Originally published at: Weber, Miriam S; Eibauer, Matthias; Sivagurunathan, Suganya; Magin, Thomas M; Goldman, Robert D; Medalia, Ohad (2021). Structural heterogeneity of cellular K5/K14 filaments as revealed by cryo-electron microscopy. eLife, 10:e70307.t RESEARCH ARTICLE Miriam S Weber1, Matthias Eibauer1, Suganya Sivagurunathan2, Thomas M Magin3, Robert D Goldman2, Ohad Medalia1* 1Department of Biochemistry, University of Zurich, Zurich, Switzerland; 2Department of Cell and Developmental Biology, Northwestern University Feinberg School of Medicine, Chicago, United States; 3Institute of Biology, University of Leipzig, Leipzig, Germany Abstract Keratin intermediate filaments are an essential and major component of the cytoskeleton in epithelial cells. They form a stable yet dynamic filamentous network extending from the nucleus to the cell periphery, which provides resistance to mechanical stresses. Mutations in keratin genes are related to a variety of epithelial tissue diseases. Despite their importance, the molecular structure of keratin filaments remains largely unknown. In this study, we analyzed the structure of keratin 5/keratin 14 filaments within ghost mouse keratinocytes by cryo-electron microscopy and cryo-electron tomography. By averaging a large number of keratin segments, we have gained insights into the helical architecture of the filaments. Two-dimensional classification revealed profound variations in the diameter of keratin filaments and their subunit organization. Computational reconstitution of filaments of substantial length uncovered a high degree of internal heterogeneity along single filaments, which can contain regions of helical symmetry, regions with less symmetry and regions with significant diameter fluctuations. Cross-section views of filaments revealed that keratins form hollow cylinders consisting of multiple protofilaments, with an electron dense core located in the center of the filament. These findings shed light on the complex and remarkable heterogenic architecture of keratin filaments, suggesting that they are highly flexible, dynamic cytoskeletal structures. *For correspondence: omedalia@bioc.uzh.ch Competing interests: The authors declare that no competing interests exist. Funding: See page 19 Received: 12 May 2021 Preprinted: 14 May 2021 Accepted: 23 July 2021 Published: 29 July 2021 Reviewing editor: Andrew P Carter, MRC Laboratory of Molecular Biology, United Kingdom Copyright Weber et al. This article is distributed under the terms of the Creative Common Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited. Competing interests: The authors declare that no competing interests exist. Funding: See page 19 Received: 12 May 2021 Preprinted: 14 May 2021 Accepted: 23 July 2021 Published: 29 July 2021 Introduction Keratin Intermediate Filaments (KIFs) are an essential component of the cytoskeleton of epithelial cells. KIFs are classified as type I and type II Intermediate Filament (IF) proteins, according to their sequence (Szeverenyi et al., 2008; Bragulla and Homberger, 2009; Toivola et al., 2015). Keratins form a highly flexible and dynamic filamentous network in the cytoplasm (Etienne-Manneville, 2018; Pora et al., 2020; Ko¨lsch et al., 2010; Robert et al., 2016; Windoffer et al., 2004; Yoon et al., 2001). Their main known function is to protect the cell from external stresses by providing mechani- cal stability and ensuring the integrity of tissues through cell-cell and cell-matrix contacts. Point mutations in keratin genes are associated with cell and tissue instabilities and severe diseases, termed keratinopathies (Toivola et al., 2015; Haines and Lane, 2012). For example, the skin blister- ing disease Epidermolysis Bullosa Simplex (EBS) is caused by point mutations in the Keratin 5 (K5) and 14 (K14) genes (Jacob et al., 2018; Coulombe et al., 1991; Coulombe and Lee, 2012). A key to understanding the function of KIFs in both normal and diseased cells is to unveil the structural organization of the filaments. Structural heterogeneity of cellular K5 K14 filaments as revealed by cryo- electron microscopy Miriam S Weber1, Matthias Eibauer1, Suganya Sivagurunathan2, Thomas M Magin3, Robert D Goldman2, Ohad Medalia1* Reviewing editor: Andrew P Carter, MRC Laboratory of Molecular Biology, United Kingdom Reviewing editor: Andrew P Carter, MRC Laboratory of Molecular Biology, United Kingdom Reviewing editor: Andrew P Carter, MRC Laboratory of Molecular Biology, United Kingdom Copyright Weber et al. This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited. Keratin proteins are composed of three domains: a highly conserved a-helical central rod doma known to facilitate filament assembly, and intrinsically disordered head and tail domains. The lat 1 of 23 Weber et al. eLife 2021;10:e70307. DOI: https://doi.org/10.7554/eLife.70307 Research article Research article Research article Cell Biology Structural Biology and Molecular Biophysics Cell Biology Structural Biology and Molecular Biophysics are highly post-translationally modified and are regarded as essential for regulatory functions and fil- ament stability (Wilson et al., 1992; Parry and Steinert, 1999; Chernyatina et al., 2015). Keratins are obligatory heterodimers, formed by the parallel and in-register assembly of an acidic type I and a basic type II keratin protein. Two dimers assemble into antiparallel tetramers, which can further align laterally and longitudinally to build mature keratin filaments (Steinert et al., 1993; Lee et al., 2020; Herrmann and Aebi, 2016). Longitudinally elongated tandem arrays of tetrameric subunits are termed protofilaments. Although KIFs have been studied intensely for many years, details of their molecular architecture remain largely unknown (Eldirany et al., 2021). On the level of keratin monomers and dimers, crys- tallographic studies have provided high-resolution insights into the organization of small regions of the central rod domain (Lee et al., 2020; Lee et al., 2012; Bunick and Milstone, 2017; Eldirany et al., 2019) and molecular dynamics simulations have presented a 3D model of a complete K1/K10 dimer (Bray et al., 2015). Four different modes of tetramer assembly have been identified by cross-linking studies, describing how higher order oligomers form during filament assembly (Steinert et al., 1993). It is expected that filament assembly starts by the formation of A11 tetramers, where the 1B domains of the rod of two adjacent dimers interact in a half-staggered, antiparallel arrangement (Lee et al., 2020; Herrmann and Aebi, 2016). Tetramers can elongate longitudinally by formation of A22 interactions, where the 2B domains of adjacent rods overlap (Lee et al., 2020; Herrmann and Aebi, 2016). Laterally, neighboring tetramers interact via A12 bindings to form 10 nm wide filaments (Lee et al., 2020; Herrmann and Aebi, 2016). Reviewing editor: Andrew P Carter, MRC Laboratory of Molecular Biology, United Kingdom However, little is known about the 3D high-resolution structure of mature keratin filaments. It is generally accepted that keratin fila- ments are helical assemblies consisting of multiple protofilaments (Astbury, 1939; Crick, 1952; Crick, 1953), which form a cylindrical tube (Parry and Steinert, 1999). However, the exact number of protofilaments per filament and therefore the number of keratin monomers per cross section is still debated and may vary with respect to the specific type I/type II keratin pairs expressed. Mass- Per-unit-Length (MPL) measurements of recombinant in vitro assembled K8/K18 filaments have sug- gested that they are built from 16 to 25 monomeric protein chains in cross section, depending on the ionic strength of the buffer and the assembly time (Herrmann et al., 1999). Interestingly, MPL measurements of KIFs assembled in vitro from keratins extracted from human epidermis have revealed that the majority of them contain 13–16 polypeptides in cross section, with fewer numbers of KIF comprised of either 20–26 or 28–35 polypeptides (Engel et al., 1985). These variations have been attributed to structural polymorphism of KIFs and apparently occur by varying the number of protofilaments along and among keratin filaments (Engel et al., 1985). In addition, there is a lack of consensus as to whether KIFs are hollow or filled tubes and whether they contain an internal struc- ture (Parry and Steinert, 1999; Bruce Fraser et al., 2003; Parry, 1996; Fraser and Parry, 2017; Watts et al., 2002; Herrmann and Aebi, 2004). Here, we studied the structure of native cellular K5/K14 filaments by Cryo-Electron Microscopy (cryo-EM) and Cryo-Electron Tomography (cryo-ET) (Weber et al., 2019). Since the expression of keratin isoforms is variable and complex in cultured cells, we prepared a cell line expressing fila- ments composed of K5/K14 only and studied their architecture within cells that were grown and lysed on EM grids, that is ghost cells. This process avoids potential structural artifacts due to in vitro assembly of KIFs. Our cryo-EM analysis revealed the remarkably heterogenic nature of keratin fila- ments and uncovered changes in the diameter and the helical pattern propagating along the fila- ment. Cryo-ET and analysis of filament cross-sections revealed that the K5/K14 filaments are composed of a hollow cylinder with an internal electron dense core. The wall of the cylinder is con- structed of a ring of six protofilaments. Reviewing editor: Andrew P Carter, MRC Laboratory of Molecular Biology, United Kingdom Our results quantify the flexibility of keratin filaments and uncover the immense structural heterogeneity of individual K5/K14 filaments. Cellular keratin filaments revealed by cryo-electron microscopy Cellular keratin filaments revealed by cryo-electron microscopy K5/14_1 cells were cultured on cryo-EM grids and subjected to cytoskeleton extraction buffer that permeabilizes the cells and removes soluble cytoplasmic components and nuclear structures, pro- ducing IF-enriched ghost cells (Material and Methods section) (Hu et al., 2019; Turgay et al., 2017; Kronenberg-Tenga et al., 2021; Svitkina and Borisy, 1998; Svitkina et al., 1995; Sailer et al., 2010). The ghost cells were instantly plunge frozen and imaged by cryo-EM and cryo-ET (Figure 1B–D). Keratin filaments could be easily identified in cryo-EM micrographs (Figure 1C, blue arrows, Figure 1—figure supplement 1D), while the 3D organization of the keratins within the ghost cells was revealed by cryo-ET (Figure 1D light blue, Video 1). Actin filaments were detected in the sample as well (Figure 1C, orange arrows and D, red, Figure 1—figure supplement 1D) and used as an internal quality control for structural preservation by the extraction protocol and for cryo-EM image quality. Under these conditions, the structure of cellular F-actin was resolved to 6.1 A˚ using single particle image processing (Figure 1—figure supplement 2). Moreover, images were acquired from variety of cellular positions, encompassing internal to peripheral regions to ensure that the ker- atin IF analysis is not biased by cellular location. y y Keratins form a complex filamentous network, including thick bundles containing numerous fila- ments, meshworks, in which filaments are often crossing and interacting with each other, as well as long stretches of individual filaments (Figure 1C,E, Figure 1—figure supplement 1D). Keratin fila- ments exhibit a wide range of shapes suggesting a high degree of flexibility. While some filaments are straight over long distances (several hundreds of nm), others exhibit a wavy appearance (Figure 1E, arrow). Additionally, highly bent keratin filaments are frequently detected (Figure 1F, Figure 1—figure supplement 1D). We traced 65 of these highly bent filaments in cryo-EM micro- graphs and determined their minimal apparent persistence length (contour length upon 90˚ turn, Figure 1H). Keratin 5/14 filaments are able to undergo a 90˚ turn within 118.4 ± 39.2 nm (Figure 1G), similar to distances observed for nuclear lamins (Turgay et al., 2017; Tenga and Med- alia, 2020). These measurements were conducted only on the sub-population of highly bent fila- ments and not on the full range of shapes detected (e.g. straight filaments), as their varying behavior prohibited us to describe all of them with a single measure (Block et al., 2015). Cellular keratin filaments revealed by cryo-electron microscopy Interest- ingly, some filaments undertake even 180˚ turns within a few hundred nanometers without breaking and change directions multiple times within the inspected field of view (Figure 1F). In agreement with previous in vitro assembled keratin analyses, the remarkable flexibility of keratin filaments sup- ports their ability to maintain filament integrity even under extreme conditions (Ma et al., 2001; Coulombe and Fuchs, 1990; Herrmann et al., 2002; Ko¨ster et al., 2015). ation of mouse keratinocytes expressing only K5/K14 filaments Generation of mouse keratinocytes expressing only K5/K14 filaments Heterogeneity is a challenging problem that hampers structural determination (Scheres, 2016). Thus, the occurrence of multiple keratin pairs in most epithelial cells hinders the structural analysis of keratin filaments in their native environment, due to intrinsic structural heterogeneity (Moll et al., 2008). In this study, we set to gain insights into the architecture of cellular K5/K14 filaments. Weber et al. eLife 2021;10:e70307. DOI: https://doi.org/10.7554/eLife.70307 2 of 23 2 of 23 Research article Research article Cell Biology Structural Biology and Molecular Biophysics Therefore, we utilized the murine keratinocyte cell line KtyI KO K14, which expresses the K5, K6 and K14 proteins as their only keratin isoforms, and the only IF proteins (Homberg et al., 2015; Kumar et al., 2015; Kumar et al., 2016). To reduce the keratin expression to K5 and K14 only, CRISPR/Cas9 was used to knock out the Krt6a and the Krt6b gene, which share 92.6% sequence identity (Materials and methods section). Although a small amount of wild-type Krt6b DNA was retained (Figure 1—figure supplement 1A,B), immunostaining revealed that no filaments containing K6 assemble in the resulting K5/14_1 cell line, and therefore do not affect the structural analysis car- ried out in our study (Figure 1—figure supplement 1C). We therefore conclude that the keratin fila- ments in this cell line consist only of K5/K14 protein pairs. A careful analysis of the KIF network after the CRISPR/Cas9 knockout procedure by confocal fluorescence microscopy indicated no obvious impact on the K5/K14 filament network (Figure 1A, Figure 1—figure supplement 1C). Heterogeneity in filament diameter and helical pattern g y p To obtain deeper insights into the architecture of keratin filaments, we extracted 55 nm long straight keratin segments picked along filaments in ~1700 cryo-EM micrographs and subjected them to structural averaging by single particle data processing (Scheres, 2012; He and Scheres, 2017). A helical pattern that spirals along the long filament axis can be detected in several class averaged structures (Figure 2A, Figure 2—figure supplement 1A). While several structural classes show a clear helical pattern, others reveal elongated, rather straight sub-structures without an apparent heli- cal symmetry (Figure 2A, bottom, Figure 2—figure supplement 1A). Transition regions between the two distinguished patterns can also be detected (Figure 2A, arrows). The mean diameter of Weber et al. eLife 2021;10:e70307. DOI: https://doi.org/10.7554/eLife.70307 3 of 23 Research article Cell Biology Structural Biology and Molecular Biophysics 100 nm 100 nm 100 nm 10 µm H G E C Minimal persistence length (nm) 50 100 150 200 250 0 2 4 6 8 10 12 14 16 st n e m alfi f o r e b m u N -50 50 100 150 200 250 x (nm) -150 -100 -50 0 50 100 y (nm) 0 10 µm A D 100 nm B F Figure 1. Cellular K5/K14 filaments as revealed by light and cryo-electron microscopy. (A) The murine keratinocyte cell line K5/K14_1 expressing only K5 and K14 filaments forms a complex KIF meshwork, as revealed by confocal immunofluorescence. Cells were stained for K14 (green) and chromatin (blue). (B) Ghost cells were analyzed by cryo-EM and cryo-ET. Low-magnification image of a cell grown on an EM-grid and treated with detergent prior to vitrification. Cell boundaries (dashed white line) are detected as well as a neighboring cell (dashed black line). Research article Research article Cell Biology Structural Biology and Molecular Biophysics Cell Biology Structural Biology and Molecular Biophysics Figure 1 continued cryo-EM are marked (yellow circles). (C) A typical cryo-EM micrograph of a ghost cell imaged at a higher magnification allows the detection of keratin filaments and other cytoskeletal elements (n=1860). Keratin filaments (blue arrows) and actin filaments (orange arrows) are distinguished by their characteristic diameter. A large keratin bundle is visible in the top right corner. (D) Surface rendering view of a cryo-tomogram of a ghost cell (n=44). Keratin filaments (light blue), actin filaments (red), vesicles (dark blue), and cellular debris (yellow) were manually segmented. (E) Different organizations of keratin filaments observed in the cryo-EM micrographs (n=1860), including straight filaments (middle), curved (top, green arrows) and bundled filaments (bottom left). Scale bars: 100 nm. (F) Highly bent keratin filaments are found within cryo-EM micrographs of ghost cells. Scale bars: 100 nm. (G) Quantification of the minimal apparent persistence length measurements performed on (n=65) highly bent keratin filaments extracted from cryo-EM micrographs. (H) A plot combining 65 contours of filaments that were used for the minimal apparent persistence length measurements in (G). Individual filaments, shown in different colors, are aligned at their origins for visualization purposes. Th li i f thi ti l i l d th f ll i fi l t( ) f fi 1 g g p p The online version of this article includes the following figure supplement(s) for figure 1: Figure supplement 1. Knockout of K6 isoforms by CRISPR/Cas9. Figure supplement 2. Validation that the sample preparation allows us to retrieve data at sub-nanometer resolution. Figure supplement 1. Knockout of K6 isoforms by CRISPR/Cas9. Figure supplement 2. Validation that the sample preparation allows u Figure supplement 1. Knockout of K6 isoforms by CRISPR/Cas9. Figure supplement 2. Validation that the sample preparation allows us to retrieve data at sub-nanometer resolution. supplement 1. Knockout of K6 isoforms by CRISPR/Cas9. supplement 2. Validation that the sample preparation allows us to retrieve data at sub-nanometer resolution. keratin filaments, as determined by direct measurement of intensity line-profiles through the class averages, is 10.1 ± 0.5 nm (Figure 2B,C), in agreement with previous observations (Herrmann and Aebi, 2016). A mean intensity line-profile through a lateral average of the most populated classes defined the edges of the filaments as well as a central density peak (Figure 2C). Research article The outer bound- aries of the filaments show the highest electron density values and therefore are their most pro- nounced structural features (i.e. the filament diameter), while a central density peak with slightly lower intensity is also apparent. This analysis further revealed a subset of structural classes with a much larger diameter than the majority of filaments (Figure 2D). Intensity line profiles of a thicker class (black asterisk) and a more frequently detected class (blue asterisk) indicate a 30% difference in filament diameter, 13.2 nm vs 10.1 nm, respectively (Figure 2E). Moreover, the internal structure of the thicker classes diverges from the classes shown in Figure 2A. Specifically, some classes reveal two distinct linear electron densities within the filament (Figure 2D, arrowheads), indicating a less dense packing of the individual protofilaments as compared to the compact classes. Others capture transitions between a thinner and a thicker region along an individual filament (Figure 2D, arrows). These findings indicate deviations in the organization of protofilaments, reflecting structural hetero- geneity along individual filaments. g y g In order to determine the repeating unit, that is the pitch of the helical patterns observed in the class averages we calculated autocorrelation spectra for each helical class (Figure 2F, Figure 2—fig- ure supplement 1D). Using this approach, the helical pitch can be determined, which is an intrinsic parameter of helical assemblies and reflects a 360˚ rotation of the helix (Diaz et al., 2010). We found that the pitch of the helical pattern varies dramatically among different classes, ranging from ~132 A˚ to ~163 A˚ (Figure 2F). Between these two extremes numerous distinct values for the pitch of the helical pattern can also be iden- tified (Figure 2—figure supplement 1D). There are two possible scenarios that can explain a In order to determine the repeating unit, that class averages we calculated autocorrelation spec ure supplement 1D). Using this approach, the he parameter of helical assemblies and reflects a 360˚ rotation of the helix (Diaz et al., 2010). We found that the pitch of the helical pattern varies dramatically among different classes, ranging from ~132 A˚ to ~163 A˚ (Figure 2F). Between these two extremes numerous distinct values for the pitch of the helical pattern can also be iden- tified (Figure 2—figure supplement 1D). Heterogeneity in filament diameter and helical pattern Typical regions that were analyzed by Figure 1 continued on next page Research article Cell Biology Structural Biology and Molecular Biophysic 100 nm 100 nm 100 nm 10 µm E C 10 µm A D 100 nm B F 100 nm C 10 µm A 10 µm B B C A A 100 nm 100 nm 100 nm 10 µm H G E 100 10 µm D 100 nm F 100 nm F 100 nm 100 nm E D 100 nm F 100 nm E D 100 nm D E H -50 50 100 150 200 250 x (nm) -150 -100 -50 0 50 100 y (nm) 0 G Minimal persistence length (nm) 50 100 150 200 250 0 2 4 6 8 10 12 14 16 st n e m alfi f o r e b m u N G G H Minimal persistence length (nm) 50 100 150 200 250 -50 50 100 150 200 250 x (nm) -150 -100 -50 0 50 100 y (nm) 0 Figure 1. Cellular K5/K14 filaments as revealed by light and cryo-electron microscopy. (A) The murine keratinocyte cell line K5/K14_1 expressing only K5 and K14 filaments forms a complex KIF meshwork, as revealed by confocal immunofluorescence. Cells were stained for K14 (green) and chromatin (blue). (B) Ghost cells were analyzed by cryo-EM and cryo-ET. Low-magnification image of a cell grown on an EM-grid and treated with detergent prior to vitrification. Cell boundaries (dashed white line) are detected as well as a neighboring cell (dashed black line). Typical regions that were analyzed by Figure 1 continued on next page Weber et al. eLife 2021;10:e70307. DOI: https://doi.org/10.7554/eLife.70307 4 of 23 Research article On the y-axis, the number of individual particles that constituted the 2D classes is plotted. (C) Mean intensity line-profile through all classes used in (B). The mean filament diameter (10.1 nm) is indicated and was measured between the zero-crossings of the curve. (D) Subset of keratin class averages showing larger filament diameters. Two individual filamentous densities are often detected within a filament (yellow arrowheads). Additionally, transition regions between thinner and thicker filament regions are detected (white arrows). (E) Intensity line-profiles through a narrow and a wide class indicated by blue and black asterisk (in (A) and (D)), respectively. Diameters of 10.1 nm and 13.2 nm (arrow) were detected. (F) Autocorrelation spectra of the displayed keratin classes (insets). Research article Peaks of the autocorrelation function corresponding to the distance between repetitive elements along the filament (pitch) are indicated (arrows) (G) To show that out-of-plane tilting of KIFs can shift the autocorrelation peaks D B A 80 90 100 110 120 130 140 150 160 170 -0.15 -0.1 -0.05 0 0.05 0.1 0.15 0.2 0.25 Pixel density 10.1 nm C * * 20 nm 20 nm Diameter (nm) 9.2 9.4 9.6 9.8 10.0 10.2 10.4 10.6 10.8 11.0 Number of particles B C Diameter (nm) 9.2 9.4 9.6 9.8 10.0 10.2 10.4 10.6 10.8 11.0 Number of particles A B A Diameter (nm) C 80 90 100 110 120 130 140 150 160 170 Mean filament profile (pix) -0.15 -0.1 -0.05 0 0.05 0.1 0.15 0.2 0.25 Pixel density 10.1 nm C ( ) D Pixel density 80 90 100 110 12 Mean filam 0.15 80 90 100 110 12 -0.2 -0.15 -0.1 -0.05 0 0.05 0.1 0.15 0.2 0.25 Filament Pixel density 13 0 50 100 150 200 250 0.7 0.75 0.8 0.85 0.9 0.95 1 163 Å Autocorrelation Distance (pix) 0 50 100 150 200 250 0.55 0.6 0.65 0.7 0.75 0.8 0.85 0.9 0.95 1 132 Å Autocorrelation Distance (pix) E F Class 10 Class 24 0 100 110 120 130 140 150 1 Mean filament profile (pix) E p (p ) 80 90 100 110 120 130 140 150 160 170 -0.2 -0.15 -0.1 -0.05 0 0.05 0.1 0.15 0.2 0.25 Filament profiles (pix) Pixel density 13.2 nm E ** Filament profiles (pix) Distance (pix) Tilt angle (°) 100 150 200 250 0.5 0.6 0.7 0.8 0.9 1 Autocorrelation Distance (pix) 0 50 132 Å H I H (p ) Class 24 34° 132 Å Class 10 0° G 132 Å H I 90 100 110 120 130 140 150 160 170 Apparent pitch length (Å) 0 5 10 15 20 25 30 35 40 45 50 G Tilt angle (°) Figure 2. The architecture and heterogeneity of keratin filaments. (A) Ten of the most populated 2D class averages of keratin segments. High electron density is shown in white. Arrows indicate transition regions between helical and straight-line patterns. In total, 50 classes containing keratin segments (n=305,495) were collected (Figure 2—figure supplement 1A). (B) Distribution of filament diameters as measured in 50 2D class averages (Figure 2— figure supplement 1A). Research article There are two possible scenarios that can explain a varying pitch distance: Either, a variable pitch is an intrinsic property of keratin filaments, which would add to the structural heterogeneity observed throughout this study, or the diverging pitch originates from filaments that lay tilted and not flat in the ice layer. Since keratin filaments are very flexible and form a 3D network in cells, we exploited the possibility that the different pitch lengths reflect filaments that are oriented out of plane. The projection of a tilted filament in our cryo-EM micrographs would therefore yield classes with an apparent shorter repeating pattern. In this case, the class with the longest Video 1. Cryo-tomogram of a keratin network in a ghost cell. https://elifesciences.org/articles/70307#video1 Video 1. Cryo-tomogram of a keratin network in a ghost cell. https://elifesciences.org/articles/70307#video1 Weber et al. eLife 2021;10:e70307. DOI: https://doi.org/10.7554/eLife.70307 Research article Cell Biology Structural Biology and Molecular Biophysics D B A 80 90 100 110 120 130 140 150 160 170 Mean filament profile (pix) -0.15 -0.1 -0.05 0 0.05 0.1 0.15 0.2 0.25 Pixel density 10.1 nm 80 90 100 110 120 130 140 150 160 170 -0.2 -0.15 -0.1 -0.05 0 0.05 0.1 0.15 0.2 0.25 Filament profiles (pix) Pixel density 13.2 nm 0 50 100 150 200 250 0.7 0.75 0.8 0.85 0.9 0.95 1 163 Å Autocorrelation Distance (pix) 0 50 100 150 200 250 0.55 0.6 0.65 0.7 0.75 0.8 0.85 0.9 0.95 1 132 Å Autocorrelation Distance (pix) C E F * * ** 20 nm 20 nm Class 10 0° Class 24 34° Class 10 Class 24 90 100 110 120 130 140 150 160 170 Apparent pitch length (Å) 0 5 10 15 20 25 30 35 40 45 50 Tilt angle (°) 100 150 200 250 0.5 0.6 0.7 0.8 0.9 1 Autocorrelation Distance (pix) 0 50 132 Å G H I 132 Å 132 Å Diameter (nm) 9.2 9.4 9.6 9.8 10.0 10.2 10.4 10.6 10.8 11.0 Number of particles Figure 2. The architecture and heterogeneity of keratin filaments. (A) Ten of the most populated 2D class averages of keratin segments. High electron density is shown in white. Arrows indicate transition regions between helical and straight-line patterns. In total, 50 classes containing keratin segments (n=305,495) were collected (Figure 2—figure supplement 1A). (B) Distribution of filament diameters as measured in 50 2D class averages (Figure 2— figure supplement 1A). Weber et al. eLife 2021;10:e70307. DOI: https://doi.org/10.7554/eLife.70307 Computationally reconstituting keratin filaments from the class averages g The 2D class averages allowed us to identify structural differences in 55 nm long keratin segments. To understand how these structural features are organized at the level of long keratin filaments, it was important to determine how the class averages are arranged along keratin filaments which are up to several hundreds of nanometers in length. For this purpose, we utilized a back-mapping strat- egy that permits the computational reconstitution of the original filament out of 2D class averages (Figure 2—figure supplement 1A,B; Kronenberg-Tenga et al., 2021; Martins et al., 2021). There- fore, every segment was represented by its corresponding 2D class image, which was inversely trans- formed, so that it matched the original orientation of the raw segment. Then it was plotted at the original coordinate position, where the raw segment was selected from the electron micrographs. In this fashion, we assembled the original keratin filaments made out of the respective 2D class aver- ages (Figure 3—figure supplement 1A), which were subsequently extracted and straightened. Since these computationally reconstituted filaments are assembled from class averages, their signal- to-noise ratio is drastically improved compared to the raw filaments. This approach allowed us to study long stretches of keratin filaments with improved resolution up to ~12 A˚ (Figure 3, Figure 2— figure supplement 1C). g pp The appearance of these computationally reconstituted keratin filaments is very heterogenous (Figure 3, Figure 3—figure supplement 1). Overall, they consist of patches of helical regions with clear repetitive patterns (Figure 3A, red, E), which are frequently interrupted by straight patterned stretches with less defined features (Figure 3A, yellow, E). The helical as well as the straight-line stretches are variable in length and frequency. While some filaments consist of mostly helical stretches, others are mixed or exhibit a mostly straight-line appearance (Figure 3E, Figure 3—fig- ure supplement 1B). Additionally, the diameter fluctuates along a single filament (Figure 3B,D). For example, KIFs have regions of increased width up to 13.2 nm that often allow the identification of individual sub-chains (Figure 3B,E), as well as thinner regions with widths down to 9.2 nm (Figure 3D,E). The thinner regions usually display a straight pattern, whereas not all straight regions show a decrease in diameter. Interestingly, the computationally reconstituted filaments revealed that helical regions with differ- ent pitch lengths, identified in the 2D class averages (Figure 2), can co-exist along a single filament (Figure 3C). Research article Research article Cell Biology Structural Biology and Molecular Biophysics Cell Biology Structural Biology and Molecular Biophysics Figure 2 continued shown in (G). After tilting of Class 24 by 34˚, both classes show an autocorrelation peak at the same marked position, an indicator that filament tilting might be the reason for the different pitches observed in the 2D classes. Green and black dots indicate which curve belongs to which class in (G). (I) Dependence of the apparent pitch length (autocorrelation peaks) on the filament tilt angle, measured by tilting Class 24 from 0˚ to 50˚ and calculating corresponding autocorrelation spectra. The gray area indicates the range of pitches found in keratin 2D classes. The online version of this article includes the following figure supplement(s) for figure 2: Figure supplement 1. 2D structural analysis of keratin segments. pitch length would reflect the untilted filament, while all other repeating patterns would originate from different degrees of tilting. Based upon this reasoning, we tilted and projected the class with a pitch of 163 A˚ in silico and retrieved similar pitch lengths as seen in the real classes (Figure 2I). A tilt of up to ~34˚ can induce shortening of the pitch length from 163 A˚ to 132 A˚ (Figure 2G,H). There- fore, tilting between 0˚ - 34˚ would explain the variations that were detected in the repeating pattern of the keratin classes (Figure 2I). With ice thicknesses of up to ~300 nm and a minimal apparent per- sistence length of ~118 nm, this amount of tilting can be expected, and further analysis by cryo-ET revealed that even higher degrees of tilting are possible (see below). A helical pitch of ~163 A˚ agrees well with previous studies that suggested a pitch of ~162 A˚ for keratin filaments (Aebi et al., 1983; Parry et al., 2001). Research article On the y-axis, the number of individual particles that constituted the 2D classes is plotted. (C) Mean intensity line-profile through all classes used in (B). The mean filament diameter (10.1 nm) is indicated and was measured between the zero-crossings of the curve. (D) Subset of keratin class averages showing larger filament diameters. Two individual filamentous densities are often detected within a filament (yellow arrowheads). Additionally, transition regions between thinner and thicker filament regions are detected (white arrows). (E) Intensity line-profiles through a narrow and a wide class indicated by blue and black asterisk (in (A) and (D)), respectively. Diameters of 10.1 nm and 13.2 nm (arrow) were detected. (F) Autocorrelation spectra of the displayed keratin classes (insets). Peaks of the autocorrelation function corresponding to the distance between repetitive elements along the filament (pitch) are indicated (arrows). (G) To show that out-of-plane tilting of KIFs can shift the autocorrelation peaks, Class 24 was tilted in silico by 34˚, while Class 10 is untilted. The apparent pitch of both classes is indicated. (H) Autocorrelation spectra of the classes Figure 2 continued on next page Weber et al. eLife 2021;10:e70307. DOI: https://doi.org/10.7554/eLife.70307 6 of 23 6 of 23 Computationally reconstituting keratin filaments from the class averages Short repeating atterns (purple) indicate higher tilt angles in comparison to longer repeating patterns of in-plane filament stretches (green). (D) Keratin filament isplaying diameter fluctuations. Areas of decreased diameter are indicated (orange arrows). (E) A collage of six computationally reconstituted keratin aments showing structural diversity (total n=4460 filaments). he online version of this article includes the following figure supplement(s) for figure 3: igure supplement 1. Reconstitution of keratin filaments E 20 nm E Figure 3. Structural polymorphism along keratin filaments. Computationally reconstituted filaments provide a realistic view of the KIFs at higher resolution (see Materials and methods). (A – D) Scale bar: 20 nm. (A) A typical keratin filament consisting of various regions with helical and straight-line patterns, indicated by red and yellow lines, respectively. (B) Keratin filament displaying diameter fluctuations. Areas of increased diameter are indicated (blue arrows). (C) Keratin filament showing helical patterns exhibiting different pitches, indicating modulations within the ice layer. Short repeating patterns (purple) indicate higher tilt angles in comparison to longer repeating patterns of in-plane filament stretches (green). (D) Keratin filament displaying diameter fluctuations. Areas of decreased diameter are indicated (orange arrows). (E) A collage of six computationally reconstituted keratin filaments showing structural diversity (total n=4460 filaments). The online version of this article includes the following figure supplement(s) for figure 3: 8 of 23 Computationally reconstituting keratin filaments from the class averages This indicates that individual filaments changed their tilt angle along the course of the filament and ran through different z-heights of the ghost cell volume. Helical patterns with different pitches, indicating different tilt angles, lie in close proximity along KIFs, where they appear to transi- tion smoothly into each other. These structural transitions reveal that keratin filaments constantly Weber et al. eLife 2021;10:e70307. DOI: https://doi.org/10.7554/eLife.70307 7 of 23 A B Helical region Straight region Increased width Short repeating pattern Long repeating pattern C Decreased width D E 20 nm 20 nm igure 3. Structural polymorphism along keratin filaments. Computationally reconstituted filaments provide a realistic view of the KIFs at higher esolution (see Materials and methods). (A – D) Scale bar: 20 nm. (A) A typical keratin filament consisting of various regions with helical and straight-li atterns, indicated by red and yellow lines, respectively. (B) Keratin filament displaying diameter fluctuations. Areas of increased diameter are indicate blue arrows). (C) Keratin filament showing helical patterns exhibiting different pitches, indicating modulations within the ice layer. Short repeating atterns (purple) indicate higher tilt angles in comparison to longer repeating patterns of in-plane filament stretches (green). (D) Keratin filament isplaying diameter fluctuations. Areas of decreased diameter are indicated (orange arrows). (E) A collage of six computationally reconstituted keratin aments showing structural diversity (total n=4460 filaments). he online version of this article includes the following figure supplement(s) for figure 3: igure supplement 1. Reconstitution of keratin filaments. Research article Cell Biology Structural Biology and Molecular Biophys Research article Research article Cell Biology Structural Biology and Molecular Biophysics A B Helical region Straight region Increased width Short repeating pattern Long repeating pattern C Decreased width D 20 nm Research article Cell Biology Structural Biology and Molecular Biophy A B Short repeating pattern Long repeating pattern C Decreased width D D C Short repeating pattern Long repeating pattern E 20 nm igure 3. Structural polymorphism along keratin filaments. Computationally reconstituted filaments provide a realistic view of the KIFs at higher esolution (see Materials and methods). (A – D) Scale bar: 20 nm. (A) A typical keratin filament consisting of various regions with helical and straight-line atterns, indicated by red and yellow lines, respectively. (B) Keratin filament displaying diameter fluctuations. Areas of increased diameter are indicated blue arrows). (C) Keratin filament showing helical patterns exhibiting different pitches, indicating modulations within the ice layer. Weber et al. eLife 2021;10:e70307. DOI: https://doi.org/10.7554/eLife.70307 Weber et al. eLife 2021;10:e70307. DOI: https://doi.org/10.7554/eLife.70307 Research article Research article Cell Biology Structural Biology and Molecular Biophysics Cell Biology Structural Biology and Molecular Biophysics fluctuate in the z-direction (perpendicular to the plane of the EM grid) and thus appear to be as flex- ible in the z-direction as they are in the xy plane (parallel to the EM grid). Overall, computationally reconstituted keratin filaments reveal an enormous amount of structural heterogeneity (Figure 3E, Figure 3—figure supplement 1C). Every filament examined displays a unique phenotype, which demonstrates that keratin filaments are as versatile as the challenges they encounter in a living cell. Keratin filaments are cylindrical tubes with an internal electron dense core revealed several cryo-EM micrographs and multiple cryo-tomo- KIFs that undergo a 90˚ turn along the thickness of the sample n of direct perpendicular cross sections of the filaments (Video 2). as it was never seen in tomograms (n = 225) of cellular vimentin within detergent extracted mouse embryonic fibroblasts (MEF) gure supplement 1A,B). Analysis of cross-sections from cryo-EM revealed that keratin filaments are cylindrical tubes, in which an d (Figure 4A–E). This finding agrees with previous studies, which ernal mass, but less than anticipated for a completely filled fila- Engel et al., 1985; Bruce Fraser et al., 2003; Parry, 1996; n and Aebi, 2004; Watts, 2002) and was previously reported for s of human epidermis and the stratum corneum (Norle´n and Al- ld identify individual sub-filaments, which form a hexameric ring B). Based on this geometry and previous literature, it is likely that meric protofilaments and therefore the mature filament would be to yield ~24 polypeptides in cross-section (Parry and Steinert, mass-per-unit-length analyses of epidermal keratins and keratins et al., 1999; Engel et al., 1985). To study this phenomenon in ss-sectional views detected in cryo-tomograms. We found that ent and tomographic slice, the number of visible protofilaments orphism, that is a variable number of protofilaments building the imaging artifact originating from the low signal-to-noise ratio of s the identification of the thin protofilaments. Nevertheless, the nique insights into filament arrangement. When following a fila- omographic volume, in certain slices densities of neighboring pro- ne continuous are positions ofilaments are could not be Figure 4—fig- sitions reveal ch may reflect ers along the mpression of ns containing y the position ratin tube, we (labeled with cisely into 90˚ m-thick slices e central den- center of the ted closer to e supplement entral density s not fixed to Video 2. Cryo-tomogram showing the modulations of the keratin filaments within the ice layer. https://elifesciences.org/articles/70307#video2 7 9 of 23 A careful analysis of our datasets revealed several cryo-EM micrographs and multiple cryo-tomo- grams (21 out of 44) which contain KIFs that undergo a 90˚ turn along the thickness of the sample and therefore allow the observation of direct perpendicular cross sections of the filaments (Video 2). This behavior is quite remarkable, as it was never seen in tomograms (n = 225) of cellular vimentin intermediate filaments, imaged within detergent extracted mouse embryonic fibroblasts (MEF) (Eibauer et al., 2021, Figure 4—figure supplement 1A,B). Keratin filaments are cylindrical tubes with an internal electron dense core Analysis of cross-sections from cryo-EM micrographs and cryo-tomograms revealed that keratin filaments are cylindrical tubes, in which an internal electron dense core is found (Figure 4A–E). This finding agrees with previous studies, which predicted that keratins contain internal mass, but less than anticipated for a completely filled fila- ment (Parry and Steinert, 1999; Engel et al., 1985; Bruce Fraser et al., 2003; Parry, 1996; Fraser and Parry, 2017; Herrmann and Aebi, 2004; Watts, 2002) and was previously reported for keratin filaments of the living layers of human epidermis and the stratum corneum (Norle´n and Al- Amoudi, 2004). Moreover, we could identify individual sub-filaments, which form a hexameric ring structure in cross-section (Figure 4B). Based on this geometry and previous literature, it is likely that the sub-structures represent tetrameric protofilaments and therefore the mature filament would be composed of ~six protofilaments to yield ~24 polypeptides in cross-section (Parry and Steinert, 1999). This agrees with previous mass-per-unit-length analyses of epidermal keratins and keratins from simple epithelia (Herrmann et al., 1999; Engel et al., 1985). To study this phenomenon in more detail, we analyzed the cross-sectional views detected in cryo-tomograms. We found that depending on the individual filament and tomographic slice, the number of visible protofilaments varies, which might indicate polymorphism, that is a variable number of protofilaments building the KIF. However, it might also be an imaging artifact originating from the low signal-to-noise ratio of individual sections, that aggravates the identification of the thin protofilaments. Nevertheless, the tomographic slices yielded some unique insights into filament arrangement. When following a fila- ment in cross section through the tomographic volume, in certain slices densities of neighboring pro- tofilaments seem to merge into one continuous structure, indicating that there are positions along the filament where the protofilaments are interacting so tightly that they could not be resolved individually (Figure 4E, Figure 4—fig- ure supplement 1C). Other positions reveal more than six protofilaments, which may reflect overlap regions between tetramers along the keratin filaments, yielding the impression of more strains, or filament regions containing additional protofilaments. To study the position of the central density within the keratin tube, we extracted a cross-sectional filament (labeled with E in Figure 4D) and rotated it precisely into 90˚ cross-section. Keratin filaments are cylindrical tubes with an internal electron dense core Projection of 4.2-nm-thick slices along the filament revealed that the central den- sity is not always found in the exact center of the filament, but is sometimes detected closer to the filament walls (Figure 4—figure supplement 1E,F). This indicates, that the central density moves within the keratin tube and is not fixed to the center of the filament. Video 2. Cryo-tomogram showing the modulations of the keratin filaments within the ice layer. https://elifesciences.org/articles/70307#video2 Video 2. Cryo-tomogram showing the modulations of the keratin filaments within the ice layer. https://elifesciences.org/articles/70307#video2 Video 2. Cryo-tomogram showing the modulations of the keratin filaments within the ice layer. https://elifesciences.org/articles/70307#video2 Weber et al. eLife 2021;10:e70307. DOI: https://doi.org/10.7554/eLife.70307 9 of 23 Research article Cell Biology Structural Biology and Molecular Biophysics 100 nm C B 25 nm 10 nm 10 nm D E F D E 10 nm A B G 5 nm Figure 4. Multiple protofilaments and an internal electron dense core are canonical components of keratin filaments. (A) Cross-section views of keratin ilaments detected within the cryo-EM micrographs of ghost cells. An electron dense core is visible in the center of the keratin tube. Scale bars: 25 nm. B) Zoomed-in view of the area boxed in (A). The cross section view reveals an internal core surrounded by six protofilaments as constituents of the tub yellow arrowheads). (C) A surface rendered tomogram of a ghost cell was rotated in order to show the modulation of the keratin filaments within the Figure 4 continued on next page 100 nm C B 25 nm 10 nm 10 nm D F D E A B G 10 nm B B 25 nm A B 25 nm 10 nm A B B A 100 nm C D E C 10 nm G 10 nm D E F 10 nm G 5 nm F 5 nm 10 nm D E G F D Figure 4. Multiple protofilaments and an internal electron dense core are canonical components of keratin filaments. (A) Cross-section views of keratin filaments detected within the cryo-EM micrographs of ghost cells. An electron dense core is visible in the center of the keratin tube. Scale bars: 25 nm. (B) Zoomed-in view of the area boxed in (A). The cross section view reveals an internal core surrounded by six protofilaments as constituents of the tube (yellow arrowheads). Discussion Keratin intermediate filaments are major components of the cytoskeleton that are involved in many cellular processes (Redmond and Coulombe, 2021; Gordon, 2020; Danielsson et al., 2018). How- ever, due to their flexibility, heterogeneity and yet to be resolved symmetry, a high-resolution struc- ture of keratin filaments in their native state has not been obtained to date. In this study, we describe novel insights into the architecture of in vivo assembled K5/K14 filaments by imaging them directly within ghost cells. This approach has enabled us to study native KIFs containing all their post-translational modifications, which are known to play an important role in their assembly and function (Snider and Omary, 2014). In addition, this preparation circumvents the need for denatur- ing and renaturing the keratin proteins prior to in vitro assembly, a step which likely increases struc- tural polymorphism (Parry and Steinert, 1999). Since the preparation of ghost cells involves a short detergent exposure of 15–20 s, we showed that the well-established structure of F-actin is retained and can be resolved to 6.1 A˚ . Similarly, the structure of lamin filaments is also preserved using this approach (Kronenberg-Tenga et al., 2021; Turgay and Medalia, 2017). Therefore, we concluded that the structure of keratin IFs is unlikely to be affected by the sample preparation procedure. The K5/K14 IFs filaments are known to assemble in the cell periphery (Windoffer et al., 2004) to form bundles, networks and single IFs in all cellular domains (Ma et al., 2001; Yamada et al., 2002; Lee and Coulombe, 2009). In this study, we have focused on individual KIFs located both in periph- eral and more central regions as KIF bundles are dense, highly complex structures and would be unsuited for our averaging procedures (Sigworth, 2016). Individual filaments were found to be very flexible, which agrees with previous studies (Ma et al., 2001; Coulombe and Fuchs, 1990; Herrmann et al., 2002; Ko¨ster et al., 2015), and showed a high degree of bending within a few hundred nanometers. We determined the minimal apparent persistence length of highly bent KIFs to be 118.4 ± 39.2 nm, indicating that intact keratin filaments can undergo a 90˚ turn within a dis- tance of 2–3 dimer lengths (dimer length ~44 nm Bray et al., 2015; Quinlan et al., 1984). This value is slightly lower than the persistence length determined for K8/K18 filaments (300–650 nm) Weber et al. eLife 2021;10:e70307. Figure 4 continued ice layer (n=44). The three-dimensional keratin network is visualized (light blue). The level of the support is shown as a gray colored slice. Tomographic slices through vertically oriented filaments showing cross section views are indicated by boxes. (D) - (E) 7 nm thick xy-slices of the areas indicated in (C), showing KIFs as tube-like structures with a central density. Individual protofilaments can be identified. Scale bars: 10 nm. (F) A 2D class average of cross-section views extracted from individual regions of vertically oriented filaments (n=19), revealing the six individual protofilaments constituting the keratin filament tube (yellow arrowheads). (G) Low-resolution 3D model indicating the overall dimensions of a keratin filament and the presence of the central density. The structure was calculated template-free by randomizing the rotation angle of extracted 55 nm long keratin segments. Left: Side view. Right: Tilted cross section view revealing internal electron dense core. g g The online version of this article includes the following figure supplement(s) for figure 4: Figure supplement 1. Keratin filaments show a unique flexibility and contain a central density. To study the number of protofilaments in more detail, we selected 710 cross-sectional views of fil- aments found within the cryo-tomograms, extracted central slices and subjected them to 2D classifi- cation, revealing a symmetric hexameric class (Figure 4F). Six individual protofilaments can be clearly distinguished in the ring (arrowheads), which agrees with our studies of the raw data. As expected, additional classes were found showing deviations from this hexameric structure (Fig- ure 4—figure supplement 1D). These structural differences may represent actual changes in sym- metry and a polymorphic composition of KIFs, but also deviations from a perfect perpendicular cross-section may account for this observation. Finally, to get an impression of a mature keratin filament in three dimensions, we generated a low-resolution 3D model of a keratin filament from our single particle dataset of 55 nm long seg- ments using the Relion software package (Scheres, 2012; He and Scheres, 2017). By randomizing the rotational angle along the filament axis, a template-free unbiased model was generated (Figure 4G). The 3D model strengthens our findings that keratin filaments are formed as hollow cyl- inders with a central electron density. Due to randomization of the rotation angle, the individual pro- tofilaments are not resolved in this structure, however, it provides a view of the central density and the overall dimensions of the K5/14 intermediate filaments. Keratin filaments are cylindrical tubes with an internal electron dense core (C) A surface rendered tomogram of a ghost cell was rotated in order to show the modulation of the keratin filaments within the Figure 4 continued on next page Weber et al. eLife 2021;10:e70307. DOI: https://doi.org/10.7554/eLife.70307 Weber et al. eLife 2021;10:e70307. DOI: https://doi.org/10.7554/eLife.70307 10 of 23 Research article Research article Cell Biology Structural Biology and Molecular Biophysics Cell Biology Structural Biology and Molecular Biophysics Figure 4 continued Figure 4 continued Cell Biology Structural Biology and Molecular Biophysics Cell Biology Structural Biology and Molecular Biophysics (Block et al., 2015; Lichtenstern et al., 2012; Pawelzyk et al., 2014), which can be attributed to the fact that the K5/K14 filaments were still embedded into the cellular filament network. (Block et al., 2015; Lichtenstern et al., 2012; Pawelzyk et al., 2014), which can be attributed to the fact that the K5/K14 filaments were still embedded into the cellular filament network. The high flexibility and modulation of keratin filament orientation is also apparent as filaments can span through the entire thickness of the ice on a cryo-EM grid. The filaments often do not lie flat on the grid, but are frequently tilted out of the xy plane and form a wavy network in all three dimen- sions. Cryo-tomograms of ghost cells allowed us to follow individual filaments through different heights of the cell and show that the filaments can undergo 90˚ turns within a thickness of <300 nm (Figure 4). Interestingly, other IFs such as vimentin seem to be fluctuating less through the different heights of the ghost cells (Figure 4—figure supplement 1A,B). The bending ability of keratin filaments has enabled us to analyze perpendicular cross-section views and therefore directly reveal that they are built from six sub-filaments surrounding an electron dense core. A hexameric filament arrangement accompanied by a central core has been previously observed in keratin filaments of the human stratum corneum (Norle´n and Al-Amoudi, 2004). This supports a structure comprised of six tetrameric protofilaments, yielding 24 polypeptides in cross section. In support of this finding, mass-per-unit-length studies identified 21–25 polypeptides per cross-section of reassembled epidermal keratin extracts and in recombinant K8/K18 IFs prepared in vitro (Herrmann et al., 1999; Engel et al., 1985). It is unlikely that the identified sub-filaments rep- resent protofibrils, that is octameric assemblies, as this would yield 48 polypeptides in cross section, which does not agree with the MPL studies. The proposed model of a ‘universal IF’, consisting of 32 polypeptides in cross section, is not supported by our data. While this model agrees well with previ- ous studies of other IFs, for example vimentin (Herrmann et al., 1999; Goldie et al., 2007; Steven et al., 1982; Kooijman et al., 1997), it does not fit with studies of keratin filaments. There- fore, we conclude that different members of the IF superfamily show substantial diversity with respect to their structure (Goldie et al., 2007). Cell Biology Structural Biology and Molecular Biophysics Although MPL studies reveal that a small number of keratin filaments may contain ~32 polypeptides in cross-section, both MPL and cryo-EM studies including our own show that the majority of KIFs contain ~24 polypeptides in cross-section, orga- nized into ~six protofilaments (Herrmann et al., 1999; Engel et al., 1985; Norle´n and Al-Amoudi, 2004). Early studies of Aebi et al proposed a model of KIFs consisting of 8 protofilaments, arranged in four protofibrils, based on EM studies of unraveled filaments (Aebi et al., 1983). It is noteworthy that keratin filaments are not completely filled, but possess a distinct density in the center that is separated from the protofilaments forming the filament tube A central core in KIF cross-sections has also been reported in the viable cell layers and stratum corneum of human epider- mis (Norle´n and Al-Amoudi, 2004). As the viable cell layers of the epidermis express mostly K5/K14 filaments, this lends further support that the central core is a canonical structure of K5/K14 IFs. An internal core has also been identified in trichocyte keratins found in wool (Bruce Fraser et al., 2003; Rogers, 1964; Kadir et al., 2017). Previous cryo-ET studies of in vitro assembled vimentin filaments indicated no central core subunit (Goldie et al., 2007), however, a recent cryo-ET analysis of cellular assembled vimentin filaments also identified a frequently occurring central density (Eibauer et al., 2021). The central density described in the present study may correspond to an additional tetra- meric protofilament (Bruce Fraser et al., 2003) or another cellular component. Interestingly, this density is not fixed to the exact center of the keratin tube, but small changes in its position were identified along the keratin filaments. The 2D class averaging of keratin segments revealed structural variations that can be divided into multiple unique phenotypes. This is a remarkable property, that becomes even more apparent when comparing keratins to other cytoskeletal elements: While all analyzed actin class averages highly resemble each other, for example, showing the same helical pattern, filament organization and diameter, each keratin class average seems to be unique and to reflect a different structural arrange- ment of keratin filaments. The most pronounced feature is the helical repeating pattern in some 2D classes, while other classes show no apparent helical symmetry. Computational reconstitution of long keratin filaments revealed that these regions alternate along the same filaments, indicating that both patterns are crucial components of the keratin structure. Research article Discussion DOI: https://doi.org/10.7554/eLife.70307 11 of 23 Research article Research article Research article Cell Biology Structural Biology and Molecular Biophysics Previous studies showed that upon stretching beyond 200%, keratins adopt a plastic behavior that is accompanied by strain hard- ening and a significant reduction in diameter (Block et al., 2015; Kreplak et al., 2005; Fudge et al., 2003), which is thought to be mainly a result of a-helix to b-sheet transitions of the coiled-coil domains (Kreplak et al., 2005; Fudge et al., 2003; Pinto et al., 2014). Our experiments were con- ducted in ghost cells after forces were relaxed, therefore, only irreversible unfolding can be detected. Thus, thinner regions in keratin filaments may reflect local domains where extensive forces were applied to the filament network and the filaments adapted by irreversibly unfolding their coiled-coil domains, leading to a reduction in diameter. Overall, our results demonstrate that structural polymorphism is an intrinsic property of keratin fil- aments that were assembled within epithelial cells, and not only occurs in filaments that were assem- bled in vitro. When compared to the other components of the cytoskeleton, microtubules and actin filaments, which adapt a highly uniform arrangement, the structural heterogeneity of keratin fila- ments is clearly exceptional. Structural flexibility and changes in helical packing of the filament may provide structural support for the elastic nature of the keratin network and would help to explain their high resistance to breakage (Etienne-Manneville, 2018; Block et al., 2015). This feature of ker- atin filaments would coincide with their task to adapt to different mechanical stresses while maintain- ing a stable network. Our findings demonstrate the importance of determining a high-resolution structure of keratin fil- aments in order to understand the details of their assembly states and the functional significance of their heterogeneity in cells. Analyzing in vivo assembled filaments provides an approach to study the structure of keratins in their native state with their original post-translational modifications. Under- standing the high-resolution structure of keratin filaments would also provide a foundation for deter- mining how keratin mutations affect their structure and how they interact with binding partners. However, with reference to the findings of the current study, we want to emphasize that it is likely that there is not a single 3D structure that can describe keratin filaments in their entirety. In contrast, it is more likely that the conformational range needs to be described by a multitude of structures. Cell Biology Structural Biology and Molecular Biophysics This finding demonstrates that the heterogeneity and potential polymorphism does not lie between filaments, but within filaments. 2D classification and computational filament reconstitution additionally revealed large heterogeneities in the filament thickness. Although the most prevalent diameter detected in keratin filaments is 10.1 nm, the diameter can fluctuate between 9.2 and 13.2 nm. This type of heterogeneity has been previ- ously described for several types of IFs (Herrmann et al., 1999; Engel et al., 1985; Steven et al., Weber et al. eLife 2021;10:e70307. DOI: https://doi.org/10.7554/eLife.70307 12 of 23 Research article Research article Research article Cell Biology Structural Biology and Molecular Biophysics 1983) and is thought to reflect a varying number of subunits per cross-section. However, our results show that regions of increased diameter frequently yield insights into the subunit organization of the filament, indicating that the individual protofilaments are more loosely packed. The increased widths may therefore also reflect regions that are in a state of assembly or subunit exchange (C¸ olakog˘lu and Brown, 2009; Ngai et al., 1990; Miller et al., 1991; Vikstrom et al., 1989). Fur- ther, they might reflect areas of local post-translational modifications or regions where filaments were locally damaged. Interestingly, these regions are not restricted to the edges of filaments, but can occur in the mid regions of already assembled filaments. 1983) and is thought to reflect a varying number of subunits per cross-section. However, our results show that regions of increased diameter frequently yield insights into the subunit organization of the filament, indicating that the individual protofilaments are more loosely packed. The increased widths may therefore also reflect regions that are in a state of assembly or subunit exchange (C¸ olakog˘lu and Brown, 2009; Ngai et al., 1990; Miller et al., 1991; Vikstrom et al., 1989). Fur- ther, they might reflect areas of local post-translational modifications or regions where filaments were locally damaged. Interestingly, these regions are not restricted to the edges of filaments, but can occur in the mid regions of already assembled filaments. Filament stretches with diameters smaller than 10 nm might reflect either supercoiling or regions that have experienced a greater degree of localized mechanical stress. Cell Biology Structural Biology and Molecular Biophysics Cryo-EM and cryo-ET are the methods of choice for unravelling the complexities of the 3D structure of mature keratin filaments, as the coordinated use of these techniques can resolve both their flexi- bility and heterogeneity. Given the rapid advances in cryo-EM imaging, sample preparation and image processing, we anticipate that the structural analysis of keratin intermediate filaments will con- tinue to provide new insights into their cellular structure and functions. Materials and methods Materials and methods Materials and methods Key resources table Reagent type (species) or resource Designation Source or reference Identifiers Additional information Gene (Mus musculus) Krt6a UniProtKB - P50446 Targeted by CRISPR/ Cas9 Knockout Gene (Mus musculus) Krt6b UniProtKB - Q9Z331 Targeted by CRISPR/ Cas9 Knockout Strain, strain background (E. coli) DH5a ThermoFisher Scientific Cat# 18265017 Chemically competent cells Continued on next page Weber et al. eLife 2021;10:e70307. DOI: https://doi.org/10.7554/eLife.70307 13 of 23 Weber et al. eLife 2021;10:e70307. DOI: https://doi.org/10.7554/eLife.70307 13 of 23 Continued Reagent type (species) or resource Designation Source or reference Identifiers Additional information Cell line (Mus musculus) KtyI KO K14 Homberg et al., 2015. DOI: 10.1038/jid.2015.184 T. Magin lab, Institute of Biology, University of Leipzig, Germany. Cell line used to generate K5/ K14_1 cell line Cell line (Mus musculus) K5/K14_1 This paper Clonal knockout cell line of Krt6a and Krt6b, maintained in the Medalia lab Antibody Anti-mouse Keratin 14 (mouse monoclonal) ThermoFisher Scientific MA5-11599, Clone LL002 (1:100 – 1:10) Antibody Anti-mouse Keratin 5 (rabbit polyclonal) BioLegend Cat# 905503 (1:500) Antibody Anti-mouse Keratin 6a (rabbit polyclonal) BioLegend Cat# 905702 (1:500) Antibody Cy3 AffiniPure anti-rabbit (donkey polyclonal) Jackson Immuno Research Cat# 711-165-152 (1:100) Antibody FITC AffiniPure anti-mouse (donkey polyclonal) Jackson Immuno Research Cat# 715-095-150 (1:100) Recombinant DNA reagent pX458 (pSpCas9(BB)2A-GFP) (plasmid) Addgene Cat# 48138 CRISPR/Cas9 knockout Recombinant DNA reagent pGEM-T Easy (plasmid) Promega Cat# A1360 Sequence- based reagent guideRNA insert targetting Krt6a and Krt6b gene This paper guideRNA sequence: GAGCCACCGCTG CCCCGGGAG. guideRNA cloned Research article Cell Biology Structural Biology and Molecular Biophysics Cell Biology Structural Biology and Molecular Biophysics Cell Biology Structural Biology and Molecular Biophysics Continued Reagent type (species) or resource Designation Source or reference Identifiers Additional information Cell line (Mus musculus) KtyI KO K14 Homberg et al., 2015. DOI: 10.1038/jid.2015.184 T. Magin lab, Institute of Biology, University of Leipzig, Germany. Cell line used to generate K5/ K14_1 cell line Cell line (Mus musculus) K5/K14_1 This paper Clonal knockout cell line of Krt6a and Krt6b, maintained in the Medalia lab Antibody Anti-mouse Keratin 14 (mouse monoclonal) ThermoFisher Scientific MA5-11599, Clone LL002 (1:100 – 1:10) Antibody Anti-mouse Keratin 5 (rabbit polyclonal) BioLegend Cat# 905503 (1:500) Antibody Anti-mouse Keratin 6a (rabbit polyclonal) BioLegend Cat# 905702 (1:500) Antibody Cy3 AffiniPure anti-rabbit (donkey polyclonal) Jackson Immuno Research Cat# 711-165-152 (1:100) Antibody FITC AffiniPure anti-mouse (donkey polyclonal) Jackson Immuno Research Cat# 715-095-150 (1:100) Recombinant DNA reagent pX458 (pSpCas9(BB)2A-GFP) (plasmid) Addgene Cat# 48138 CRISPR/Cas9 knockout Recombinant DNA reagent pGEM-T Easy (plasmid) Promega Cat# A1360 Sequence- based reagent guideRNA insert targetting Krt6a and Krt6b gene This paper guideRNA sequence: GAGCCACCGCTG CCCCGGGAG. guideRNA cloned into pX458 plasmid and transfected into KtyI KO K14 cells. Weber et al. eLife 2021;10:e70307. DOI: https://doi.org/10.7554/eLife.70307 Commercial assay or kit P3 primary cell 4D-Nucleofector kit Lonza Cat# V4XP-3032 Transfection of KtyI KO K14 cells Commercial assay or kit jetPRIME Polyplus transfection Cat# 114–07 Transfection of KtyI KO K14 cells Commercial assay or kit GenElute Mammalian Genomic DNA kit Sigma-Aldrich Cat# G1N70-1KT Genomic DNA extraction Chemical compound, drug 99.9% anhydrous methanol Alfa Aesar Cat# 41838 Fixation of mammalian cells Chemical compound, drug Hoechst 33342 Sigma-Aldrich Cat# B2261 (1:10000) Chemical compound, drug Dako mounting medium Agilent Cat# S3023 Chemical compound, drug Prolong Glass Anti-Fade ThermoFisher Scientific Cat# P36980 Continued on next page Weber et al. eLife 2021;10:e70307. DOI: https://doi.org/10.7554/eLife.70307 14 of 23 14 of 23 Cell Biology Structural Biology and Molecular Biophysics Cell Biology Structural Biology and Molecular Biophysics Continued Reagent type (species) or resource Designation Source or reference Identifiers Additional information Software, algorithm TIDE webtool Netherlands Cancer Institute https://tide.nki.nl/ CRISPR/Cas9 knockout analysis Software, algorithm FIJI Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany RRID:SCR_002285 For data analysis Software, algorithm Illustrator Adobe Inc RRID:SCR_010279 For data analysis Software, algorithm OriginPro OriginLab RRID:SCR_014212 For data analysis Software, algorithm MATLAB MathWorks RRID:SCR_001622 For data analysis Software, algorithm RELION Scheres, 2012. DOI: 10.1016/j.jsb. 2012.09.006 RRID:SCR_016274 For data analysis Software, algorithm IMOD University of Colorado Boulder, Colorado, USA RRID:SCR_003297 For data analysis Software, algorithm Amira ThermoFisher Scientific RRID:SCR_007353 For data analysis Software, algorithm SerialEM University of Colorado Boulder, Colorado, USA RRID:SCR_017293 For data acquisition Software, algorithm UCSF Chimera University of California, California, USA RRID:SCR_004097 For structure visualization Research article Cell Biology Structural Biology and Molecular Biophysics Generation of the K5/K14_1 cell line Generation of the K5/K14_1 cell line Generation of the K5/K14_1 cell line Generation of the K5/K14_1 cell line Mouse keratinocytes lacking the entire type I keratin cluster (KtyI KO) but stably transfected with K14 (Homberg et al., 2015) were authenticated using transcriptome profiling and checked for mycoplasma contamination in the T. Magin lab (Institute of Biology, University of Leipzig, Germany). KtyI KO K14 cells were cultured on Collagen I (bovine, CellSystems) coated dishes at 32˚C and 5% CO2 in calcium-depleted FAD medium in the presence of puromycin (LabForce, 8 mg/ml medium). Confluent cells were trypsinized using 2.5 x trypsin/EDTA solution (Sigma-Aldrich, T4174) and re- seeded at a maximum splitting ratio of 1:2. To knock-out the Krt6a and Krt6b genes, cells were transfected with the pX458 (pSpCas9(BB)2A-GFP) plasmid (Addgene) carrying a GFP-tagged Cas9 and a guideRNA insert targeting both the Krt6a and Krt6b gene (guideRNA sequence: GAGC- CACCGCTGCCCCGGGAG). Transfection using electroporation was performed according to the manufacturer’s protocol using a P3 primary cell 4D-Nucleofector kit (Lonza) and program 138 for human keratinocytes, followed by another round of transfection using jetPRIME (Polyplus transfec- tion). Next, genomic DNA was extracted from clonal cell lines using the GenElute Mammalian Geno- mic DNA kit (Sigma-Aldrich) and the Krt6a and Krt6b gene fractions where the indel mutations were expected were amplified by PCR. PCR fragments were sequenced (Microsynth) and the indel muta- tion spectrum was analyzed using the TIDE webtool (https://tide.nki.nl/). The K5/K14_1 clone was identified as homogenous K6a knockout and heterogenous K6b knockout and was therefore used for all studies. To further verify the knockout, PCR fragments were cloned into the pGEM-T Easy vec- tor (Promega, A1360) and amplified in DH5a cells. Bacterial clones carrying individual gene sequen- ces of Krt6a or Krt6b were picked and amplified, plasmids were extracted and sequenced (Microsynth). By analyzing 19 Krt6a and 22 Krt6b sequences, the homogenous K6a and heteroge- nous K6b knockout were verified. 15 of 23 Weber et al. eLife 2021;10:e70307. DOI: https://doi.org/10.7554/eLife.70307 Research article Research article Cell Biology Structural Biology and Molecular Biophysics Cell Biology Structural Biology and Molecular Biophysics Sample preparation for cryo-EM and cryo-ET p p p y y K5/K14_1 cells were seeded on glow-discharged Collagen I coated holey carbon gold EM grids (Au R2/1, 200 mesh, Quantifoil) and incubated overnight at 32˚C and in 5% CO2. The grids were rinsed in washing buffer (1x PBS, 2 mM MgCl2), cells were permeabilized for 15–20 s in permeabilization buffer (1x PBS, 0.1% Triton X-100, 600 mM KCl, 10 mM MgCl2 and protease inhibitors), and rinsed again in washing buffer. Next, the grids were incubated with 2.5 units/ml benzonase (Merck, 71206– 3) in washing buffer for 30 min and washed in PBS before vitrification in liquid ethane using a manual plunge freezing device. For cryo-ET samples, 10 nm gold fiducial markers (Aurion, Netherlands) were added to the grids right before freezing. Immunostaining The KtyI KO K14 and K5/K14_1 cells were seeded on Collagen I coated glass cover slips in cell cul- ture dishes and incubated overnight at 32˚C and in 5% CO2. For staining with keratin antibodies, cells were fixed for 5 min using ice-cold 99.9% anhydrous methanol (Alfa Aesar, 41838). Non-specific antibody binding sites were blocked by incubating the cover slips for 30 min in blocking buffer (1% BSA, 22.52 mg/ml glycine in PBS with 0.1% Tween (PBS-T)). Next, cover slips were incubated for 1 hr at room temperature with mouse anti-mouse Keratin 14 (LL002, Thermo Fisher, MA5-11599, 1:100 – 1:10), rabbit anti-mouse Keratin 5 (BioLegend, 905503, 1:500) or rabbit anti-mouse Keratin 6a (BioLegend, 905702, 1:500) in 1% BSA in 0.1% PBS-T. It should be noted here that the K6a anti- body used is a polyclonal antibody, which is suspected to bind to the K6b protein as well, due to their high sequence identity (92.6%). After 3x 5 min washing steps in PBS, cover slips were incubated with Cy3 donkey anti-rabbit (Jackson Immuno Research, 711-165-152, 1:100) or FITC donkey anti- mouse (Jackson Immuno Research, 715-095-150, 1:100) secondary antibodies in 1% BSA in 0.1% PBS-T. Cells were washed 3x for 5 min in PBS, before nuclei were stained with Hoechst 33342 (Sigma-Aldrich, B2261, 1:10,000) for 10–20 min at room temperature. After a final wash step 3x for 5 min in PBS, the cover slips were mounted on glass slides with Dako mounting medium (Agilent, S3023) or Prolong Glass Anti-Fade (Thermo Fisher, P36980). Confocal imaging for Figure 1 was car- ried out with a laser scanning confocal microscope (Nikon A1R confocal microscope, Nikon) using an oil immersion objective lens (Plan Apo 60X Oil objective, 1.4 NA, Nikon). Keratin was excited with a 488 nm wavelength laser and the optical sections were imaged at 100 nm intervals. Maximum inten- sity projections of the Z-stacks are presented. Keratin networks for Figure 1—figure supplement 1C were imaged using a spinning disk confocal laser scanning microscope (Olympus IXplore SpinSR10 with YOKOGAWA CSU-W1 spinning disk). 3D confocal stacks were acquired with a UPLSAPO UPlan S Apo 60x/1.3 OIL objective (Olympus). Fluorescent proteins were excited at 405 nm (50 mW, 10% laser power), 488 nm (100 mW, 15% laser power) and 561 nm (100 mW, 5% laser power). Cryo-EM data processing 1860 cryo-EM micrographs acquired from ~20 cells, at a magnification of 22,665 were processed with RELION 2.1 and RELION 3.0 using the helical toolbox (Scheres, 2012; He and Scheres, 2017; Zivanov et al., 2018). At the initial stages of analysis, individual micrographs were excluded when no individual keratin filaments could be identified. Frame-based motion correction and dose-weight- ing were performed using MotionCor2 (Zheng et al., 2017). The contrast transfer function was esti- mated using CTFFIND4 (Rohou and Grigorieff, 2015). Low-quality micrographs showing high defocus, high astigmatism or low resolution (assessed by CTF estimation, information below 7.5 A˚ ) were excluded, resulting in 1763 high-quality micrographs used for further processing steps. Keratin filaments were either picked manually or automatically using the RELION helical toolbox. To gener- ate a template for autopicking, 55,073 keratin particles were picked manually as start-to-end helices, extracted with a box size of 250 pixels (~55 nm) and 2D classified twice to create classes with straight keratin segments. These classes served as a reference for automated picking of 505,211 par- ticles. For manual picking, 298,056 particles were selected as start-to-end helices. Particles were extracted in boxes of 250 pixels, corresponding to ~55 nm, or 164 pix, corresponding to ~36 nm, with an inter-box distance of 50 A˚ . Iterative 2D classification procedures were performed, using a spherical mask of 500 A˚ or 356 A˚ , respectively. Keratin filament segments, 55 nm in length, were classified to yield 305,495 particles in straight classes. Autocorrelation spectra were calculated with MATLAB (2019a, MathWorks). The filament diameter was measured by plotting vertical intensity line-profiles of all classes using MATLAB and measuring the area where the intensity lies above zero. Intensity line-profiles of each class were generated by averaging all lateral sections through the segment. OriginPro 2018 software (OriginLab Corporation) was used to plot the diameter distribu- tion. Instead of plotting the number of classes, which was set to a fixed value, the number of par- ticles in each class was plotted, as it reflects how many particles with a certain diameter are present in the dataset. A mean intensity line-profile for Figure 2C was generated by averaging all classes from Figure 2—figure supplement 1A. Segments with a box size of 36 nm were used for computational filament reconstitution. Filament reconstitution was performed as previously reported (Kronenberg-Tenga et al., 2021) and described below with classes of automatically, as well as manually, picked particles. Minimal apparent persistence length measurements Highly bent keratin filaments were identified in electron micrographs and traced with Fiji using the segmented line tool including spline fit (Schindelin et al., 2012). Minimal filament contour lengths that undergo a 90˚ turn were traced. The persistence length is defined as the distance along a fila- ment at which the tangent-tangent correlation function along the contour length decays, this occurs after a 90˚ turn (Reisner et al., 2012). However, since our sample is out of equilibrium, as individual filaments are entangled in a network and absorbed to the EM grid, and filaments are imaged in 2D, only an apparent persistence length is described. Further, only highly bent filaments were consid- ered in this analysis, yielding a minimal apparent persistence length, as the whole filament popula- tion is diverse and cannot be described as a single state. Cryo-EM and cryo-ET data acquisition y y q The grids were analyzed using a 300 kV Titan Krios electron microscope (Thermo Fisher) equipped with a K2 Summit direct electron detector (Gatan) mounted on a post-column energy filter (Gatan). Cryo-EM micrographs were acquired in zero-loss energy mode using a 20 eV slit. Data were recorded with SerialEM 3.5.8 in low-dose mode (Mastronarde, 2005). Overview images were acquired, in which the filaments were identified prior to data acquisition. Micrographs were acquired at nominal magnifications of 46,511 with a pixel size of 1.075 A˚ , 28,571 with a pixel size of 1.75 A˚ , and 22,665 with a pixel size of 2.206 A˚ . A defocus range between 0.5 and 3.5 mm was cho- sen. Dose-fractionation was used with a frame exposure of 0.2 s with a total exposure time of 10 s (50 frames in total). This corresponds to a total electron dose of ~20 e/A˚ 2 for the 22,665 dataset, ~41 e/A˚ 2 for the 28,571 and ~84 e/A˚ 2 for the 46,511 dataset. In total, data were collected from ~60 ghost cells. Micrographs acquired at a magnification of 22,665 x were used for most experi- ments conducted in this study, as stated below. The datasets acquired at 46,551 and 28,571 magnification were used to visualize the keratin cytoskeleton in closer detail and micrographs from these datasets are shown in Figure 1, Figure 1—figure supplement 1 and Figure 4. g g g pp g Tilt series were acquired in zero-loss energy mode with a 20 eV slit at a nominal magnification of 28,571, resulting in a pixel size of 1.75 A˚ and a defocus of 3 mm. A bidirectional tilt scheme with a tilt range of ± 60˚ and an increment of 3˚ was chosen, corresponding to 41 projections per tilt Weber et al. eLife 2021;10:e70307. DOI: https://doi.org/10.7554/eLife.70307 16 of 23 Research article Research article Research article series and a total accumulative electron dose of ~89 e/A˚ 2. SerialEM 3.5.8 in low dose mode was used for data acquisition (Mastronarde, 2005). series and a total accumulative electron dose of ~89 e/A˚ 2. SerialEM 3.5.8 in low dose mode was used for data acquisition (Mastronarde, 2005). Cryo-EM data processing Actin filaments were processed identically to keratin filaments to guarantee comparability. To generate a template for autopicking, 22,228 actin particles were picked manually as start-to-end helices, extracted with a box size of 164 pixels (~36 nm) and 2D classified twice to create classes with straight actin segments. These classes served as a reference for automated picking of 693,903 particles. Particles were extracted in boxes of 164 pixels, corresponding to ~36 nm, with an inter- box distance of 50 A˚ . Multiple rounds of 2D classification were performed, using a spherical mask of 356 A˚ . A total of 405,044 particles from the highest resolved 2D classes were used for 3D classifica- tion into five classes. The highest resolved 3D class, containing 174,954 particles, was subjected to 3D refinement. 17 of 23 Weber et al. eLife 2021;10:e70307. DOI: https://doi.org/10.7554/eLife.70307 Research article Research article Cell Biology Structural Biology and Molecular Biophysics The final unmasked map showed a resolution of 7.38 A˚ , based on the gold standard Fourier shell correlation (FSC) 0.143 criterion (Rosenthal and Henderson, 2003; Scheres, 2012). The structure was sharpened to 6.13 A˚ using an isotropic B-factor of 276 A˚ 2. Cryo-ET data processing Tilt series were processed using the IMOD workflow, including contrast transfer function (CTF) cor- rection (Kremer et al., 1996). For visualization purposes, a SIRT-like filter according to 10 iterations was applied during tomogram reconstruction. Cellular structures present in the tomograms were manually segmented and visualized using the Amira 5.6.0 software package (Thermo Fisher Scien- tific). Tomogram movies were created using Amira 5.6.0 and Fiji (Schindelin et al., 2012). 710 cross section views of keratin filaments were picked in 21 tomograms and reconstructed as sub-tomo- grams using IMOD. Central 2D slices were extracted from the sub-tomograms and utilized for 2D classification in RELION. To assess the position of the central density within the keratin filament for Figure 4—figure supplement 1E,F, the filament labeled with E in Figure 4D was extracted in silico and rotated into precise 90˚ cross section. Afterwards, 4.2 nm thick slices were projected along the length of the filament. The resulting cross-sectional views were filtered with a gaussian blur using Fiji and analyzed for the position of the central density within the filament tube. Positions were repre- sented by circles using Adobe Illustrator (Adobe Inc). Computational reconstitution of keratin filaments p To generate computationally reconstituted filaments a back-mapping strategy was pursued in MAT- LAB, using the ~36 nm long keratin segments which were used for 2D classification. First, all particles belonging to the same filament were grouped. Filament assignments were made based on the heli- cal tube ID defined by RELION for every particle. Next, all particles belonging to the same filament were sorted in ascending order based on their picking coordinates. Then, their corresponding 2D class images were inversely transformed, so that their orientation matches the original orientation of the raw segments in the cryo-EM micrographs. Next, the 2D class images were plotted at the origi- nal coordinates of the particles. To remove background noise, the classes were masked in the y-direction and only the central 132 A˚ were plotted. Since particles were picked with inter-box dis- tances of 50 A˚ , while 2D classes have a box size of 360 A˚ , neighboring segments would strongly overlap. To avoid this, classes were cropped to not extend into neighboring particle positions, and only a small amount of overlap of <four classes with soft edges was allowed to avoid cropped edges in slightly bent filaments. Reconstituted filaments were normalized to equal intensity. Next, a straightening procedure was applied as previously described to extract, align and straighten the reconstituted filaments (Steinert et al., 1985; Kocsis et al., 1991). Validity of this approach was ensured by using high-resolution actin classes as a control. Analyzing helical and straight-line patterns in individual filaments Plots representing the order of helical and straight segments along individual filaments, represented by colored circles, were generated in MATLAB as previously described (Martins et al., 2021). 2D classes were grouped into helical or straight clusters based on their appearance. Next, each particle within a filament was represented by red or blue circles, depending on whether its corresponding 2D class belonged to the helical or straight cluster. In the analysis seen in Figure 3—figure supple- ment 1B, segments that originate from the same filament are plotted as columns of circles. Seg- ments are sorted in ascending order based on their coordinates along the filament. Funding Funder Grant reference number Author reconstruction of a keratin filament 305,495 uniform keratin segments from 55 nm boxes were selected by 2D classification (Figure 2— figure supplement 1A) and used for 3D reconstruction. To generate a low-resolution 3D filament model, the rotation angle along the filament axis of all particles was randomized to prevent pre- ferred orientations. Next, a filament was reconstructed using relion_reconstruct. The 3D model was visualized using Chimera (Pettersen et al., 2004). Weber et al. eLife 2021;10:e70307. DOI: https://doi.org/10.7554/eLife.70307 18 of 23 Research article Research article Cell Biology Structural Biology and Molecular Biophysics Cell Biology Structural Biology and Molecular Biophysics Acknowledgements The authors thank the Center for Microscopy and Image Analysis at the University of Zurich for pro- viding support and equipment. Funding This research was funded by the Swiss National Science Foundation Grant (31003A_179418). MSW was supported by the Forschungskredit of the University of Zurich (FK-18–041). The Goldman laboratory is supported by grants 5PO1 GM096971 and RO1GM140108 from the National Institutes of Health. Additional information Author contributions Miriam S Weber, Data curation, Formal analysis, Validation, Investigation, Visualization, Methodol- ogy, Writing - original draft, Writing - review and editing; Matthias Eibauer, Data curation, Method- ology, Writing - review and editing; Suganya Sivagurunathan, Data curation; Thomas M Magin, Resources, Writing - review and editing; Robert D Goldman, Ohad Medalia, Conceptualization, Funding acquisition, Project administration, Writing - review and editing Author ORCIDs Author ORCIDs Miriam S Weber https://orcid.org/0000-0003-2125-8222 Ohad Medalia https://orcid.org/0000-0003-0994-2937 Author ORCIDs Miriam S Weber https://orcid.org/0000-0003-2125-8222 Ohad Medalia https://orcid.org/0000-0003-0994-2937 Decision letter and Author response Decision letter https://doi.org/10.7554/eLife.70307.sa1 Author response https://doi.org/10.7554/eLife.70307.sa2 Decision letter and Author response Decision letter https://doi.org/10.7554/eLife.70307.sa1 Author response https://doi.org/10.7554/eLife.70307.sa2 Additional information Funding Funder Grant reference number Author Schweizerischer Nationalfonds zur Fo¨ rderung der Wis- senschaftlichen Forschung 31003A_179418 Ohad Medalia NIH Office of the Director 5PO1 GM096971 Robert D Goldman NIH Office of the Director RO1GM140108 Robert D Goldman The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. Additional files . Transparent reporting form Data availability Data availability Representative cryo-ET data have been deposited in the Electron Microscopy Data Bank under accession codes EMD-12958 and EMD-12959. In addition, data was uploaded to https://doi.org/10. 5061/dryad.gqnk98sn4. The following datasets were generated: The following datasets were generated: The following datasets were generated: Author(s) Year Dataset title Dataset URL Database and Identifier Goldman RD 2021 Structural heterogeneity of cellular K5/K14 filaments as revealed by cryo-electron microscopy https://doi.org/10.5061/ dryad.gqnk98sn4 Dryad Digital Repository, 10.5061/ dryad.gqnk98sn4 Weber MS, Eibauer M, Medalia O 2021 Cryo-tomogram of the K5/K14 keratin network in a keratinocyte ghost cell https://www.ebi.ac.uk/ emdb/entry/EMD-12959 EMBD, EMD- 12959 Weber et al. eLife 2021;10:e70307. DOI: https://doi.org/10.7554/eLife.70307 19 of 23 19 of 23 Research article Research article Research article Cell Biology Structural Biology and Molecular Biophysics Cell Biology Structural Biology and Molecular Biophysics https://www.ebi.ac.uk/ emdb/entry/EMD-12958 EMBD, EMD-12958 Weber MS, Eibauer M, Medalia O 2021 Cryo-tomogram showing the modulation of the K5/K14 keratin network in a keratinocyte ghost cell https://www.ebi.ac.uk/ emdb/entry/EMD-12958 EMBD, EMD-12958 Weber MS, Eibauer M, Medalia O 2021 Cryo-tomogram showing the modulation of the K5/K14 keratin network in a keratinocyte ghost cell bi U, Fowler WE, Rew P, Sun TT. 1983. The fibrillar substructure of keratin filaments unraveled. Journal of Cell iology 97:1131–1143. DOI: https://doi.org/10.1083/jcb.97.4.1131 Aebi U, Fowler WE, Rew P, Sun TT. 1983. The fibrillar substructure of keratin filaments unraveled. Journal of Cell Biology 97:1131–1143. DOI: https://doi.org/10.1083/jcb.97.4.1131 gy g j Astbury WT. 1939. 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Optimal determination of particle orientation, absolute hand, and contrast loss in single-particle electron cryomicroscopy. Journal of Molecular Biology 333:721–745. DOI: https://doi.o 10.1016/j.jmb.2003.07.013, PMID: 14568533 j j Sailer M, Ho¨ hn K, Lu¨ ck S, Schmidt V, Beil M, Walther P. 2010. Novel electron tomographic methods to study the morphology of keratin filament networks. Microscopy and Microanalysis 16:462–471. DOI: https://doi.org/10. 1017/S1431927610093657, PMID: 20598205 Scheres SHW. 2012. RELION: Implementation of a Bayesian approach to cryo-EM structure determination. Journal of Structural Biology 180:519–530. DOI: https://doi.org/10.1016/j.jsb.2012.09.006 Scheres SHW. 2012. RELION: Implementation of a Bayesian approach to cryo-EM structure determination. Journal of Structural Biology 180:519–530. DOI: https://doi.org/10.1016/j.jsb.2012.09.006 Scheres SH. 2016. Processing of structurally heterogeneous Cryo-EM data in RELION. 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Nucleus 8:475–481. DOI: https://doi.org/10.1080/19491034.2017.1337622 Turgay Y, Medalia O. 2017. The structure of lamin filaments in somatic cells as revealed by cryo-electron tomography. Nucleus 8:475–481. DOI: https://doi.org/10.1080/19491034.2017.1337622 Vikstrom KL Borisy GG Goldman RD 1989 Dynamic aspects of intermediate filament networks in BHK 21 cell g p y p g Vikstrom KL, Borisy GG, Goldman RD. 1989. Dynamic aspects of intermediate filament networks in BHK-21 ce PNAS 86:549–553. DOI: https://doi.org/10.1073/pnas.86.2.549 , y , y p PNAS 86:549–553. DOI: https://doi.org/10.1073/pnas.86.2.549 Watts A. 2002. Direct studies of ligand-receptor interactions and ion channel blocking (Review). Molecular Membrane Biology 19:267–275. DOI: https://doi.org/10.1080/0968768021000035113 Watts NR, Jones LN, Cheng N, Wall JS, Parry DA, Steven AC. 2002. Cryo-electron microscopy of trichocyte (hard alpha-keratin) intermediate filaments reveals a low-density core. 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Supplementary Table 2 from Unphosphorylated STAT1 Promotes Sarcoma Development through Repressing Expression of Fas and Bad and Conferring Apoptotic Resistance
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Supplemental Table 2. PCR Primers Bcl2l1-F 5'- TCCACTCACCTCTCACCTCCC -3' Bcl2l1-R 5'- TCCCTCACACACCCCTCTCTG -3' Ccnd1-F 5'- CCTCACTCCCTGGCTTGCTC -3' CCnd1-R 5'- GTCACTATTTCATCCCTACCGCTG -3' Fas-F 5'- AACCAGACTTCTACTGCGATTCTCC -3 Fas-R 5'- CCTTTTCCAGCACTTTCTTTTCCG -3 JAK1-F 5'- CAAGTCCCTGAAGCCTGAGAGTG -3' JAK1-R 5'- GAGAACCCAAGTAGTCCATCCCC -3' Bad-F 5'- AAGGATGAGCGATGAGTTTGAGG -3' Bad-B 5'- ACAGGACAGCACCCAGACCC -3' Bcl-xL-F 5'- GCAGGTAGTGAATGAACTCTTTCGG -3' Bcl-xL-R 5'- CAGAACCACACCAGCCACAGTC -3' STAT3-F 5'- CGAGAGCAGCAAAGAAGGAGGG -3' STAT3-R 5'- GGGACATCGGCAGGTCAATG -3'b -Actin-F 5'-ATTGTTACCAACTGGGACGACATG-3'b -Actin-B 5'-CTTCATGAGGTAGTCTGTCAGGTC-3' STAT1-ChIP-F 5'-TTAGGAAGAAAAGGAAAAAAATTAG-3' STAT1-ChIP-R 5'-CTAATTTTTTTCCTTTTCTTCCTAA-3' Supplemental Table 2. PCR Primers Bcl2l1-F 5'- TCCACTCACCTCTCACCTCCC -3' Bcl2l1-R 5'- TCCCTCACACACCCCTCTCTG -3' Ccnd1-F 5'- CCTCACTCCCTGGCTTGCTC -3' CCnd1-R 5'- GTCACTATTTCATCCCTACCGCTG -3' Fas-F 5'- AACCAGACTTCTACTGCGATTCTCC -3 Fas-R 5'- CCTTTTCCAGCACTTTCTTTTCCG -3 JAK1-F 5'- CAAGTCCCTGAAGCCTGAGAGTG -3' JAK1-R 5'- GAGAACCCAAGTAGTCCATCCCC -3' Bad-F 5'- AAGGATGAGCGATGAGTTTGAGG -3' Bad-B 5'- ACAGGACAGCACCCAGACCC -3' Bcl-xL-F 5'- GCAGGTAGTGAATGAACTCTTTCGG -3' Bcl-xL-R 5'- CAGAACCACACCAGCCACAGTC -3' STAT3-F 5'- CGAGAGCAGCAAAGAAGGAGGG -3' STAT3-R 5'- GGGACATCGGCAGGTCAATG -3'b -Actin-F 5'-ATTGTTACCAACTGGGACGACATG-3'b -Actin-B 5'-CTTCATGAGGTAGTCTGTCAGGTC-3' STAT1-ChIP-F 5'-TTAGGAAGAAAAGGAAAAAAATTAG-3' STAT1-ChIP-R 5'-CTAATTTTTTTCCTTTTCTTCCTAA-3' Supplemental Table 2. PCR Primers Supplemental Table 2. PCR Primers
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Right ventricular (RV) velocity measurements using high resolution spiral myocardial phase velocity mapping (PVM)
Journal of cardiovascular magnetic resonance
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Simpson et al. Journal of Cardiovascular Magnetic Resonance 2013, 15(Suppl 1):P133 Simpson et al. Journal of Cardiovascular Magnetic Resonance 2013, 15(Suppl 1):P133 http://www.jcmr-online.com/content/15/S1/P133 Background PVM is capable of accurately and reproducibly measur- ing myocardial velocities in the LV [1]. However, analyz- ing RV motion is more difficult because of the thinness of the free wall and its asymmetrical geometry. Cur- rently, the most commonly used technique for assessing RV velocities and function is Tissue Doppler Imaging (TDI) where the high temporal resolution allows the detailed analysis of fine features of motion (small peaks in velocity during isovolumic contraction (IC) and isovo- lumic relaxation (IR) [2], for example). However TDI is restricted by inadequate acoustic windows and cannot comprehensively assess velocities over the entire RV. This study aims to establish that high resolution spiral PVM is capable of reproducibly measuring RV free wall velocities and that, consequently, it has a future role in assessing RV function. Results The high spatial resolution allowed the analysis of the thin RV free wall in all slices and in all volunteers on both occasions - example data are shown in Figure 1. Mean+/-SD velocities and TTP velocities for systolic, early diastolic and late diastolic peaks in the basal, mid and apical short axis slices are shown in Table 1, and are highly consistent between subjects. Inter-study reproducibilities of peak systolic, early diastolic and late diastolic velocities were excellent (0.21±1.31, 0.15 ±1.13and 0.69±2.39cm/s respectively) as were the inter- study reproducibilities of the corresponding TTPs (11.6 ±78.2, -13.1±30.6 and -6.67±19.9 ms respectively). The high temporal resolution of the sequence allowed detec- tion of IC and IR (Figure 1) in 22 out of 40 velocity- time curves. © 2013 Simpson et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Conclusions PVM can be used to measure RV free wall velocities with a high degree of reproducibility. PVM is potentially a more flexible and comprehensive modality for asses- sing regional RV motion than TDI. Methods K-space is covered with 13 spiral interleaves (12 ms duration, TR 21ms). Navigator-gated reference and velo- city-encoded data (25 cm/s through-plane) are acquired in consecutive cardiac cycles following a single dummy cycle. The acquired spatial resolution is 1.4 x 1.4 x 8 mm (reconstructed to 0.7 x 0.7 mm). Retrospective gat- ing allows full coverage of the cardiac cycle with 60 phases per RR-interval (reconstructed temporal resolu- tion 14-20 ms). Basal, mid and apical short-axis slices were acquired in 10 healthy volunteers on a Siemens Skyra 3 Tesla scanner. The mid slice was also acquired on a second day to assess inter-study reproducibility. Longitudinal velocities averaged over the RV free wall Right ventricular (RV) velocity measurements using high resolution spiral myocardial phase velocity mapping (PVM) Robin Simpson1,2*, Jennifer Keegan1,2, David N Firmin1,2 From 16th Annual SCMR Scientific Sessions San Francisco, CA, USA. 31 January - 3 February 2013 Robin Simpson1,2*, Jennifer Keegan1,2, David N Firmin1,2 were extracted and peak velocities and times to those peak (TTP) velocities were measured and normalized to a fixed systolic (350 ms) and diastolic length (650 ms). The reproducibility of mid slice values was determined as the mean (+/- SD) of the signed differences of the two measurements made on different days. © 2013 Simpson et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 1NIHR Cardiovascular Biomedical Research Unit, Royal Brompton Hospital, London, UK Full list of author information is available at the end of the article Author details 1 Published: 30 January 2013 Funding h h The authors acknowledge the support of Heart Research UK, Imperial College London and NIHR Royal Bromp- ton Cardiovascular Biomedical Research Unit. Page 2 of 2 Simpson et al. Journal of Cardiovascular Magnetic Resonance 2013, 15(Suppl 1):P133 http://www.jcmr-online.com/content/15/S1/P133 Figure 1 Peak velocities are at the lower end of the healthy subject range defined by previous TDI studies [2]; this is likely to be due to reduced temporal resolution and to averaging the longitudinal velocities over the entire free wall instead of in a small region of interest. (a) Example magnitude images (left) and velocity maps (right) at the time of peak systolic (S, top) and peak early diastolic (ED, bottom) velocity. (b) Example RV wall average velocity-time curve. The 5 phases of right ventricular motion (isovolumic contraction (IC), systole (S), isovolumic relaxation (IR), early diastole (ED) and late diastole (LD)) observed with TDI [2] can clearly be seen. Author details 1NIHR Cardiovascular Biomedical Research Unit, Royal Brompton Hospital, London, UK. 2Imperial College, London, UK. Published: 30 January 2013 References 1. Foll : . Circ 2010. 2. Horton : . JASE 2009. doi:10.1186/1532-429X-15-S1-P133 Cite this article as: Simpson et al.: Right ventricular (RV) velocity measurements using high resolution spiral myocardial phase velocity mapping (PVM). Journal of Cardiovascular Magnetic Resonance 2013 15 (Suppl 1):P133. Table 1 Mean +/- SD peak and TTP values for the ten volunteers (Continued) Peak early diastolic -5.67±1.29 487.3±24.3 Peak late diastolic -2.42±2.52 884.6±24.7 APEX Peak systolic 2.98±0.82 188.6±115.0 Peak early diastolic -3.34±0.89 508.6±35.8 Peak late diastolic -1.87±0.64 900.1±28.2 Table 1 Mean +/- SD peak and TTP values for the ten volunteers (Continued) Peak early diastolic -5.67±1.29 487.3±24.3 Peak late diastolic -2.42±2.52 884.6±24.7 APEX Peak systolic 2.98±0.82 188.6±115.0 Peak early diastolic -3.34±0.89 508.6±35.8 Peak late diastolic -1.87±0.64 900.1±28.2 Table 1 Mean +/- SD peak and TTP values for the ten volunteers (Continued) References doi:10.1186/1532-429X-15-S1-P133 Cite this article as: Simpson et al.: Right ventricular (RV) velocity measurements using high resolution spiral myocardial phase velocity mapping (PVM). Journal of Cardiovascular Magnetic Resonance 2013 15 (Suppl 1):P133. Figure 1 Peak velocities are at the lower end of the healthy subject range defined by previous TDI studies [2]; this is likely to be due to reduced temporal resolution and to averaging the longitudinal velocities over the entire free wall instead of in a small region of interest. (a) Example magnitude images (left) and velocity maps (right) at the time of peak systolic (S, top) and peak early diastolic (ED, bottom) velocity. (b) Example RV wall average velocity-time curve. The 5 phases of right ventricular motion (isovolumic contraction (IC), systole (S), isovolumic relaxation (IR), early diastole (ED) and late diastole (LD)) observed with TDI [2] can clearly be seen. Table 1 Mean +/- SD peak and TTP values for the ten volunteers Parameter Mean+/-SD velocity for 10 volunteers (cm/s) Mean+/-SD normalized TTP for 10 volunteers (ms) BASE Peak systolic 7.29±1.65 146.6±50.3 Peak early diastolic -7.83±2.00 495.6±22.4 Peak late diastolic -4.61±2.10 903.1±19.5 MID Peak systolic 5.08±1.67 152.7±72.2 Table 1 Mean +/- SD peak and TTP values for the ten volunteers Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of:
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EVALUATION OF HYPOLIPIDEMIC ACTIVITY OF ETHANOLIC EXTRACT OF LEAVES OF MORINGA CONCANENSIS NIMMO IN GUINEA PIGS
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ISSN: 2320-5407 ISSN: 2320-5407 Int. J. Adv. Res. 10(01), 859-872 Journal Homepage: -www.journalijar.com Article DOI:10.21474/IJAR01/14117 DOI URL: http://dx.doi.org/10.21474/IJAR01/14117 Dr. Archana S. Patel, Dr. Viren Naik and Dr. Prashant M. Parmar ……………………………………………………………… Objective & Scope: To evaluate the hypolipidemic activity of ethanolic extract of leaves of Moringa concanensisnimmo in guinea pigs p g Methods: 64 Guinea pigs of either gender were randomly divided into 8 groups. Hyperlipidemia was produced by giving cholesterol powder (500 mg/kg) for 60 days. Rosuvastatin 1.5 mg/kg served as active control group. While Moringa was given as 200 mg/kg and 400 mg/kg for 30 days as preventive and curative groups for 0 to 30 days and 31 to 60 days respectively. Distilled water with normal diet for 30 dys was served as Normal control group. The preventive and curative effect of ethanolic extract of Moringa were assessed by serological and histopathological parameter. Results: Cholesterol administration produced increased level of Lipids and liver enzymes along with histopathological alteration of liver and thoracic aorta. Preventive and therapeutic administration of 200 mg/kg and 400 mg/kg ethanolic extract of Moringa resulted into a significant decrease of lipid profile and restoration of histopathological architecture of liver and thorasic aorta as compared to disease control group. Conclusion: Because of antioxidant properties, curative and preventive administration of 400 and 200 mg/kg ethanolic extract of Moringa exerts lipid lowering effect and can be used to treat and prevent dyslipidemia. Introduction:- Introduction:- Hyperlipidemia is a medical condition characterized by an increase in one or more of the plasma lipids, including triglycerides, cholesterol, cholesterol esters, phospholipids and or plasma lipoproteins including very low-density lipoprotein and low-density lipoprotein along with reduced high-density lipoprotein levels. This elevation of plasma lipids is among the leading risk factors associated with cardiovascular diseases (Phogat, P et al., 2010). Hyperlipidemia is an important risk factor in development of atherosclerosis and heart disease. Risk factors for hyperlipidemia may be male > 45 years old, female > 55 old, family history of CHD (coronary heart disease), hypertension, low HDL-C (<40 mg/dl), Smoking genetic factors or by generalized metabolic disorders like diabetes mellitus, excessive alcohol intake, hypothyroidism, or primary biliary cirrhosis. (VERMA, N et al., 2013). Cardiovascular diseases, especially coronary heart disease (CHD), are epidemic in India. The Registrar General of India reported that CHD led to 17% of total deaths and 26% of adult deaths in 2001-2003, which increased to 23% 859 Corresponding Author: -Dr. Prashant M. Parmar Int. J. Adv. Res. 10(01), 859-872 Int. J. Adv. Res. 10(01), 859-872 ISSN: 2320-5407 of total and 32% of adult deaths in 2010-2013. The World Health Organization (WHO) and Global Burden of Disease Study also have highlighted increasing trends in years of life lost (YLLs) and disability-adjusted life years (DALYs) from CHD in India. (DBIDC, 2018). The association between CVD and higher low-density lipoprotein cholesterol (LDL-C) is well known and it plays a major role in the development of CVD, independently (Mizuno, 2012; Li M, 2013). Dyslipidaemia induces oxidative stress in liver, heart, and kidney by reactive oxygen species which plays a significant role in the etiology of atherosclerosis and CVD (Suanarunsawat, 2011). Health Organization (WHO) of life lost (YLLs) and disab on between CVD and higher n the development of CVD, i liver, heart, and kidney by r and CVD (Suanarunsawat, 20 ompounds that shown their va peutically effective as compar ngs to the family Moringacea attumurungai or Peyimurunga intense horseradish odor has d in different types of ailme esic, antimicrobial, reduce c calories (Vidya T J et al., 2 nd hyperlipidemia. The studie y done but the study on hypo study to evaluate hypolipidem macology, Government Medic Animal Ethics Committee of s dy. Guinea pigs were housed i al animals were kept under no le) throughout the study perio ments. Introduction:- Herbal products have many biological active phytochemical compounds that shown their valuable affects on CVD by hypolipidemic action. It is also cost effective, safe and therapeutically effective as compared to present allopathic drugs (Stephen Leeder et al., 2004). Moringa concanensis belongs to the family Moringaceae. It is abundantly seen in Perambalur district of Tamilnadu commonly known as Kattumurungai or Peyimurungai. This small tree with strong central trunk, thick bark, bipinnate leaves and with an intense horseradish odor has vast therapeutic uses in traditional medicine. The different parts of the plants are used in different types of ailments and various human diseases such as anti inflammatory, antifertility agent, analgesic, antimicrobial, reduce cholesterol, skin tumor, diabetes, and eye care etc.( Joyet al., 2013). Herbal products may be useful as a safest way to burn excess calories (Vidya T J et al., 2002). In India, roots of Moringa concanensis are used as a folk medicine for obesity and hyperlipidemia. The studies on anti-inflammatory, analgesic, antimicrobial effect of Moringa concanensis already done but the study on hypolipidemic action of the leaves are lacking. On the basis of these, we have planned this study to evaluate hypolipidemic activity of Leaves of Moringa concanensis Nimmo in guinea pigs. Normal diet: Mixtures of cereals and pulses (60% wheat plus 35% bengal gram plus 15% peanuts) in the morning- Total 50 grams/animal In evening- Green leafy vegetables -30 grams / animal. Material &Methodology:- The study was conducted at Animal room, Department of Pharmacology, Government Medical College, Bhavnagar, Gujarat, after approval (IAEC No. 49/2016) from Institutional Animal Ethics Committee of same institute. CPCSEA guidelines were followed during animal experiments of our study. Guinea pigs were housed in a stainless steel cages and acclimatization for 15 days before experiment. Experimental animals were kept under normal light, temperature and humidity conditions (i.e. 24 ± 2°C; 12-hour light/dark cycle) throughout the study period. Standard laboratory feed and water ad libitum were provided throughout the experiments. Animal groups: g p 64 healthy adult guinea pigs of 400- 800 grams, either sex were taken. Weighing and sex determination was done of each animal. After selecting the animals, they were kept in the animal room of the department of pharmacology, Government Medical College, Bhavnagar, Gujarat for 15 days for acclimatization. After that, the guinea pigs were divided in to eight groups as following: g g p g 1. Group 1 (control - Normal diet) were receive mixtures of cereals and pulses in the morning –total 50 grams/animal and evening-Green leafy vegetables-30 grams/animal.(Distilled Water for 60 days). g g p g 1. Group 1 (control - Normal diet) were receive mixtures of cereals and pulses in the morning –total 50 grams/animal and evening-Green leafy vegetables-30 grams/animal.(Distilled Water for 60 days). g g y g g y 2. Group 2: (High fat diet ) were receive cholesterol powder (500 mg/kg, 60 days) mixed in the wheat and green gram flour by 40 grams of the above mixtures of the normal diet /animal in morning and in evening green leafy vegetables-30 grams/animal. 2. Group 2: (High fat diet ) were receive cholesterol powder (500 mg/kg, 60 days) mixed in the wheat and green gram flour by 40 grams of the above mixtures of the normal diet /animal in morning and in evening green leafy vegetables-30 grams/animal. g g 3. Group 3: (Extract Control): will receive normal diet(for 60 days) + 400 mg/kg ethanolic extract of leaves of Moringa concanensis Nimmo ( for 31 to 60 days). 4. Group 4: (Low dose): were receive high fat diet(for 60 days) + 200 mg/kg ethanolic extract of leaves of Moringa concanensis Nimmo(for 31 to 60 days). 5. Group 5: (Active Control): were receive high fat diet(for 60 days) + 1.5 mg/kg, Rosuvastatin (for 31 to 60 days). y 6. Group 6: (Preventive low dose ) : were receive high fat diet + 200 mg/kg ethanolic extract of leaves of Moringa concanensis Nimmo (Both for 30 days). In evening: Green leafy vegetables – 30 gm/kg animal. During the acclimatization period all the animals were given normal diet and water ad libitum. In addition animals of the high fat diet groups (group- 2, 4, and 6), also given flour of the mixtures of wheat and bengal gram (70% wheat plus 30% bengal gram, 10 g/animal) to acclimatized the flour. After overnight fasting, baseline blood sample and blood sample on day 30 was collected from lateral saphenous vein of hind paw of each animal. Blood samples were analyzed for Liver function test serum lipid profile and cardiac enzymes in the Clinical Biochemistry Laboratory of our institute which is accredited by National Accreditation Board for Testing and Calibration Laboratories (NABL). After baseline blood collection, the animals were divided as in above mentioned groups. Diet was given according torespective group diet throughout study period of 60 days in 1, 2, 3, 4, and 5 group and 30 days in group 8 as mentioned above. During the last 30 days of the experiment, distilled water was daily fed to animals of group 1. Animals of group 3, 4, 5and were given ethanolic extract of leaves of Moringa concanensis Nimmo in the dose of 200 mg/kg (low dose), respectively for 30 days. Group 5 animals were given rosuvastatin calcium in the dose of 1.5 mg/kg for last 30 days. Distilled water and above all the drugs were given orally by gavages feeding tube daily in the morning in the fasting state to ensure maximum absorption. Animals of all groups were sacrificed after blood collection from the saphenous vein in the overnight fasting stat at the end of 60 days. Blood was sent for the analysis of the Liver function test serum lipid profile and cardiac enzymes. We obtained the liver and thoracic aorta from each animal of above seven groups for histopathological analysis which was done by senior faculty from Pathology department of our institute. S li id fil S l l d f t t l h l t l HDL C t i l id LDL C d VLDL • Biochemical Evaluation of Liver and Cardiac Injury: Alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) levels in serum were analyzed to evaluate any functionalabnormality of liver. Cardiac function was assessed by measuring serum level of lactate dehydrogenase (LDH). Statistical analysis y All parameters were expressed as Mean ± SEM. One-way Analysis of Variance (ANOVA) followed by Tukey- Kramer Multiple comparison test was used to compare inter group differences of lipid profile, liver enzymes, cardiac enzymes and extent of body weight gain at the end of 60 days. Mean differences of lipid profile, liver enzymes, cardiac enzymes and extent of body weight gain at the end of 60 days in each group of animals were compared by paired t-test. The value of P< 0.05 was considered significant. The statistical calculations were done using GraphPad InStat, Demo version 3.06. In evening: • Weighing of the animals: Weight of each animal was recorded before and after study to confirm any effect of Ethanolic extract of leaves of Moringa concanensis Nimmo on the weight of the animals. High fat diet: In morning: In morning: Cholesterol powder (500 mg/kg) mixed in the wheat and bengal gram flour followed by 40 grams of the above mixtures of the normal diet/animal Diet composition: Normal diet: Diet composition: Normal diet: Normal diet: Mixtures of cereals and pulses (60% wheat plus 35% bengal gram plus 15% peanuts) in the morning- Total 50 grams/animal In evening- Green leafy vegetables -30 grams / animal. 860 Int. J. Adv. Res. 10(01), 859-872 ISSN: 2320-5407 High fat diet: In morning: Cholesterol powder (500 mg/kg) mixed in the wheat and bengal gram flour followed by 40 grams of the above mixtures of the normal diet/animal Discussion:- In the present study, we evaluate the hypolipidemic action of Moringa concanensis Nimmo. in guinea pig because the metabolism of lipoproteins in guinea pig is closer to the human. Various research shows that guinea pigs are commendable models to evaluate hypolipidemic activity and lipoprotein metabolism (Fernandez, 2006). Result of present study showed that 60 day feeding of high fat diet resulted in increased serum lipid profile and histopathological changes like mid zone and peripheral diffuse ballooning degeneration and fatty changes like fat deposition (Figure 1 B) and liver cell degeneration in liver and aorta. This increase and change in the histology of aorta and liver were in accordance with the previous experimental studies with high fat diet (Wang Y, 1866; Suanarunsawat, 2011). We select study period sixty days which was sufficient to produce fatty changes in guinea pigs and it’s also supported by previous studies (Ahmad, 2001). Various pre-clinical, clinical and epidemiological studies had proved that high level of plasma cholesterol is a major risk factor for development of coronary heart disease. Atherosclerosis is a preliminary lipid disorder that affects the major arteries and many factors contributing to its etiology, among them diabetes, hypertension, smoking, glucocorticoid, diet and psychological factors (V.V. Pande et al., 2008) and sedentary life style are the major one (Stephanie E et al., 2006). Foam cells and fatty streaks were formed in vessels by LDL-C oxidation which is trademark of early atherosclerosis (An S J et al., 2013). Several studies have shown that the improvement and incidence of CVD are associated with lowering the levels of total cholesterol and LDL-C (Nohara R et al., 2013). So, agents who have free radical scavenging and serum cholesterol lowering activity, they would be given importance in management of hyperlipidemia. Because of modern lifestyle, hypercholesterolemic patients could not be successfully managed in spite of much intervention. Thus, herbal medicines have got attention to treat hyperlipidemia, because of safe and cost-effective alternative (Suanarunsawat T et al., 2011). In the present study, administrations of ethanolic extract of leaves of Moringa concanensis Nimmo (400 mg/kg) in guinea pigs restored serum total cholesterol, serum triglyceride, LDL-C and VLDL-C in treatment group as compared to disease control group at 60 days (Table 1) and reduced decreased in serum total cholesterol, Triglyceride levels, LDL-C, VLDL-C levels as compared to Disease Control group at 30 days. Effect of Moringa concanensis Nimmo on Weight: g g ncrease in mean body weight of all groups at the end of 60 days was shown in table 3 but, extent of not statistically significant among the groups. body weight of all groups at the end of 60 days was shown in table 3 but, extent of weight gain was ignificant among the groups. g y p g Cholesterol Powder administration for 60 days in disease control group liver showed diffuse areas ballooning degeneration and macrovascular and microvascular fat deposition in hepatocytes of varying degrees (grade 3+, 4+), fatty changes (midzone and periportal) and congestion of central vein and hepatic sinusoids in all animals, histological study of aorta shows focal areas of foamy changes of varying degrees (grade 2+, 3+ ) in tunica media and tunica intima compared to normal control group. Restoration of macrovascular and microvascular fat deposition in liver and focal areas of foamy changes in aorta were noted in active control groups; Moringa concanensis Nimmo treatment group (Low dose and high dose) as well as in Moringa concanensis Nimmo preventive group (Low dose and high dose). as compared to disease control group.(Figure 1 A - G) Effect of Moringa concanensis Nimmo on Serum lipid profile: Effect of Moringa concanensis Nimmo on Serum lipid profile: d d d 6 d l f li id fil i h di Effect of Moringa concanensis Nimmo on Serum lipid profile: 0 day, 30 day and 60 days values of serum lipid profile in each diet treatment group were shown in table 1. In high fat diet fed group significant increase in the serum total cholesterol, serum triglyceride, LDL-C, VLDL-C level (P< 0.05) at 30 and 60 days as compared to 0 days, no significant changes in serum HDL-C level in disease control group (Table 1). In treatment low dose group, serum LDL-C level (P<0.05) significantly restored but level of serum total cholesterol (P<0.05), Triglyceride, HDL-C and VLDL-C, levels were restored but did not reach to statistical significant, as compared to Disease Control group at 60 days (Table 1). In treatment high dose group significantly restored the serum Total Cholesterol, Triglyceride, LDL-C and VLDL-C (P<0.05),but serum HDL-C level was restored but did not reach to statistical significant as compared to disease control group at 60 days (Table 1). In preventive low dose group levels of total cholesterol, Triglyceride, LDL-C, and VLDL-C were not significantly decreased and level of HDL-C was not significantly increased as compared to Disease Control group at 30 days (Table 1). Active Control group shown significantly restored in the serum total cholesterol, Triglyceride and LDL-C, VLDL-C, levels (P< 0.05) but serum HDL-C levels are restored but did not reach to statistical significant (P< 0.05) as compared to Disease Control group at 60 days (Table 1). 861 Int. J. Adv. Res. 10(01), 859-872 ISSN: 2320-5407 Int. J. Adv. Res. 10(01), 859-872 Effect of Moringa concanensis Nimmo on Liver Enzymes: g y There was significant increased in AST, ALP, LDH and ALT (P< 0.05) in disease control group at 60 days compared to 30 days figure (Table 2). In treatment low dose group LDH level (P<0.05) significantly restored and levels of AST, ALP, and ALT were restored but did not reach to statistical significant, as compared to disease control group at 60 days (Table 2). In preventive low dose group, levels of AST, ALP, LDH and ALT were not significantly decreased, as compared to disease control group at 30 days (Table 2). Active Control group shown significantly restored in AST, ALP, LDH and ALT levels (P<0.05) as compared to disease control group at 60 days (Table 2). Discussion:- Ethanolic extract of leaves of Moringa concanensis Nimmo shown dose dependent action on lipid profile more restoring effect on serum total cholesterol, serum triglyceride, LDL-C, VLDL-C in 200 mg/kg dose of ethanolic extract of leaves of Moringa concanensis Nimmo in treatment group compared to disease control group at 60 days. 200 mg/kg of ethanolic 862 Int. J. Adv. Res. 10(01), 859-872 Int. J. Adv. Res. 10(01), 859-872 ISSN: 2320-5407 extract of leaves of Moringa concanensis Nimmo has decreased in serum total cholesterol, serum triglyceride, LDL- C, VLDL-C in preventive group. Hypercholesterolemia endangers primary organs like liver and heart. High fat diet significantly increases retention of lipid in liver, followed by hepatic steatosis and reduce hepatic functions (T. Suanarunsawatet al., 2010). Our study results show that high fat diet suppressed hepatic functions which were expressed as augmentation of serum levels of AST, ALT, LDH, and ALP in disease control group as compared to normal control group significantly ethanolic extract of leavesof Moringa concanensis Nimmo (200 mg/kg) significantly reduce AST, ALT, LDH, and ALP level. where as compared to high fat diet group. Histology of liver show improvement in hepatocytes and decreased lipid content in high fat diet fed groups treated with ethanolic extract of leaves of Moringa concanensis Nimmo (Table 1). Medicinal plants with Ethanolic compound have the ability of scavenging hydroxyl radical, superoxide anion radicals and lipid peroxyradicals. compounds and flavanoid were present in our extract ethanolic extract of leaves of Moringa concanensis Nimmo. Saponins from plants have long been employed for their detergent properties. They are used as mild detergents and in intracellular histochemistry staining to allow antibody access to intracellular proteins The ethanolic leaves extract showed precipitation followed by emulsion formation. In medicine, they are used in hyperglycaemia, antioxidant, anti-cancer, anti-inflammatory and weight loss etc. (Bhamadevi . D et al.,2015). Rosuvastatin in the dose of 1.5 mg/kg for thirty days in high fat diet fed group animals also counteracts the rise in serum level of total cholesterol, triglycerides, LDL-C, VLDL-C and resist the fatty changes in the liver induced by high fat diet. It also increases level of HDL-C. High fat diet group treated with rosuvastatin has increased AST and ALP level at the end of sixty days as compared to baseline level. Discussion:- Thus, the present study shows significant lipid lowering and antioxidant activities of ethanolic extract of leaves of Moringa concanensis Nimmo that might be due to their flavanoid and Saponins compounds. We did not know types of mechanism and ethanolic compounds responsible for lipid lowering actions which were the limitations of our study. Conclusion:- Treatment and preventive administration of 200 mg/kg ethanolic extract of leaves of Moringa concanensis Nimmo resulted in to a significant decreased of lipid profile and liver enzymes parameters and histopathological architecture of liver and thoracic aorta as compared to disease control group. the present study shows significant lipid lowering and antioxidant activities of ethanolic extract of leaves of Moringa concanensis Nimmo that might be due to their flavanoid and Saponins compounds and can be use to prevent and treat cholesterol powder induced hypercholesterolemia in guinea pigs. 8. Mizuno K, Nakaya N, Teramoto T, et al. 2012. Usefulness of LDLC- related parameters to predict cardiovascular risk and effect of pravastatin in mild-to-moderate hypercholesterolemia. J AtherosclerThromb, 19(2): 176-185. 7. Li M, Li Y, Liu J., 2013 Metabolic syndrome with hyperglycemia and the risk of ischemic stroke. Med J, 54(2), pp. 283-287. 6. Joy, A.E., Kunhikatta, S.B. and Manikkoth, S., 2013. Anti-convulsant activity of ethanolic extract of Moringa concanensis leaves in Swiss albino mice. Archives of Medicine and Health sciences, 1(1), pp.6. References:- 1. Ahmad-Raus RR, Elda-Surhaida ES, Abdul-Latif, Mohammad JJ, 2001. Lowering of lipid composition in aorta of guinea pigs by Curcuma Domestica. BMC Complementary and Alternative Medicine, 1, pp.6 1. Ahmad-Raus RR, Elda-Surhaida ES, Abdul-Latif, Mohammad JJ, 2001. Lowering of lipid composition in aorta of guinea pigs by Curcuma Domestica. BMC Complementary and Alternative Medicine, 1, pp.6 2. An S J, Jung U J, Choi M S, et al., 2013. Functions of monocyte chemotactic protein-3 in transgenic mice fed a high-fat, high-cholesterol diet. J Microbiol Biotechnol, 23(3): pp.405-413. 2. An S J, Jung U J, Choi M S, et al., 2013. Functions of monocyte chemotactic protein-3 in transgenic mice fed a high-fat, high-cholesterol diet. J Microbiol Biotechnol, 23(3): pp.405-413. 3. Bhamadevi D, 2015. Screening of Bioactive Compounds From The Leaves of Moringa Concanensis Nimmo. International Journal of Innovative Research in Science Engineering and Technology. (10), pp.9702-9. 3. Bhamadevi D, 2015. Screening of Bioactive Compounds From The Leaves of Moringa Concanensis Nimmo. International Journal of Innovative Research in Science Engineering and Technology. (10), pp.9702-9. 4. Fernandez ML, Volek JS, 2006. Guinea pigs: a suitable animal model to study lipoprotein metabolism, atherosclerosis and inflammation. NutrMetab (Lond), 3(3), pp.17-23. 5. India State-Level Disease Burden Initiative Diabetes Collaborators. The increasing burden of diabetes and variations among the states of India: the Global Burden of Disease Study 1990-2016. Lancet Glob Health. 2018 Dec;6(12):e1352-e1362. 5. India State-Level Disease Burden Initiative Diabetes Collaborators. The increasing burden of diabetes and variations among the states of India: the Global Burden of Disease Study 1990-2016. Lancet Glob Health. 2018 Dec;6(12):e1352-e1362. 6. Joy, A.E., Kunhikatta, S.B. and Manikkoth, S., 2013. Anti-convulsant activity of ethanolic extract of Moringa concanensis leaves in Swiss albino mice. Archives of Medicine and Health sciences, 1(1), pp.6. 6. Joy, A.E., Kunhikatta, S.B. and Manikkoth, S., 2013. Anti-convulsant activity of ethanolic extract of Moringa concanensis leaves in Swiss albino mice. Archives of Medicine and Health sciences, 1(1), pp.6. 7. Li M, Li Y, Liu J., 2013 Metabolic syndrome with hyperglycemia and the risk of ischemic stroke. Med J, 54(2), pp. 283-287. 7. Li M, Li Y, Liu J., 2013 Metabolic syndrome with hyperglycemia and the risk of ischemic stroke. Med J, 54(2), pp. 283-287. 8. Mizuno K, Nakaya N, Teramoto T, et al. 2012. 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Lipid lowering and antioxidative activities of aqueous extracts of Ocimum sanctum L. leaves in rats fed with a high-cholesterol diet . Oxid Med Cell Longev. q g g 15. VERMA, N., 2013. Introduction to hyperlipidemia and its treatment: A review. International journal of current pharmaceutical research, 9(1), pp.6-14. p , ( ), pp 16. Vidya TJ, Kulkarni KS, 2002. Using herbal tea in the treatment modality: special reference to slirntea in overweight individuals .Anc Sci Life, 21(3),pp. 202-204. g ( ) pp 17. Wang Y., Ausman L.M., Russell R.M., Greenberg A.S., Wang X.D., 2008: J. Nutr. 138, pp.186 864 Int. J. Adv. Res. 10(01), 859-872 ISSN: 2320-5407 Table 1: Comparison of lipid parameters within (0 day vs. 30 day and 30 day vs. 60 day) and between study groups (at 60 day). References:- Groups Time Period Day Total Cholesterol (mg/dl) Triglycerides (mg/dl) HDL Cholesterol (mg/dl) LDL Cholesterol (mg/dl) VLDL Cholesterol (mg/dl) Group 1 0 46.6 ± 2.99 55.87 ± 3.74 4.5 ± 0.46 22.87±1.47 18.42 ± 0.95 30 46.3 ± 3.00 56.5 ± 3.68 4.5 ± 0.53 22.5 ± 1.45 18.77 ± 1.00 60 45.8 ± 2.95 57.00 ± 3.73 4.37 ± 0.32 22.62 ± 1.58 18.45 ± 0.94 Group 2 0 41.27 ± 2.09 58 ± 3.49 4.87 ± 0.61 22.62 ± 1.58 17.23 ± 0.84 30 58.7 ± 0.89* 67 ± 4.07* 4.50± 0.56 43.00± 1.87* 24.13 ± 0.94* 60 85.2 ± 2.85# 80.37± 3.59# 5.12 ± 0.54 61.37± 1.55# 27.63 ± 0.92# Group 3 0 39.09 ± 1.61 59 ± 1.29 5.12 ± 0.44 22.12± 1.39 21.28 ± 1.30 30 39.67 ± 1.62 58.87 ± 1.30 4.75 ± 0.36 21.5 ± 1.58 20.71 ± 1.34 60 39.35 ± 1.55 57.87 ± 1.18 5.12 ± 0.47 20.87 ±1.48 20.38 ± 1.37 Group 4 0 38.17 ± 1.31 57.62 ± 2.17 4.87 ± 0.39 25.25± 2.00 18.46 ± 0.49 30 51.00 ± 1.29* 71.12 ± 2.48* 5.12 ± 0.58 48.25± 2.16* 22.41 ± 0.93 * 60 48.20 ± 1.60$ 69.87 ± 2.36$ 5.55 ± 0.70 46.25± 2.27#$ 21. 67 ± 0.75$ Group 5 0 40.97 ± 1.84 54.5 ± 1.95 5.25 ± 0.41 22.37 ± 1.94 17.80 ± 0.86 30 40.72 ± 1.78$ 53.75 ± 1.43$ 5.62 ± 0.49 21.75 ± 1.64$ 17.10± 0.85$ 60 39.63 ±1.71 55.0 ± 1.72 4.37± 0.49 26.12 ± 1.67 17.21 ± 0.30 Group 6 0 57.55 ± 1.09* 68.12 ± 1.68* 5.37 ±0.62* 51.87 ± 3.13* 22.53 ± 0.66* 30 43.00± 1.06#$ 60.00 ± 1.73#$ 7.37 ±0.62#$ 43.5 ± 2.73#$ 18.02 ± 0.34#$ Table 1: Comparison of lipid parameters within (0 day vs. 30 day and 30 day vs. 60 day) and between study groups (at 60 day). Data are expressed as Mean ± SEM. *P< 0.05, paired t-test, intra group comparison between 0 and 30 days. #P< 0.05 paired t-test, intra group comparison between 30 and 60 days. $P< 0.05: as compared to disease control group using ANOVA followed by Tukey-Kramer Multiple comparison test (inter group comparison), LDL = low density lipoprotein, VLDL = very low- density lipoprotein, HDL = high density lipoprotein 865 Int. J. Adv. Res. 10(01), 859-872 ISSN: 2320-5407 Table 2: Comparison of Liver enzyme parameters within (0 day vs. References:- 30 day and 30 days vs. 60 days) and between study groups (at 60day). Groups Time period Groups Time period Groups Time period Group 1 0 60 ± 6.19 86.87 ± 4.02 86.25 ± 2.81 53.25 ± 1.98 30 61.37 ± 6.27 86.88 ± 4.12 86.75 ± 2.61 53.87 ± 2.31 60 61 ± 6.21 86.89 ± 3.92 87.25 ± 2.58 54.25 ± 2.28 Group 2 0 59.37 ± 4.41 86.37 ± 5.15 90 ± 3.74 56.12 ± 2.19 30 97.37 ± 3.19* 125.62 ± 1.97* 134 ± 2.43* 92.37 ± 2.83* 60 124.8 ± 2.76# 150.12 ± 4.81# 159 ± 2.40# 110 ± 3.52# Group 3 0 57.50 ± 3.30 89.25 ± 6.13 89.25 ± 3.70 55.25 ± 1.55 30 57 ± 2.24 88.87 ± 6.50 88.00 ± 3.80 56 ± 1.72 60 58.75 ± 3.07 89.12 ± 6.26 88.87 ± 3.60 55.37± 1.47 Group 4 0 58.7 ± 2.64 83.50 ± 4.92 92.00 ± 3.16 56.62 ± 3.32 30 92.87 ± 4.17* 134.50 ± 2.66* 145 ± 2.59* 93.87 ± 3.46* 60 92.00 ± 4.32$ 133.12 ± 2.51$ 148 ± 2.73$ 92.87± 3.61$ Group 5 0 57.7 ± 4.28 86.85 ± 5.70 91.62±4.50 56.37 ± 2.84 30 93.25 ± 3.52* 133.12 ± 3.38* 137.12±4.40 95.87 ± 1.46 60 79.25 ± 3.32#$ 107 ± 2.65#$ 121.12 ± 3.19#$ 79.81 ± 1.73#$ Group 6 0 56.75 ± 3.63 86.75 ± 5.62 88.62 ± 3.60 52.25 ± 1.77 30 56.25 ± 4.06$ 86 ± 5.10$ 87.75 ± 3.70$ 51.37 ± 1.91$ Table 2: Comparison of Liver enzyme parameters within (0 day vs. 30 day and 30 days vs. 60 days) and between study groups (at 60day). Data are expressed as Mean ± SEM. *P< 0.05: paired t-test, intra group comparison between 0 and 30 days. #P< 0.05 paired t-test, intra group comparison between 30 and 60 days. $P< 0.05: ANOVA followed by Tukey-Kramer Multiple comparison test (inter group comparison) ALT: Alanine aminotransferase, AST: Aspartate aminotransferase, ALP: Alkaline phosphatase, LDH: Lactate dehydrogenase. Data are expressed as Mean ± SEM.pairedt-test for intra group comparison between 0 and 30 days. paired t-test for intra group comparison between 30 and 60 days. No statistically significant. Data are expressed as Mean ± SEM. $P<0.05 as compared to normal control and ٭P<0.05 as compared to disease control group using Kruskal Wallis followed by Dunn’s multiple comparison test. Figure A to E: Histopathology evaluations: Figure A: Group. 1 Normal Diet Group A M I L Guinea pig normal Aorta A: Adventitia tunica M: Media tunica I : Intima tunica L : Lumen Guinea pig normal liver C: Central vein H: Hepatocyte S : Sinusoid C I H S Figure A: Group. 1 Normal Diet Group A M I L Guinea pig normal Aorta A: Adventitia tunica M: Media tunica I : Intima tunica L : Lumen Figure A: Group. 1 Normal Diet Group C I H S References:- Table 3: Comparison of Weight in grams parameters Groups Weight of animal in grams 0 Day 30 Day 60 Day Group 1 566.3 ±11.6 567.5 ± 11.29 568 ± 11.20 Group 2 681.7 ± 15.06 692 ± 15.87 700.8 ± 16.02 Group 3 682.5 ± 17.87 679 ± 18.23 689.5 ± 18.04 Group 4 676.75 ± 20.2 682 ± 19.06 690.3 ± 19.28 Group 5 563.7 ± 20.26 580.3 ± 20.08 588.87 ± 19.44 Group 6 608.75 ± 21.58 611.82 ± 21.54 --- Table 3: Comparison of Weight in grams parameters Table 3: Comparison of Weight in grams parameters Data are expressed as Mean ± SEM.pairedt-test for intra group comparison between 0 and 30 days. paired t-test for intra group comparison between 30 and 60 days. No statistically significant. 866 Int. J. Adv. Res. 10(01), 859-872 ISSN: 2320-5407 Table 4: Comparison of histopathological grading of Liver and Aorta between study groups at the end of experiment: Study Group Liver Aorta Microvascular or Macrovascular fat disposition Grade Degeneration Grade Fat deposition in aorta Group 1 (Normal control) 0 0 0 Group 2 ( Disease Control) 4.06 ± 0.26$ 3.37 ± 0.18$ 3.25 ± 0.16$ Group 3 (Extrect Control) 0 0 0 Group 4 (Treatment Low Dose) 1.87 ± 0.22* 1.82 ± 0.22* 1.62 ± 0.18* Group 5 (Active Control) 0.25 ± 0.16* 0.75 ± 0.25* 0.5 ± 0.18* Group 6 (Preventive low dose) 1.86 ± 0.28* 1.26 ± 0.46* 1.37 ± 0.18* Table 4: Comparison of histopathological grading of Liver and Aorta between study groups at the end of experiment: Data are expressed as Mean ± SEM. $P<0.05 as compared to normal control and ٭P<0.05 as compared to disease control group using Kruskal Wallis followed by Dunn’s multiple comparison test. 867 Int. J. Adv. Res. 10(01), 859-872 ISSN: 2320-5407 Guinea pig Aorta Black arrows shows foamy changes in intima and media (Grade 3+) Guinea pig Liver Round shows diffuse areas of ballooning degeneration (Grade 4+) Guinea pig Liver Guinea pig normal liver C: Central vein H: Hepatocyte S : Sinusoid 868 ISSN: 2320-5407 Int. J. Adv. Res. 10(01), 859-872 Figure B: Group 2. High Fat diet group Guinea pig Aorta Black arrows shows foamy changes in intima and media (Grade 3+) Guinea pig Liver Round shows diffuse areas of ballooning degeneration (Grade 4+) Figure B: Group 2. High Fat diet group Guinea pig Aorta Black arrows shows foamy changes in intima and media (Grade 3+) Figure B: Group 2. High Fat diet group Guinea pig Liver Black round shows fatty changes seen only Ballooning degeneration (Grade 2+) Guinea pig Liver Guinea pig Liver Guinea pig Liver Black arrows shows foamy changes in intima and media (Grade 1+) Black arrows shows foamy changes in intima and media (Grade 1+) Round shows diffuse areas of ballooning degeneration (Grade 4+) 869 ISSN: 2320-5407 Int. J. Adv. Res. 10(01), 859-872 Figure C: Group 4. Treatment low dose Guinea pig Aorta Black arrows shows foamy changes in intima and media (Grade 1+) Guinea pig Liver Black round shows fatty changes seen only Ballooning degeneration (Grade 2+) Figure C: Group 4. Treatment low dose Guinea pig Aorta Black round shows fatty changes seen only Ballooning degeneration (Grade 2+) 870 ISSN: 2320-5407 Int. J. Adv. Res. 10(01), 859-872 Figure D: Group 5. Active control group Guinea pig liver No histological changes seen Guinea pig Aorta No histological Changes seen Guinea pig Liver Black round no fatty changes seen only degeneration (Grade 2+) with repaired and regeneration process. Guinea pig Liver Guinea pig Aorta Black arrows shows foamy changes in intima and media (Grade 1+) Black arrows shows foamy changes in intima and media (Grade 1+) Black arrows shows foamy changes in intima and media (Grade 1+) Figure D: Group 5. Active control group Figure D: Group 5. Active control group Figure D: Group 5. Active control group Guinea pig liver No histological changes seen Guinea pig Aorta No histological Changes seen Guinea pig Aorta No histological Changes seen Guinea pig liver No histological changes seen G N se 871 : 2320-5407 Int. J. Adv. Res. 10(01), 859-872 Figure E: Group 6. Preventive Low dose Guinea pig Aorta Black arrows shows foamy changes in intima and media (Grade 1+) Guinea pig Liver Black round no fatty changes seen only degeneration (Grade 2+) with repaired and regeneration process. SN: 2320-5407 Int. J. Adv. Res. 10(01), 859-872 Figure E: Group 6. Preventive Low dose Guinea pig Aorta Black arrows shows foamy changes in intima and media (Grade 1+) Guinea pig Liver Black round no fatty changes seen only degeneration (Grade 2+) with repaired and regeneration process. Int. J. Adv. Res. 10(01), 859-872 ISSN: 2320-5407 Figure E: Group 6. Preventive Low dose Guinea pig Aorta Black arrows shows foamy changes in intima and media (Grade 1+) Gui Bla foam and Black round no fatty changes seen only degeneration (Grade 2+) with repaired and regeneration process. 872
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A Digital Solution for an Advanced Breast Tumor Board: Pilot Application Cocreation and Implementation Study
JMIR cancer
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5,695
Abstract Background: Cancer treatment is constantly evolving toward a more personalized approach based on clinical features, imaging, and genomic pathology information. To ensure the best care for patients, multidisciplinary teams (MDTs) meet regularly to review cases. Notwithstanding, the conduction of MDT meetings is challenged by medical time restrictions, the unavailability of critical MDT members, and the additional administrative work required. These issues may result in members missing information during MDT meetings and postponed treatment. To explore and facilitate improved approaches for MDT meetings in France, using advanced breast cancers (ABCs) as a model, Centre Léon Bérard (CLB) and ROCHE Diagnostics cocreated an MDT application prototype based on structured data. Objective: In this paper, we want to describe how an application prototype was implemented for ABC MDT meetings at CLB to support clinical decisions. Methods: Prior to the initiation of cocreation activities, an organizational audit of ABC MDT meetings identified the following four key phases for the MDT: the instigation, preparation, execution, and follow-up phases. For each phase, challenges and opportunities were identified that informed the new cocreation activities. The MDT application prototype became software that integrated structured data from medical files for the visualization of the neoplastic history of a patient. The digital solution was assessed via a before-and-after audit and a survey questionnaire that was administered to health care professionals involved in the MDT. Results: The ABC MDT meeting audit was carried out during 3 MDT meetings, including 70 discussions of clinical cases before and 58 such discussions after the implementation of the MDT application prototype. We identified 33 pain points related to the preparation, execution, and follow-up phases. No issues were identified related to the instigation phase. Difficulties were grouped as follows: process challenges (n=18), technological limitations (n=9), and the lack of available resources (n=6). The preparation of MDT meetings was the phase in which the most issues (n=16) were seen. A repeat audit, which was undertaken after the implementation of the MDT application, demonstrated that (1) the discussion times per case remained comparable (2 min and 22 s vs 2 min and 14 s), (2) the capture of MDT decisions improved (all cases included a therapeutic proposal), (3) there was no postponement of treatment decisions, and (4) the mean confidence of medical oncologists in decision-making increased. Hodroj et al Hodroj et al JMIR CANCER Original Paper Corresponding Author: Corresponding Author: Pierre Heudel, MD Centre Leon Berard 28 avenue Laënnec Lyon, 69008 France Phone: 33 0478782958 Email: pierreetienne.heudel@lyon.unicancer.fr Corresponding Author: Pierre Heudel, MD Centre Leon Berard 28 avenue Laënnec Lyon, 69008 France Phone: 33 0478782958 Email: pierreetienne.heudel@lyon.unicancer.fr https://cancer.jmir.org/2023/1/e39072 A Digital Solution for an Advanced Breast Tumor Board: Pilot Application Cocreation and Implementation Study Khalil Hodroj1, MD; David Pellegrin2; Cindy Menard1; Thomas Bachelot1, MD; Thierry Durand1; Philippe Toussaint1, MD; Armelle Dufresne1, MD; Benoite Mery1, MD; Olivier Tredan1, MD; Thibaut Goulvent2, PhD; Pierre Heudel1, MD 1Centre Leon Berard, Lyon, France 2Roche Healthcare Development Division - Roche Diagnostics France, Meylan, Meylan, France The MDT Application Prototype The MDT application prototype serves as a platform that optimizes the presentation of patient cases for the purposes of MDT discussion and decision-making. The application allows for the importing and exporting of structured data based on the local EMR, imaging, and genomic pathology information. The data within the MDT application conforms to international terminologies. In order to better facilitate patient case review during MDT meetings, ROCHE Diagnostics and Centre Léon Bérard (CLB) coimagined a new MDT application prototype. This digital application was tested during ABC MDT meetings, beginning in January 2021. In this paper, we discuss how this application was implemented within ABC MDT meetings at CLB and how the MDT application prototype supported clinical decisions based on accurate clinical histories. Two factors—authentication and a personal Répertoire Partagé des Professionnels de Santé (RPPS) number (shared directory of health care professionals)—are needed to access MDT application. KEYWORDS digital health; multidisciplinary meeting; advanced breast cancer; cancer; breast cancer; tumor; clinician; confidence; treatment; pathology; genomic; care; patient; software; data; neoplastic; pain; follow-up; electronic medical records; records digital health; multidisciplinary meeting; advanced breast cancer; cancer; breast cancer; tumor; clinician; confidence; treatment; pathology; genomic; care; patient; software; data; neoplastic; pain; follow-up; electronic medical records; records patient is discussed. Medical histories are shared by the medical oncologist in charge of the patients, and data are exposed thanks to the electronic medical record (EMR). MDT advice is audio-recorded. After the MDT meeting (follow-up phase), the medical assistant transcribes the medical advice based on the recording, and a report is added to the EMR after a final medical validation. Abstract Conclusions: The introduction of the MDT application prototype at CLB to support the ABC MDT seemed to improve the quality of and confidence in clinical decisions. The integration of an MDT application with the local electronic medical record and the utilization of structured data conforming to international terminologies could enable a national network of MDTs to support sustained improvements to patient care. (JMIR Cancer 2023;9:e39072) doi: 10.2196/39072 (JMIR Cancer 2023;9:e39072) doi: 10.2196/39072 (JMIR Cancer 2023;9:e39072) doi: 10.2196/39072 JMIR Cancer 2023 | vol. 9 | e39072 | p. 1 (page number not for citation purposes) JMIR Cancer 2023 | vol. 9 | e39072 | p. 1 (page number not for citation purposes) https://cancer.jmir.org/2023/1/e39072 https://cancer.jmir.org/2023/1/e39072 XSL•FO RenderX Hodroj et al JMIR CANCER Introduction Cancer care has been improved by many new therapeutic approaches in the last decade, with the emergence of immune checkpoint blockade treatment and new targeted therapies [1-4]. The wide spread of new treatments can be seen for advanced breast cancer (ABC), with the use of cyclin-dependent kinase 4 and 6 inhibitors in hormone receptor–positive ABC [5-7] and the development of many new drugs that target human epidermal growth factor receptor 2 (HER2) [8,9], resulting in an update to the classification of HER2-positive ABC [10]. These new approaches are implemented in clinical routines, and to ensure that all patients receive timely care, multidisciplinary team (MDT) meetings have been introduced in Europe, the United States of America, and most high-income countries [11-14]. Since the French law of March 4, 2002, the MDT approach in oncology has been structured with quality point requirements. MDT work is generally associated with better adherence to updated clinical guidelines [15], and to conduct such work, a detailed medical history of all presented patients should be highlighted to make the best clinical decision. However, the conduction of MDT meetings can be challenged by time restrictions, the unavailability of all members, and increased administrative work [16,17]. The development and uses of new applications have already been tested for daily health decisions [18-21]. Cocreation of the MDT Application Version 1 of the MDT application was prototyped based on the challenges and needs identified during the ABC MDT audit. Subsequently, version 1 of the MDT application was used routinely for 2 months during the weekly ABC MDT meetings. After each MDT meeting, a debriefing session was held by the medical team and application development team to refine the prototype based on continuous user evaluations. After the initial 2 months, the cybersecurity for version 1 of the MDT application was evaluated before implementing version 2 of the MDT application for an ongoing routine use test. The application was developed by an external company, in collaboration with medical oncologists of a French comprehensive cancer center. Assessment Usual MDT Meeting Organization for ABC JMIR Cancer 2023 | vol. 9 | e39072 | p. 2 (page number not for citation purposes) https://cancer.jmir.org/2023/1/e39072 Usual MDT Meeting Organization for ABC A questionnaire was sent to 15 health care professionals involved in the ABC MDT. The items assessed precisely were the level of satisfaction, the level of confidence in decision-making, and an open question on what would have driven any level change. and determine the types of pain points (those linked to problems related to the organization of the process, those linked to the technological limits of the tools used, or those linked to a lack of human resources). Similarly, a before-and-after survey was used to assess the user experience among the health care professionals involved in the ABC MDT. A questionnaire was sent to 15 health care professionals involved in the ABC MDT. The items assessed precisely were the level of satisfaction, the level of confidence in decision-making, and an open question on what would have driven any level change. Preparation Phase In the preparation phase, a nurse navigator uses structured data from the MDT application to systematically prepare a patient case. The Pain Points and Needs Identified for the ABC MDT An audit was carried out prospectively before the implementation of version 2 of the digital solution during 3 MDT meetings, including 70 clinical case discussions. The first audit of the original ABC MDT approach identified 33 discrete pain points related to the preparation (n=16), execution (n=11), and post–tumor board (n=6) phases. No issues were identified related to the instigation phase; however, for the other three phases (the preparation, execution, and post-MDT meeting phases), multiple difficulties were identified and subsequently classified as process, technology, or resource issues. In the preparation phase, 8 difficulties were identified with processes (eg, the lack of a systematic approach to informing the medical question and the overbooking of cases with a lack of transparency on time available), 5 pain points were related to technology, and 3 pain points concerned resources. In the execution phase, 6 pain points were related to processes, 3 were related to technology, and 2 were related to resources. In the follow-up phase, there were 4 pain points related to processes, 1 was related to technology, and 1 was related to resources (Table 1). Post–Tumor Board Phase An autogenerated MDT report in PDF format is added to the EMR at the conclusion of the meetings. Subsequently, call-to-action notifications are sent to accountable individuals. Patient data remain in the MDT application in a structured format for potential use in future meetings and for audit and reporting purposes. An overview of the new process is shown in Figure 1. Instigation Phase No issues were identified for this phase. The process for this phase remained the same; a medical doctor informs the medical assistant office of the need for a patient to be registered on UltraGenda for discussion at an upcoming MDT meeting. Usual MDT Meeting Organization for ABC An audit was carried out prospectively before and after the implementation of version 2 of the digital solution. The cases discussed before and after using the application prototype consisted of ABC cases only and had the same complexity level. The items assessed precisely were the total duration of an MDT meeting, the estimated time lost searching for information in the EMR, the average discussion time per patient, the percentage of clinical cases that were postponed due to a lack of information, the percentage of clinical cases that were registered but already discussed previously, the percentage of clinical cases that were registered but postponed due to a lack of time, the percentage of files processed, and the number of clinical cases that were discussed but not recorded. Based on this audit, a detailed assessment was carried out in order to determine in which phases of the process pain points were identified (the instigation, preparation, execution, or post–tumor board phase) MDT meetings are mandatory for all patients with cancer in France. ABC MDT meetings were selected as a model for evaluation and cocreation activities. The following four distinct phases were identified: (1) the instigation phase, (2) the preparation phase, (3) the execution phase, and (4) the follow-up phase. In the first phase, a medical oncologist informs the medical assistant office that an MDT discussion is needed for a patient, who is then registered on UltraGenda (UltraGenda; instigation phase). UltraGenda is a medical appointment scheduling software used at CLB [22]. Based on time availability, the patient’s medical history is ideally prepared by medical oncologists or residents (preparation phase). The completion of this task may facilitate an MDT decision. In the execution phase of the MDT meeting, based on the UltraGenda list, each XSL•FO RenderX JMIR CANCER Hodroj et al application. The MDT application was implemented in accordance with current regulations. application. The MDT application was implemented in accordance with current regulations. application. The MDT application was implemented in accordance with current regulations. and determine the types of pain points (those linked to problems related to the organization of the process, those linked to the technological limits of the tools used, or those linked to a lack of human resources). Similarly, a before-and-after survey was used to assess the user experience among the health care professionals involved in the ABC MDT. Ethics Approval This study was approved in October 2019 by the local data protection officer, on behalf of French regulatory authorities (Commission Nationale de l’Informatique et des Libertés), in accordance with the MR004 methodology (reference number: H001 – 002). This study adhered to the European laws for the protection of personal data (General Data Protection Regulation). All patients were informed of the possibility of their health data being used for research purposes, and none expressed an opposition to this possibility. The implementation of the MDT application in the ABC MDT meetings did not result in changes to the rules for the application’s use; at least three different medical specialists are required to discuss each case and share the conclusions of the MDT, and a personal RPPS number must be used to access the Table 1. Pain point distribution by tumor board phase. Total, N Follow-up phase Execution phase Preparation phase Instigation phase Pain point type 18 4 6 8 0 Process, n 6 1 2 3 0 Resources, n 9 1 3 5 0 Technology, n 33 6 11 16 0 All pain points, N a single-slide timeline visualization of the medical history, patient characteristics, and previous treatments, superseding the use of the EMR for case presentation. The documentation of case decisions is now captured, structured, and validated within the MDT application, through which an autogenerated MDT report is created as a permanent record. This replaces the use of audio-recorded case decisions. a single-slide timeline visualization of the medical history, patient characteristics, and previous treatments, superseding the use of the EMR for case presentation. The documentation of case decisions is now captured, structured, and validated within the MDT application, through which an autogenerated MDT report is created as a permanent record. This replaces the use of audio-recorded case decisions. New Approach for Tumor Boards Involving a Cocreated MDT Application JMIR Cancer 2023 | vol. 9 | e39072 | p. 3 (page number not for citation purposes) Execution Phase In the execution phase, patients are discussed by the MDT based on lists generated by UltraGenda. The MDT application presents JMIR Cancer 2023 | vol. 9 | e39072 | p. 3 (page number not for citation purposes) https://cancer.jmir.org/2023/1/e39072 XSL•FO RenderX XSL•FO RenderX XSL•FO RenderX Hodroj et al JMIR CANCER Figure 1. Diagram of the new process for MDT meeting organization via the MDT application. EMR: electronic medical record; MD: medical doctor; MDT: multidisciplinary team; TB: tumor board. MDT: multidisciplinary team; TB: tumor board. MDTs require time and coordination for a specialist group of health care professionals to meet regularly, as well as additional time to prepare cases [15]. Considering the increasing number of patients and the increasing complexity of the clinical cases discussed, it appears that the average discussion time for a clinical case is around 5 minutes [23]. This proves the need for intelligent computing systems that integrate and analyze clinical data from the EMR to enable better clinical decision-making. User Feedback Assessments A before-and-after survey was used to assess user experience. The web-based questionnaire was sent to 15 health care professionals involved in the ABC MDT meetings, and of these 15, 8 (53%) responded. After the introduction of the MDT application, the mean level of satisfaction (a score out of 5) improved from 3.4 to 4. In addition, the mean confidence in decision-making (a score out of 10) improved from 5.6 to 8. The main drivers for this were the standardized presentation of cases and patient history preparation by an oncology nurse navigator or by oncology residents. The main change enabled by the new process was the systematic preparation of patient medical histories by the oncology nurse navigator or by oncology residents in a structured format within the MDT application. Further, the automatic generation of the MDT decisions removed the need for audio recordings of decisions and additional work to manually record the conclusions. Improvements Made by the ABC MDT After the Implementation of the New Process Involving the MDT Application A second audit was carried out prospectively after the implementation of version 2 of the digital solution during 3 MDT meetings, including 58 clinical cases. After the implementation of the MTD application, the time dedicated to patient case discussion slightly decreased, and the percentage of cases for which a therapeutic recommendation was made improved. The mean discussion times per patient were comparable (legacy approach: 2 min and 22 s; new approach: 2 min and 14 s). The total time per meeting dedicated to case discussions fell from 53 minutes and 20 seconds to 42 minutes and 40 seconds; however, this was predominantly driven by the lower average case numbers per meeting (22.7 cases vs 16.3 cases). Most interestingly, no case postponements occurred after the introduction of the MDT application, whereas the legacy process had an average case postponement rate of 31%. In CLB, the ABC MDT conducted an audit to optimize its functioning. This assessment identified 33 pains points that were used to inform the development of a new process for ABC MDT work. Difficulties concerned the process (18/33, 55%), the technology (9/33, 27%), and the lack of available resources (6/33, 18%; Table 1). Based on these observations, ROCHE Diagnostics and CLB imagined a new process for the ABC MDT meetings that would be enabled by a dedicated MDT digital application. The introduction of the MDT application into the MDT meetings improved the likelihood of reaching a decision, as this resulted in discussions only for cases where all the required information was available. Moreover, user feedback showed that participants had increased confidence in the decisions made. It is likely that this was due to the improved presentation of data on the MDT application dashboard, as it displays a single-slide timeline visualization of previous treatments, tumors, and patient characteristics, and its use replaces the time-consuming and frustrating process of searching for various key information within distributed reports that the EMR may or may not contain. https://cancer.jmir.org/2023/1/e39072 Discussion Optimal decisions for patients with cancer have been related to MDT care [11]. Since its implementation as a regular practice, MDT meetings have shown an impact on management plans, patients, and process outcomes [14]. Nevertheless, successful The main limitation of our results is that the MDT application has only been tested in ABC MDT meetings, limiting its https://cancer.jmir.org/2023/1/e39072 JMIR Cancer 2023 | vol. 9 | e39072 | p. 4 (page number not for citation purposes) JMIR Cancer 2023 | vol. 9 | e39072 | p. 4 (page number not for citation purposes) XSL•FO RenderX XSL•FO RenderX JMIR CANCER Hodroj et al implementation for localized breast cancer boards or other metastatic histology boards. Digital solutions for MDT meetings have already been shown to significantly reduce the overall case preparation time [24]. Moreover, our study shows that an MDT application has the potential to improve MDTs’ confidence in making the best decisions for patients. Further work is needed to assess whether the use of an MDT application improves the implementation of decisions and results in better clinical outcomes. implementation for localized breast cancer boards or other metastatic histology boards. Digital solutions for MDT meetings have already been shown to significantly reduce the overall case preparation time [24]. Moreover, our study shows that an MDT application has the potential to improve MDTs’ confidence in making the best decisions for patients. Further work is needed to assess whether the use of an MDT application improves the implementation of decisions and results in better clinical outcomes. medical reports. It is envisioned that over time, an MDT application could serve a national network for rare tumors, such as the one supported by the French National Cancer Institute (Institut National du Cancer). This network provides diagnostic expertise and aims to improve the care of patients with rare tumors by using referral MDT boards. It can also facilitate recruitment for clinical trials that are dedicated to only rare cancers and involve international efforts. MDT meetings are important elements in the management of patients with cancer. However, the number and complexity of the clinical cases treated make organizational and technological development necessary for being able to meet medical and administrative needs. A precise evaluation of ABC MDT practices allowed for the coconstruction of an MDT application that improved the confidence of clinicians in their decisions while structuring health data. Data Availability Data Availability The data sets generated and analyzed during this study are available from the corresponding author on reasonable request. The data sets generated and analyzed during this study are available from the corresponding author o Discussion A benefit of an MDT application that collects structured clinical data and conforms to internationally accepted terminologies is its ability to generate a real-world data set, which could be used to answer additional research questions in the future [25]. 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JMIR CANCER [doi: 10.1016/j.bulcan.2016.10.001] [Medline: 27816168] Abbreviations ABC: advanced breast cancer CLB: Centre Léon Bérard ConSoRe: Continuum Soins Recherche EMR: electronic medical record HER2: human epidermal growth factor receptor 2 MDT: multidisciplinary team RPPS: Répertoire Partagé des Professionnels de Santé Abbreviations ABC: advanced breast cancer CLB: Centre Léon Bérard ConSoRe: Continuum Soins Recherche EMR: electronic medical record HER2: human epidermal growth factor receptor 2 MDT: multidisciplinary team RPPS: Répertoire Partagé des Professionnels de Santé JMIR Cancer 2023 | vol. 9 | e39072 | p. 6 (page number not for citation purposes) JMIR Cancer 2023 | vol. 9 | e39072 | p. 7 (page number not for citation purposes) Abbreviations JMIR Cancer 2023 | vol. 9 | e39072 | p. 6 (page number not for citation purposes) https://cancer.jmir.org/2023/1/e39072 XSL•FO RenderX JMIR CANCER Hodroj et al Edited by A Mavragani; submitted 27.04.22; peer-reviewed by L Guo, K Le Du; comments to author 05.01.23; revised ve 25.01.23; accepted 16.02.23; published 18.05.23 Please cite as: Hodroj K, Pellegrin D, Menard C, Bachelot T, Durand T, Toussaint P, Dufresne A, Mery B, Tredan O, Goulvent T, Heudel P A Digital Solution for an Advanced Breast Tumor Board: Pilot Application Cocreation and Implementation Study JMIR Cancer 2023;9:e39072 URL: https://cancer.jmir.org/2023/1/e39072 doi: 10.2196/39072 PMID: ©Khalil Hodroj, David Pellegrin, Cindy Menard, Thomas Bachelot, Thierry Durand, Philippe Toussaint, Armelle Dufresne, Benoite Mery, Olivier Tredan, Thibaut Goulvent, Pierre Heudel. Originally published in JMIR Cancer (https://cancer.jmir.org), 18.05.2023. This is an open-access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work, first published in JMIR Cancer, is properly cited. The complete bibliographic information, a link to the original publication on https://cancer.jmir.org/, as well as this copyright and license information must be included. JMIR Cancer 2023 | vol. 9 | e39072 | p. 7 (page number not for citation purposes) JMIR Cancer 2023 | vol. 9 | e39072 | p. 7 (page number not for citation purposes) https://cancer.jmir.org/2023/1/e39072 XSL•FO RenderX XSL•FO RenderX
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Supplementary Figure 2 from SOCS3 Transactivation by PPARγ Prevents IL-17–Driven Cancer Growth
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Supplemental Figure 2 30 40 DHA-enriched diet Control diet r % 10 20 Molar 16:0 16:1 18:0 18:1 18:2N-6 20:4N-6 20:5N-3 22:6N-3 EPA DHA Supplemental Figure 2 Supplemental Figure 2 30 40 DHA-enriched diet Control diet r % 10 20 Molar 16:0 16:1 18:0 18:1 18:2N-6 20:4N-6 20:5N-3 22:6N-3 EPA DHA Supplemental Figure 2 30 40 DHA-enriched diet Control diet r % 10 20 Molar 16:0 16:1 18:0 18:1 18:2N-6 20:4N-6 20:5N-3 22:6N-3 EPA DHA
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A Little Good Is Good Enough: Ethical Consumption, Cheap Excuses, and Moral Self-Licensing
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RESEARCH ARTICLE A little good is good enough: Ethical consumption, cheap excuses, and moral self- licensing a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 Jannis Engel1‡, Nora SzechID1,2,3‡* 1 Department of Economics, Karlsruhe Institute of Technology (KIT), Karlsruhe,Germany, 2 Berlin Social Science Center (WZB), Berlin, Germany, 3 CESifo, Munich, Germany Jannis Engel1‡, Nora SzechID1,2,3‡* Jannis Engel1‡, Nora SzechID1,2,3‡* 1 Department of Economics, Karlsruhe Institute of Technology (KIT), Karlsruhe,Germany, 2 Berlin Social Science Center (WZB), Berlin, Germany, 3 CESifo, Munich, Germany Jannis Engel ‡, Nora SzechID , , ‡* 1 Department of Economics, Karlsruhe Institute of Technology (KIT), Karlsruhe,Germany, 2 Berlin Social Science Center (WZB), Berlin, Germany, 3 CESifo, Munich, Germany 1 Department of Economics, Karlsruhe Institute of Technology (KIT), Karlsruhe,Germany, 2 Berlin Social Science Center (WZB), Berlin, Germany, 3 CESifo, Munich, Germany ‡ These authors are co-first authors on this work. * nora.szech@kit.edu ‡ These authors are co-first authors on this work. ‡ These authors are co-first authors on this work. * nora.szech@kit.edu * nora.szech@kit.edu a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 RESEARCH ARTICLE Editor: Baogui Xin, Shandong University of Science and Technology, CHINA Received: August 13, 2019 Accepted: December 10, 2019 Published: January 15, 2020 Copyright: © 2020 Engel, Szech. 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. Abstract a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 This paper explores the role of cheap excuses in product choice. If agents feel that they fulfill one ethical aspect, they may care less about other independent ethical facets within product choice. Choosing a product that fulfills one ethical aspect may then suffice for maintaining a high moral self-image in agents and render it easier to ignore other ethically relevant aspects they would otherwise care about more. The use of such cheap excuses could thus lead to a “static moral self-licensing” effect, and this would extend the logic of the well-known dynamic moral self-licensing. Our experimental study provides empirical evidence that the static counterpart of moral self-licensing exists. Furthermore, effects spill over to unrelated, ethi- cally relevant contexts later in time. Thus, static moral self-licensing and dynamic moral self- licensing can exist next to each other. However, it is critical that agents do not feel that they fulfilled an ethical criterion out of sheer luck, that is, agents need some room so that they can attribute the ethical improvement at least partly to themselves. Outsiders, although monetarily incentivized for correct estimates, are completely oblivious to the effects of moral self-licensing, both static and dynamic. OPEN ACCESS Citation: Engel J, Szech N (2020) A little good is good enough: Ethical consumption, cheap excuses, and moral self-licensing. PLoS ONE 15 (1): e0227036. https://doi.org/10.1371/journal. pone.0227036 OPEN ACCESS Citation: Engel J, Szech N (2020) A little good is good enough: Ethical consumption, cheap excuses, and moral self-licensing. PLoS ONE 15 (1): e0227036. https://doi.org/10.1371/journal. pone.0227036 Editor: Baogui Xin, Shandong University of Science and Technology, CHINA Editor: Baogui Xin, Shandong University of Science and Technology, CHINA Introduction The production of green, environmentally sustainable products has been increasing. This phe- nomenon includes products from organic agriculture [1, 2]and sustainably produced elec- tronic items [3, 4]. However, even though consumers buy more green products, purchases seem to fall short compared with the stated intents that consumers express. Experts have argued that the ratio is one to ten: Spending on ethically improved products amounts to approximately 10% of what consumers claimed they would be willing to spend [5]. Moreover, data suggests that many consumers attempt to ignore ethically questionable production stan- dards when possible [6]. This finding suggests that although many consumers have some awareness of ethical problems in production processes and some willingness to pay (WTP), cheap excuses may be welcome when regarding opting for less-ethical items. Data Availability Statement: We made the data available on our homepage, under the following link: http://polit.econ.kit.edu/downloads/. Funding: The authors received no specific funding for this work. Competing interests: The authors have declared that no competing interests exist. 1 / 19 PLOS ONE | https://doi.org/10.1371/journal.pone.0227036 January 15, 2020 A little good is good enough: Ethical consumption, cheap excuses, and moral self-licensing Some brands, such as Wholefoods, foster a universal approach toward production that adheres to high, broad ethical standards. By contrast, many companies have focused on one ethical facet of their products while remaining rather silent on others. For example, fashion- producer H&M advertises that it replaces conventional cotton in its “conscious collection” with organic or recycled cotton [7]. In a similar manner, Apple publicly announced to audit of all its suppliers regarding the use of conflict minerals [8]. However, even though H&M and Apple have included the well-being of their workers in their codes of conduct [9–11], empha- sizing its importance, reports of problematic working conditions have been repeatedly observed. For example, articles by Duhigg and Barboza [12], Burke [13]and Fullerton [14] have highlighted problematic working conditions in the production of Apple products, and Preston and Leffler [15] reported workers’ rights violations among some of the best-ranked suppliers of H&M. We follow Bandura [16] and Gert [17] and define moral and ethical behavior as avoiding harm to other people and/or the environment. For a company, focusing on improving one eth- ical facet of their production process compared with many might be easier. Introduction For example, textile producers might limit environmental damage from growing cotton but ignore other ethically relevant aspects of their production processes. What might also be is that some facets such as environmental impact are easier to address than other facets such as labor and safety stan- dards. This may partly explain why many companies stress specific ethical aspects to increase their ethical reputation. Another reason for such practices could be that they serve customers’ moral interests. Maybe, for customers, fulfilling one ethical aspect is sufficient to ease their moral conscience when buying a product. Potentially, for many product decisions, “a little good is good enough.” If so, the need to improve other ethical facets may become irrelevant for companies as soon as they address one aspect. In three experiments, we explore whether subjects care comparatively less about another ethical, unrelated dimension as soon as one ethical aspect of a product appears to be fulfilled. For this purpose, in so-called Product treatments, we randomize subjects into different condi- tions and elicit their WTP for improved manufacturing standards of textile products. Subjects make decisions on towels in neutral colors and of a comparable size and weight (grammage of the cotton used). If they opt for a towel manufactured under certified conditions, they receive less money for themselves. Subjects make real economic decisions, that is, they receive a towel and money in the end, depending on their decisions in the study. Thus, in line with Levitt and List [18], we employ a real item to increase the outside validity of our economic experiments. We also elicit beliefs on how other subjects may behave in an incentivized manner. In all experiments, we follow the standards of economic experiments that have been described in Charness and Fehr [19] and Falk and Heckman [20]. In all treatments, the instructions inform subjects that manufacturing (i.e., sewing towels) is a production step unrelated to generating the raw material, in our case cotton, and that the two production steps often occur in different countries. Thus, whether a towel is made from conventional versus organic cotton has nothing to do with the manufacturing standards in the sewing step. Nevertheless, our data shows that the WTP for secure working standards in the sewing process is highly significantly smaller if subjects know that they decide between towels made from organic cotton instead of conventional cotton. PLOS ONE | https://doi.org/10.1371/journal.pone.0227036 January 15, 2020 Introduction Therefore, we run two additional treatments identical to the original product choice treatments, respectively, with one difference: The instructions stress and visually illustrate that it is perfectly random whether a subject makes decisions about towels made from conventional or from organic cotton. In this manner, organic cotton may become a less valid, self-attribut- able excuse to not care about sewing conditions and refugees, and this is indeed what we find. Studies of economic institutions in which agents make morally relevant decisions are a pressing and strongly growing field within economics. Recent contributions from economics include Sobel [27], Falk and Szech [28], Kirchler et al. [29], and Pigors and Rockenbach [30] on market trading and morals. Falk and Szech [31] and Rothenha¨usler et al. [32] have studied morals in group voting. Kerschbamer et al. [33], Bartling et al. [34], and Friedrichsen and Engelmann [35] have considered ethical consumption and/or morally relevant credence goods. Our results indicate that consumers’ moral excuses should receive specific attention in follow-up research. The question arises as to whether self-attribution is critical for the small moral excuses we identify. Therefore, we run two additional treatments identical to the original product choice treatments, respectively, with one difference: The instructions stress and visually illustrate that it is perfectly random whether a subject makes decisions about towels made from conventional or from organic cotton. In this manner, organic cotton may become a less valid, self-attribut- able excuse to not care about sewing conditions and refugees, and this is indeed what we find. Studies of economic institutions in which agents make morally relevant decisions are a pressing and strongly growing field within economics. Recent contributions from economics include Sobel [27], Falk and Szech [28], Kirchler et al. [29], and Pigors and Rockenbach [30] on market trading and morals. Falk and Szech [31] and Rothenha¨usler et al. [32] have studied morals in group voting. Kerschbamer et al. [33], Bartling et al. [34], and Friedrichsen and Engelmann [35] have considered ethical consumption and/or morally relevant credence goods. Our results indicate that consumers’ moral excuses should receive specific attention in follow-up research. Introduction The organic cotton is observed operate as a type of moral excuse to care less about the workers’ conditions in the sewing sector. Next, we explore whether fulfilling one ethical dimension (organic cotton) also has spill- overs to another, unrelated ethical context later in time and find the following. In our study, approximately half an hour after the main part of the experiment, in which the subjects’ focused on towels, they are offered the opportunity to share money with refugees from a local PLOS ONE | https://doi.org/10.1371/journal.pone.0227036 January 15, 2020 2 / 19 A little good is good enough: Ethical consumption, cheap excuses, and moral self-licensing refugee camp. We find that subjects who know they will receive a towel made from organic cotton after the experiment donate less than subjects who know their towel will be made from conventional cotton. Again, organic cotton is observed to serve as an excuse to not care about another, ethically relevant context. We observe these effects even though subjects most likely knew that the refugee crisis was drastic when the experiments occurred, and that all help was needed to cope with it [21, 22]. Potentially, we find these static and dynamic moral spillovers because for the subjects, all ethical behaviors load on one and the same personality factor: “being an ethical person.” Aker- lof and Kranton [23] argue that identity may play a critical role from which people derive posi- tive utility. Falk and Szech [24] demonstrate that people care about their moral identity. However, maybe fulfilling one ethical aspect is sufficient for a decent moral identity and a posi- tive self-image[25, 26]. Knowledge of these effects could be a critical first step to discussing social implications. Therefore, we incentivize new, independent subjects to predict the behavior of subjects in our original product choice treatments. Our data shows that these outsiders are completely unaware of the effects of static moral self-licensing, and they underestimate moral spillover effects in time. Potentially, outsiders follow a different moral compass and consider different product facets and contexts over time as what they are, namely, unrelated regarding conse- quences. Thus, outsiders do not anticipate that subjects, when weighing self-interest against something morally relevant, may prefer to find some type of moral excuse for self-oriented behavior. The question arises as to whether self-attribution is critical for the small moral excuses we identify. PLOS ONE | https://doi.org/10.1371/journal.pone.0227036 January 15, 2020 Literature review Our results relate to the classic moral self-licensing effect. However, we explore a static deci- sion context, whereas the classical literature on moral self-licensing has focused on dynamic effects only. Researchers have documented that ethical behavior in the past serves as a justifica- tion to act less ethically later. Such dynamic moral self-licensing was first described by Monin and Miller [36] in the contexts of racism and sexism in a two-stage experiment. Subjects had to make a hypothetical job decision in a neutrally framed environment. In one treatment group, the best applicant was African American, and in the other group, the best applicant was white. After making their decision, all subjects were confronted with another hypothetical hiring scenario: A chief of police had to hire a new deputy in a racially charged job environ- ment. All subjects were asked to rate whether the job was better suited for a white or a black person. Subjects with the opportunity to present themselves as non-prejudiced before were PLOS ONE | https://doi.org/10.1371/journal.pone.0227036 January 15, 2020 3 / 19 A little good is good enough: Ethical consumption, cheap excuses, and moral self-licensing significantly more likely to prefer a white person now. Thus, subjects who could demonstrate they were not racist in the first step, tended to prefer the white police applicant later. A second study by Monin and Miller [36] yielded similar findings in the field of sexism. Sachdeva et al. [37] found that subjects use dynamic moral self-licensing to justify selfish behavior. Subjects asked to write positive short stories on their good past deeds were later observed to be more selfish than others regarding making a real donation to charity. In a related study, Mazar and Zhong [38] demonstrated that subjects who acquired “green” prod- ucts in the first step are more likely to cheat for personal gain and steal later compared with subjects who could only acquire conventional products in the first step in time. For more examples of dynamic moral self-licensing, see Merritt et al. [39]. Another closely related concept from the field of behavioral economics is conscience accounting, proposed in Gneezy et al. [40]. In the first stage of their experiment, subjects could lie to increase their profit at the expense of their fellow players. In stage two, subjects had the opportunity to make a small donation to charity. Literature review The authors observed that the share of dona- tions was significantly higher among the subjects who lied in stage one compared with those who told the truth. This finding suggests that by donating, subjects atone for past moral norm violations. Furthermore, the share of donations among the liars decreases significantly if the time delay between the two stages increases. A possible explanation for this finding is that the memory of an individual’s own unethical behavior fades over time. Another finding is that subjects are more likely to lie if they know that they have the opportunity to donate later. This leads to the conclusion that people set off their future ethical behavior against their current ethical behavior, and this conclusion is in line with the idea that all types of moral behavior may load on one aspect of personality if subjects can attribute those moral improvements to themselves [24, 25, 26]. The authors also documented that a comparatively cheap future possi- bility of “compensation” might be sufficient for subjects to maintain a high moral self-image. What differentiates this approach from ours is that in our Product treatments, subjects make decisions on the static moral improvement (the upgrade to the fair wear towel at the dis- pense of different monetary amounts) in a price list. They do not know which of their deci- sions the computer will randomly select when they decide to donate money to refugees a half an hour later. Moreover, the subjects are unaware of the opportunity to make a donation beforehand. This design enables the subjects to focus on whether deciding about towels made from organic instead of conventional cotton is sufficient to care less about other ethically rele- vant aspects—static and dynamic. PLOS ONE | https://doi.org/10.1371/journal.pone.0227036 January 15, 2020 Belief treatments In Belief Control and in Belief Organic, we elicited in an incentivized manner outsiders’ beliefs about the behavior of the subjects in Product Control and in Product Organic, respec- tively. The Belief treatments provided information on what the uninvolved third parties, who did not make the decisions, predicted about the individuals involved. Subjects were randomly assigned to the two Belief treatments. For organizational reasons, randomization in this study was at the session level. In all treatments, the instructions informed subjects of the different ethically relevant dimensions in the production of textiles such as towels. We focused on two important, sepa- rate aspects. First, the cotton used could be conventional or certified “organic.” Here, we relied on the well-known Global Organic Textile Standard (GOTS) and the Organic Content Stan- dard (OCS), which certify the organic origin of cotton. All subjects knew from the instructions that the certified organic cotton was produced without the use of agrochemicals, such as syn- thetic pesticides, herbicides, and fertilizers commonly used in conventional production and that pollute the soil. Second, the manufacturing conditions under which workers must work when sewing the towel can be certified by the well-established Fair Wear Foundation (FWF), or not. The FWF is a well-established, independent non-profit organization that aims to improve labor standards in factories based on the conventions of the International Labour Organization (ILO). Subjects were informed that under these FWF conditions, safety, eco- nomic living standards, and political rights of workers are monitored. These benefits are not guaranteed in conventional production. Indeed, on the topic of ethics, the textile and garment industry is one most frequently discussed industries, e.g., Danzer and Grundke [45]. The instructions also provided information on the fragmentation of production chain in the garment industry, that is, the cotton is typically grown and manufactured in different countries. Thus, subjects knew that these two aspects, growing organic cotton and ensuring FWF-controlled manufacturing standards, could be considered separate from each other. To analyze moral self-licensing within product decisions, we elicited the monetary value subjects assigned to the FWF manufacturing standards in Product Control and in Product Organic. In Product Organic, subjects knew that the towel already fulfilled another indepen- dent, ethically relevant criterion (i.e., organic cotton). Decisions were elicited in a price list. Design of the study We explore the effects of moral self-licensing across different ethical facets in purchase deci- sions and across different points in time. To this end, we implement a product choice regard- ing different towels and a subsequent donation situation. In addition to behavior, we study beliefs of outsiders about the behavior of decision-makers to analyze whether beliefs of outsid- ers are in line with real choice behavior. Therefore, we conduct four treatments: Product Con- trol, Product Organic, Belief Control, and Belief Organic. In our experiment, we employed towels for the following three reasons. First, the value of the fashion industry is USD trillions, encompassing a wide variety of companies [41] and employs over 57 million people worldwide [42]. Thus, this sector is a critical industrial sector. Second, the bulk of the production occurs in developing and newly industrialized countries, such as China, India, Vietnam, Bangladesh [43, 44], and the textile industry has often been the focus of consumers’ demand for improved production standards. Third, we specifically used a product that we hoped most subjects found useful, independent of personal characteristics PLOS ONE | https://doi.org/10.1371/journal.pone.0227036 January 15, 2020 4 / 19 A little good is good enough: Ethical consumption, cheap excuses, and moral self-licensing such as gender or taste in fashion. Therefore, the subjects made decisions about towels in neu- tral colors. Product treatments In Product Control and in Product Organic, subjects made ethically relevant product deci- sions. Subjects were randomized into the two treatments. They choose between ethically differ- ent yet otherwise comparable towels. In a price list, they weighed money and receiving a more conventionally produced towel against receiving no money and a towel that fulfills a more eth- ical set of production standards. The subjects choose between towels in neutral colors; in a standard, medium size of 100 cm x 50 cm, and with a standard surface weight of approximately 450 g/m2. PLOS ONE | https://doi.org/10.1371/journal.pone.0227036 January 15, 2020 Belief treatments Subjects choose between a towel that was manufactured under the FWF-controlled standards and no additional money versus a towel without FWF-controlled manufacturing standards and additional money. Monetary amounts varied from 0.25 to 12 euro in steps of 25 cents. Subjects knew that one of their decisions would be randomly selected at the individual level and implemented. That is, subjects received—in any case—a towel with the respective ethical attributes from their choice in the price list and, depending on the choice, additional money. PLOS ONE | https://doi.org/10.1371/journal.pone.0227036 January 15, 2020 5 / 19 A little good is good enough: Ethical consumption, cheap excuses, and moral self-licensing Fig 1. Price list as used in product organic. In the Organic Product treatment, subjects choose from a price list between a towel without an FWF certificate and money versus a towel with an FWF certificate and no additional money. The FWF certificate ensured controlled manufacturing standards for workers when sewing the towel. In any case, the towel is made from organic cotton. https://doi.org/10.1371/journal.pone.0227036.g001 Fig 1. Price list as used in product organic. In the Organic Product treatment, subjects choose from a price list between a towel without an FWF certificate and money versus a towel with an FWF certificate and no additional money. The FWF certificate ensured controlled manufacturing standards for workers when sewing the towel. In any case, the towel is made from organic cotton. Fig 1. Price list as used in product organic. In the Organic Product treatment, subjects choose from a price list between a towel without an FWF certificate and money versus a towel with an FWF certificate and no additional money. The FWF certificate ensured controlled manufacturing standards for workers when sewing the towel. In any case, the towel is made from organic cotton. https://doi.org/10.1371/journal.pone.0227036.g001 We defined the lowest monetary amount for which subjects still preferred no FWF certifi- cate over the FWF certificate as the WTP for FWF certification. The lower the switch point, the less a subject cared about FWF-certified working standards. In cases of multiple switching, we used the mean switch point for our analysis. We observed that the results were robust to multiple switching. Fig 1 illustrates the price list in Product Organic. Product Control was similar except that the cotton was conventional (see Appendix B in S1 Appendix. for details). PLOS ONE | https://doi.org/10.1371/journal.pone.0227036 January 15, 2020 Hypotheses The insights gained from the experiments on moral self-licensing [36, 37, 38] and conscience accounting [40] show that people use acquired moral credentials to justify less-ethical behavior at a later point in time. In a similar fashion, they morally redeem themselves from unethical behavior in the past by performing subsequent ethical deeds. Even if the ethical deeds are small and cheap later on, they seem to serve as a good justification for rather selfish behavior at an earlier point in time [40]. Gneezy et al. [40] asserted that a similar logic may have been behind the sale of indulgences, as practiced by the Catholic Church in medieval times. Yet in that case, moral redemption was often costly. Moral cleansing may provoke a reduction in moral behavior in the first place, specifically if subjects know about the possibility to morally cleanse themselves afterward [40]. Such a mech- anism could also exist in the framework of a static decision across different ethically relevant facets of one product. The satisfaction of fulfilling one ethical dimension could lead to a decreased valuation of a second—though unrelated—ethical dimension at the same point in time. This would imply that the time dimension is not what renders moral cleansing relevant; instead, it is the opportunity to have some type of “moral excuse.” This logic would be specifi- cally attractive if subjects did not care about consequences but instead considered all these decisions relevant for one specific aspect about themselves, that is, a positive self-image [24, 25, 26]. Our first and main hypothesis is therefore that subjects in Product Organic have a lower WTP for controlled manufacturing conditions than subjects in Product Control; thus, static moral self-licensing exists. Therefore, we expect that WTPProduct Organic < WTPProduct Control: Therefore, we expect that In addition to the static product decision, subjects later had to make another unrelated ethi- cal decision by choosing whether to share their show-up fee with local refugees. Because our subjects do not receive any feedback on their payoff from the price list until after the experi- ment, they know whether their towel is ethical in one dimension (the material) but cannot be sure whether it was produced under controlled manufacturing conditions. Belief treatments After this main deci- sion, subjects completed questionnaires on their political preferences, personal characteristics, and socioeconomic background. We used standard tests of personality, such as the Big Five [46], and of Machiavellianism [47] and the rather new Preference Survey Module [48]. At the end of the study, approximately half an hour later, the instructions confronted subjects with another ethically relevant decision. Subjects did not know this in advance, and therefore, com- pared with the study of Gneezy et al. [40], subjects could not use the later sharing decisions as a justification for selfish behavior in the towel decision. Through the computer screen, subjects had to decide whether they wanted to share their show-up fee (i.e., 2 euro) with refugees from a local refugee camp. We chose to implement a binary decision instead of a price list to distin- guish this choice, also regarding its framing, from the previous decision on towels. The dona- tion amount was substantial but not exceedingly high compared with the payout that subjects could earn in the study, which comprised the show-up fee, a towel, and a potential payoff from the price list. At this point, subjects had not received feedback on whether they would receive a towel produced under the FWF or conventional production standards. In the Belief treatments, that is, in Belief Control and in Belief Organic, subjects did not make any ethically relevant product or donation decision themselves. Instead, subjects indi- cated their expectations of subjects in the respective Product treatments behaved. We incentiv- ized these estimates through quadratic scoring rules. The respective estimation for the mean willingness to pay, given in cents, had the following payout: max {500 - 1 4 (true—guess)2, 0} cent. In Belief Control, subjects guessed the average WTP for controlled manufacturing stan- dards in Product Control. Subjects in Belief Control knew the instructions from the Product PLOS ONE | https://doi.org/10.1371/journal.pone.0227036 January 15, 2020 6 / 19 A little good is good enough: Ethical consumption, cheap excuses, and moral self-licensing Control treatment for the towel decision. Thus, they had the exact same information as the subjects in Product Control. After indicating their estimate, they also filled out the same ques- tionnaires as subjects in the Product treatments. Then, the instructions confronted them with a second estimation task, requiring them to guess what percentage of subjects in Control opted to share their show-up fee with refugees. Belief treatments Again, we employed a quadratic scoring rule to incentivize this task. The payout for the estimated willingness to share was max {5 - 1 5 (true– guess)2, 0} euro. Subjects had to type in integer percentage values. In Belief Organic, subjects accordingly guessed the behavior from Product Organic. In total, subjects in Belief Control and in Belief Organic could earn up to 10 euro for good esti- mates, in addition to a show-up fee of 7 euro. The study took place at the Karlsruhe Decision and Design Laboratory (KD2 Lab) at the Karlsruhe Institute of Technology. We used ORSEE [49] and hroot [50] to recruit 200 subjects (50 subjects per treatment). Subjects were placed in separate cubicles. Instructions regarding the towels were presented on paper, and questionnaires were presented on a computer screen by using SoSci Survey [51]. See Appendix B in S1 Appendix for details. Hypotheses (The exception is subjects willing to pay the maximal amount or unwilling to pay the minimal amount for the controlled manufacturing conditions.) In line with the findings on dynamic moral self-licens- ing, the certainty of having acquired a product that fulfills (at least) one ethical facet in the first PLOS ONE | https://doi.org/10.1371/journal.pone.0227036 January 15, 2020 7 / 19 A little good is good enough: Ethical consumption, cheap excuses, and moral self-licensing stage should “free” the subjects to behave less ethically afterward. This leads to our second hypothesis: Subjects in Product Organic will be less likely to share their show-up fee than sub- jects in Product Control, that is, static moral self-licensing and dynamic moral self-licensing can exist next to each other: DonationProduct Organic < DonationProduct Control: Exploratively, we also investigate the incentivized beliefs of outsiders about the behavior of subjects in the respective Product treatments. Thus, we conduct two Belief treatments: Belief Control and Belief Organic. In these treatments, subjects do not face trade-offs between more ethical decisions and money. Instead, they are incentivized to predict the decisions of other subjects from the respective Product treatment. It may be that subjects in the Belief treatments predict that moral self-licensing will take place in the Product treatments. This is not so clear, however, for several reasons. First, sub- jects in the Belief treatments do not face a trade-off between more ethical decisions and money. In the literature, hypothetical questions and real behavior have often correlated regarding altruistic versus selfish behavior. However, typically, in real behavior, subjects care much more about their self-interest than when hypothetically asked. Accordingly, the litera- ture on ethical consumption speaks of the famous 30:3 ratio [5] or attitude behavior gap[52, 53], comparing stated intent from questionnaires to real decisions in the market. Furthermore, the literature has shown that context can affect morally relevant judgments (see e.g.,[16, 28, 29, 31, 34, 54, 55, 56]). Therefore, we expect that Therefore, we expect that Therefore, we expect that Belief WTP Product Organic  Belief WTP Product Control; Belief WTP Product Organic  Belief WTP Product Control; and accordingly and accordingly Belief Donation Product Organic  Belief Donation Product Control: If subjects do not predict that moral self-licensing will take place, that is, if both formulas hold with equality, emphasis on the importance of moral self-licensing in social and public debate might be relevant. PLOS ONE | https://doi.org/10.1371/journal.pone.0227036 January 15, 2020 Beliefs about static moral self-licensing Do uninvolved people expect static moral self-licensing to take place? To consciously counter the effects of static moral self-licensing, people must be aware of it. We hypothesized, that for at least three reasons, this phenomenon may not be the case. Our data from Belief Organic demonstrates that subjects are not aware of the effects of static moral self-licensing (Fig 3). Estimates for switch points are at any conventional level and are virtually identical to those from Belief Control (4.39 vs. 4.33, p = 0.89, two-sided t test). Static moral self-licensing We hypothesized that static moral self-licensing takes place, that is, subjects treat orthogonal, ethically relevant aspects as if they were substitutes in product choice. Therefore, we expected that subjects in Product Organic would pay less money to ensure FWF-controlled manufactur- ing standards than in Product Control, as reflected in a lower switch point in the price list. The hypothesis was validated. In Product Control, the average switch point was 5.73 euro. We have seven multiple switchers in the sample. Our findings remain robust if we exclude them or use the first switch point, last switch-point, or median switch-point to approximate their willing- ness to pay. (See the robustness checks in Appendix A in S1 Appendix. for details.) In Product Organic, the average switch point and therefore WTP was 4.00 euro (Fig 2). This is a decrease of 30% (p = 0.002, one-sided t test), which is highly significant. We will speak of “highly signifi- cant” if the p value is below 0.01. Subjects know from the instructions that sewing the towel is a very different production step than growing the cotton because the people involved and ecological consequences differ. Nevertheless, they attribute less value to guaranteeing minimum working standards when they PLOS ONE | https://doi.org/10.1371/journal.pone.0227036 January 15, 2020 8 / 19 A little good is good enough: Ethical consumption, cheap excuses, and moral self-licensing Fig 2. WTP product treatments. In Product Organic, subjects pay highly significantly less money to ensure controlled manufacturing standards compared with Product Control. Thus, static moral self-licensing takes place. https://doi.org/10.1371/journal.pone.0227036.g002 Fig 2. WTP product treatments. In Product Organic, subjects pay highly significantly less money to ensure controlled manufacturing standards compared with Product Control. Thus, static moral self-licensing takes place. https://doi.org/10.1371/journal.pone.0227036.g002 https://doi.org/10.1371/journal.pone.0227036.g002 know that their towel is made from organic cotton. Potentially, subjects already have a fairly good moral conscience in the latter case and accordingly care less about the workers in manufacturing. Thus, we find that static moral self-licensing exists. PLOS ONE | https://doi.org/10.1371/journal.pone.0227036 January 15, 2020 Dynamic moral self-licensing At the end of the study, approximately half an hour later, subjects in the Product treatments were asked whether they wanted to share their show-up fee of 2 euros with refugees from a local refugee camp. Subjects did not know beforehand that they would be confronted with this decision. If static moral self-licensing has spillovers in time, subjects in Product Organic should less frequently share their show-up fee with refugees than subjects in Product Control, and this is what we observed (Fig 4): 72% of subjects are willing to share in Product Control, and 56% of subjects are willing to share in Product Organic (p = 0.048, one-sided test of pro- portions). As subjects in Product Organic could expect to leave with a higher income than sub- jects in Product Control because of the higher costs of organic textiles, they may be even more inclined to donate. The data display the opposite effect, and this demonstrates that the effects of dynamic moral self-licensing may be stronger than this potential income effect. Looking into the estimates from the two Belief treatments (Fig 5), there is again no signifi- cant difference in estimates of willingness to share with refugees for the two Product PLOS ONE | https://doi.org/10.1371/journal.pone.0227036 January 15, 2020 9 / 19 A little good is good enough: Ethical consumption, cheap excuses, and moral self-licensing Fig 3. Estimated WTP belief treatments. In the Belief treatments, subjects guess the average WTP for controlled manufacturing in Product Organic, resp. Product Control. The data displays no significant difference in estimates at any conventional level. Thus, subjects in Belief Organic are not aware that static moral self-licensing may take place. https://doi.org/10.1371/journal.pone.0227036.g003 Fig 3. Estimated WTP belief treatments. In the Belief treatments, subjects guess the average WTP for controlled manufacturing in Product Organic, resp. Product Control. The data displays no significant difference in estimates at any conventional level. Thus, subjects in Belief Organic are not aware that static moral self-licensing may take place. https://doi.org/10.1371/journal.pone.0227036.g003 treatments (65% in Belief Organic vs. 67% in Belief Control, p = 0.68, two-sided t test). Outsid- ers therefore neither expect static nor dynamic moral self-licensing. In summary, although the effects of moral self-licensing with regard to specific ethical dimensions are pronounced within decisions and over time, uninvolved parties do not antici- pate their effects. This occurs even though uninvolved parties were monetarily incentivized Fig 4. Willingness to share product treatments. Dynamic moral self-licensing Subjects in Product Organic are significantly less likely to share their show-up fee of euro with local refugees than subjects in Product Control. https://doi.org/10.1371/journal.pone.0227036.g004 Fig 4. Willingness to share product treatments. Subjects in Product Organic are significantly less likely to share their show-up fee of euro with local refugees than subjects in Product Control. https://doi org/10 1371/journal pone 0227036 g004 Fig 4. Willingness to share product treatments. Subjects in Product Organic are significantly less likely to share their show-up fee of euro with local refugees than subjects in Product Control. 10 / 19 PLOS ONE | https://doi.org/10.1371/journal.pone.0227036 January 15, 2020 A little good is good enough: Ethical consumption, cheap excuses, and moral self-licensing Fig 5. Estimated willingness to share belief treatments. Estimates from subjects in the Belief treatments for average willingness to share in the respective Product treatment. https://doi.org/10.1371/journal.pone.0227036.g005 Fig 5. Estimated willingness to share belief treatments. Estimates from subjects in the Belief treatments for average willingness to share in the respective Product treatment. Fig 5. Estimated willingness to share belief treatments. Estimates from subjects in the Belief treatments for average willingness to share in the respective Product treatment. https://doi.org/10.1371/journal.pone.0227036.g005 https://doi.org/10.1371/journal.pone.0227036.g005 https://doi.org/10.1371/journal.pone.0227036.g005 and could earn much more money from making accurate guesses than from the flat payment they received for participating in the study. Many subjects’ beliefs were far from the correct answer such that they did not earn additional money for this task. 27% of subjects earned money for their estimates. Potentially, subjects in the Belief treatments merely indicate what they consider morally appropriate and do not figure in the effects of moral self-attribution. Subjects in Belief Control underestimated the willingness to pay in Product Control (p = 0.018, two-sided t test) but do not underestimate subjects’ willingness to donate to refugees. and could earn much more money from making accurate guesses than from the flat payment they received for participating in the study. Many subjects’ beliefs were far from the correct answer such that they did not earn additional money for this task. 27% of subjects earned money for their estimates. Potentially, subjects in the Belief treatments merely indicate what they consider morally appropriate and do not figure in the effects of moral self-attribution. Subjects in Belief Control underestimated the willingness to pay in Product Control (p = 0.018, two-sided t test) but do not underestimate subjects’ willingness to donate to refugees. PLOS ONE | https://doi.org/10.1371/journal.pone.0227036 January 15, 2020 A little good is good enough: Ethical consumption, cheap excuses, and moral self-licensing Table 1. Comparison of subjects in product control and product organic. Product Control Product Organic p value Age † 21.82 21.06 0.22 Proportion of males ‡ 76% 72% 0.65 Disposable income after rent (in euro) † 410 398 0.77 Financial concern (7-point Likert-scale) † 2.58 2.80 0.20 Machiavellianism † 56.88 58.06 0.47 Agreeableness † 12.9 12.4 0.37 The sociodemographics and the ethically relevant personality traits do not differ significantly between Product Control and Product Organic. † two-sided t test, n = 100 ‡ two-sided test of proportions, n = 100. Table 1. Comparison of subjects in product control and product organic. hics and the ethically relevant personality traits do not differ significantly between Product Control and Product Organic. 100 The sociodemographics and the ethically relevant personality traits do not differ significantly between Product Control and Product Organic. † two-sided t test, n = 100 d d f (p = 0.65, two-sided t test), hunger, insufficient nourishment or natural disasters (p = 0.39, two-sided t test), or unemployment (p = 082, two-sided t test). Regarding personality traits, we elicit measures that might influence ethical decision-mak- ing: Machiavellianism and agreeableness. Machiavellianism encompasses a variety of world views and actions characterized by a general disregard for other people to increase personal gain. Machiavellianism has been found to correlate negatively with ethical orientation and decision-making [54]. Machiavellianism has furthermore been positively linked to love for money [57] and economic opportunism[58]. Sacrificing money to improve the working condi- tions of unknown people without personal benefit would therefore not constitute Machiavel- lian behavior. The second trait of interest is agreeableness, and it is elicited in the Big Five. Agreeableness encompasses positive personal attitudes and behaviors, such as generosity, trustworthiness, and compassion [59, 60]. We find that subjects in Product Control and Prod- uct Organic show similar levels of Machiavellianism (p = 0.47, two-sided t test) and agreeable- ness (p = 0.37, two-sided t test). These similarities in sociodemographics and personality traits suggest that differences across Product treatments are based on other factors, that is, differ- ences in decision contexts. Second, a question could be whether self-attribution is critical for the treatment effect we observe in the Product treatments. An argument could be made for an alternative driver, that is, subjects’ utility functions are concave regarding fulfilling separate ethical dimensions of products. Discussion In the following, we discuss our results. First, we demonstrate robustness by controlling for sociodemographic variables, personality traits, and attitudes toward refugees across Product treatments. Second, we discuss whether self-attribution is critical to our results. For this pur- pose, we run two new treatments that demonstrate that self-attribution seems to matter signifi- cantly. Third, we discuss the potential effects of decreasing attention in subjects and an alternative interpretation of why subjects pay less for certified working conditions in Product Organic. Fourth, we stress the need for follow-up research. First, subjects were randomly assigned to treatments. This was also the case for the Product treatments. Nevertheless, a concern might be that treatment effects may be driven by differ- ences in age or demographic background; reassuringly, Table 1 demonstrates that this is not the case. Subjects in Product Control and Product Organic do not significantly differ in age (p = 0.22, two-sided t test) or gender (p = 0.65, two-sided test of proportions). Furthermore, subjects have comparable amounts of money at their monthly disposal (p = 0.77, two-sided t test) and state no significant differences in their concern for their financial situation (p = 0.20, two-sided t test). In addition, subjects have similar opinions on whether Germany should sup- port refugees fleeing war (p = 0.66, two-sided t test), political, religious or ethnic persecution PLOS ONE | https://doi.org/10.1371/journal.pone.0227036 January 15, 2020 11 / 19 PLOS ONE | https://doi.org/10.1371/journal.pone.0227036 January 15, 2020 This could imply a lower marginal utility in the organic treatment and lead to a lower WTP in Product Organic compared with Product Control. A related argument could be that subjects in Product Organic bunched the ethical attributes differently and therefore became less willing to pay for the certified sewing conditions (we thank an anonymous referee for pointing this out). If subjects in Product Organic consider the organic cotton and certified sewing conditions as part of one ethical factor, this could reduce their willingness to pay for the individual certificates, although they address very diffferent ethical aspects from a conse- quentialist viewpoint. Thus, a question arises as to whether self-attribution significantly affected our results. Do people indeed need self-attribution to find some minimal yet plausible excuse to pay less in Product Organic? This question motivated us to run two additional treatments: Lottery Con- trol and Lottery Organic. These treatments are similar to the original product choice treat- ments, respectively, with only one difference: much less room for self-attribution. The instructions stress that it is perfectly random regarding whether a subject decides about towels made from conventional or organic cotton. In this manner, subjects deciding about organic towels may attribute the fulfillment of this ethically relevant criterion less to themselves. 12 / 19 PLOS ONE | https://doi.org/10.1371/journal.pone.0227036 January 15, 2020 A little good is good enough: Ethical consumption, cheap excuses, and moral self-licensing Accordingly, fulfilling this criterion may not serve as a morally relevant excuse anymore, such that subjects may care more about the upgrade to the fair wear certificate and about donating to refugees. These two treatments were run 19 months after the original Product treatments with 50 subjects per treatment. Unfortunately, we had to exclude one subject (in Lottery Con- trol) due to incorrect handling of the price list. Accordingly, fulfilling this criterion may not serve as a morally relevant excuse anymore, such that subjects may care more about the upgrade to the fair wear certificate and about donating to refugees. These two treatments were run 19 months after the original Product treatments with 50 subjects per treatment. Unfortunately, we had to exclude one subject (in Lottery Con- trol) due to incorrect handling of the price list. In Lottery Control and Lottery Organic, the randomness is made salient to subjects as fol- lows. Blue and yellow cards are alternatingly handed to subjects. Next, all subjects within a ses- sion receive the same information on the chain of production of textile products, on FWF standards, and on organic cotton production (as in the original Product treatments). Subjects learn that a random coin toss and the color of their card determine whether they subsequently decide about towels made of conventional or organic cotton. Thus, the design renders the fol- lowing salient: subjects are randomly distributed to one of the two treatments: Lottery Control or Lottery Organic. After the coin toss, subjects receive their respective price lists and instruc- tions (see Appendix II for details). In contrast, in the original Product treatments, assignment to the specific treatment was random. This randomness was neither stressed nor visually demonstrated. If self-attribution matters, we expect that both moral self-licensing effects should be less pronounced in the Lottery treatments. The following simple model describes the approach. We capture the self-attribution from fulfilling the ethically relevant cotton criterion in the Product Organic treatment through α. In Product Control, in which towels are never made from ethically produced cotton, we set α to 0. In the Lottery treatments, we assume that it is a 2 because subjects know that fulfilling the organic cotton criterion is random and has a probabil- ity of 50%. In Product Organic, α is 1. We assume that utility is separable between the moral and the monetary dimensions, and quasilinear in monetary payments. Utility from receiving a monetary amount m and having a moral value of α is thus given by u(α)+m. We assume that u is three times continuously differ- entiable. The monetary amount m(α) that renders the agent indifferent between receiving money or receiving an additional moral value of f is given by m(α) = u(α+f)−u(α) Writing this as Writing this as mðaÞ ¼ uða þ f Þ uðaÞ ¼ R f 0 u0ða þ tÞdt; we find that we find that m0 ðaÞ ¼ R f 0 u00ða þ tÞdt and m00 ðaÞ ¼ R f 0u000ða þ tÞdt: In particular, under risk aversion, u00<0, m is decreasing in α. Moreover, if the agent is prudent in the sense of Kimball [61] in the moral dimension, u000>0, then m is convex. Con- versely, imprudence, u000<0 implies the concavity of m. PLOS ONE | https://doi.org/10.1371/journal.pone.0227036 January 15, 2020 Thus, for a prudent agent, we have by Jensen’s inequality m a 2   < 1 2 m a ð Þ þ 1 2 m 0 ð Þ; thus, thus, m a 2   m a ð Þ < m 0 ð Þ m a 2   ; m a 2   m a ð Þ < m 0 ð Þ m a 2   ; that is, m a 2  is closer to m(α) than to m(0). In the imprudent case, m a 2  is closer to m(0) than to m(α). that is, m a 2  is closer to m(α) than to m(0). In the imprudent case, m a 2  is closer to m(0) than to m(α). 13 / 19 PLOS ONE | https://doi.org/10.1371/journal.pone.0227036 January 15, 2020 PLOS ONE | https://doi.org/10.1371/journal.pone.0227036 January 15, 2020 A little good is good enough: Ethical consumption, cheap excuses, and moral self-licensing Fig 6. WTP lottery treatments. In the Lottery treatments, the static moral self-licensing effect disappears because there are no significant differences in the WTP for controlled manufacturing conditions. If anything, the pattern reverses based on a tendency. https://doi.org/10.1371/journal.pone.0227036.g006 Fig 6. WTP lottery treatments. In the Lottery treatments, the static moral self-licensing effect disappears because there are no significant differences in the WTP for controlled manufacturing conditions. If anything, the pattern reverses based on a tendency. Fig 6. WTP lottery treatments. In the Lottery treatments, the static moral self-licensing effect disappears because there are no significant differences in the WTP for controlled manufacturing conditions. If anything, the pattern reverses based on a tendency. https://doi.org/10.1371/journal.pone.0227036.g006 https://doi.org/10.1371/journal.pone.0227036.g006 In the Lottery treatments, self-attribution of the morally relevant organic cotton should amount to the same value a 2. Therefore, the WTP for a second, morally relevant criterion should be comparable across the two Lottery treatments. Indeed, the data shows that WTP is statistically comparable. In Lottery Organic, the average WTP for controlled manufacturing conditions is 5.49 euro, and it is 4.86 euro (p = 0.30, two-sided t test) in Lottery Control (Fig 6). As hypothesized, the moral self-licensing effect disappears. Analogously to the Product treatments, all subjects in the Lottery treatments were offered to share their show-up fee of 2 euro with local refugees. PLOS ONE | https://doi.org/10.1371/journal.pone.0227036 January 15, 2020 The share of subjects willing to donate is 66% in Lottery Organic and 62% in Lottery Control (Fig 7). In accordance with the hypothe- sis, the difference is not statistically significant (p = 0.68, two-sided test of proportions). The results suggest that moral self-attribution differs if the role of randomness in the experiment is emphasized. A direct comparison to the original Product treatments, elicited 19 months earlier, must be used with some caution because, of course, ethical values in the population of interest may change over time. Notably, the general attitude toward refugees could have changed substan- tially between the sessions, which is why we refrain from conducting comparisons between the willingness to share between the Product and the Lottery treatments. Reassuringly though, when we ask our subjects whether Germany should support refugees fleeing from different causes, comparing the Product and the Lottery treatments, we find no significant differences. This is valid whether refugees are fleeing war (p = 0.96, two-sided t test), political, religious or ethnic persecution (p = 0.30, two-sided t test), hunger, insufficient nourishment or natural disasters (p = 0.37, two-sided t test), or unemployment (p = 0.21, two-sided t test).We find that compared with the original Product treatments, the average WTP for controlled manufactur- ing conditions does not change (p = 0.47, two-sided t test). When examining at the organic PLOS ONE | https://doi.org/10.1371/journal.pone.0227036 January 15, 2020 14 / 19 A little good is good enough: Ethical consumption, cheap excuses, and moral self-licensing Fig 7. Willingness to share lottery treatments. Compared with the Product treatments, the share of donations is statistically comparable between subjects in the organic and the control treatment. The effects of dynamic moral self- licensing disappear in the Lottery treatments. https://doi org/10 1371/journal pone 0227036 g007 Fig 7. Willingness to share lottery treatments. Compared with the Product treatments, the share of donations is statistically comparable between subjects in the organic and the control treatment. The effects of dynamic moral self- licensing disappear in the Lottery treatments. https://doi.org/10.1371/journal.pone.0227036.g007 Fig 7. Willingness to share lottery treatments. Compared with the Product treatments, the share of donations is statistically comparable between subjects in the organic and the control treatment. The effects of dynamic moral self- licensing disappear in the Lottery treatments. https://doi.org/10.1371/journal.pone.0227036.g007 https://doi.org/10.1371/journal.pone.0227036.g007 treatments only, however, subjects in Lottery Organic are willing to pay significantly more than subjects in Product Organic (5.49 euro vs. PLOS ONE | https://doi.org/10.1371/journal.pone.0227036 January 15, 2020 Acknowledgments We would like to thank Noemi Christensen, Anke Greif-Winzrieth, Sibille Hackel, and Tim Walter for their excellent administrative support. For helpful comments and suggestions, we thank Leonie Ku¨hl, Nikolaus Schweizer, Erik Snowberg, Joachim Weimann, and audiences at the Asian Meeting of the Econometric Society, CREST, EEA Annual Congress, ESA World Meeting, GEABA, HeiKaMaX, HeiKaMaXY, Karlsruhe Institute of Technology, University of Mannheim, University of Tu¨bingen, and VfS Annual Meeting. 4.00 euro, p = 0.01, two-sided t test). This find- ing is another indication that subjects care more about additional, ethically relevant facets if it is more difficult to attribute the fulfillment of the ethically relevant criterion regarding organic cotton to themselves. Third, an argument could be that other drivers could play a critical role in the original Product treatments or that the effects in the Product treatments could be driven by decreasing attention when reading the instructions. Maybe, subjects no longer focus on what other ethical upgrades are possible for a towel once they learn of the first aspect (cotton production). Like- wise, subjects may have not paid attention to the later donation decisions. However, as a driver of the differences across the Product treatments, this seems unlikely. Notably, instructions were identical in length across Product treatments. All subjects learned about cotton produc- tion first, then about the conditions in the sewing sector, and then (approximately half an hour later) about the donation opportunity. Fourth, further research in different contexts is necessary to explore how general the moral self-licensing effects are that we documented in our study. Of course, as the first step, further research could also vary the design we used, that is, allow subjects to decide about certified cot- ton instead of the Fair Wear certification. Another important follow-up focus could be market analyses because maybe for firms, offering products that fulfill exactly one ethical criterion, for example, ecological cotton in textile items, is a smart strategy for selling products to customers with easy-to-ease ethical concerns. A further possibility is that leaving moral wiggle room (see Dana et al. [62], van der Weele [63], Bartling et al. [64], Grossman [65], Grossman and van der Weele [66], Freddi [67], Serra-Garcia and Szech [68], and Golman et al. [69] for a recent over- view) and/or room for motivated reasoning [70, 71] could further decrease morally responsible behavior in customers. This potential interaction of moral self-licensing and other well-known mechanisms providing moral excuses is also a topic for further research. PLOS ONE | https://doi.org/10.1371/journal.pone.0227036 January 15, 2020 15 / 19 A little good is good enough: Ethical consumption, cheap excuses, and moral self-licensing Conclusion Our data documents that people behave as if there was strong substitutive relations between ethically relevant, non-correlated product dimensions. Our data thus suggest that static moral self-licensing exists. Fulfilling one ethical dimension in a product choice seems to ease moral conscience in a later, unrelated yet morally relevant context. Thus, different aspects of moral self-licensing and static (across morally relevant dimensions at one point in time) over differ- ent points in time seem to coexist. Outsiders were not observed to infer that moral self-licensing could occur. Although mone- tarily incentivized to make accurate estimates, subjects were observed to be completely oblivi- ous to effects of moral self-licensing, both static and dynamic. Therefore, it may have value for social and political debates on the potential effects of moral self-licensing. Firms know better than ordinary customers about effects of moral self-licensing and other mechanisms of moral excuses. Providing such excuses to customers may be a profitable approach. If so, political and societal debate should be aware of these mechanisms. PLOS ONE | https://doi.org/10.1371/journal.pone.0227036 January 15, 2020 References 1. The World of Organic Agriculture–Statistics and Emerging Trends 2016. FiBL & IFOAM. 2017 [Cited 2019 Dec 14]. 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González Aja, Teresa: Historia del fútbol. De juego simple a espectáculo complejo. Madrid, Catarata, 2023. 208 pp.
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Cuadernos de Historia Contemporánea ISSN: 0214-400X ISSN-e 1988-2734 RESEÑAS González Aja, Teresa: Historia del fútbol. De juego simple a espectáculo complejo. Madrid, Catarata, 2023. 208 pp. Noemi García-Arjona Université Rennes 2 https://dx.doi.org/10.5209/chco.91142 Pocos temas han suscitado más ríos de tinta en el mundo del deporte que el fútbol y su historia. El impacto social, económico y cultural del fenómeno futbolístico es incuestionable, lo que lo convierten en un objeto de estudio que necesita de una continua revisión y crítica. Así, en la mayoría de las publicaciones sobre la historia del fútbol orientadas al gran público prevalecen aproximaciones más o menos literarias, firmadas en muchos casos por periodistas y narradores deportivos, o por los propios protagonistas (entrenadores, jugadores, dirigentes) en forma de crónicas y memorias; nos encontramos en muchos casos con una versión mitificada y anecdótica de los hechos y fuertemente condicionada por defender los colores del club o de la selección nacional a la que se quiere loar. Por otro lado, la historiografía científica sobre los orígenes y evolución del fútbol, en su mayor parte francesa (Dietschy, 2010; Wahl, 1997) y anglosajona (Alegi y Bolsmann, 2013; Eisenberg et al. 2004), es abundante, pero adolece de un inevitable doble sesgo, ya que en la mayoría de los casos la investigación se limita a criterios cronológicos y geográficos muy precisos de su historia, o bien no se consigue divulgar los resultados de dichos estudios al público de masas como se debiera. Pero el libro de Teresa González Aja consigue un raro equilibrio: abordar con rigor la historia de un fenómeno cultural tan supuestamente conocido, amado y denostado a partes iguales, como es el fútbol. La empresa es ambiciosa. Con una extensa trayectoria investigadora en la historia social y política del deporte en España reconocida a nivel internacional (formada en Francia en L’école des Annales, sus publicaciones se han traducido al inglés, alemán, italiano o japonés), la catedrática emérita Teresa González Aja propone aquí una mirada audaz sobre la historia del fútbol, abarcando una narración genealógica y geográfica de más de siglo y medio en poco más de 200 páginas que componen un todo legible y estructurado hábilmente. Recorremos así un relato historiográfico que arranca en los inicios del juego balompédico tal y como lo conocemos hoy en día, nacido en Inglaterra en la segunda mitad del siglo XIX, hasta la última victoria de la selección española en el Mundial de 2010 en Sudáfrica. Basándose en una miríada de fuentes, la autora cita documentos oficiales de federaciones, revistas y prensa de la época, conjugándolo con trabajos de historiadores eminentes tales como Wahl (1997), Andrade de Melo (2017), Dietschy (2010) o Bahamonde (2002). Su aproximación panorámica no cae en lo superficial; es más, no elude el compromiso del historiador de rebatir y desmitificar ciertos datos que han alimentado las leyendas futbolísticas. Así, el libro es conciso y abundante en referentes, pero no en referencias bibliográficas que harían de su lectura un ejercicio áspero para el gran público. González Aja nos proporciona una densa lista de referencias al final de la obra. Dividido en 10 capítulos, que siguen un orden cronológico, pero sobre todo temático, el libro dedica una especial atención al nacimiento y expansión del fútbol, desde su gestación en Europa a su impacto global, primero en Latinoamérica, pasando por África, Norteamérica y Asia más tarde, para Cuad. his. cont. 46(1), 2024: 299-300 299 300 Reseñas. Cuad. his. cont. 46(1), 2024: 299-300 convertirse en el producto de consumo de masas en todo el mundo que conocemos hoy en día. El relato historiográfico que presenta González Aja resulta ameno y esclarecedor por su alternancia de lo minucioso con una encomiable capacidad de síntesis. Por ejemplo, dedica un tiempo a esclarecer ciertos datos poco contrastados sobre el origen mismo del fútbol en Inglaterra en torno a la figura de Thomas Arnold, la Escuela de Rugby y el paso a la profesionalización, sin renunciar a un agudo análisis de la importancia de la división social de clases, tanto en el proceso del fin del amateurismo, como en el propio juego: el dribbling game como símbolo aristocrático, materializado en un juego individual, virtuoso y elegante con el balón, enfrentado al passing game, propio de la llegada de jugadores de clases populares, y basado en el pase, empujado por “el espíritu colectivo de los obreros que suplanta el individualismo burgués” (p. 35). Es interesante comprobar la importancia política que la autora impregna en su explicación histórica de este fenómeno deportivo, como puede verse en la descripción de la evolución del fútbol europeo y sus estructuras federativas ligada inexorablemente a la historia misma de Europa y de su identidad cultural, o en la manipulación de las victorias futbolísticas por parte de los regímenes totalitarios, como el fascismo de Mussolini o el comunismo de la Unión Soviética. Del contexto español tampoco se eluden temas controvertidos y González Aja da cuenta de la importancia de las victorias del Real Madrid y la leyenda de Alfredo Di Stefano, la saeta rubia, durante el franquismo, la revolución del fútbol total en el FC Barcelona de Johan Cruyff en los años 70 y la influencia holandesa de un nuevo estilo de juego, o la evolución de una selección española que se encontraba a rebufo de otras grandes potencias cuando organizó el Mundial de 1982, hasta la unión identitaria de la sociedad española que supuso el gol de Iniesta en la final de Sudáfrica. Como fenómeno cultural que es, la autora atraviesa las diferentes etapas del desarrollo del fútbol ligándolo al desarrollo de los medios de comunicación. La llegada de la televisión y los adelantos técnicos permitieron hacer llegar a millones de espectadores las imágenes de futbolistas como Pelé, identificarse con ellos por sus orígenes humildes y soñar con “amplios espacios de movilidad social” (p. 138). Una mención aparte merece la atención dedicada al fútbol femenino, y en especial al de España, ahora que por primera vez en su historia la selección ha conseguido el título de campeonas del mundo en agosto de 2023. Los hechos históricos que se recogen en el libro permiten conjeturar que todavía quedan muchas etapas por franquear, si comparamos el desarrollo del fútbol femenino en países como Inglaterra, Italia, Alemania, Estados Unidos o los países escandinavos. En estos contextos, las mujeres también tuvieron que hacer frente a fuertes prejuicios que evolucionaron con el tiempo, teniendo un mayor empuje a mediados de los años 60. A los estereotipos vinculados exclusivamente por la pertenencia al género femenino (salvo excepciones como, por ejemplo, la figura de las munitionettes en la Primera Guerra Mundial en Gran Bretaña, cuando se valorizó las capacidades físicas de la mujer, obligada a remplazar al soldado en las fábricas), se añadían intereses económicos de las federaciones y clubes por evitar gestionar una filial deportiva que consideraban poco rentable. No será hasta los 80 y principios de los 90 cuando la UEFA y la FIFA por fin se involucran en el desarrollo y expansión del fútbol jugado por mujeres, desde el deporte practicado en categorías inferiores hasta la culminación de una élite que necesita aún de apoyo económico, mediático y político. Pero esa historia está aún por escribir. Bibliografía Alegi, Peter y Bolsmann, Chris (2013): South Africa and the Global Game. Football, Apartheid and beyond, Londres, Routledge. Andrade de Melo, Víctor (2017): “Evidência e especulaçao: ‘a origem’ do futebol no Rio de Janeiro (1898-1902)”, Movimento: Revista da Escola de Educaçao Física, 23(3), pp. 919-934. Bahamonde, Ángel (2002): El Real Madrid en la historia de España, Madrid, Taurus. Dietschy, Paul (2010): Histoire du football, Paris, Perrin. Eisenberg, Christiane, Pierre Lanfranchi, Tony Mason y Alfred Wahl (2004): 100 years of football: the FIFA centennial book, London, Weidenfeld and Nicholson. Wahl, Alfred (1997): Historia del fútbol, del juego al deporte, Barcelona, Ediciones B.
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Factors that Influence Farmer’s Behavior Towards Risk
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Factors that Influence Farmer's Behavior Towards Risk Muhamad Nurdin Yusuf, Agus Yuniawan Isyanto, and Sudradjat Sudradjat Muhamad Nurdin Yusuf, Agus Yuniawan Isyanto, and Sudradjat Sudradjat Department of Agribusiness, Faculty of Agriculture, Galuh University, Jl. R. E. Martadinata No.150, Ciamis 46274, Indonesia partment of Agribusiness, Faculty of Agriculture, Galuh University, Jl. R. E. Martadinata No.150, mis 46274, Indonesia Abstract. The research was carried out with the aim to find out the behavior of farmers towards risk and the factors that influence it. The research sample was 100 paddy farmers in flood-prone area paddy fields in Pangandaran District, West Java Province, Indonesia. Farmer's behavior towards risk was analyzed using quadratic utility functions, while the factors that influence farmer's behavior towards risk were analyzed using logistic regression. The results showed farmers 87 was risk neutral, while 13 farmer risk takers were farmers. Education, familys size and income significantly influence farmer's behavior towards risk; while age, experience, land area, production risk, price risk, income risk and group did not significantly influence farmer's behavior towards risk. Keywords: Land area, logistic regression, risk taker, quadratic utility. © 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 226, 00030 (2021) ICoN BEAT 2019 E3S Web of Conferences 226, 00030 (2021) ICoN BEAT 2019 https://doi.org/10.1051/e3sconf/202122600030  Coresponding author: muhamadnurdinyusuf@gmail.com 2 Research methodology Amount ten villages in Padaherang and Kalipucang Subdistricts in Pangandaran District were taken purposively as a sample area with consideration of being areas that have flood-prone rice fields. From each village a sample of ten farmers was taken, so that the total number of samples was 100 farmers. 1 Introduction The impact of global climate change has an effect on paddy production in Indonesia in the form of flooding which results in crop failure or a decrease in paddy production [1]. Climate change has occurred in Indonesia with indications of an increase in temperature and changes in rainfall patterns. Agriculture is very vulnerable to the effects of climate change with an indication of the high level of danger in decreasing paddy production as a result of increasing temperatures and changes in rainfall patterns. The average decline in paddy production is 1.37 % yr–1 which has the potential to cause a decline in national food production [2]. The threat of flooding in paddy fields can lead to reduced harvest area and paddy production [3]. Production is related to the nature of farming which is always dependent on nature supported by risk factors [4]. The risk of failure in farming comes from the use of new technology, prices of agricultural production, capital, government policies and individual behavior of farmers in dealing with outsiders [5], as well as climate change and weather that are not in accordance with crop needs [6]. The main risks farming include flooding [7]. Farmer's behavior towards risk consists of risk averter, risk neutral, and risk taker. Farmer behavior is the basis of farmers' decision making in carrying out their farming [1]. Farmer's behavior towards risk has an important role in influencing the productivity of agricultural products which has an impact on production efficiency [8]. Factors that E3S Web of Conferences 226, 00030 (2021) ICoN BEAT 2019 https://doi.org/10.1051/e3sconf/202122600030 influence farmer behavior towards risk are the area of planting, age, education, experience, family size, income and productivity risk [8]. influence farmer behavior towards risk are the area of planting, age, education, experience, family size, income and productivity risk [8]. 2 Research methodology rmer's behavior towards farming risk was analyzed using the quadratic utility Equation (1): U = τ1 + τ2 M + τ3 M2 (1) (1) U = τ1 + τ2 M + τ3 M2 Where: U : utility for expected income (in util) τ1 : intercept M : expected income at the balance point (rupiah value from certainty equivalent (CE) τ2 : indifference income coefficient (CE) τ3 : farmer risk coefficient The risk preference coefficient shows the farmer's attitude to risk, namely: τ3 = 0 : Risk neutral τ3 < 0 : Risk averse τ3 > 0 : Risk taker The factors that influence farmers' behavior towards risk are analyzed using ordinal logit regression with the following Equation (2): Pi = F(Yi)= F(α + β1X1 + β2X2 + β3X3 + β4X4 + β5X5 + β6X6 + β8D8+ β9D9 + β10D) (2) Yi values are calculated using the following Equation (3): Yi = Log [Pi/(1-Pi)] = (α + β1X1 + β2X2 + β3X3 + β4X4 + β5X5 + β6X6 + β7X7 + β8X8 +β9X9+ β10D + e) (3) Pi = F(Yi)= F(α + β1X1 + β2X2 + β3X3 + β4X4 + β5X5 + β6X6 + β8D8+ β9D9 + β10D) (2) Yi values are calculated using the following Equation (3): Yi = Log [Pi/(1-Pi)] = (α + β1X1 + β2X2 + β3X3 + β4X4 + β5X5 + β6X6 + β7X7 + β8X8 +β9X9+ β10D + e) (3) (3) Where: Yi = Opportunities for farmers to make decisions, where: Yi = Opportunities for farmers to make decisions, where: Y1 = 1 for the farmer who are risk averse Y2 = 2 for the farmer who are risk neutral Y3 = 3 for the farmer who are risk taker α = Intercept βi = Parameter regression coefficient (i = 1, 2, 3,….10) X1 = Age (year) X2 = Education (year) X3 = Family size (person) X4 = Experience (year) X5 = Land area (ha) X6 = Farm income (IDR) X7 = Production risk X8 = Price risk X9 = Income risk D = Membership in groups (1 if being a member of a group, 0 if not) e = error term 2 https://doi.org/10.1051/e3sconf/202122600030 E3S Web of Conferences 226, 00030 (2021) ICoN BEAT 2019 3.1 Farmer's behavior towards risk Farmer's behavior towards risk in paddy farming in flood-prone paddy fields can be seen in Table 1. Table 1. Farmer's behavior towards risk Table 1. Farmer's behavior towards risk Table 1. Farmer's behavior towards risk Farmer's behavior towards risk Number of people Percentage Risk Neutral 87 87.00 Risk Taker 13 13.00 Total 100 100.00 Table 1 shows that most farmers (87 %) are risk neutral, while the rest (13 %) are risk takers. There are no risk averse farmers. According to [9], the absence of risk avers farmers show that there are no farmers who are willing to sacrifice their income or potential income to reduce opportunities for loss or low income. According to [1], farmers will try to avoid failure and not get big profits by taking risks. Such behavior is called safety first, which is characteristic of most farmers. Farmers who are risk neutral are farmers who have a rational attitude in facing risks [9]. Risk neutral farmers tend to cultivate based on hereditary habits. They only seek income that can meet their family's needs [10]. If there is additional capital for risk neutral farmers, then they might add input to get higher income [11]. The availability of capital for farmers is a risk factor that is considered to affect farmers to the farming they are doing [12]. Risk taker farmers are farmers who are willing to allocate and use their production factors to the maximum, even though there are risks that must be faced with the aim of obtaining optimal results. [8] states that conceptually farmers are able to reduce production risk and price risk by improving their productivity, the use of diversification, the use of appropriate cropping patterns, strengthening farmer institutions, and bargaining position of farmers can increase farmers' production and income [7]. (Continued on next page) (Continued on next page) 3.2 Factors that influence farmer's behavior toward risk The results of the analysis of factors that influence farmer behavior on risk in paddy farming in flood-prone paddy fields can be seen in Table 2. Table 2. Factors that influence farmer's behavior towards risk Table 2. Factors that influence farmer's behavior towards risk Variabel B Wald Exp(B) Age 0.788 0.020 2.199 Education 7.139 3.452** 1.260E3 Family size 4.983 4.303* 145.958 Experience -0.149 0.003 0.861 Land area 0.687 0.279 1.988 Farm income 2.896 4.313* 18.103 (Continued on next page) 3 https://doi.org/10.1051/e3sconf/202122600030 E3S Web of Conferences 226, 00030 (2021) ICoN BEAT 2019 Table 2. Continued Variabel B Wald Exp(B) Production risk 1.029 0.407 2.799 Price risk -0.834 1.016 0.434 Income risk -0.134 0.014 0.875 Group 1.795 0.946 6.020 Constant -69.854 3.703** 0.000 Model Summary -2 Log likelihood Cox & Snell R Square Nagelkerke R Square Chi-square 19.158 0.441 0.819 18.307* *,** = significant at 5 %, 10 % Table 2 shows that age is not significant influence farmers' behavior towards risk. This shows that the difference in age of farmers does not affect the behavior of farmers in dealing with risk. Education significantly influences farmer's behavior towards risk. The results of this study are in accordance with the results of research from [13]. The higher the level of education of farmers, the more courageous farmers face risks [5, 7]. According to [13], the level of education of farmers who are still low makes the main cause of the majority of farmers who choose safety first (zero risk) behavior in developing their farming. Family size significantly influences farmer's behavior towards risk. The results of this study are in accordance with the results of research by [14]. According to [9], family size affects the outpouring of time that can be allocated for farming. According to [14], the more number of members of the farmer's family, the higher the capacity of the workforce owned by the farmer in facing the risk. Experience does not significantly influence farmers' behavior towards risk. The longer the farmer's experience, the more careful it will be in carrying out farming so that it tends to be more neutral to risk. The results of this study are in accordance with the results of research from [7]. Land area does not significantly affect farmer's behavior to risk. 4 Conclusion Amount 87 farmers are risk neutral and 13 farmers are risk takers. Education, family size and income significantly influence farmer's behavior towards risk; while age, experience, land area, production risk, price risk, income risk and group did not significantly influence farmer's behavior towards risk. 3.2 Factors that influence farmer's behavior toward risk The results of this study are consistent with the results of a study from [5] which shows that the addition or reduction of land area will not reduce risk aversion or that farmers are neutral towards risk. Revenue significantly influences farmer's behavior towards risk. The greater the income the farmer receives from the farming that is carried out, the farmer will be more courageous in accepting the risk. The results of this study are in accordance with the results of research from [8, 14, 5]. According to [8], the higher the farm income received by farmers, the farmers will be more willing to accept the risk. Production risk does not significantly influence farmer's behavior towards risk. According to [7], the magnitude of the risk of production faced by farmers due to the uncertainty of results as a result of natural factors and income as a result of price fluctuations, causes farmers to tend to reject the possibility of accepting the risks and uncertainties of the business. According to [15], efforts to handle production risk can be done by implementing a diversification and agricultural insurance program. Price risk does not significantly influence farmer's behavior towards risk. According to [16], farmers' household attitudes in carrying out production activities can be seen from variations in prices as a measure of price risk. According to [17], the behavior of farmers who accept price risk is caused by the expectation of expected price reductions compared to the actual prices that make farmers continue to carry out farming as long as they provide benefits. According to [18], the behavior of farmers does not dare to risk due to fluctuations 4 https://doi.org/10.1051/e3sconf/202122600030 E3S Web of Conferences 226, 00030 (2021) ICoN BEAT 2019 in production and selling prices which will have an impact on farmers' income. Income risk does not significantly influence farmer behavior towards risk. The higher the income risk faced by farmers, the more farmers will behave neutrally against risk. Membership in groups does not significantly influence farmers' behavior towards risk. According to [19], group membership is one of the efforts of farmers to reduce the risk of farming by cooperating with each other, exchanging information in managing their farming with fellow farmers. References 1. C. Gardebroek, M.D. Chavez, A.O. Lansink, J. Agric. Econ. 61,1:60–75(2010). https://doi.org/10.1111/j.1477-9552.2009.00222.x 1. C. Gardebroek, M.D. Chavez, A.O. Lansink, J. Agric. Econ. 61,1:60–75(2010). https://doi.org/10.1111/j.1477-9552.2009.00222.x 2. 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Risk Manag. 16:1–37(2017). https://www.researchgate.net/publication/310815962_Knowledge_And_Passive_Adapt ation_to_Climate_Change_an_Example_From_Indian_Farmers/fulltext/583a4d6308ae 3a74b49ea7e4/Knowledge-And-Passive-Adaptation-to-Climate-Change-an-Example-F rom-Indian-Farmers.pdf?origin=publication_detail 6
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Extruded Tessellations: A novel structural ceramic system at the intersection of industrial ceramic extrusion and CNC fabrication
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Extruded Tessellations: A novel structural ceramic system at the intersection of industrial ceramic extrusion and CNC fabrication Juan Pablo Ugarte-Urzúa Material Processes + Systems Group, Harvard GSD | USA | jugarte@gsd.harvard.edu Saurabh Mhatre Material Processes + Systems Group, Harvard GSD | USA | smhatre@gsd.harvard.edu Martin Bechthold Material Processes + Systems Group, Harvard GSD | USA | mbechthold@gsd.harvard.edu Sarah Norman Material Processes + Systems Group, Harvard GSD | USA | snorman@gsd.harvard.edu Sarah Norman Sarah Norman Material Processes + Systems Group, Harvard GSD | USA | snorman@gsd.harvard.edu Abstract This research explores the customization potential of ceramic extrusion by means of integrating CNC fabrication tools into current industrial ceramic extrusion lines. In order to support this approach, we designed and built two wall prototypes made of 700 extruded ceramic pieces. The pieces were produced using a single extrusion die and were cut to custom lengths and angles using CNC disk cutters to produce a total of 38 unique pieces. We introduce the motivation behind our work, present a three-stage design workflow for the design of this type of ceramic system, and show our built prototype. Keywords: Ceramic extrusion; CNC customization; Design workflow; Prototype; Tessellation. INTRODUCTION INTRODUCTION appearance. In academia, the emphasis has been on the formal customization of construction components. A variety of production methods have been explored, including slip- casting (Weston, 2013) and form-pressing (Bechthold et al., 2013). However, no method has received as much attention as additive manufacturing technologies have (Khoshnevis, 2004; Seibold et al., 2018). In spite of academia’s widespread interest in AMTs, this technology has not yet been embraced by the industry, mainly due to cost and precision limitations (Bechthold, 2016). Ceramic is one of the oldest materials known to man. In fact, it is thought to be the first material system designed by humans (Bechthold et al. 2015). Notable for its structural and thermal properties, and its aesthetic versatility, ceramic has been the object of renewed interest in mainstream architecture in recent decades. New design and fabrication technologies have greatly expanded the formal potential and scope of application of this material, catalyzing this rediscovery (Bechthold 2016). Our research takes another direction and explores the customization potential of ceramic extrusion, a different and comparatively underexplored production technique. Ceramic extrusion is the process where ceramic objects of fixed cross-sectional profile are produced by means of Today, an active area of research is the customization of ceramic building components. This is true both in industry and academia. For industry, the main effort has been on low-cost customization of ceramic tiles’ surface Figure 1: Top row: examples of work in ceramics and digital design and fabrication done by the Material Processes and Systems Group at the Harvard Graduate School of Design. Left to right: Suspended Ceramic Shell (2014), Protoceramics (2015), Ceramic morphologies (2017), Ceramic Tile Grid Shell (2019), Ceramic Hypar Tower (2020), Bottom row: examples of work in ceramics and AMTs. Left to right: Woven Clay (2014), Spatial Print Trajectory (2019), Janus Printing (2019). Figure 1: Top row: examples of work in ceramics and digital design and fabrication done by the Material Processes and Systems Group at the Harvard Graduate School of Design. Left to right: Suspended Ceramic Shell (2014), Protoceramics (2015), Ceramic morphologies (2017), Ceramic Tile Grid Shell (2019), Ceramic Hypar Tower (2020), Bottom row: examples of work in ceramics and AMTs. Left to right: Woven Clay (2014), Spatial Print Trajectory (2019), Janus Printing (2019). 326 Figure 2: Left: Typical industrial ceramic extrusion setup. INTRODUCTION Right: Vision of the integration of a ceramic extrusion line with an industrial robot equipped with a wire cutter to customize individual components. ure 2: Left: Typical industrial ceramic extrusion setup. Right: Vision of the integration of a ceramic extrusion line with an industrial robot equipped with a wire cutter to customize individual components. pushing clay through an extrusion die. A typical industrial ceramic extrusion line consists of a helical extruder that shoves clay through the die and a conveyor belt that receives the extruded clay (Händle, 2007). These pieces are then usually cut into smaller parts as needed. Ceramic extrusion allows for the creation of high quality, geometrically complex components at a competitive cost per unit in medium- to high-volume production (Bechthold, 2016). METHODS To assess the design possibilities latent in the integration of ceramic extrusion and CNC customization, we designed a structural ceramic system and tested it by building a wall prototype at a ceramics fair. This effort included the development of a three-stage design workflow to systematize the design of ceramic systems of this kind. A THREE-STAGE DESIGN WORKFLOW We created a parametric design model using Rhino Grasshopper (McNeel 2016) to efficiently design and test different components and wall assemblies. In this workflow, the design of the ceramic component is split into a three- stage iterative process. At each stage, we approach the design of the extrusion at both the component and the aggregation levels. We chose this approach to simultaneously address the individual component’s design requirements and the effects that design decisions made on that level have on the aggregation’s performance. Our process starts with the design of a simplified cross-section, continues with the refinement of the extrusion profile, and concludes with the CNC customization of each component, tying together design performance, material behavior and production constraints. We created a parametric design model using Rhino Grasshopper (McNeel 2016) to efficiently design and test different components and wall assemblies. In this workflow, the design of the ceramic component is split into a three- stage iterative process. At each stage, we approach the design of the extrusion at both the component and the aggregation levels. We chose this approach to simultaneously address the individual component’s design requirements and the effects that design decisions made on that level have on the aggregation’s performance. Our process starts with the design of a simplified cross-section, continues with the refinement of the extrusion profile, and concludes with the CNC customization of each component, tying together design performance, material behavior and production constraints. One drawback of ceramic extrusion is that it does not lend itself easily to individual differentiation of the extruded components. Given that an extrusion die will impose a unique and continuous cross-section on the extruded element, the only possible variations to the component’s geometry include bending the clay extrusion and cutting it into pieces at different lengths and/or cutting angles. In typical industrial settings, this customization can increase production costs proportionally to the amount of individually differentiated parts. GEOMETRIC STAGE The first step to design the ceramic extrusion is to produce a diagrammatic representation of the cross-section to be extruded —i.e. the component’s base geometry. This simplified polygon broadly determines the component’s behavior during production and the geometric rules for the aggregation of multiple modules. Because of our decision The first step to design the ceramic extrusion is to produce a diagrammatic representation of the cross-section to be extruded —i.e. the component’s base geometry. This simplified polygon broadly determines the component’s behavior during production and the geometric rules for the aggregation of multiple modules. Because of our decision Our work explores the integration of CNC fabrication tools into current industrial production lines to overcome these limitations and exploit the possibilities inherent to ceramic extrusion. While some CNC capabilities such as automated disk cutting are part of many extrusion lines today, they are not typically employed in the customization of structural ceramic components. We describe our approach to achieving the above result in the following section. Figure 3: Different examples of monohedral tessellations: triangular (a); quadrilateral (b); pentagonal (c); hexagonal (d-i). PROOF-OF-CONCEPT WALL PROTOTYPE We set out to design a ceramic system based on a single component manufactured using a single extrusion die. We decided the component’s cross-section —as opposed to its boundary surface— would be the locus to resolve most if not all the system’s performative aspects. Furthermore, we determined that the ceramic system should maximize design effect and component variability while keeping production costs and logistical complexity to a minimum. Figure 3: Different examples of monohedral tessellations: triangular (a); quadrilateral (b); pentagonal (c); hexagonal (d-i). 327 Figure 4: Adjustments made to the base geometery: a) original tile; b) interior offset to account for non-homogeneous shrinking; c) scaling operation to account for homogeneous shrinking. to use a single extrusion die, we used monohedral tessellations as the foundation for our base geometry. to use a single extrusion die, we used monohedral tessellations as the foundation for our base geometry. to use a single extrusion die, we used monohedral tessellations as the foundation for our base geometry. In two-dimensional Euclidean geometry, a tessellation is the tiling of the plane using one or more geometric shapes, called tiles. A monohedral tessellation is a special type of tiling in which all tiles are the same shape and size. The fact that they can produce complex repeating patterns using a single base unit makes these tessellations relevant to the design of extruded ceramic components (fig.3). Strictly speaking, our base geometry is a polyrhomb that generates a rhombille tessellation. However, it can also be thought of as a concave hexagon that produces a hexagonal tiling, which is a better description design-wise. Figure 4: Adjustments made to the base geometery: a) original tile; b) interior offset to account for non-homogeneous shrinking; c) scaling operation to account for homogeneous shrinking. incorporating a buffer distance between the components in the final aggregation. This is done by offsetting the component’s outer perimeter (Fig. 4). It is important to note that offsets maintain angular but not dimensional relationships. This means that after the offset operation, the extrusion profile will not tessellate the plane if the tiles are aggregated in direct contact with each other. When selecting the base geometry, we looked for specific attributes at the component and aggregation levels. At the component level, we looked for a tile with bilateral and rotational symmetry. The former results in more stability during extrusion while the latter maximizes the efficiency of the CNC cuts downstream. PROOF-OF-CONCEPT WALL PROTOTYPE At the aggregation level, we prioritized tilings that had co-planar coincident sides —i.e. tilings where adjacent or nearby tiles have sides that are aligned on a straight line— and interlocking features. From a mechanical standpoint, ceramic materials are brittle and lack tensile strength (Bechthold et al. 2015). The component’s cross-section must account for these shortcomings. As a result, most non-solid ceramic extrusions will need some kind of interior substructure in order to be self-supporting. Tessellations often present interlocking characteristics in which tiles are tightly coupled with their neighboring units. From a geometric standpoint, a tessellation can be said to be interlocking when no group of tiles (e.g. a row) can be moved on the plane of the tiling without affecting all the other tiles. Literature in masonry systems recognizes several types of interlocking between adjacent units, such as tongue and groove, dovetail, and geometry and stacking pattern (Ramamurthy and Nambiar, 2004). Our system falls within the last category. Interlocking tessellations may offer superior structural performance and reduce the need of bondage compared to non-interlocking tessellations (Dyskin et al., 2003). However, they may also result in intricate and time-consuming assembly sequences. The two most important parameters to consider when designing the component’s interior substructure are the minimum wall thickness and maximum slab span. The former refers to the lowest possible cross-sectional width of the component’s walls (both interior and exterior) while the latter refers to the distance the walls can span without needing additional supporting substructure. The mechanical properties of a ceramic material depend on the particular clay body employed and the circumstances surrounding the production of the ceramic part. Therefore, the component’s minimum wall thickness and maximum slab span need to be determined in collaboration with a ceramic producer, who should also provide other relevant Figure 5: Different cross-section and substructure alternatives. MATERIAL STAGE The support structure is temporary and prevents deformations caused by self-weight while the clay is wet. It is removed after firing, and often leaves marks on the component’s surface. Figure 6: Tessellations resulting from the aggregation of different cross-sections. The characters in italic refer to the different component designs presented in Fig. 5. Figure 7: FE analysis of the component’s performance at the aggregation level. The model includes modules in all possible orientations. Resulting stresses are well below material limits. Another important aspect to consider in the cross-section design is the connections between adjacent components. This is particularly relevant when the ceramic system uses mechanical connectors, as opposed to mortar. In such cases, the cross-section may include formal features such as channels, notches or rails to work in tandem with the connectors. The fixation system must take into account the distance buffer between units described earlier. Tolerance errors will rapidly and unpredictably increase in this gap due to components’ non-uniform deformations and the rotations involved in the assemblies. The fixation system needs to be designed with this issue in mind. Figure 7: FE analysis of the component’s performance at the aggregation level. The model includes modules in all possible orientations. Resulting stresses are well below material limits. material parameters. In the case of our extrusion, the minimum wall thickness and the maximum slab span were 10mm and 200mm before shrinking, respectively. material parameters. In the case of our extrusion, the minimum wall thickness and the maximum slab span were 10mm and 200mm before shrinking, respectively. We designed a novel fixation system consisting of off-the- shelf plastic profiles and zip ties. The cross-section of the ceramic piece has six notches distributed across its perimeter. These notches correspond with the notches of the neighboring units such that a plastic profile can be inserted on the corresponding notches of adjacent units and tied together using a zip tie to produce a clamp-like mechanism. To deal with accumulated tolerance between pieces, we use neoprene padding that can be placed incrementally between adjacent units to compensate for larger or smaller dimensional errors. The fixation system was primarily informed by the need to quickly assemble and disassemble the structure, before and after the ceramics fair. In that regard, we followed general design for Regarding the substructure design, our workflow allows us to operate at the unit scale and preview the results at the aggregation scale. MATERIAL STAGE After the component’s base geometry is defined, its actual cross-section has to be designed. Doing so entails distributing material within the bounds of the base geometry to produce solid regions, voids, and other features on the unit’s perimeter and interior substructure. These features will determine the component’s performative capacities —structural, thermal, aesthetic, and so forth— and must simultaneously address material, manufacturing, and assembly constraints. Before jumping into designing the component’s interior substructure, the base geometry needs to be adjusted to compensate for ceramic shrinking. Clay shrinks during the drying and firing processes at a rate between 8–12% depending on the clay body’s particle size and water content (Bechthold et al, 2015). Shrinkage works in two ways. First, the entire component shrinks uniformly until a given point, after which some areas continue shrinking, causing deformation in the component. This differential shrinking is more common in complexly shaped parts with unevenly distributed solid and void regions. Our design workflow addresses uniform shrinkage by upscaling the base geometry to produce an extrusion die larger than the desired final size of the component. The differential shrinkage, on the other hand, is counteracted by Figure 5: Different cross-section and substructure alternatives. 328 328 Figure 6: Tessellations resulting from the aggregation of different cross-sections. The characters in italic refer to the different component designs presented in Fig. 5. After evaluating different substructure design alternatives, we selected one (l in figures 5 and 6) and conducted a finite element analysis in Solidworks (Dassault Systemes, 2016). The analysis estimated the internal stresses in the ceramics from the wall’s self-weight only. The study assumed a component weight of 14 kg based on density of ceramics of 2,300 kg/m3, and a wall height of 2.25m. Bending stresses were in the order of 3 MPa, well below even the lowest allowable bending stress of ceramics, which typically ranges from 15-25 MPa. Furthermore, the analysis aimed to determine how exactly the module’s substructure was carrying the loads from unit to unit. The visual inspection described earlier is only referential; due to the formal complexity of the unit’s substructure and the multiple orientations they adopt in space, understanding the system’s structural behavior requires computational simulation. It is important to note that the component’s substructure and overall cross-section also needs to provide stability during the extrusion process. This often requires creating additional substructure outside the base geometry, i.e. on the exterior of the component. MATERIAL STAGE Designing at these two levels is crucial given that monohedral tessellations often involve rotational transformations. This means that once they are assembled into a wall aggregation, the components will have different orientations and thus will be subject to different structural solicitations. Seeing the units assembled in their final orientation allows us to better understand this issue and visually assess the continuity of load transmission from unit to unit. Figure 8: Fixation system detail: a) 80mm long and 2mm thick plastic profile with one perforation on each end; b) zip tie to secure the profiles; c) tolerance gap between components; d) neoprene strips for separation between modules; e) notch to insert the connectors. Figure 8: Fixation system detail: a) 80mm long and 2mm thick plastic profile with one perforation on each end; b) zip tie to secure the profiles; c) tolerance gap between components; d) neoprene strips for separation between modules; e) notch to insert the connectors. 329 329 Figure 9: Left: Final iterations of the cross section design. Changes include small adjustments to the interior substructure (4, 6, 7, 10, 12), and sizing of the fixation notches (1, 2, 12). Right: Final component’s cross section, in its extrusion orientation with support walls. Figure 9: Left: Final iterations of the cross section design. Changes include small adjustments to the interior substructure (4, 6, 7, 10, 12), and sizing of the fixation notches (1, 2, 12). Right: Final component’s cross section, in its extrusion orientation with support walls. assembly and disassembly recommendations outlined in Boothroyd and Alting (1992). aggregation spaces. The cutting-type taxonomy identifies three relevant dimensions. First, the geometry of the cut: cuts may be planar or ruled. Second, the effect of the cut on the component: cuts may either alter the shape of a component (e.g. length) or change its topology (e.g. exposing its inner substructure on its outer perimeter). And third, the purpose of the cut: cuts may add an aesthetic or performative effect at the aggregation level, or may change the aggregation logics of the system. Our component was customized with planar cuts that changed the shape of the units to produce an aesthetic effect in the walls. CNC CUSTOMIZATION STAGE The third and final stage in the extruded component’s design involves customizing the different pieces to achieve a desired design and/or performative effect at the aggregation level. Our research identifies two possible technical implementations: using industrial robots equipped with wire cutters to cut the clay while it is still wet, and using CNC disk cutters to cut it once it has been fired. Robotic arms offer the greatest customization potential due to their degrees of freedom and the fact that the clay is still wet and malleable. However, they are not commonly used in industrial ceramic production today. CNC disk cutters, on the other hand, are not uncommon in industrial contexts and still offer great potential for component customization. Although using them makes the customization process more cumbersome and potentially more expensive, those concerns can be mitigated by making design decisions that make the most out of each cut. Another aspect our workflow brings forth is the relationship between the ceramic extrusion, the CNC cutting space, and the unit at the aggregation level. Monohedral tilings often present rotational symmetry such that different units will have different orientations in space once they are part of the wall assembly. This means that three units placed with the same orientation on the CNC bed and cut with the same disk cutter angle will produce different surface angles on the aggregation space after being rotated along their longitudinal axis (figure 11 wall 1). Alternatively, different pieces can be placed in different orientations on the CNC bed and be cut using the same disk angle. If the piece To better understand all the above, our workflow lays out a basic taxonomy of cut types and draws connections between cutting operations at the unit, CNC, and Figure 10: Examples of different cuts classified based on our cutting-type taxonomy: a) no cut; b) planar formal cut for performative effect; c) ruled planar cut for performative effect; d) ruled topological cut for performative effect. Figure 10: Examples of different cuts classified based on our cutting-type taxonomy: a) no cut; b) planar formal cut for performative effect; c) ruled planar cut for performative effect; d) ruled topological cut for performative effect. RESULTS orientation on the CNC bed matches its final orientation in the wall assembly, the result is a co-planarity between adjacent units (figure 11 wall 2). Finally, a single cut can be used to produce two pieces (i.e. splitting a larger piece in half). This means that a single cutting angle in the CNC space becomes two surface angles on the aggregation space. Taken together, all the above means that a ceramic unit made from a tessellation with a three-fold rotational symmetry (such as ours), can be cut using a single disk- cutter angle to produce six different surface angles on the aggregation space (figure 11 wall 3). This minimizes the number of unique cutting operations and may result in lower production costs. We designed and built two ceramic wall prototypes made from 700 elements of 38 unique types. Wall A consisted of 350 pieces of 10 different types while wall B consisted of the same number of pieces of 28 different types (Fig. 12 and 13). The walls were 2.10m tall; the pieces ranged in length from 15-60cm and were cut in three different angles ranging from 15 to 25º for a total of 9 different orientations on the wall. We designed and built two ceramic wall prototypes made from 700 elements of 38 unique types. Wall A consisted of 350 pieces of 10 different types while wall B consisted of the same number of pieces of 28 different types (Fig. 12 and 13). The walls were 2.10m tall; the pieces ranged in length from 15-60cm and were cut in three different angles ranging from 15 to 25º for a total of 9 different orientations on the wall. The walls were erected following a specific assembly sequence and the units were dry stacked and clipped together using perforated plastic profiles and zip ties. On top of each wall, a 5mm steel plate connected the top row of modules together using the same zip tie connectors employed in the rest of the wall. The steel bar acted as a beam, making the whole wall work together structurally. In our wall design, we followed the strategy above and combined it with cutting the pieces at different lengths to produce a staggered effect on the wall surface. Additionally, we left one of the component’s sides flat for ease of transportation from factory to site. Figure 12: Top: Artistic rendering of our wall design. CNC CUSTOMIZATION STAGE 330 Figure 11: Diagram showing how a single cutting angle on the CNC space (either a robot or CNC disk cutter) can produce up to six different suface angles on the aggregation space in the case of a base geometry with a three-fold rotational symmetry. Pieces (a), (b), and (c) are cut in different orientations in space (120º rotation along longitudinal axis), while pieces a’, b’, and c’, are the other half resulting from the cutting operation. Figure 11: Diagram showing how a single cutting angle on the CNC space (either a robot or CNC disk cutter) can produce up to six different suface angles on the aggregation space in the case of a base geometry with a three-fold rotational symmetry. Pieces (a), (b), and (c) are cut in different orientations in space (120º rotation along longitudinal axis), while pieces a’, b’, and c’, are the other half resulting from the cutting operation. Figure 11: Diagram showing how a single cutting angle on the CNC space (either a robot or CNC disk cutter) can produce up to six different suface angles on the aggregation space in the case of a base geometry with a three-fold rotational symmetry. Pieces (a), (b), and (c) are cut in different orientations in space (120º rotation along longitudinal axis), while pieces a’, b’, and c’, are the other half resulting from the cutting operation. DISCUSSION There are two aspects of our work that we will unpack further in this section: the methods and tools used in the CNC customization process, and our design workflow and its implications for practice. This last point connects to our second topic, namely the need for actionable knowledge, such as computational design tools and design guidelines and recommendations, to facilitate the design of structural ceramic extrusions. Our three-stage workflow, although limited, streamlines the extrusion’s cross-section and wall assembly design processes. Each stage has a narrow design scope and deals with a well-defined number of design and technical considerations. This makes the task of designing the structural ceramic system structured and manageable yet flexible and generative. Regarding the first topic, the potential of using robotic tools to customize ceramic extrusions has been the focus of great work in recent years (e.g. Andreani and Bechthold, 2014, Schmidt, 2014). Much of that work was experimental in scope and did not involve real-world, industrial-scale production logistics, which are in turn crucial aspects in our project. Robotic tools have not been widely adopted in the ceramic industry and thus, using them in our research — situated in that context and directed towards that audience— did not seem justified. Furthermore, while CNC disk cutters and similar technologies are commonplace in industrial ceramic extrusion facilities, they are not regularly employed in the customization of ceramic components such as the one showcased here. One possible explanation is the increase in costs that these customization processes bring to the table, even when carried out by automated machines. The cost increase is caused by the difficult and time consuming additional machine and piece setup processes that automated customization entails —something often exacerbated by the industry’s lack of know-how in this regard. Furthermore, Our work has several limitations. First, we did not exhaustively explore the CNC customization cut space described earlier. We believe there is great potential in topological cuts and in cutting operations that transform the assembly logics of the components. Second, the extruded units presented structural problems at weak points during transportation and handling. Having had the chance to test different cross-section designs (i.e. multiple die designs) would have helped; however, it was cost prohibitive in this Our work has several limitations. First, we did not exhaustively explore the CNC customization cut space described earlier. RESULTS The two walls face each other, and are L-shaped to increase their stability. Bottom: Frontal elevation of the two walls. The different colors depict different unique pieces. Figure 12: Top: Artistic rendering of our wall design. The two walls face each other, and are L-shaped to increase their stability Bottom: Frontal elevation of the two walls. The different colors depict different unique pieces. 331 Figure 13: Series of images summarizing the design and production process of this project: a) initial formal explorations using 3d printed scaled blocks; b) test conducted by the contractor to evaluate our proposed connection system; c) our own initial test of the connector system —the image was used to communicate with the contractor overseas; d) component being extruded; e) component being cut with the CNC disk cutters; f) components being transported from factory to site; g) neoprene padding/separator; h) components being assembled; i) fastening of the zip tie connector; j) wall B almost finished. Figure 13: Series of images summarizing the design and production process of this project: a) initial formal explorations using 3d printed scaled blocks; b) test conducted by the contractor to evaluate our proposed connection system; c) our own initial test of the connector system —the image was used to communicate with the contractor overseas; d) component being extruded; e) component being cut with the CNC disk cutters; f) components being transported from factory to site; g) neoprene padding/separator; h) components being assembled; i) fastening of the zip tie connector; j) wall B almost finished. Figure 13: Series of images summarizing the design and production process of this project: a) initial formal explorations using 3d printed scaled blocks; b) test conducted by the contractor to evaluate our proposed connection system; c) our own initial test of the connector system —the image was used to communicate with the contractor overseas; d) component being extruded; e) component being cut with the CNC disk cutters; f) components being transported from factory to site; g) neoprene padding/separator; h) components being assembled; i) fastening of the zip tie connector; j) wall B almost finished. designers and architects in academia and practice do not seem to push the industry in this direction either. This lack of interest may be partially explained by the belief that ceramic extrusions are limited and inherently difficult to customize, and by the complexities involved in their design, as shown in the methods section. FUTURE WORK Boothroyd, G., & Alting, L. (1992). Design for Assembly and Disassembly. CIRP Annals, 41(2), 625–636. https://doi.org/10.1016/S0007-8506(07)63249-1 To continue this work, we plan to develop a computational design tool to fully streamline the design of this type of extruded component. Furthermore, we believe a set of formalized, actionable design guidelines that build upon our three-stage design workflow could be a long-lasting contribution to this research niche. As of now, our workflow does not take into account the whole breadth of design considerations that stem from the use of monohedral tessellations, nor does it comprehensively tackle the complexities of the cross-section design. Ceramic Hypar Tower (2020). Retrieved from https://research.gsd.harvard.edu/maps/ Dyskin, A. V., Estrin, Y., Pasternak, E., Khor, H. C., & Kanel-Belov, A. J. (2003). Fracture Resistant Structures Based on Topological Interlocking with Non-planar Contacts. Advanced Engineering Materials, 5(3), 116–119. https://doi.org/10.1002/adem.200390016 Friedman, J., Kim, H., & Mesa, O. (2014). Experiments in additive clay depositions. In Robotic Fabrication in Architecture, Art and Design 2014 (pp. 261-272). Springer, Cham. ACKNOWLEDGEMENTS We thank ASCER Tile of Spain for their continuing support, Javier Mira from the Instituto de Tecnología Cerámica, Pepe Castellano from Decorativa (ceramic producer), and Jordi Font de Mora from Grupo On Market (wall installation). We are also thankful to Leire Asensio Villoria for her early contributions to the design of the ceramic component, and Eva Pirker and Jared Friedman for their help with the detailing strategy. Seibold, Z. H. (2018). Ceramic Morphologies. Precision and control in paste-based additive manufacturing. ACADIA // 2018: Recalibration. On Imprecision and Infidelity. [Proceedings of the 38th Annual Conference of the Association for Computer Aided Design in Architecture (ACADIA)]. Pp. 350-357. http://papers.cumincad.org/cgi- bin/works/paper/acadia18_350 Seibold, Z., Mesa, O., Stavric, M., & Bechthold, M. (2018, July). Ceramic Tectonics: Tile Grid Shell. In Proceedings of IASS Annual Symposia (Vol. 2018, No. 8, pp. 1-8). International Association for Shell and Spatial Structures (IASS) DISCUSSION We believe there is great potential in topological cuts and in cutting operations that transform the assembly logics of the components. Second, the extruded units presented structural problems at weak points during transportation and handling. Having had the chance to test different cross-section designs (i.e. multiple die designs) would have helped; however, it was cost prohibitive in this Figure 14: Photographs of the finalized wall prototype on site. Figure 14: Photographs of the finalized wall prototype on site. Figure 14: Photographs of the finalized wall prototype on site. Figure 14: Photographs of the finalized wall prototype on site. 332 332 Andreani, S., & Bechthold, M. (2014). [Re]volving Brick: Geometry and Performance Innovation in Ceramic Building Systems Through Design Robotics. In Gramazio F., Kohler M., & Langenberg S. (Authors), Fabricate 2014: Negotiating Design & Making (pp. 182-191). London: UCL Press. https://doi.org/10.2307/j.ctt1tp3c5w.26 context. These are aspects of our design we will address in future work. context. These are aspects of our design we will address in future work. context. These are aspects of our design we will address in future work. Finally, an interesting aspect of the project’s design, construction and assembly process was that the design team and the manufacturer and contractor were in different countries and spoke different languages. This forced all actors involved to develop a shared language around these rather complicated geometries and entailed finding inventive ways to share knowledge about the novel ceramic system. In that regard, while this work was conducted pre- COVID, the problems arising from the geographical spread of the teams resonate with the challenges many design researchers face today to carry on their work. Bechthold, M. (2016). Prototipos cerámicos – diseño, computación y fabricación digital. Informes de La Construcción, 68(544), 167. https://doi.org/10.3989/ic.15.170.m15 Bechthold, Martin, Kane, A., & King, N. (2015). Ceramic Material Systems: In Architecture and Interior Design. In Ceramic Material Systems. Birkhäuser. https://www.degruyter.com/view/title/301948 Bechthold, Martin, King, J., Kane, A., Niemasz, J., & Reinhart, C. (2011). Integrated Environmental Design and Robotic Fabrication Workflow for Ceramic Shading Systems. ISARC Proceedings, 70–75. CONCLUSION This project investigated the design space at the intersection of industrial ceramic extrusion and CNC fabrication. We used a single extrusion die to design a ceramic module able to produce planar, folding and curved wall assemblies that feature interlocking, ornamental patterns. The extrusions were cut at different lengths and angles using CNC disk cutters after the clay was fired, producing 700 pieces of 38 different types. These customized pieces were used to create a unique surface texture on every wall surface. The pieces were then dry stacked for ease of assembly and disassembly, using an ad-hoc mechanical connection made of off-the-shelf plastic profiles and zip ties. We outlined a three-stage design workflow where the extruded component was iteratively refined in three stages and at two different levels. We believe that the tessellated extruded wall succeeds in demonstrating the potential for the creation of novel structural systems at the intersection of industrial ceramic extrusion and CNC fabrication. García del Castillo y López, J. L. B. (2019). Janus Printing. ACADIA 19: UBIQUITY AND AUTONOMY [Proceedings of the 39th Annual Conference of the Association for Computer Aided Design in Architecture (ACADIA)]. Pp. 576-585. http://papers.cumincad.org/cgi- bin/works/paper/acadia19_576 Händle, F. (Ed.). (2007). Extrusion in Ceramics. Springer-Verlag. https://doi.org/10.1007/978-3-540-27102-4 Khoshnevis, B. (2004). Automated construction by contour crafting—Related robotics and information technologies. Automation in Construction, 13(1), 5–19. https://doi.org/10.1016/j.autcon.2003.08.012 Protoceramics (2015). Retrieved from https://research.gsd.harvard.edu/maps/ Ramamurthy, K., & Nambiar, E. K. K. (2004). Accelerated masonry construction review and future prospects. Progress in Structural Engineering and Materials, 6(1), 1–9. https://doi.org/10.1002/pse.162 Schmidt, P. (2014). Architect manufacturer collaboration through the design of a ruled surface cutter for extruded terracotta (Order No. 1566983). Available from ProQuest Dissertations & Theses Global. (1627753367). Retrieved from http://search.proquest.com/docview/1627753367 Alothman, S., Im, H. C., Jung, F., & Bechthold, M. (2019). Spatial Print Trajectory. In J. Willmann, P. Block, M. Hutter, K. Byrne, & T. Schork (Eds.), Robotic Fabrication in Architecture, Art and Design 2018 (pp. 167–180). Springer International Publishing. https://doi.org/10.1007/978-3-319-92294-2_13 REFERENCES Alothman, S., Im, H. C., Jung, F., & Bechthold, M. (2019). Spatial Print Trajectory. In J. Willmann, P. Block, M. Hutter, K. Byrne, & T. Schork (Eds.), Robotic Fabrication in Architecture, Art and Design 2018 (pp. 167–180). Springer International Publishing. https://doi.org/10.1007/978-3-319-92294-2_13 Suspended Ceramic Shell (2014). Retrieved from https://research.gsd.harvard.edu/maps 333
https://openalex.org/W2314828738
https://europepmc.org/articles/pmc3642804?pdf=render
English
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Prehospital risk stratification following out-of-hospital cardiac arrest
Critical care
2,013
cc-by
263,567
P1 Protective role of autophagy in mouse cecal ligation and puncture-induced sepsis model Methods After profound anesthesia with pentothal, the Wistar rats were killed by exsanguination. After sternotomy, the heart was taken and connected to the Langendorf column. The apex of the heart was hooked to a strength sensor. Biopac student laboratory software was used to record and analyse heart contractions. Contractions were recorded every 5  minutes during periods of 20  minutes. Control measurements were fi rst recorded. We measured four parameters: heart rate, contraction force, speeds of contraction and relaxation for control, during TNFα (20  ng/ml) exposure and after removal of TNFα. We express the variations of parameters as percentage of the control  ±  SEM. A paired t test was used to compare heart rate, contraction amplitude, speeds of contraction and relaxation with TNFα and control measurements and after removal of TNFα. pu ctu e duced seps s ode W Takahashi1, H Hatano2, H Hirasawa1, S Oda1 1Graduate School of Medicine, Chiba University, Chiba, Japan; 2Biomedical Research Center, Chiba University, Chiba, Japan Critical Care 2013, 17(Suppl 2):P1 (doi: 10.1186/cc11939) Introduction Autophagy is well known as one of the biogenic responses against various stresses, which possesses the benefi cial roles for survival, but little is known about the dynamics and its signifi cance during the septic condition. We hypothesized that autophagy is induced during the septic condition, and contributes to protect from tissue damage which subsequently leads to organ dysfunction. We confi rm whether the autophagic process is accelerated or sustained in an acute phase of sepsis and we also determine its physiological role. Results Eight rat hearts Wistar (weight = 325 ± 23 g) were studied. See Table 1. y g Methods Sepsis was induced by cecal ligation and puncture (CLP) in mice. We examined the kinetics of autophagosome and auto lysosome formation which may explain the status of autophagy by western blotting, immunohistochemistry, and electron microscopy. To investigate a precise role of autophagy in CLP-induced sepsis, chloroquine, an autophagy inhibitor, was administered to the CLP-operated mice, and blood chemistry, pathology of the liver and survival were evaluated. Table 1 (abstract P2) TNFα Removal of TNFα Heart rate 78 ± 6* 91 ± 5 Contractile 62 ± 8* 91 ± 4 Speed of contraction 72 ± 6* 93 ± 2 Speed of relaxation 53 ± 10* 89 ± 4 Results expressed as percentage of control ± SEM. *P <0.05. MEETING ABSTRACTS MEETING ABSTRACTS P1 Protective role of autophagy in mouse cecal ligation and puncture-induced sepsis model y p gy Results Autophagy demonstrated by the ratio of LC3-II/LC3-I was induced over the time course up to 24 hours after CLP. The ratio was particularly increased in the liver, heart and spleen. Autophagosome formation became maximal at 6 hours and declined by 24 hours after CLP. Autolysosome formation as evaluated by both fusion of GFP-LC3 dots with LAMP1 immunohistochemistry and electron microscopy was also increased after the procedure. Furthermore, inhibition of autophagy by chloroquine during the CLP procedure resulted in elevation of serum AST levels, and signifi cantly increased mortality in mice. Conclusion TNFα decreases signifi cantly the heart rate, contractile force, speeds of contraction and relaxation on isolated perfused rat heart. TNFα probably plays a role in the pathophysiology of cardiomyopathy during septic shock. The partial reversibility of these eff ects could explain why left ventricular hypokinesia in patients with septic shock is reversible. Conclusion Autophagy was induced in several organs over the time course of the CLP sepsis model and then the process was gradually completed to degradation of the components. Our data suggest autophagy plays a protective role in organ dysfunction in sepsis. Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 P1 Protective role of autophagy in mouse cecal ligation and puncture-induced sepsis model W Takahashi1, H Hatano2, H Hirasawa1, S Oda1 1Graduate School of Medicine, Chiba University, Chiba, Japan; 2Biomedical Research Center, Chiba University, Chiba, Japan Critical Care 2013, 17(Suppl 2):P1 (doi: 10.1186/cc11939) P1 Protective role of autophagy in mouse cecal ligation and puncture-induced sepsis model W Takahashi1, H Hatano2, H Hirasawa1, S Oda1 1Graduate School of Medicine, Chiba University, Chiba, Japan; 2Biomedical Research Center, Chiba University, Chiba, Japan Critical Care 2013, 17(Suppl 2):P1 (doi: 10.1186/cc11939) References References 1. Vieillard-Baron A, et al.: Actual incidence of global left ventricular hypokinesia in adult septic shock. Crit Care Med 2008, 36:1701-1706. 2. Hunter JD, et al.: Sepsis and the heart. Br J Anaesth 2010, 104:3-11. 1. Vieillard-Baron A, et al.: Actual incidence of global left ventricular hypokinesia in adult septic shock. Crit Care Med 2008, 36:1701-1706. yp p 2. Hunter JD, et al.: Sepsis and the heart. Br J Anaesth 2010, 104:3-11. 2. Hunter JD, et al.: Sepsis and the heart. Br J Anaesth 2010, 104:3-11. 33rd International Symposium on Intensive Care and Emergency Medicine Brussels, Belgium, 19-22 March 2013 Published: 19 March 2013 The aim of our study was to observe the eff ects of TNFα on the model of perfused rat heart. P3 Eff ect of cdp-choline on microcirculatory alterations during endotoxemia BV Nguyen1, M Guillouet2, MA Giroux-Metges2, G Gueret3, M Ould-Ahmed1, JP Pennec2 1Hôpital d’Instruction des Armées Clermont Tonnerre, Brest, France; 2Laboratory of Physiology, Faculty of Medicine, Brest, France; 3University Hospital, Brest, France Critical Care 2013, 17(Suppl 2):P2 (doi: 10.1186/cc11940) K Schmidt1, M Doerr1, T Brenner1, S Hofer1, A Walther2 1Universitätsklinikum Heidelberg, Germany; 2Klinikum Stuttgart, Klinik für Anästhesiologie u. operative Intensivmedizin, Stuttgart, Germany Critical Care 2013, 17(Suppl 2):P3 (doi: 10.1186/cc11941) K Schmidt1, M Doerr1, T Brenner1, S Hofer1, A Walther2 1Universitätsklinikum Heidelberg, Germany; 2Klinikum Stuttgart, Klinik für Anästhesiologie u. operative Intensivmedizin, Stuttgart, Germany Critical Care 2013, 17(Suppl 2):P3 (doi: 10.1186/cc11941) Introduction Acute myocardial depression in septic shock is common [1]. Myocardial depression is mediated by circulating depressant substances, which until now have been incompletely characterized [2]. Introduction The cholinergic anti-infl ammatory pathway (CAP) is a physiological mechanism that inhibits cytokine production and minimizes tissue injury during infl ammation. CAP-mediated anti- infl ammatory signals in vagal eff erent nerve fi bers result in the release of acetylcholine, which interacts with innate immune cells that express the nicotinic acetylcholine receptor subunit α7 (α7nAChR). Introduction The cholinergic anti-infl ammatory pathway (CAP) is a physiological mechanism that inhibits cytokine production and minimizes tissue injury during infl ammation. CAP-mediated anti- infl ammatory signals in vagal eff erent nerve fi bers result in the release of acetylcholine, which interacts with innate immune cells that express the nicotinic acetylcholine receptor subunit α7 (α7nAChR). © 2010 BioMed Central Ltd © 2013 BioMed Central Ltd Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Endothelial dysfunction during sepsis is responsible for increased endothelial permeability, leukocyte–endothelial interaction and functional breakdown of microvascular perfusion. Endotoxemia- induced endothelial dysfunction can be reduced by cholinergic CAP activation [1]. The aim of this study was to determine the eff ects of the α7nAChR-agonist cdp-choline on microcirculatory alterations during experimental endotoxemia. response (a fi vefold to ninefold rise compared with other groups who had an increase of 14-fold to 36-fold). Conclusion Severely ill patients and secondarily hemodialysis and diabetic patients are in a proinfl ammatory state. The response of all examined groups to provocation by LTA was suffi cient, with a diff erential expression of severely ill patients, a fact that refl ects their diff erent immunologic status. P5 Correlation of the oxygen radical activity and antioxidants and severity in critically ill surgical patients: preliminary report J Lee1, H Shim2, JY Jang1 , , g 1Yonsei University College of Medicine, Seoul, South Korea; 2Wonju Severance Christian Hospital, Yonsei University Wonju College of Medicine, Wonju, Korea Critical Care 2013, 17(Suppl 2):P5 (doi: 10.1186/cc11943) Introduction In septic patients, the oxygen radical (OR) showed toxic eff ect to induce infl ammation and antioxidant activity could aff ect organ dysfunction. This study was designed to determine the relationship between antioxidant level and severity of organ dysfunction. Results There were no signifi cant diff erences in venular wall shear rate between the groups after 120 minutes. There was no signifi cant diff erence in the number of adhering leukocytes between the cdp- choline/LPS groups (iii, iv) and the LPS group after 120  minutes. Macromolecular effl ux signifi cantly increased in all groups over 120  minutes. All groups (i, ii, iii, iv) showed a signifi cantly reduced macromolecular effl ux compared with the LPS group after 120 minutes. Conclusion Cdp-choline has no eff ect on leukocyte–endothelial interaction and microhemodynamic alterations during endotoxemia. By activating the CAP, cdp-choline reduces capillary leakage. Thus cdp- choline might have a prophylactic and therapeutic anti-infl ammatory eff ect on LPS-induced endothelial permeability. These fi ndings identify the endothelium as a target of anti-infl ammatory cholinergic mediators and cdp-choline as a potential therapeutic substance in sepsis treatment. Methods The medical records of adult patients managed in a surgical ICU from August 2012 to December 2012 were reviewed prospectively. Abstracted data included age, body weight (with BMI), APACHE II scores, SOFA scores, MODS scores, fl uid intake, fl uid output, nutritional support, shock, antioxidant levels, OR activities, zinc and selenium levels, complication and mortality. In addition, length of stay (LOS) in the ICU and in hospital, and in-hospital mortality were collected. These data were investigated on the fi rst, the third and the seventh day, respectively. p y Results A total of 13 patients were enrolled. The in-hospital mortality rate was 7.7% and mean LOS in the ICU and hospital was 6.5 and 27.6, respectively. Mean APACHE II score was 20.2. On the fi rst day of ICU, the mean antioxidant level and OR were 1.5 (± 0.5) mmol/l and 1.6 (± 0.5) mmol/l, respectively. At the same time, SOFA and MODS scores were 7.3 and 5.0, respectively, and zinc and selenium were 32.6 μg/dl and 68.4 ng/ml. P5 Correlation of the oxygen radical activity and antioxidants and severity in critically ill surgical patients: preliminary report J Lee1, H Shim2, JY Jang1 On the third day, mean antioxidant and OR were 1.5 (± 0.4) and 1.8 (± 0.7) respectively (SOFA 6.6, MODS 4.9, zinc 50.0, selenium 70.7). On the seventh day, mean antioxidant and OR were 1.4 (± 0.5) and 1.9 (± 0.7), respectively (SOFA 4.3, MODS 3.1, zinc 62.8, selenium 77.3). In the correlation analysis, MODS scores and antioxidant level had signifi cant correlations on the fi rst and seventh days of ICU (P = 0.001, P = 0.009). p Reference 1. Peter C, et al.: Shock 2010, 33:405-411. References 1. Noveanu M, Mebazaa A, Mueller C: Cardiovascular biomarkers in the ICU. Curr Opin Crit Care 2009, 15:377-383. y Methods Blood sample was obtained from 10 healthy volunteers, 10 hemodialysis patients with end-stage chronic renal failure (CRF), 10 patients with type II diabetes mellitus (DM) and 10 ICU patients on the second day of hospitalization, who suff ered nonseptic SIRS and had an APACHE II score >25. After suitable treatment the samples were incubated with 1 mg LTA for 8 hours and maintained at –20°C until the measurement of cytokines TNFα, IL-6, IL-1β, and IL-10, using the ELISA method. The results are presented as mean values ± SEM. Graph Pad 4.0 was used, applying a t test to test the variation of each cytokine in each group, and ANOVA to assess the diff erences between the four groups. Methods Blood sample was obtained from 10 healthy volunteers, 10 hemodialysis patients with end-stage chronic renal failure (CRF), 10 patients with type II diabetes mellitus (DM) and 10 ICU patients on the second day of hospitalization, who suff ered nonseptic SIRS and had an APACHE II score >25. After suitable treatment the samples were incubated with 1 mg LTA for 8 hours and maintained at –20°C until the measurement of cytokines TNFα, IL-6, IL-1β, and IL-10, using the ELISA method. The results are presented as mean values ± SEM. Graph Pad 4.0 was used, applying a t test to test the variation of each cytokine in each group, and ANOVA to assess the diff erences between the four groups. Results Baseline cytokine values in the three groups were increased compared with the control group, but the diff erence was signifi cant only for the ICU group (Table  1, data only for IL-6 and IL-10). The quotient IL-10/IL-6 of baseline values was between 0.23 and 0.96 among healthy, ESRD and DM persons, and 1.32 among ICU patients. In all examined groups the levels of cytokines increased signifi cantly after stimulation with LTA, although ICU patients showed a diff erential 2. Piechota M, Banach M, Irzmanski R, Barylski M, Piechota-Urbanska M, Kowalski J, et al.: Plasma endothelin-1 levels in septic patients. J Intensive Care Med 2007, 22:232-239. 3. Kotsovolis G, Kallaras K: The role of endothelium and endogenous vasoactive substances in sepsis. Hippokratia 2010, 14:88-93. P3 Eff ect of cdp-choline on microcirculatory alterations during endotoxemia Methods Using fl uorescent intravital microscopy, we determined venular wall shear rate, macromolecular effl ux and leukocyte adhesion in mesenteric postcapillary venules of male Wistar rats. Endotoxemia was induced over 120 minutes by intravenous infusion of lipopolysaccharide (LPS). Control groups received an equivalent volume of saline. Cdp-choline was applied as an i.v. bolus in treatment groups. Animals received either (i)  saline alone, (ii)  cdp-choline 10 minutes prior to saline administration, (iii) cdp-choline 10 minutes prior to LPS administration, (iv)  cdp-choline 30  minutes after LPS administration or (v) LPS alone.if Immune response after stimulation with wall components of Gram-positive bacteria S Aloizos1, E Tsigou2, P Myrianthefs2, S Gourgiotis1, A Tsakris3, G Baltopoulos2 1NIMTS Hospital, Athens, Greece; 2A. Anargiroi Hospital, Athens, Greece; 3M di l S h l Ath G S Aloizos1, E Tsigou2, P Myrianthefs2, S Gourgiotis1, A Tsakris3, G Baltopoulos2 1NIMTS Hospital, Athens, Greece; 2A. Anargiroi Hospital, Athens, Greece; 3Medical School Athens Greece S Aloizos1, E Tsigou2, P Myrianthefs2, S Gourgiotis1, A Tsakris3, G Baltopoulos2 1NIMTS Hospital, Athens, Greece; 2A. Anargiroi Hospital, Athens, Greece; 3Medical School, Athens, Greece Critical Care 2013, 17(Suppl 2):P4 (doi: 10.1186/cc11942) Critical Care 2013, 17(Suppl 2):P4 (doi: 10.1186/cc11942) Critical Care 2013, 17(Suppl 2):P4 (doi: 10.1186/cc11942) Conclusion Antioxidant level had a correlation with organ dysfunction which might be used as a prognostic factor in critically septic patients. To prove this, large-scale data collection is required. R f Introduction The purpose of this study was to evaluate the immune response of patients susceptible to infection by Gram-positive bacteria after ex vivo provocation with lipoteichoic acid (LTA) and to compare the reaction with the one of healthy adults. Prevention of sepsis by correcting apoptosis M Puhtinskaya, V Estrin y Research Institute of Obstetrics and Pediatrics, Rostov-on-Don, Russia Critical Care 2013, 17(Suppl 2):P8 (doi: 10.1186/cc11946) Research Institute of Obstetrics and Pediatrics, Rostov-on-Don, Russia Critical Care 2013, 17(Suppl 2):P8 (doi: 10.1186/cc11946) Research Institute of Obstetrics and Pediatrics, Rostov-on-Don, Russ Critical Care 2013, 17(Suppl 2):P8 (doi: 10.1186/cc11946) Introduction Activation of apoptosis in lymphocytes determines the development of neutropenia and of sepsis [1,2]. We investigated prevention of sepsis and correction of lymphocyte apoptosis by recombinant human granulocyte colony-stimulating factor (hr-GCSF, fi lgrastim) [1,2]. Methods With the permission of the ethics committee, a controlled, randomized, blind clinical trial included 69 term newborns on mechanical ventilation, without neutropenia and clinical signs of infection, with a content of lymphocytes in early apoptosis (AnnexinV– FITC+PI–) of >9.59%, and in late (AnnexinV–FITC+PI+) of 0.56%. Lymphocytes in apoptosis were detected using antibodies to AnnexinV and propidium iodide staining method of immunophenotyping (fl ow cytometry; Beckman Coulter Epics XL, USA). The survey was conducted at admission, at 3 to 5 days, and 20 days. The method of random numbers in Group I included 39 newborns who on admission (with written parental consent) received an intravenous infusion of hr-GCSF dose of 10 μg/kg, 3 days. Newborns of Group II (n = 30) did not receive hr-GCSF. Power of the study was 80% (α ≤0.05).i Conclusion In this study we noticed that during sepsis some signifi cant diff erences in the status of activation of immune cells exist between peripheral blood and lung resident cells. The lung milieu seems to promote activation of monocytes while neutrophil activation is more dependent on the site of infection. However, these observations require further studies in a larger group of patients. Acknowledgements This study was supported by the Centre of Postgraduate Medical Education grant no 501-01-02-012 and by the sources of the Medical University of Warsaw. P7 Anti-infl ammatory eff ects of Kupff er cells through α7-nicotinic acetylcholine receptors Y Li, X Shi Changzheng Hospital, Second Military Medical University, Shanghai, China Critical Care 2013, 17(Suppl 2):P7 (doi: 10.1186/cc141945) Results For 3 to 5 days, Group I signifi cantly decreased apoptosis of lymphocytes in the early from 16.1% to 7.8%, and in late from 1.3% to 0.1%. The development of sepsis and neutropenia have been reported. We observed no clinical or laboratory signs of adverse eff ects of the drug. Fatal outcomes (n = 4) are not associated with hr-GCSF, which was confi rmed postmortem. Prevention of sepsis by correcting apoptosis M Puhtinskaya, V Estrin Decreased duration of mechanical ventilation (P <0.05). In Group II, 27 patients at 3 to 5 days developed neutropenia and increased lymphocytes in apoptosis (P <0.05). Sepsis was diagnosed in 19 children; eight fatal outcomes. Introduction Nicotine exerts anti-infl ammatory eff ects in several cell types. α7-nicotinic acetylcholine receptor (α7-nAChR), which has high permeability to calcium, is believed to contribute signifi cantly to nicotinic anti-infl ammatory eff ects. However, the molecular mechanism is largely unknown. Kupff er cells in the liver play an important role in infl ammatory response to pathogens invading, but whether there is α7-nAChR expression in Kupff er cells or cholinergic anti-infl ammatory pathway involved in this process remains unclear.f Conclusion hr-GCSF reduces the incidence of septic complications and one of the mechanisms of its clinical eff ectiveness is the reduction of apoptotic factors aff ecting the development of neutropenia. References 1. Gillan ER, Christensen RD, Suen Y, et al.: A randomized, placebo-controlled trial of recombinant human granulocyte colony-stimulating factor administration in newborn infants with presumed sepsis: signifi cant induction of peripheral and bone marrow neutrophilia. Blood 1994, 84:1427-1433. 1. Gillan ER, Christensen RD, Suen Y, et al.: A randomized, placebo-controlled trial of recombinant human granulocyte colony-stimulating factor administration in newborn infants with presumed sepsis: signifi cant induction of peripheral and bone marrow neutrophilia. Blood 1994, 84:1427-1433. Methods (1) Kupff er cells, isolated by collagenase digestion and diff erential centrifugation from mice and labeled with FITC-aBGT, were observed under laser scanning confocal microscope to test the expression of α7-nAChR. Protein level was also tested by western blotting, with RAW264.7 as positive control; (2) 100  nM LPS was given to Kupff er cells, with or without 1 mM nicotine. TNFα, IL-10 and HMGB-1 were tested at 4 hours, 12 hours or 24 hours, respectively; (3) 100 BALB/c mice were randomly divided into four group: Group I (only lethal dose of LPS was given), Group II (nicotine and LPS were given), Group III (LPS, nicotine and GdCl3 were given), and Group IV (LPS and nicotine were given and the left cervical vagus nerve was cut off ). The mortality of mice was observed for 72 hours.fi 2. Pukhtinskaya MG, Estrin VV, Gulova ES: Clinical and diagnostic value of apoptosis markers in the pathogenesis of neutropenia and bacterial complications in newborns with respiratory distress syndrome. Cytokines Infl amm 2011, 10:66-69. Prevention of sepsis by correcting apoptosis M P h k V E g y Results The mortality of sepsis reached 70%. Nonsurvivors had signifi cantly (P = 0.001) elevated expression of CD64 on neutrophils. Expression of HLA-DR was higher in monocytes from BAL than PB GMF (1,032 vs. 342; P = 0.02) and this tendency was present in sepsis originating from both pneumonia and peritonitis. Percentage of HLA- DR-positive T cells was lower in PB than in BAL (2.9% vs. 6.5%; P = 0.07), but the GMF values for HLA-DR were higher in the circulating T cells (1,904 vs. 1,346; P = 0.004). The expression of CD64 on neutrophils was not signifi cantly diff erent in PB and BAL, but there was a trend towards its higher expression in BAL from patients with pneumonia while its expression was higher in PB of patients with peritonitis.i Simultaneous analysis of the expression of CD64 and HLA-DR in the peripheral blood and bronchoalveolar lavage fl uid in sepsis It was verifi ed that left cervical vagus nerve was essential for the anti- infl ammatory eff ect of nicotine and α7 acetylcholine receptors might play a critical role. anti-infl ammatory compensatory response. However, controversies exist regarding the status of the immune system in local tissue compartments during sepsis. The aim of this study was to compare selected markers of activation between the systemic circulation and local lung environment. Methods Twenty patients with severe sepsis were included into this study. Peripheral blood (PB) samples and bronchoalveolar lavage fl uid (BALF) samples were obtained on the day of diagnosis (D1). BALF was collected from 11 patients. Samples were stained with antibodies: CD15/CD64 and CD3/CD14/HLA-DR and isotypic control. Cells were analysed by fl ow cytometry. Expression of markers of activation was analysed as the geometric median of fl uorescence (GMF). All values are expressed as median values. Comparisons between groups were performed using Mann–Whitney and Wilcoxon tests. References 1. Wang H, et al.: Nicotinic acetylcholine receptor α7 subunit is an essential regulator of infl ammation. Nature 2003, 421:384-388. 1. Wang H, et al.: Nicotinic acetylcholine receptor α7 subunit is an essential regulator of infl ammation. Nature 2003, 421:384-388. 2. Wang H, et al.: Cholinergic agonists inhibit HMGB1 release and improve survival in experimental sepsis. Nat Med 2004, 10:1216-1221. 2. Wang H, et al.: Cholinergic agonists inhibit HMGB1 release and improve survival in experimental sepsis. Nat Med 2004, 10:1216-1221. Simultaneous analysis of the expression of CD64 and HLA-DR in the peripheral blood and bronchoalveolar lavage fl uid in sepsis f Results Baseline cytokine values in the three groups were increased compared with the control group, but the diff erence was signifi cant only for the ICU group (Table  1, data only for IL-6 and IL-10). The quotient IL-10/IL-6 of baseline values was between 0.23 and 0.96 among healthy, ESRD and DM persons, and 1.32 among ICU patients. In all examined groups the levels of cytokines increased signifi cantly after stimulation with LTA, although ICU patients showed a diff erential l T Skirecki1, M Mikaszewska-Sokolewicz2, G Hoser1, U Zielińska-Borkowska1 1The Centre of Postgraduate Medical Education, Warsaw, Poland; 2 Medical University of Warsaw, Poland T Skirecki1, M Mikaszewska-Sokolewicz2, G Hoser1, U Zielińska-Borkowska1 1The Centre of Postgraduate Medical Education, Warsaw, Poland; 2 Medical University of Warsaw, Poland y Critical Care 2013, 17(Suppl 2):P6 (doi: 10.1186/cc11944) Introduction The core pathophysiological changes in sepsis involve systemic activation of the immune system followed by the Table 1 (abstract P4). Levels of cytokines before and after stimulation with LTA Control baseline LTA ESRD baseline LTA DM baseline LTA ICU baseline LTA IL-6 8.90 ± 0.76, 245.30 ± 26.68 86.60 ± 45.55, 1,310.00 ± 154.80 15.90 ± 1.89, 252.00 ± 35.52 372.40 ± 120.60, 3,659.00 ± 485.20 IL-10 3.00 ± 1.08, 40.90 ± 7.45 19.20 ± 7.14, 273.10 ± 126.50 15.30 ± 2.08, 350.50 ± 89.42 492.60 ± 66.72, 2,822.00 ± 432.70 Values in pg/ml. Table 1 (abstract P4). Levels of cytokines before and after stimulation with LTA Table 1 (abstract P4). Levels of cytokines before and after stimulation with LTA Control baseline LTA ESRD baseline LTA S3 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Conclusion Kupff er cells played a crucial rule in modulating infl ammation and the anti-infl ammatory eff ect of nicotine was partially weakened after left cervical vagus nerve cut off or aBGT was given. It was verifi ed that left cervical vagus nerve was essential for the anti- infl ammatory eff ect of nicotine and α7 acetylcholine receptors might play a critical role. Conclusion Kupff er cells played a crucial rule in modulating infl ammation and the anti-infl ammatory eff ect of nicotine was partially weakened after left cervical vagus nerve cut off or aBGT was given. Immune paralysis in trauma patients; implications for prehospital interventionf Results (1) Expression of α7-nAChR in Kupff er cells was confi rmed by confocal microscope and western blotting; (2) after nicotine was administered, the level of TNFα and HMGB-1 increased and the level of IL-10 decreased. Given left cervical vagus nerve cut off or aBGT, the eff ect of nicotine was weakened; (3) Group I had the highest mortality rate, while in Group II nicotine did reduce the mortality rate dramatically. After the left cervical vagus nerve was cut off or aBGT was given, the eff ects of nicotine were weakened. Diff erence for the mortality rate between Group III and Group IV was not signifi cant. M Kox, K Timmermans, M Vaneker, GJ Scheff er, P Pickkers Radboud University Nijmegen Medical Center, Nijmegen, the Netherlands Critical Care 2013, 17(Suppl 2):P9 (doi: 10.1186/cc11947) Introduction Multi-trauma is one of the major indications for intensive care admission. Recovery is frequently complicated by post-injury immunological complications, caused by a dysfunctional immune system; for example, sepsis and multiple organ failure. In order to treat S4 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 hematologic or metastasized malignancies, cardiac arrest, and use of bone marrow suppressive drugs. Laboratory data were collected from blood samples, prior to in-hospital i.v. fl uid therapy. In order to detect a diff erence in Hb concentration of 0.2 mmol/l, we anticipated a sample size of 283 per group, based on a standard deviation (SD) of 1.2, α = 0.05 and β = 0.8. Data are expressed as mean ± SD. or prevent this immune paralysis, knowledge on the time course of immune paralysis in vivo and the pathophysiological mechanisms of immune paralysis is essential. The aim of this study is to determine factors that could predict and/or induce immunological complications in these patients to ultimately fi nd a suitable target and timeframe for intervention. hematologic or metastasized malignancies, cardiac arrest, and use of bone marrow suppressive drugs. Laboratory data were collected from blood samples, prior to in-hospital i.v. fl uid therapy. In order to detect a diff erence in Hb concentration of 0.2 mmol/l, we anticipated a sample size of 283 per group, based on a standard deviation (SD) of 1.2, α = 0.05 and β = 0.8. Data are expressed as mean ± SD. β p Results We included 296 patients in the S-group and 320 in the C-group. Do changes in red blood cell deformability in patients with septic shock correlate with changes in SOFA scores? T Clark1, S Jewell2, M Sair1, P Petrov2, P Winlove2 T Clark1, S Jewell2, M Sair1, P Petrov2, P Winlove2 1Derriford Hospital, Plymouth, UK; 2University of Exeter, UK C l C (S l ) P (d / ) Derriford Hospital, Plymouth, UK; 2University of Exeter, UK Critical Care 2013 17(Suppl 2):P11 (doi: 10 1186/cc11949) Conclusion Immune paralysis can be established within hours after trauma. Production of anti-infl ammatory IL-10 in the prehospital phase could play a crucial role in the pathogenesis. Patients with a higher injury severity score are more prone to produce excessive IL-10 in this phase. Immune stimulatory strategies applied by the HEMS or early after hospital admission could form a potential future approach to prevent immune paralysis in multitrauma patients in the intensive care ward. p , y , ; y , Critical Care 2013, 17(Suppl 2):P11 (doi: 10.1186/cc11949) Introduction Traditional whole blood experiments suggest that sepsis causes abnormal red blood cell (RBC) deformability. To investigate this at the cellular level, we employed a novel biophysical method to observe individual RBC membrane mechanics in patients with septic shock. Methods We collected blood samples from patients with septic shock until either death or day 5 of admission. Thermal fl uctuations of individual RBCs were recorded allowing a complete analysis of RBC shape variation over time. Mean elasticity of the cell membrane was then quantifi ed for each sample collected. Is hemoglobin concentration aff ected by sepsis in the acute phase? G Jansma, H Buter, RT Gerritsen, EC Boerma Medical Centre Leeuwarden, the Netherlands Critical Care 2013, 17(Suppl 2):P10 (doi: 10.1186/cc11948) Immune paralysis in trauma patients; implications for prehospital interventionf The diff erence in Hb between the S-group and C-group was not signifi cant (8.76 ± 1.18 mmol/l vs. 8.93 ± 1.16 mmol/l, P = 0.07). After correction for a number of confounders, using a multivariate regression analysis, we observed a signifi cant diff erence in Hb of –0.23 mmol/l in the S-group in comparison with the C-group (P = 0.01).il Methods Blood was drawn from adult multi-trauma patients (n = 94) admitted to the emergency room (ER) of the Radboud University Nijmegen Medical Center. Blood was drawn at the trauma scene by the helicopter emergency medical services (HEMS), at arrival in the ER and at days 1, 3, 5, 7, 10 and 14 after trauma. Plasma concentrations of TNFα, IL-6, IL-10, IFNγ, IL-8 and MCP-1 were determined by Luminex. Ex vivo 24-hour whole blood stimulations with LPS or pam3cys were performed and produced TNFα, IL-6 and IL-10 was measured using ELISA to determine the level of immune paralysis. Clinical data – for example, Injury Severity Scores, trauma mechanism, medication and survival – were collected from electronic patient fi les. g p p g p Conclusion At fi rst presentation, prior to in-hospital i.v. fl uid therapy, Hb concentration in patients with sepsis is signifi cantly lower in comparison with controls; however, the diff erence is very small, without the existence of anemia. R f Is hemoglobin concentration aff ected by sepsis in the acute phase? G Jansma, H Buter, RT Gerritsen, EC Boerma Medical Centre Leeuwarden, the Netherlands Critical Care 2013, 17(Suppl 2):P10 (doi: 10.1186/cc11948) i Results We recruited nine patients with septic shock. Table 1 shows mean RBC thermal fl uctuation and SOFA scores.l Introduction In the acute phase of sepsis several potential mechanisms may change the hemoglobin (Hb) concentration. On the one hand, endothelial activation may lead to increased vascular permeability and fl uid sequestration to the interstitium, leading to hemoconcentration. On the other hand, degradation of the glycocalyx has been reported [1]. Shedding of this carbohydrate-rich layer with an estimated thickness of 0.2 to 0.5 μm may lead to a substantial increase of the intravascular space, and thus to decrease of Hb concentration [2]. The aim of this study is to determine whether there is a decrease in Hb in the acute phase of sepsis.i l Conclusion RBC thermal fl uctuation analysis allows variations in RBC elasticity during sepsis to be quantifi ed at a cellular level. We could not identify any specifi c trend between sepsis severity and erythrocyte elasticity. Cells demonstrated both increases and decreases in fl uctua- tion independent of SOFA score. This is contrary to current evidence that suggests RBC deformability is reduced during sepsis. Reference 1. Piagnerelli M, et al.: Intensive Care Med 2003, 29:1052-1061. References i Results The plasma IL-10 concentration at ER was 16.5-fold increased in comparison with time-point HEMS (P  <0.01). Similar but less pronounced eff ects were found for IL-8 and MCP-1. A signifi cant correlation (P  = 0.03, R  = 0.53) was found between injury severity scores and IL-10 plasma concentration at time-point ER. Time-courses of ex vivo produced cytokines suggest that LPS-induced IL-6 and TNFα production is already decreased in the fi rst few hours after trauma and recovering from day 5. Ex vivo IL-10 production shows an inverse pattern. 1. Steppan J, et al.: Sepsis and major abdominal surgery lead to fl aking of the endothelial glycocalyx. J Surg Res 2011, 165:136-141. 2. van den Berg BM, et al.: The endothelial glycocalyx protects against myocardial edema. Circ Res 2003, 92:592-594. 2. van den Berg BM, et al.: The endothelial glycocalyx protects against myocardial edema. Circ Res 2003, 92:592-594. Reference Reference 1. Strauss R, et al.: Thrombocytopenia in patients in the medical intensive care unit: bleeding prevalence, transfusion requirements, and outcome. Crit Care Med 2002, 30:1765-1771. Methods We collected blood samples from elective cardiac surgery patients pre (at induction) and post (immediately, each day until CICU discharge) CPB. Thermal fl uctuations of individual RBCs were recorded using a high-frame-rate camera allowing a complete analysis of RBC shape variation over time. Mean elasticity of the cell membrane was then quantifi ed for each sample collected. P14 N t i Results Fifteen patients were recruited. Table  1 displays the results. RBC thermal fl uctuation is measured relative to pre-bypass values. An increase in RBC fl uctuation marks a decrease in stiff ness. CPB caused two distinct changes in RBC elasticity; pre-fi x A indicates samples where stiff ness increases or shows no change, B those where stiff ness decreases. Data on day 2 were not collected in patients discharged from the CICU. CPB type or time had no apparent impact on RBC response to CPB. Introduction Neutrophil gelatinase-associated lipocalin (NGAL)/ lipocalin2, known as a sensitive biomarker of acute kidney injury, prevents bacterial iron uptake, resulting in the inhibition of its overgrowth [1]. We previously demonstrated that this protein was discharged into gut lumen from crypt cells in septic conditions, and inhibited the growth of Escherichia coli [2]. However, it remains unclear which pathway is associated with the upregulation of NGAL. We therefore designed the present study to reveal whether the pattern- recognition receptor of bacteria, the Toll-like receptor (TLR) family, plays a pivotal role for NGAL secretion from gut crypt cells. Conclusion RBC thermal fl uctuation analysis quantifi es the impact of CPB on erythrocyte membrane elasticity. We clearly identifi ed two separate RBC elasticity responses to CPB. This fi nding is contrary to traditional fl ow measurement techniques that suggest CPB impairs whole blood fl ow and reduces RBC deformability. Reference 1. Lindmark et al.: J Thoracic Cardiovasc Surg 2002, 123:381-383. p y p g yp Methods With our institutional approval, the ileum and colon of male C57BL/6J mice (6 to 7 weeks) were everted and washed by Ca2+ and Mg2+ free PBS buff er fi ve times. Tissues were incubated with Ca2+ and Mg2+ free PBS containing 30 mM EDTA for 1 hour to isolate crypt cells of gut. Platelet-associated oxidative stress and ADAMTS-13 levels are inversely associated with a poor prognosis in septic shock Introduction Sepsis causes widespread microvascular injury and thrombosis. Some hemostatic factors mediate the mechanisms involved in sepsis-related organ ischemia and failure. Oxidative stress is also increased in sepsis and reactive oxygen species (ROS) favor secretion of von Willebrand factor (vWF) multimers from endothelium and inhibit vWF proteolysis by ADAMTS-13. Moreover, the enzyme indoleamine-2,3-dioxygenase, an important immune regulator, is activated in sepsis and, through generation of kynurenins, promotes antioxidative and anti-infective activities. This study evaluated the relative role of ADAMTS-13, vWF and fi brinogen in the morbidity and mortality of patients with septic shock (SS). The above hemostatic factors were measured together with kynurenine and plasma protein carbonyls, marker of oxidative stress. Results More than 70 to 80% of collected cells were stained by amido black. LPS signifi cantly upregulated the expression of NGAL and TLR4 mRNA in ileum and colon crypt cells (P <0.05). Although the CpG-DNA did not upregulate NGAL and TLR9 mRNA in ileum crypt cells, the apparent expression of NGAL and TLR9 mRNA was found in colon crypt cells (P <0.05). Conclusion Bacterial stimulation of TLR4 and TLR9 pathways plays a pivotal role in the expression of NGAL mRNA in gut, suggesting that NGAL, derived from gut crypt cells, could contribute to the regulation of the intraluminal microfl ora in the critically ill. y Methods One group of 12 patients with SS, defi ned using standard criteria, was enrolled in the ICU of the ‘A. Gemelli’ Hospital (Rome, Italy). Biochemical, hematologic and hemodynamic parameters were measured on days 1 to 4, 7, 14 and 21. A group of 12 age-matched and gender-matched healthy subjects was used as controls. References References 1. Nature 2004, 432:917. 2. Crit Care Med 2011, 39:46. 1. Nature 2004, 432:917. 2. Crit Care Med 2011, 39:46. Reference The cell suspension was fi ltered through a cell strainer (40 μm) twice, and deposited the crypt cells by centrifugation at 700×g. The isolated crypt cells were resuspended in PBS and stained with 0.25% amido black for labeling paneth cells. The 5×105 crypt cells were resuspended in 50 ml HBSS containing 2.5% fetal bovine serum and 1% penicillin–streptomycin. The crypt cells were incubated at 37°C with or without TLR ligands: lipopolysaccharide (TLR4 ligand, 10 μg/ml) and CpG-DNA (TLR9 ligand, 8 μg/ml). After a 2-hour incubation period, the crypt cells were deposited and eluted mRNA to measure the expression of both NGAL and TLR mRNA using real-time PCR. P13 Platelet-associated oxidative stress and ADAMTS-13 levels are inversely associated with a poor prognosis in septic shock L Montini1, G De Pascale1, MA Pennisi1, ES Tanzarella1, SL Cutuli1, A Occhionero1, R De Cristofaro2, M Antonelli1 1Catholic University School of Medicine, Rome, Italy; 2Haemostasis and Thrombosis Center, Catholic University School of Medicine, Rome, Italy Critical Care 2013, 17(Suppl 2):P13 (doi: 10.1186/cc11951) P15 y Results Low ADAMTS-13 activity was observed in SS patients (268 ± 123 ng/ml vs. 760 ± 80 ng/ml in controls). vWF levels (antigen and activity) were increased ~3-fold compared with controls. Likewise, plasma protein carbonyls and kynurenine were globally increased in patients (2.1 ± 1.5 nmol/mg vs. 0.3 ± 02 nmol/mg and 14.4 ± 9.7 μM vs. 2.3 ± 1.3 μM, respectively). Intra-ICU mortality (3 of 15) was strongly and inversely correlated with carbonyl levels (P = 0.04) and platelets (P = 0.022). Lethal infl uenza virus A H1N1 infection in two relatives with autosomal dominant GATA-2 defi ciency J Sole-Violan1, I Sologuren1, E Betancor2, S Zhang3, C Pérez1, E Herrera-Ramos1, M Martínez-Saavedra1, M López-Rodríguez1, J Pestano2, J Ruiz-Hernández1, J Ferrer1, F Rodríguez de Castro1, J Casanova3, C Rodríguez-Gallego1 1Hospital GC Dr Negrín, Las Palmas de GC, Spain; 2Universidad Las Palmas de GC, Spain; 3The Rockefeller University, New York, NY, USA Critical Care 2013, 17(Suppl 2):P15 (doi: 10.1186/cc11953) Lethal infl uenza virus A H1N1 infection in two relatives with autosomal dominant GATA-2 defi ciency P12 Do erythrocytes subjected to cardiopulmonary bypass exhibit changes in their membrane mechanical properties? T Cl k1 S J ll2 M S i 1 P P 2 P Wi l 2 p p Methods In this single-center retrospective analysis we identifi ed patients with sepsis as the primary reason for non-elective ICU admission from a standard patient database. Patients who fulfi lled the international criteria of sepsis and organ failure during ICU admission were included in the sepsis group (S-group). The control group was formed by patients with other non-elective reasons for ICU admission (C-group). Exclusion criteria were (recent) bleeding, surgery in the last 6 weeks, chronic renal failure (creat >177  μmol/l, or hemodialysis), untreated chronic anemia, pregnancy, polytrauma, age <18, T Clark , S Jewell , M Sair , P Petrov , P Winlove 1Derriford Hospital, Plymouth, UK; 2University of Exeter, UK Critical Care 2013, 17(Suppl 2):P12 (doi: 10.1186/cc11950) Introduction Whole blood experiments suggest that cardiopulmonary bypass (CPB) causes red blood cell (RBC) trauma and changes in deformability that may contribute to postoperative microcirculatory Table 1 (abstract P11). Mean RBC fl uctuation (daily SOFA score) Day A B C D E F G H I 1 4.8 (10) 5.2 (9) – 4.8 (12) 4.6 (16) 4.9 (13) 5.1 (16) 4.6 (18) 5.1 (15) 2 4.9 (9) 5.0 (10) 4.6 (13) 5.1 (11) 4.8 (17) 5.1 (13) 5.0 (16) 4.9 (19) – (16) 3 4.0 (6) 5.1 (9) 4.7 (12) 4.8 (11) 4.9 (18) 4.8 (13) 5.0 (16) 4.7 (21) 5.3 (16) 4 – 4.8 (7) 4.6 (11) 5.0 (9) 5.9 (19) – – (18) – 5.0 (15) 5 – – – 5.1 (6) 5.2 (18) – – (19) – 5.0 (10) Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 S5 Table 1 (abstract P12). Change in RBC thermal fl uctuation relative to baseline: two distinct groups seen Patient A1 A2 A3 A4 A5 A6 A7 B8 B9 B10 B11 B12 B13 B14 B15 Post CPB +0.1 –0.4 –0.1 –0.5 –0.1 –0.2 –0.1 +0.5 0 +0.4 +0.1 +0.2 0 +0.2 +0.1 Day 1 0 0 +0.1 –0.1 –0.1 +0.1 +0.1 0 +0.5 +0.4 +0.7 +0.4 +0.6 +0.5 +0.7 Day 2 –0.2 +0.1 0 NA NA 0 NA +0.5 –0.1 +0.4 NA +0.1 NA NA NA Table 1 (abstract P12). Change in RBC thermal fl uctuation relative to baseline: two distinct groups seen dysfunction. We used a novel fl uctuation microscopy technique to quantify the eff ects of CPB on RBC elasticity at a cellular level. Lethal infl uenza virus A H1N1 infection in two relatives with autosomal dominant GATA-2 defi ciency Conclusion Hence, we hypothesize that, in the SS setting, platelets contribute to oxidative stress that counteracts the organ failure- associated mortality. Thus, low platelet count, irrespective of bleedings, may favor mortality in SS patients by generating lower ROS amounts. Introduction Most individuals infected with the 2009 pandemic H1N1 infl uenza A virus (IAV) (H1N1pdm) experienced uncomplicated fl u. Introduction Most individuals infected with the 2009 pandemic H1N1 infl uenza A virus (IAV) (H1N1pdm) experienced uncomplicated fl u. S6 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 isolated and incubated for 24 hours in the presence of 10 ng/gl LPS of Escherichia coli O55:B5 and of 5 μg/ml phytohemagglutin (PHA); TNFα was measured in supernatants by a bioassay on L929 fi broblasts.i isolated and incubated for 24 hours in the presence of 10 ng/gl LPS of Escherichia coli O55:B5 and of 5 μg/ml phytohemagglutin (PHA); TNFα was measured in supernatants by a bioassay on L929 fi broblasts. Results Fifty percent of rabbits died early; that is, within the fi rst 48  hours. Mean  ±  SE log10 bacteria in the liver and lung of animals that died early was 2.27 ± 0.62 and 3.16 ± 0.78 cfu/g; respective values of rabbits that started dying late (that is, after 72 hours) were below the limit of detection. Mean circulating LPS at 24 hours was 2.09 EU/ ml and 1.99 EU/ml respectively (P = NS). Mean LPS of the portal vein of the sham and of the injury groups were 1.25 and 5.62 EU/ml (P = 0.047). Concentrations of TNFα in splenocyte supernatants are shown in Figure 1. However, in a small subset of patients the infection rapidly progressed to primary viral pneumonia (PVP) and a minority of them developed ARDS. Inherited and acquired variability in host immune responses may infl uence susceptibility and outcome of IAV infection. However, the molecular nature of such human factors remains largely elusive. p y yi Results Fifty percent of rabbits died early; that is, within the fi rst 48  hours. Mean  ±  SE log10 bacteria in the liver and lung of animals that died early was 2.27 ± 0.62 and 3.16 ± 0.78 cfu/g; respective values of rabbits that started dying late (that is, after 72 hours) were below the limit of detection. Lethal infl uenza virus A H1N1 infection in two relatives with autosomal dominant GATA-2 defi ciency Mean circulating LPS at 24 hours was 2.09 EU/ ml and 1.99 EU/ml respectively (P = NS). Mean LPS of the portal vein of the sham and of the injury groups were 1.25 and 5.62 EU/ml (P = 0.047). Concentrations of TNFα in splenocyte supernatants are shown in Figure 1. y Methods We report three adult relatives with the autosomal dominant GATA-2 defi ciency. P1 and his son P2 had a history of myelodysplastic syndrome and a few episodes of mild respiratory infections. They developed PVP by H1N1pdm which rapidly evolved to ARDS. They died at the age of 54 and 31, respectively. y Results Patients were heterozygous for a novel R396L mutation in GATA2. Like other patients with GATA-2 defi ciency, the three relatives had absence of peripheral NK and B cells and monocytopenia. However a high number of plasma cells, which were found to be pauciclonal, were observed in peripheral blood from P1 during H1N1pdm infection. P1 and P2 had normal levels of immunoglobulins and IgG antibodies against common viruses. Microneutralization test showed that P1 produced normal titers of neutralizing antibodies against H1N1pdm and against the previous annual H1N1 strain. Our results suggest that a few clones of long-living memory B cells against IAV expanded in P1; and that these cells produced cross-reactive antibodies against H1N1pdm, similar to those recently described. During the fl u episode P1 had a strong increase of IFNγ-producing T cells and of IFNγ production. The Th1-related chemokines CXCL10 and CXCL9, as well as IFNγ, MCP-1 and IL-8, were strongly elevated in serum from P1 and P2 in the course of H1N1pdm infection. g Conclusion Early death after injury is not related to peripheral endotoxemia and sepsis; bacterial translocation priming for enhanced proinfl ammatory responses is a likely explanation. Eff ects of the common 34C>T variant of the AMPD1 gene on immune function, multiorgan dysfunction and mortality in patients with sepsis Eff ects of the common 34C>T variant of the AMPD1 gene on immune function, multiorgan dysfunction and mortality in patients with sepsis B Ramakers1, E Giamarellos-Bourboulis2, M Coenen1, M Kox1, J Van der Hoeven1, C Routsi2, A Savva2, I Perdios3, F Diamantea4, D Sinapidis5, P Smits1, N Riksen1, P Pickkers1 1Radboud University Nijmegen Medical Centre, Nijmegen, the Netherlands; 2University of Athens, Medical School, Athens, Greece; 3Nafplion General Hospital, Nafplio, Greece; 4‘G.Gennimatas’ General Hospital, Athens, Greece; 5Alexandra General Hospital, Athens, Greece Critical Care 2013, 17(Suppl 2):P17 (doi: 10.1186/cc11955) Conclusion GATA-2 defi ciency is the fi rst described Mendelian inborn error of immunity underlying severe IAV infection. Primary immunodefi ciencies predisposing to severe IAV infections may debut, even in adults without a history of previous severe infections. The massive IFNγ-mediated cytokine storm may explain the fatal course of H1N1pdm infection in our patients. Introduction Adenosine exerts anti-infl ammatory and tissue protective eff ects during systemic infl ammation. While the anti-infl ammatory properties may induce immunoparalysis and impede bacterial clearance, the tissue protective eff ects might limit organ damage. The eff ects of a common loss-of-function variant of the adenosine monophosphate deaminase 1 gene (AMPD1), which is associated with increased adenosine formation, in patients with sepsis are unknown. Methods In a prospective cohort, genetic-association study, the eff ects of the presence of the AMPD1 gene on immune function, multiorgan dysfunction and mortality in septic patients was studied. Pneumosepsis patients (n = 402) and controls without infection (n = 101) were enrolled. P16 P16 Bacterial translocation primes proinfl ammatory responses and is connected to early death in an experimental model of lethal injury N Baxevanos, T Tsaganos, A Pistiki, D Droggiti, A Spyridaki, E Giamarellos-Bourboulis University of Athens, Medical School, Athens, Greece Critical Care 2013, 17(Suppl 2):P16 (doi: 10.1186/cc11954) P18 P18 Exploring the translational disconnect between the murine and human infl ammatory response: in vitro analysis of the dose– response relationship of LPS and NFκB activation in murine and human immune cells EP McCarron, I Welters, D Williams, D Antoine, A Kipar University of Liverpool, UK Critical Care 2013, 17(Suppl 2):P18 (doi: 10.1186/cc11956) Introduction Infl ammation, as seen in sepsis and systemic infl ammatory response, is dependent on the activation of the NFκB pathway through Toll-like receptors (TLRs) [1]. Recreating an infl ammatory response using lipopolysaccharide (LPS) can provide results that are diff erent to clinical sepsis [2]. By examining NFκB activation in murine and human cells, a species comparison can be made to investigate diff erences at the cell level that may contribute to the translational disconnect seen in vivo. Methods In the prospective, noninterventional study, we assessed the correlation between the capillary leakage marker angiopoetin-2 and serum levels of IgG and IgM in 41 patients with community-acquired severe sepsis or septic shock on admission. Blood samples were obtained during the fi rst 12 hours after admission to hospital. Results Mean age of patients (17 females) was 70  years. Median APACHE II and SOFA scores at admission were 24 and 11, respectively. The mortality rate was 45%. Thirty-four percent of all patients had level of IgG <650 mg/dl. The median concentration of angiopoietin-2 in the hypo-IgG group was 11,958 pg/ml, which was not statistically diff erent (Mann–Whitney; P >0.05) than in the rest of patients with normal levels of IgG (15,688 pg/ml). The concentration of IgM <40 mg/dl was found in only four patients (10%) and all died. Pearson’s correlation test showed that the correlation between the concentrations of angiopoietin-2 and IgG (correlation coeffi cient 0.191) or IgM (correlation coeffi cient 0.0408), respectively, were not statistically signifi cant (P <0.05). Methods THP1 human monocytes (passages 9 to 11) and RAW 264.7 murine macrophages (passages 15 to 20) were cultured in RPMI-1640 and DMEM respectively and then challenged with LPS. After settling for 24 hours, cells were dosed with six or seven doses of LPS. After 1 hour, nuclear extraction and proteins were separated by acrylamide gel electrophoresis. Membranes where then immunoblotted for actin and p65, followed by densitometric analysis in order to quantify the amount of p65 that had translocated from the cytoplasm to the nucleus (by subtraction from consistent nuclear actin). Bacterial translocation primes proinfl ammatory responses and is connected to early death in an experimental model of lethal injury N Baxevanos, T Tsaganos, A Pistiki, D Droggiti, A Spyridaki, y , , , itical Care 2013, 17(Suppl 2):P16 (doi: 10.1186/cc11954) Introduction Some cases of multiple trauma are rapidly deteriorating; the mechanism was investigated. Introduction Some cases of multiple trauma are rapidly deteriorating; the mechanism was investigated. Results In pneumosepsis patients and controls, a similar prevalence of the 34C>T (rs17602729) mutation in the AMPD1 gene was found. Univariate logistic regression analysis showed a tendency of increased mortality in patients with the CT genotype, compared with patients with the CC genotype (OR 1.53; 95% CI 0.95 to 2.5). Moreover, carriers of the CT genotype tended to suff er more from multiorgan dysfunction, OR 1.4 (0.84 to 2.3) and 3.0 (0.66 to 13.8), for CT and TT, respectively (P = 0.07). In septic carriers of the CT genotype, the ex vivo production of TNFα by LPS-stimulated monocytes was attenuated (P  = 0.005), Methods Forty-one rabbits were assigned into two groups; sham- operated and subject to crush of the right femur. Survival was recorded; peripheral blood was sampled for LPS measurement by the kinetic QCL-1000 LAL assay; quantitative tissue growth was assessed after death. Some rabbits were sacrifi ced at 48  hours; blood was sampled from the portal vein for LPS measurement; splenocytes were Figure 1 (abstract P16). Stimulation of TNFα from isolated splenocytes. y y Figure 1 (abstract P16). Stimulation of TNFα from isolated splenocytes. Figure 1 (abstract P17). Kaplan–Meier curve for the 402 sepsis patients. Figure 1 (abstract P17). Kaplan–Meier curve for the 402 sepsis patients. Figure 1 (abstract P17). Kaplan–Meier curve for the 402 sepsis patients. Figure 1 (abstract P17). Kaplan–Meier curve for the 402 sepsis patients. S7 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 indicative for more pronounced immunoparalysis in these patients. See Figure 1. P18 i Conclusion At present the hypothesis that increased microvascular leakage is responsible for hypogammaglobulinemia in septic patients could not be accepted. Studies on larger number of patients are needed. In addition, it is necessary to further explore other possible mechanisms, such as increased catabolism and consumption of antibodies or inadequate synthesis of immunoglobulins, which could also be responsible for hypogammaglobulinemia in sepsis. References y Results Murine cells required higher doses of LPS compared with human cells in order to detect p65 (human, 1 pg/ml to 100  ng/ml; murine, 30 pg/ml to 1,000 ng/ml). THP1 cells showed a greater fold increase in the p65:actin ratio compared with RAW 264.7 cells. Human cells responded to lower concentrations of LPS. Murine cells appeared to show a molecular resistance to lower doses, but their response was very sensitive at higher doses. A dose–response relationship of LPS dosing and NFκB activation was observed in both cell lines. P20 P20 Hypogammaglobulinemia in sepsis is not correlated to high circulating angiopoietin-2 levels Conclusion The presence the 34C>T variant of the AMPD1gene is not related to infection susceptibility; however, it is associated with more pronounced immunoparalysis in patients with sepsis, and shows a tendency towards increased mortality. Mechanistically, the anti- infl ammatory eff ects of adenosine may account for this and apparently overrule its tissue protective eff ects. U Kovačič1, F Starič2, M Kmet2, M Godnič2, R Kapš2 1Faculty of Medicine, University of Ljubljana, Slovenia; 2General Hospital Novo Mesto, Slovenia Critical Care 2013, 17(Suppl 2):P19 (doi: 10.1186/cc11957) Introduction Hypogammaglobulinemia has been frequently found in adult patients with severe sepsis and septic shock. Furthermore, it seems that at least a low serum level of IgM is correlated with higher mortality in sepsis. The mechanisms of hypogammaglobulinemia in septic shock have not yet been explained. It has been hypothesized that outfl ow of immunoglobulins into the extravascular space due to increased capillary permeability could reduce immunoglobulin serum concentrations. Angiopoietin-2, which directly disrupts the endothelial barrier, is markedly elevated in sepsis and other infl ammatory states and its serum level has been correlated with microvascular leakage, end-organ dysfunction and death in sepsis. TREM-1 levels are elevated in patients with liver cirrhosis SD G 1 CR G h 2 P K ll h 2 N S i1 M F t 1 S Si h 1Chelsea and Westminster Hospital, London, UK; 2Imperial College, London, UK Critical Care 2013, 17(Suppl 2):P20 (doi: 10.1186/cc11958) 1Chelsea and Westminster Hospital, London, UK; 2Imperial College, London, UK Critical Care 2013, 17(Suppl 2):P20 (doi: 10.1186/cc11958) References 1. Taccone FS, et al.: Gamma-globulin levels in patients with community- acquired septic shock. Shock 2009, 32:379-385. 1. Taccone FS, et al.: Gamma-globulin levels in patients with community- acquired septic shock. Shock 2009, 32:379-385. 2. Werdan K, et al.: Score-based immunoglobulin G therapy of patients with sepsis: the SBITS study. Crit Care Med 2007, 35:2693-2701. g Conclusion Immunoblotting for p65 is a reliable and reproducible method to determine NFκB activation in cultured cells. Macrophages are more responsive to LPS than monocytes [3] so diff erences between cell lines would have been expected to be the reverse of what was observed. The species diff erence in response to LPS may contribute to the apparent disconnect between human and murine responses to LPS and may partially explain the diffi culties of translating therapeutic interventions into clinical human sepsis. 2. Werdan K, et al.: Score-based immunoglobulin G therapy of patients with sepsis: the SBITS study. Crit Care Med 2007, 35:2693-2701. P22 Introduction Septic encephalopathy is a frequent complication in severe sepsis but its pathogenesis and mechanisms are not fully understood. Oxygen supply and utilization are critical for organ function, especially for the brain, a tissue extremely dependent on oxygen and glucose. Disturbances in oxygen utilization are common in sepsis and a number of mitochondrial dysfunctions have been described in diff erent tissues in septic animals as well as in septic patients. Our group described mitochondrial dysfunctions in the brain during experimental sepsis. l Results TREM-1 expression is signifi cantly higher in the CA group compared with HC across all monocyte subsets but not neutrophils, even in the absence of sepsis (see Figure  1). There is no correlation between cell surface TREM-1 expression and severity of cirrhosis by Child–Pugh or MELD score. sTREM and sCD14 levels were elevated in the CA group compared with HC (P = 0.0010 and 0.0016 respectively). In addition, plasma sTREM-1 levels correlated with disease severity according to MELD score (R = 0.71, CI = 0.22 to 0.92 P = 0.012) and serum bilirubin (R = 0.78,CI = 0.36 to 0.94, P = 0.004). There was no correlation with either form of TREM-1 with sCD14 levels. There was no diff erence in cell surface or soluble TREM-1 expression between blood and ascitic fl uid monocytes in culture-negative, non-neutrophilic ascites. Methods Experimental sepsis was induced by endotoxemia (LPS 10 mg/ kg i.p.) in Sprague–Dawley rats and by polymicrobial fecal peritonitis in Swiss mice. Brain glucose uptake was observed in vivo in endotoxemic rats using positron emission tomography with [18F]fl uorodeoxyglucose and autoradiography with 2-deoxy-14C-glucose. Results Mice with polymicrobial sepsis present hypoglycemia, hyperlactatemia and long-term cognitive impairment. We observed a rapid increase in the uptake of fl uorescent glucose analog 2-deoxy- 2-((7-nitro-2,1,3-benzoxadiazol-4-yl)amino)-D-glucose in brain slices from septic mice in vitro. A similar increase in brain glucose uptake was observed in vivo in endotoxemic rats. Remarkably, the increase in glucose uptake started 2 hours after LPS injection, earlier than other organs. The brains of mice with experimental sepsis presented neuroinfl ammation, mitochondrial dysfunctions and oxidative stress, but mitochondria isolated from septic brains generated less ROS in vitro in the fi rst 24 hours. This led us to investigate the role of NADPH oxidase, an enzyme induced during innate immune response, as a potential source of reactive oxygen species in experimental sepsis. 1. Olson JC, et al.: Intensive care of the patient with cirrhosis. Hepatology 2011, 54:1864-1872. P22 Inhibiting NADPH oxidase with apocynin acutely after sepsis prevented cognitive impairment in mice. l Conclusion Blood monocyte and soluble TREM-1 are elevated in cirrhotic patients even in the absence of sepsis. Soluble TREM-1 levels correlate with disease severity in cirrhosis. Further studies are ongoing to ascertain the utility of TREM-1 as a biomarker in cirrhosis. Reference 1. Olson JC, et al.: Intensive care of the patient with cirrhosis. Hepatology 2011, 54:1864-1872. g p Conclusion Our data indicate that a bioenergetic imbalance and oxidative stress is associated with the pathophysiology of septic encephalopathy. We are observing a new metabolic phenotype in the brain during sepsis, characterized by a rapid increase in glucose uptake and mitochondrial dysfunctions that may be secondary to infl ammation and hypoxia. P21 Eff ects of diff erent doses and serotypes of LPS on blood–brain barrier permeability in Sprague–Dawley rats E Senturk, F Esen, P Ergin Ozcan, G Orhun, N Orhan, N Arican, M Kucuk, M Kaya University of Istanbul, Turkey Critical Care 2013, 17(Suppl 2):P21 (doi: 10.1186/cc11959) University of Istanbul, Turkey Introduction The blood–brain barrier (BBB) is highly restrictive of the transport of substances between blood and the central nervous system. Lipopolysaccharide (LPS) from Gram-negative bacteria was reported to aff ect the permeability of the BBB. BBB disruption using a LPS is used as a model of septic encephalopathy in mice. The present study was designed to investigate the eff ects of diff erent doses and serotypes of LPS on BBB integrity in Sprague–Dawley rats. Pathophysiology of sepsis-associated brain dysfunction: an experimental study of cerebral microdialysis and mitochondrial function P Kurtz1, C Vargas-Lopes1, C Madeira1, I Mello1, R Panizzutti1, L C Azevedo2, F A Bozza1 1Fiocruz, Rio de Janeiro, Brazil; 2Hospital Sirio e Libanes, São Paulo, Brazil Critical Care 2013, 17(Suppl 2):P23 (doi: 10.1186/cc11961) P22 to the ICU have an in-hospital mortality of up to 50% [1]. Microbial translocation (MT) is the pathogenic mechanism implicated in SBP. The triggering receptor expressed by myelocytes-1 (TREM-1) modulates the immune response with resultant production of proinfl ammatory cytokines and has been used as a biomarker in the diagnosis of bacterial infection. We wish to evaluate the role of TREM-1 as a biomarker in cirrhosis. Bioenergetic imbalance and oxidative stress in the pathophysiology of septic encephalopathy J D’Avila1, R Rodrigues2, H Castro-Faria-Neto1, M Oliveira3, F Bozza4 1Oswaldo Cruz Foundation – FIOCRUZ, Rio de Janeiro, Brazil; 2Federal University of Rio de Janeiro (UFRJ) and D’Or Institute for Research and Education (IDOR), Rio de Janeiro, Brazil; 3UFRJ, Rio de Janeiro, Brazil; 4Oswaldo Cruz Foundation – FIOCRUZ and IDOR, Rio de Janeiro, Brazil Critical Care 2013, 17(Suppl 2):P22 (doi: 10.1186/cc11960) Bioenergetic imbalance and oxidative stress in the pathophysiology of septic encephalopathy J D’Avila1, R Rodrigues2, H Castro-Faria-Neto1, M Oliveira3, F Bozza4 1Oswaldo Cruz Foundation – FIOCRUZ, Rio de Janeiro, Brazil; 2Federal University of Rio de Janeiro (UFRJ) and D’Or Institute for Research and Education (IDOR), Rio de Janeiro, Brazil; 3UFRJ, Rio de Janeiro, Brazil; 4Oswaldo Cruz Foundation – FIOCRUZ and IDOR, Rio de Janeiro, Brazil Critical Care 2013, 17(Suppl 2):P22 (doi: 10.1186/cc11960) Methods Blood samples were obtained from 18 healthy controls (HC) and 29 cirrhotic patients (CA) as defi ned by clinicoradiological criteria. Disease severity was graded according to Child–Pugh class (median 10, range 5 to 13) and modifi ed end-stage liver disease (MELD) score (median 14, range 6 to 21). Simultaneous ascitic fl uid samples were taken from 10 patients in the CA group. Soluble TREM-1 and CD14 levels (a surrogate marker of MT) were measured by ELISA. Flow cytometry was used to quantify the expression of TREM-1 on monocytes and neutrophils in blood and ascitic fl uid.i Introduction Septic encephalopathy is a frequent complication in severe sepsis but its pathogenesis and mechanisms are not fully understood. Oxygen supply and utilization are critical for organ function, especially for the brain, a tissue extremely dependent on oxygen and glucose. Disturbances in oxygen utilization are common in sepsis and a number of mitochondrial dysfunctions have been described in diff erent tissues in septic animals as well as in septic patients. Our group described mitochondrial dysfunctions in the brain during experimental sepsis. References 1. Bonizzi G, Karin M: Trends Immunol 2004, 25:280-288. 1. Bonizzi G, Karin M: Trends Immunol 2004, 25:280-288. Introduction Sepsis and spontaneous bacterial peritonitis (SBP) are common sequelae in patients with cirrhosis. Cirrhotics admitted 2. Remick DG, Ward PA: Shock 2005, 24(Suppl 1):7-11. pp 3. Takashiba S, et al.: Infect Immun 1999, 67(11):5573-5578. Figure 1 (abstract P20). TREM-1 expression in healthy controls compared with cirrhotic patients. cirrhotic patients. Figure 1 (abstract P20). TREM-1 expression in healthy controls compared with cirrhotic patients. Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 S8 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Cholinergic modulation of hippocampal activity during septic encephalopathy 2 2 Cholinergic modulation of hippocampal activity during septic encephalopathy A Zivkovic1, CP Bengtson2, O Sedlaczek1, R Von Haken1, H Bading2, S Hofer1 1Universitätsklinikum Heidelberg, Germany; 2Universität Heidelberg, Germany Critical Care 2013, 17(Suppl 2):P24 (doi: 10.1186/cc11962) A Zivkovic1, CP Bengtson2, O Sedlaczek1, R Von Haken1, H Bading2, S Hofer1 1Universitätsklinikum Heidelberg, Germany; 2Universität Heidelberg, Germany Critical Care 2013, 17(Suppl 2):P24 (doi: 10.1186/cc11962) Introduction Septic encephalopathy is a sepsis-related brain dys- function with a deterioration of cortical functions. The experimental studies in the rat brain revealed a deranged neurotransmitter profi le during septic encephalopathy. Glutamatergic synapses, essential in learning and memory, undergo use-dependent changes in synaptic strength, referred to as plasticity. Permanent strengthening of synapses after a brief stimulus, termed long-term potentiation (LTP), was discovered in the hippocampus, and here it has been most thoroughly studied. Cholinergic neurotransmission plays an important role in regulating the cognitive functions of the brain. It acts as a signal-to- noise ratio modulator of sensory and cognitive inputs. The irregularities in brain functions give rise to the symptoms of delirium, including disorganized thinking and disturbances of attention and consciousness, which in turn might aff ect learning and memory. Possible mechanisms for cholinergic defi ciency include impairment of synaptic functions of acetylcholine. Imbalances in the cholinergic system during sepsis might therefore play an extensive role in the septic delirium. Results Of the 1,600 patients who gave consent to participate in the study, 205 had an ECI. Sixty-four had a positive blood culture for E. coli. Fifty of the patients met one or more criteria for severe sepsis or septic shock. The NLCR was signifi cantly higher (P <0.001) within the severe sepsis group (median = 21.1 with quartiles 11.1 to 42.4) compared with the group with no severe sepsis (median = 11.6 with quartiles 7.6 to 18.9). Conclusion The NLCR can be used as a biomarker of disease severity even in ECIs. The biomarker reacts rapidly, is cheap and needs no extra sampling. The higher the value, the higher the probability for severe sepsis. A high value can even precede the development of severe sepsis or septic shock. However, a low value never excludes neither bacteremia nor severe sepsis. The method cannot be used in patients with disturbances in neutrophil or lymphocyte levels due to other causes than sepsis. Methods By using MRI imaging we identifi ed functional changes in the hippocampal region of patients with severe sepsis. Pathophysiology of sepsis-associated brain dysfunction: an experimental study of cerebral microdialysis and mitochondrial function P Kurtz1, C Vargas-Lopes1, C Madeira1, I Mello1, R Panizzutti1, L C Azevedo2, F A Bozza1 1Fiocruz, Rio de Janeiro, Brazil; 2Hospital Sirio e Libanes, São Paulo, Brazil Critical Care 2013, 17(Suppl 2):P23 (doi: 10.1186/cc11961) y y y Methods Male Sprague–Dawley rats weighing 200 to 250  g were used in the study. Rats were given two diff erent types of LPS (026:B6- L5543 and 026:B6-L2762) in diff erent doses (3, 5, and 10 mg/kg; i.v.). Rectal temperature and arterial blood pressure measurements were recorded for sepsis severity. The changes in the BBB permeability were measured using the Evans blue (EB) and sodium fl uorescein (NaFl) dye extravasation techniques 24 hours after LPS administration. Introduction Pathophysiology of brain dysfunction associated with sepsis is still poorly understood. Potential mechanisms involve oxidative stress, neuroinfl ammation and blood–brain barrier altera- tions. Our purpose was to study the metabolic alterations and markers of mitochondrial dysfunction in a clinically relevant model of septic shock. Results Both LPS serotypes showed comparable arterial blood pressure and rectal temperature recordings and the severity of the disease increased with the increasing doses (5 mg/kg and 10 mg/kg) of LPS and the mortality rates were found to be 29% and 63% respectively. The extravasated contents of EB and NaFl tracers did not signifi cantly increase in brain parenchyma following the administration of diff erent doses of LPS with diff erent serotypes. Methods Twelve anesthetized (midazolam/fentanyl/pancuronium), invasively monitored, and mechanically ventilated pigs were allocated to a sham procedure (n = 5) or sepsis (n = 7), in which peritonitis was induced by intra-abdominal injection of autologous feces. Animals were studied until spontaneous death or for a maximum of 24 hours. In addition to global hemodynamic and laboratory assessment, intracranial pressure and cerebral microdialysis were assessed at baseline, 6, 12, 18 and 24  hours after sepsis induction. After death, brains were removed and brain homogenates were studied to assess markers of mitochondrial dysfunction. f Conclusion Our results showed no disruption to BBB by two diff erent serotypes of LPS even administered in increasing doses. These result indicate that the BBB integrity of Sprague–Dawley rats are resistant to the eff ects of two diff erent serotypes of LPS. Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 S9 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Figure 1 (abstract P23). Pathophysiology of sepsis-associated brain dysfunction: an experimental study of cerebral microdialysis and mitochondrial function Results Critically ill ICU patients diagnosed with septic delirium using the CAM-ICU method underwent diagnostic MRI scans. The serial MRI analysis revealed increased signal intensity in the hippocampal region in diff usion-weighted MRI (DWI). We used endotoxemia model to induce sepsis in the rats. Electrophysiological analysis of the hippocampal neurons in LPS-treated rats showed impaired LTP in the excitatory synapses, as compared with controls. Application of physostigmine, a blood–brain barrier permeable cholinesterase inhibitor, resulted in a partial recovery of LTP in the hippocampal synapses of LPS-treated rats. Conclusion The patients with septic delirium show functional changes in the hippocampus. Furthermore, we show that endotoxemia aff ects synaptic plasticity in the rat hippocampus, suggesting the involvement of this brain region in the pathophysiology of septic delirium. Moreover, the eff ect of the cholinergic neurotransmission onto the induction and maintenance of synaptic plasticity in the rat hippocampus during endotoxemia suggests that cholinergic neurotransmission might play a critical role in septic encephalopathy. Results All septic animals developed a hyperdynamic state associated with lower arterial pressure, fever and organ dysfunction in comparison with control animals. In the septic animals, we observed increased brain dialysate glutamin levels at 12, 18 and 24  hours after sepsis induction, as compared with control animals. Moreover, after analyzing homogenates from the frontal cortex, we found higher concentrations of glutamin and glutamate in septic as compared with control animals (85.67 ± 14.98 vs. 28.77 ± 7.0; P = 0.01 and 132.1 ± 19.72 vs. 53.33 ± 16.83; P = 0.02, respectively). See Figure 1. Results All septic animals developed a hyperdynamic state associated with lower arterial pressure, fever and organ dysfunction in comparison with control animals. In the septic animals, we observed increased brain dialysate glutamin levels at 12, 18 and 24  hours after sepsis induction, as compared with control animals. Moreover, after analyzing homogenates from the frontal cortex, we found higher concentrations of glutamin and glutamate in septic as compared with control animals (85.67 ± 14.98 vs. 28.77 ± 7.0; P = 0.01 and 132.1 ± 19.72 vs. 53.33 ± 16.83; P = 0.02, respectively). See Figure 1. P25 Cholinergic modulation of hippocampal activity during septic encephalopathy A Zivkovic1, CP Bengtson2, O Sedlaczek1, R Von Haken1, H Bading2, S Hofer 1Universitätsklinikum Heidelberg, Germany; 2Universität Heidelberg, Germany Critical Care 2013, 17(Suppl 2):P24 (doi: 10.1186/cc11962) de Jager C, et al.: The neutrophil–lymphocyte count ratio in patients with community acquired pneumonia. PLoS ONE 2012, 7:e46561. Cholinergic modulation of hippocampal activity during septic encephalopathy 2 2 This fi nding was further supported by the experimental recordings in the rat brains of lipopolysaccharide (LPS)-treated rats using the electrophysiological patch clamp technique. P25 P25 Neutrophil–lymphocyte count ratio as a biomarker of severe sepsis in Escherichia coli infections in adults LR Ljungstrom1, G Jacobsson1, R Andersson2 1Skaraborgs Sjukhus, Skövde, Sweden; 2Gothenburg University, Gothenburg, Sweden Critical Care 2013, 17(Suppl 2):P25 (doi: 10.1186/cc11963) P25 Neutrophil–lymphocyte count ratio as a biomarker of severe sepsis in Escherichia coli infections in adults LR Ljungstrom1, G Jacobsson1, R Andersson2 1Skaraborgs Sjukhus, Skövde, Sweden; 2Gothenburg University, Gothenburg, Sweden Critical Care 2013, 17(Suppl 2):P25 (doi: 10.1186/cc11963) Neutrophil–lymphocyte count ratio as a biomarker of severe sepsis in Escherichia coli infections in adults LR Ljungstrom1, G Jacobsson1, R Andersson2 Critical Care 2013, 17(Suppl 2):P25 (doi: 10.1186/cc11963) Introduction The neutrophil–lymphocyte count ratio (NLCR) is an easy to analyse biomarker reacting very early in the course of acute infl ammation. It has previously been reported to correspond to bacteremia and recently to disease severity in community-acquired pneumonia [1]. We have looked at 205 consecutive patients with Escherichia coli infections (ECI) and found the same to be true for ECIs. This may be of great clinical importance since E. coli is the most frequently isolated pathogen in patients with infections requiring in hospital care. Conclusion We found higher concentrations of glutamate and glutamin in brain tissues of septic animals as compared with control. Furthermore, glutamin concentrations increased over time in the extracellular space as measured by cerebral microdialysis. These fi ndings suggest an increased excitatory state that is potentially associated with high energy expenditure. However, associations with neuronal injury need further study. Methods This study is part of a 9-month consecutive study of community-acquired severe sepsis and septic shock in adults at Skaraborg Hospital in the western region of Sweden. The hospital serves a population of 256,000 inhabitants and has approximately 60,000 annual visits to the ED. All patients admitted to the hospital receiving intravenous antibiotic treatment within the fi rst 48  hours of admission were evaluated for severe sepsis and septic shock. Upon admission, two sets of blood cultures and other relevant cultures were obtained from each patient as well as sampling for NLCR and venous plasma lactate. The patient records were evaluated by one infectious diseases specialist. Approximately 2,300 patients were diagnosed as having a bacterial infection. From those, an informed consent to participate in the study could be obtained from approximately 1,600 patients. Reference de Jager C, et al.: The neutrophil–lymphocyte count ratio in patients with community acquired pneumonia. PLoS ONE 2012, 7:e46561. de Jager C, et al.: The neutrophil–lymphocyte count ratio in patients with community acquired pneumonia. PLoS ONE 2012, 7:e46561. S10 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Figure 1 (abstract P26). P26 Obesity and infl ammatory markers in severe sepsis P Simon1, D Thomas-Rüddel2, T Nemes1, K Reinhart2, F Bloos2, UX Kaisers1 1University Hospital of Leipzig, Germany; 2Center for Sepsis Control and Care, Jena, Germany Critical Care 2013, 17(Suppl 2):P26 (doi: 10.1186/cc11964) Introduction Chronic infl ammation has recently been recognized as an important factor in the pathophysiology of obesity and associated morbidities [1]. In this clinical study we aimed at identifying possible eff ects of obesity on infl ammatory markers in severe sepsis. fl Methods With institutional ethical committee approval, 243 consecutive patients treated for severe sepsis or septic shock in the ICUs of two university hospitals over a period of 5 months were studied. Six patients were excluded due to cachexia, syndromal disorders or missing clinical data. Diagnosis of sepsis was made according to SCCM criteria. Serum levels of C-reactive protein (CRP, mg/l) and procalcitonin (PCT, ng/ml) on day 1 of sepsis were compared among fi ve body mass index (BMI) strata according to WHO defi nitions. Two groups (BMI <30, normal weight, and BMI ≥30, obesity) were formed for further analysis, and PCT was logarithmically transformed (LogPCT), resulting in normal distribution. Statistical analysis was performed using a t test. y Results Patients with BMI ≥30 had higher values of PCT and CRP (Table 1). The diff erence in LogPCT was of borderline signifi cance (P = 0.052). However, patients with positive blood cultures had signifi cantly higher LogPCT values (P = 0.017) (Figure 1). Diff erence in CRP was not signifi cant (P = 0.09). The trends over all fi ve BMI strata (Table 1) were not signifi cant. P27 Cytokine gene expression can predict infectious complications following severe trauma HD Torrance, K Brohi, CJ Hinds, MJ O’Dwyer Bart’s Health NHS Trust, London, UK Critical Care 2013, 17(Suppl 2):P27 (doi: 10.1186/cc11965) Cytokine gene expression can predict infectious complications following severe trauma HD Torrance, K Brohi, CJ Hinds, MJ O’Dwyer Bart’s Health NHS Trust, London, UK Critical Care 2013, 17(Suppl 2):P27 (doi: 10.1186/cc11965) gi Conclusion Obesity with BMI ≥30 seems to be associated with an increase in infl ammatory markers in patients with severe sepsis, particularly in bacteraemia. The role of adipose tissue in severe sepsis should therefore be studied in more detail. 1. Wellen K, et al.: Infl ammation, stress, and diabetes. J Clin Invest 2005, 115:1111-1119. 1. Wellen K, et al.: Infl ammation, stress, and diabetes. J Clin Invest 2005, 115:1111-1119. Introduction Identifying a group of patients at high risk of developing infectious complications is the fi rst step in the introduction of eff ective pre-emptive therapies in specifi c patient groups. Quantifying cytokine gene expression also furthers our understanding of trauma-induced immunosuppression. Our group has already demonstrated that a predictive immunological signature derived from mRNA expression in elective thoracic surgical patients accurately predicts pneumonia risk [1]. Methods In total, 121 ventilated polytrauma patients were recruited. mRNA was extracted from PaxGene tubes collected within 2 hours of the initial insult, at 24 and 72 hours. T-helper cell subtype specifi c cytokines and transcription factors mRNA was quantifi ed using qPCR. Ten healthy controls served as a comparator. Table 1 (abstract P26). PCT and CRP as median (IQR) BMI n PCT (ng/ml) CRP (mg/l) 18.5 to 24.9 196 4.8 (11.9) 153 (189) ≥30.0 47 7.3 (24.6) 228 (254) 18.5 to 24.9 101 4.9 (11.7) 147 (175) 25 to 29.9 95 4.4 (12.0) 157 (218) 30 to 34.9 32 6.9 (22.6) 212 (118) 35 to 39.9 7 7.4 (38.3) 241 (264) ≥40.0 8 11.4 (32) 278 (272) y p Results The Median Injury Severity Score (ISS) was 29. Time 0 bloods demonstrated a reduction in TNFα†, IL-12§, IL-23‡, RORγT* and T bet§, and an increase in IL-10* and IL-4† mRNA levels in comparison with the control group (*P <0.0001, †P <0.001 to 0.0001, ‡P <0.01 to 0.001, §P <0.05 to 0.01). There was a positive correlation between ISS and IL-10‡ whilst both IL-23§ Figure 1 (abstract P27). Cytokine mRNA levels in trauma (time 0) and control groups. P27 Cytokine gene expression can predict infectious complications following severe trauma HD Torrance, K Brohi, CJ Hinds, MJ O’Dwyer Bart’s Health NHS Trust, London, UK Critical Care 2013, 17(Suppl 2):P27 (doi: 10.1186/cc11965) S11 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Methods From November 2010 to November 2011 we collected clinical data, drug administration, scores and PCT values of 420 consecutive patients during hospitalization. Statistical analysis was made using SPSS software. We calculated ICU mortality, 1-month mortality and 1-year mortality. Median percentage daily variation was calculated as: (PCT day after – PCT of the date value) / PCT of the date value×100. PCT variation in the last 48 hours of hospitalization was calculated as: (PCT at discharge – PCT at 48 hours before discharge) / PCT 48 hours before discharge×100. We compared peak values in dead patients versus alive patients. A logistic regression was performed in order to assess mortality odds ratio. and RORγT‡ were negatively correlated at time 0. TNFα†, IL-10* and IL-27‡ increased and IFNγ†, IL-12*, IL-17A§, RORγT* and T bet* mRNA levels decreased over the initial 24 hours. Subsequent bacteraemia (18/121 patients) was associated with a lower TNFα/IL-10 ratio‡ at baseline. Similarly, higher IL-10‡ and lower T bet‡ mRNA at 24 hours also predicted later bacteraemic episodes. Development of pneumonia followed a similar pattern. A multivariate logistical regression model proved highly accurate in predicting infectious complications from mRNA analysis of early blood samples. See Figure 1. y Conclusion Cytokine gene expression patterns indicate an immediate and sustained impairment in Th1, Th17 and innate immunity with concurrent upregulation of the Th2 response following major trauma. The magnitude of this response predicts subsequent infectious complications. y Results Of the 420 patients, 63 (15%) died in the ICU, 12 (2.86%) died 1 month after ICU discharge and 16 (3.80%) died 1 year after ICU discharge. PCT values were higher during the last day of hospitalization in dead patients versus alive patients. PCT percentage variation during the last 48 hours of hospitalization had a slower trend in patients who died than in those who survived; these diff erences are even more marked in patients who had a septic event. A slower descending trend of daily PCT values was found in patients who died than in those who survived. PCT peak levels during the ICU stay were higher in dead patients with respect to alive ones. P27 Cytokine gene expression can predict infectious complications following severe trauma HD Torrance, K Brohi, CJ Hinds, MJ O’Dwyer Bart’s Health NHS Trust, London, UK Critical Care 2013, 17(Suppl 2):P27 (doi: 10.1186/cc11965) At logistic regression analysis PCT decrease in the last 48 hours <–30% (OR 3.71), PCT peak higher than 10 ng/ml (OR 2.38), and PCT last day/PCT peak ratio >50% (OR 2.064) were ICU mortality risk factors. PCT values were a higher predictive ICU mortality risk factor than SOFA and APACHE II scores. Other prognostic factors were age and lactate values. Only age was a risk factor in 1-month and 1-year mortality. References 1. Jensen JU, Heslet L, Jensen TH, et al.: Procalcitonin increase in early identifi cation of critically ill patients at high risk of mortality. Crit Care Med 2006, 34:2596-2602. 2. Fritz HG, Brandes H, Bredle DL, et al.: Post-operative hypoalbuminaemia and procalcitonin elevation for prediction of outcome in cardiopulmonary bypass surgery. Acta Anaesthesiol Scand 2003, 47:1276-1283. Results We analyzed 150 consecutive episodes of severe sepsis (16%) or septic shock (84%) in the ICU. The median age of the patients was 64 (interquartile range, 48.7 to 71) years; the main sources of infection were intra-abdomen (45%) and respiratory (38%); 70.7% had medical diseases. The 28-day mortality was 22.7%. The profi le of death patients were men (64.7%, n =22), with signifi cantly higher average age (63 vs. 57 years; P = 0.049), as well as clinical severity scores, APACHE II (29.8 vs. 24.1; P <0.001) and SOFA (12.1 vs. 8.9; P <0.001) and major dysfunction organ number (4.6 vs. 3.6; P <0.001). Bilirubin was the best predictor of 28-day mortality with the largest AUC (0.71), followed by hemoglobin (0.69) and C3 (0.67). The multivariate logistic regression was adjusted for three risk parameters, hemoglobin (OR: 0.68; 95% CI: 0.51 to 0.94), bilirubin (OR:1.63; 95% CI: 1.08 to 2.45) and white blood cells (OR:1.04; 95% CI: 1.01 to 1.08) and with these parameters a ROC analysis was performed, giving an AUC of 0.77 (0.69 to 0.84). A cohort study of routinely used sepsis biomarkers and 28-day mortality 2 M De La Torre-Prados1, A Garcia-De la Torre2, C Trujillano-Férnández1 1Hospital Virgen de la Victoria, Málaga, Spain; 2Hospital Puerto Real, Cádiz, Spain M De La Torre-Prados1, A Garcia-De la Torre2, C Trujillano-Férnández1 1Hospital Virgen de la Victoria, Málaga, Spain; 2Hospital Puerto Real, Cádiz, Spain p Critical Care 2013, 17(Suppl 2):P28 (doi: 10.1186/cc11966) Introduction The evaluation of sepsis severity is complicated by the highly variable and nonspecifi c nature of clinical signs and symptoms. We studied routinely used biomarkers together with clinical parameters to compare their prognostic value for severe sepsis and evaluate their usefulness. Conclusion PCT is a good prognostic marker and is strongly correlated to the clinical status and gravity of the patients, so PCT seems to be a useful marker in an intensive care scenario. Methods A cohort study of 150 patients >18 years with severe sepsis according to the Surviving Sepsis Campaign, in an ICU of a university hospital. Demographic, clinical parameters and coagulation, infection and infl ammation parameters during the fi rst 24  hours from severe sepsis or septic shock onset were studied. Descriptive and comparative statistical analysis was performed using SPSS version 15.0 (SPSS Inc., Chicago, IL, USA). Changes in circulating procalcitonin versus C-reactive protein in predicting evolution of infectious disease in febrile, critically ill patients Changes in circulating procalcitonin versus C-reactive protein in predicting evolution of infectious disease in febrile, critically ill patients S Hoeboer1,2, J Groeneveld1,2 S Hoeboer1,2, J Groeneveld1,2 1VU University Medical Center, Amsterdam, the Netherlands; 2Erasmus Medical Center, Rotterdam, the Netherlands Critical Care 2013, 17(Suppl 2):P30 (doi: 10.1186/cc11968) S Hoeboer , , J Groeneveld , 1VU University Medical Center, Amsterdam, the Netherlands; 2Erasmus Medical Center, Rotterdam, the Netherlands Critical Care 2013, 17(Suppl 2):P30 (doi: 10.1186/cc11968) Introduction Although absolute values for C-reactive protein (CRP) and procalcitonin (PCT) are well known to predict sepsis in the critically ill, it remains unclear if and how changes in CRP and PCT predict evolution of infectious disease and how they compare in this respect. Introduction Although absolute values for C-reactive protein (CRP) and procalcitonin (PCT) are well known to predict sepsis in the critically ill, it remains unclear if and how changes in CRP and PCT predict evolution of infectious disease and how they compare in this respect. Conclusion The assessment of routine biomarkers (bilirubin, white blood cells and hemoglobin) may be a helpful tool in the decision- making process at the bedside, for the evaluation of early ICU admission of recoverable patients, as indicators of infl ammatory response, organ dysfunction or catabolism level, and their signifi cant predictive value on mortality. Methods In 72 critically ill patients with new-onset fever, CRP and PCT were measured on day 0, 1, 2 and 7 after inclusion, and their clinical course was documented over 1 week with follow-up to day 28. Infection was microbiologically defi ned, as was bloodstream infection; septic shock was defi ned as infection plus shock. p Reference 1. White M, et al.: Chest 2011, 139:626-632. y Reference i Results From peak at day 0 to 2 to day 7, CRP decreases most when (bloodstream) infection and septic shock (day 0 to 2) resolve and increases most when complications such as a new (bloodstream) infection or septic shock (day 3 to 7) supervene (area under the receiver operating characteristic curve 0.70 or higher, P = 0.04 or lower). PCT decreases most when septic shock resolves (AUC 0.72, P = 0.007) and increases most when a new bloodstream infection or septic shock supervenes (AUC 0.82 or higher, P <0.001). The day 7 value of PCT rather than of CRP was predictive for 28-day outcome (AUC 0.70, P = 0.005). i Results From peak at day 0 to 2 to day 7, CRP decreases most when (bloodstream) infection and septic shock (day 0 to 2) resolve and increases most when complications such as a new (bloodstream) infection or septic shock (day 3 to 7) supervene (area under the receiver operating characteristic curve 0.70 or higher, P = 0.04 or lower). PCT decreases most when septic shock resolves (AUC 0.72, P = 0.007) and increases most when a new bloodstream infection or septic shock supervenes (AUC 0.82 or higher, P <0.001). The day 7 value of PCT rather than of CRP was predictive for 28-day outcome (AUC 0.70, P = 0.005). Conclusion The data, obtained during ICU-acquired fever and infections, suggest that CRP and PCT changes predict the course of infectious disease and its complications. CRP may be favoured over PCT courses in decisions on appropriateness and duration of antibiotic treatment, whereas PCT rather than CRP courses may help predicting complications such as bloodstream infection, septic shock and mortality. 1. Glickman SW, Cairns CB, Otero RM, et al.: Disease progression in hemodynamically stable patients presenting to the emergency department with sepsis. Acad Emerg Med 2010, 17:383-390. 1. Glickman SW, Cairns CB, Otero RM, et al.: Disease progression in hemodynamically stable patients presenting to the emergency department with sepsis. Acad Emerg Med 2010, 17:383-390. Procalcitonin as prognostic marker of mortality Introduction Diff erentiation of acute heart failure from infection in patients with respiratory symptoms and a history of congestive heart failure (CHF) is challenging due to overlap of clinical symptoms and X-ray fi ndings. The BACH study found higher mortality rates if patients presenting with dyspnea were treated with antibiotics and their procalcitonin (PCT) levels were low indicating absence of bacterial infection. Yet the BACH study was observational and causal inference cannot be drawn. Herein, we analyzed the eff ects of PCT-guided antibiotic stewardship in CHF patients from a previous trial (ProHOSP). p p p Methods This is a secondary analysis of a previous randomized trial of adult ED patients with respiratory symptoms and a history of CHF. Patients were randomized to administration of antibiotics based on a PCT algorithm (PCT group) or standard guidelines without knowledge of PCT levels (control group). The primary endpoint of this analysis is the risk of adverse outcome defi ned as death or ICU admission within 30 days after ED admission. Figure 1 (abstract P32). Pro-ADM transpulmonary gradient at diff erent time points. Results A total of 233 patients met the inclusion criteria, with 116 in the PCT-guided group and 117 in the control group. In the subgroup of patients with low initial PCT levels <0.25 ng/l (n = 110), PCT-guided patients had a signifi cant reduction in antibiotic exposure (mean 3.7 vs. 6.5 days, diff erence –2.8 (95% CI –4.4, –1.2), P <0.001). Furthermore, PCT-guided patients had a signifi cant lower risk for death and ICU admission (4% vs. 20%, odds ratio 6.0 (1.3, 28.2), P = 0.02). See Figure 1. Conclusion In CHF patients with suspicion of respiratory infection, use of a PCT protocol resulted in a signifi cant decrease of antibiotic exposure and signifi cantly improved outcomes in patients with low PCT levels indicating absence of bacterial infection. Whether inadequate antibiotic therapy in these CHF patients requiring diuretic treatment explains this diff erence in clinical outcomes needs verifi cation. Methods In this prospective cohort study, 39 patients undergoing cardiac surgery using CPB were included. Blood was collected before surgery (T0), after induction of anesthesia (T1), after termination of CPB (T2), at ICU arrival (T3) and 3 hours (T4), 6 hours (T5) and 18 hours (T6) after arrival. Pro-ADM was measured with a sandwich immunoassay. Primary endpoints were length of ICU and hospital stay (ICU-LOS, hospital-LOS). Procalcitonin as prognostic marker of mortality p Results An increase of arterial and venous pro-ADM plasma con- centrations was observed after surgery. Immediately after termination of CPB the venous concentration was signifi cantly lower than arterial pro- ADM concentration, but at T6 the venous concentration was signifi cantly higher, indicating a switch from a negative to positive transpulmonary gradient (Figure 1). The pro-ADM venous–arterial diff erence at T5 was a signifi cant predictor of ICU-LOS (P = 0.032) and the diff erence at T3 was a signifi cant predictor of hospital-LOS (P = 0.001). Figure 1 (abstract P31). Time to adverse outcome according to group allocation. gi Conclusion We found that the transpulmonary gradient of pro-ADM was a predictor for ICU-LOS and hospital-LOS at T3 and T5, respectively. Pro-ADM might be a promising marker for prediction on outcome of patients undergoing cardiac surgery on CPB. The transpulmonary shift of pro-ADM might be caused by an infl ammatory response. References 1. Apostolakis E, et al.: J Card Surg 2010, 25:47-55. 1. Apostolakis E, et al.: J Card Surg 2010, 25:47-55. 2. Kamei M, et al.: Acta Anaesthesiol Scand 2004, 48:980-985. 3. Ueda S, et al.: Am J Respir Crit Care Med 1999, 160:132-136. 1. Apostolakis E, et al.: J Card Surg 2010, 25:47 55. 2. Kamei M, et al.: Acta Anaesthesiol Scand 2004, 48:980-985. 3. Ueda S, et al.: Am J Respir Crit Care Med 1999, 160:132-136. 2. Kamei M, et al.: Acta Anaesthesiol Scand 2004, 48: 2. Kamei M, et al.: Acta Anaesthesiol Scand 2004, 48:980 985. 3. Ueda S, et al.: Am J Respir Crit Care Med 1999, 160:132-136. Procalcitonin as prognostic marker of mortality Procalcitonin as prognostic marker of mortality S Zampieri1, P Bettonte2, M Ortolani1, G Frison1, V Schweiger1, L Gottin3, E Polati1 1Policlinico G.B. Rossi, Verona, Italy; 2Santa Chiara Hospital, Trento, Italy; 3Ospedale Maggiore Civile, Verona, Italy Critical Care 2013, 17(Suppl 2):P29 (doi: 10.1186/cc11967) y Conclusion The data, obtained during ICU-acquired fever and infections, suggest that CRP and PCT changes predict the course of infectious disease and its complications. CRP may be favoured over PCT courses in decisions on appropriateness and duration of antibiotic treatment, whereas PCT rather than CRP courses may help predicting complications such as bloodstream infection, septic shock and mortality. Introduction We analyze procalcitonin (PCT) as a prognostic marker, in order to assess the clinical impact of a daily PCT measure. Introduction We analyze procalcitonin (PCT) as a prognostic marker, in order to assess the clinical impact of a daily PCT measure. S12 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Figure 1 (abstract P32). Pro-ADM transpulmonary gradient at diff erent time points. P31 Procalcitonin-guided antibiotic therapy in patients with congestive heart failure and suspicion of lower respiratory tract infection: results from a randomized trial P Schuetz, E Grolimund, A Kutz, S Haubitz, B Mueller Kantonsspital Aarau, Switzerland Critical Care 2013, 17(Suppl 2):P31 (doi: 10.1186/cc11969) Introduction Diff erentiation of acute heart failure from infection in patients with respiratory symptoms and a history of congestive heart failure (CHF) is challenging due to overlap of clinical symptoms and X-ray fi ndings. The BACH study found higher mortality rates if patients presenting with dyspnea were treated with antibiotics and their procalcitonin (PCT) levels were low indicating absence of bacterial infection. Yet the BACH study was observational and causal inference cannot be drawn. Herein, we analyzed the eff ects of PCT-guided antibiotic stewardship in CHF patients from a previous trial (ProHOSP). Methods This is a secondary analysis of a previous randomized trial of adult ED patients with respiratory symptoms and a history of CHF. Patients were randomized to administration of antibiotics based on a PCT algorithm (PCT group) or standard guidelines without knowledge of PCT levels (control group). The primary endpoint of this analysis is the risk of adverse outcome defi ned as death or ICU admission within 30 days after ED admission. P33 The median age of the patients was S13 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 been standardized in early goal-oriented therapies, which may benefi t from circulating biomarkers for early risk stratifi cation. We aimed to evaluate the prognostic value of presepsin (sCD14-ST), a novel marker of bacterial infection. 64 (interquartile range, 53 to 72) years; the main sources of infection were respiratory tract (46%) and intra-abdomen (21%). The 28-day mortality was 32.5%. The profi le of death patients had a signifi cantly higher average age (64.7 vs. 57.6 years; P = 0.024), as well as clinical severity scores, APACHE II (26.6 vs. 23; P = 0.006) and SOFA (11.6 vs. 89.2; P <0.001). Kaplan–Meier survival analysis was signifi cant. P = 0.0017 for patients with pADM <1.2 nmol/l. Cox regression analysis also showed statistical signifi cance (P = 0.0033) and a likelihood ratio = 1.18 per each 1 nmol/l increase in pADM. Methods We performed a nested case–control study from the randomized controlled Albumin Italian Outcome Sepsis (ALBIOS) trial, enrolling patients with severe sepsis or septic shock from 100 ICUs in Italy. Fifty survivors and 50 nonsurvivors at ICU discharge were selected, matched for age, sex, center and time of enrollment after inclusion criteria were present. EDTA-plasma samples were collected at days 1, 2 and 7 after enrolment for presepsin (immune- chemiluminescence assay PATHFAST Presepsin, URL 320 pg/ml, CV 5%; Mitsubishi Chemicals) and procalcitonin assay (PCT, Elecsys BRAHMS Cobas® PCT, URL 0.046 ng/ml, CV 8.8%; Roche Diagnostics). p Conclusion The protein pADM is an important prognostic biomarker of survival when measured on admission of septic patients to the ICU. References 1. Pezzilli R, Barassi A, Pigna A, et al.: Time course of proadrenomedullin in the early phase of septic shock. A comparative study with other proinfl ammatory proteins. Panminerva Med 2012, 54:211-217. g g Results Clinical characteristics were similar between the two groups, except for a worse SOFA score at day 1 in decedents. Presepsin at day 1 was signifi cantly higher in decedents (2,268 (1,145 to 4,305) pg/ ml, median (Q1 to Q3)) than in survivors (1,184 (855 to 2,158) pg/ml, P = 0.001), while PCT did not diff er (18.5 (3.3 to 45.7) vs. 10.8 (2.6 to 46.4) ng/ml, P = 0.31). P33 P33 Pro-adrenomedullin as prognostic biomarker in the sepsis M De La Torre-Prados, A Garcia-De la Torre, A Enguix, M Mayor, N Zamboschi, C Trujillano-Fernández, A Garcia-Alcantara Hospital Virgen de la Victoria, Málaga, Spain Critical Care 2013, 17(Suppl 2):P33 (doi: 10.1186/cc11971) Pro-adrenomedullin as prognostic biomarker in the sepsis M De La Torre-Prados, A Garcia-De la Torre, A Enguix, M Mayor, N Zamboschi, C Trujillano-Fernández, A Garcia-Alcantara Hospital Virgen de la Victoria, Málaga, Spain Critical Care 2013, 17(Suppl 2):P33 (doi: 10.1186/cc11971) Figure 1 (abstract P31). Time to adverse outcome according to group allocation. p g , g , p Critical Care 2013, 17(Suppl 2):P33 (doi: 10.1186/cc11971) Introduction Measurement of biomarkers is a potential approach to early assessment and prediction of mortality in septic patients. The purpose of this study was to ascertain the prognostic value of pro- adrenomedullin (pADM), measured in all patients admitted to the ICU of our hospital with a diagnosis of severe sepsis or septic shock during 1 year. P32 Pro-adrenomedullin as a clinical predictor after cardiac surgery J Van Fessem, F De Graaf, J Van Paassen, S Arbous LUMC, Leiden, the Netherlands Critical Care 2013, 17(Suppl 2):P32 (doi: 10.1186/cc11970) y Methods A cohort study of 117 patients >18 years with severe sepsis according to the Surviving Sepsis Campaign, in an ICU of a university hospital. Demographic, clinical parameters and pADM, C-reactive protein and procalcitonin were studied during 1 year. Descriptive and comparative statistical analysis was performed using the statistical software packages Statistica Stat Soft Inc 7.1 and MedCalc 9.2.1.0. Results We analyzed 117 consecutive episodes of severe sepsis (15%) or septic shock (85%) in the ICU. The median age of the patients was Introduction Pulmonary complications after cardiac surgery like ARDS are frequent and linked to high mortality [1]. Pro-adrenomedullin (pro-ADM) has a possible role in the development of ARDS [2] and a positive correlation between levels of pro-ADM and infl ammation was found [3]. In this study we investigated whether intraoperative and postoperative pro-ADM transpulmonary gradient could predict postoperative morbidity. g Results We analyzed 117 consecutive episodes of severe sepsis (15%) or septic shock (85%) in the ICU. Early IL-6 response in sepsis is correlated with mortality and severity score P Srisangthong, A Wongsa, P Kittiworawitkul, A Wattanathum Phramongkutklao Hospital, Bangkok, Thailand C iti l C 2013 17(S l 2) P34 (d i 10 1186/ 11972) P Srisangthong, A Wongsa, P Kittiworawitkul, A Wattanathum Phramongkutklao Hospital, Bangkok, Thailand C iti l C 2013 17(S l 2) P34 (d i 10 1186/ 11972) g p g Critical Care 2013, 17(Suppl 2):P34 (doi: 10.1186/cc11972) Introduction IL-6, a proinfl ammatory cytokine, is synthesized from fi bro- blasts, T lymphocytes, endothelial cells and monocytes. It serves as an important mediator during the acute phase response to infl ammation in sepsis. We hypothesized that the plasma IL-6 is correlated with mortality and severity scores in critically ill patients with sepsis. Methods We conducted a prospective study of plasma IL-6 level at the initial phase of sepsis and the risk of mortality. A total of 203 patients with sepsis, who were admitted to the medical ICU at Phramongkutklao Hospital, Bangkok during January to December 2011, were analyzed. Serum IL-6, C-reactive protein (CRP), and lactate were measured within the fi rst 24 hours of ICU admission. Severity scores (APACHE II, SAP II, and SOFA scores) were measured. The primary outcome variable was 28-day all-cause mortality. Conclusion Early presepsin measurements may provide important prognostic information in patients with severe sepsis or septic shock, and may be of crucial importance for early risk stratifi cation. P36 References References 1. Pathan N, et al.: Crit Care Med 2005, 33:1839-1844. 2. Harbarth S, et al.: Am J Respir Crit Care Med 2001, 164:396-402. 1. Pathan N, et al.: Crit Care Med 2005, 33:1839-1844. 2. Harbarth S, et al.: Am J Respir Crit Care Med 2001, 164:396-402. p p y Methods This study was conducted as a single-center retrospective study. Blood samples were collected from patients admitted to the emergency room at Fukuoka University Hospital between June 2010 and October 2012. We enrolled 254 patients with suspected sepsis and other disease patients. We classifi ed the patients into an AKI group according to the RIFLE criteria (Risk n = 52, Injury n = 39, Failure n = 41, Loss of kidney function and End-stage kidney disease n = 7) and a non- AKI group (n = 115). The AKI patient group was further classifi ed into a sepsis group and a nonsepsis group in each AKI stage and we analyzed the diagnostic accuracy of presepsin in patients with sepsis. P33 Presepsin decreased over time in survivors, but remained elevated in decedents (974 (674 to 1,927) vs. 2,551 (1,438 to 5,624) pg/ml at day 7, P = 0.02 for time–survival interaction); PCT decreased similarly in the two groups (P = 0.19). Patients with early elevated presepsin had worse SOFA score, higher number of MOFs, hemodynamic instability (lower mean arterial pressure at baseline and after 6 hours), and mortality rate at 90 days (75% vs. 42%, log- rank P <0.001). The association between presepsin and outcome was more marked in patients with late enrollment (6 to 24 hours), and in septic shock. Early presepsin had better prognostic accuracy than PCT (AUROC 0.69 vs. 0.56, P = 0.07), and improved discrimination over SOFA score, especially in septic shock. pl y p 2. Wang RL, Kang FX: Prediction about severity and outcome of sepsis by proatrial natriuretic peptide and pro-adrenomedullin. Chin J Traumatol 2010, 13:152-157. Usefulness of presepsin in the diagnosis of sepsis in acute kidney injury patients Usefulness of presepsin in the diagnosis of sepsis in acute kidney injury patients Usefulness of presepsin in the diagnosis of sepsis in acute kidney injury patients Y Nakamura, H Ishikura, T Nishida, Y Kawanno, R Yuge, R Ichiki, A Murai Fukuoka University, Fukuoka, Japan Critical Care 2013, 17(Suppl 2):P36 (doi: 10.1186/cc11974) y y Results We found that the overall 28-day mortality was 46% (93 out of 203 patients). There was a signifi cantly positive correlation between mortality rate and plasma IL-6 (survivors vs. nonsurvivors; 74 (4.4 to 1,718) vs. 206 (19 to 5,000) pg/ml, P <0.05), lactate (survivors vs. nonsurvivors; 1.65 (0.7 to 11.61) vs. 2.47 (0.94 to 19.13) mmol/l, P <0.05), but not CRP levels (P = 0.24). Compared with the patients with plasma IL-6 <100 pg/ml, septic patients with IL-6 levels ≥100 were associated with an increased 28-day mortality with the odd ratio of 2.99 (95% CI 1.42 to 6.29, P <0.05). We also found that plasma IL-6 levels were well correlated with APACHE II (P <0.05), SAPS II (P <0.05), and SOFA (P <0.05) scores. Y Nakamura, H Ishikura, T Nishida, Y Kawanno, R Yuge, R Ichiki, A Murai Fukuoka University, Fukuoka, Japan Critical Care 2013, 17(Suppl 2):P36 (doi: 10.1186/cc11974) Introduction The level of presepsin is useful for diff erentiating sepsis from noninfectious systemic infl ammatory response syndrome. It has been reported that the presepsin levels in patients with chronic renal failure are abnormally high. However, there are no studies investigating the usefulness of presepsin for diagnosis of sepsis in patients with acute kidney injury (AKI). Our purpose of this study is to clarify the diagnostic accuracy of presepsin in patients with AKI and the relationship between presepsin level and AKI severity. Conclusion The initial phase plasma IL-6 levels were correlated with severity and mortality in critically ill patients with sepsis. Serum levels of presepsin refl ects the APACHE II and SOFA scores in patients with sepsis Serum levels of presepsin refl ects the APACHE II and SOFA scores in patients with sepsis R Sato, Y Suzuki, M Sato, G Takahashi, M Kojika, Y Inoue, S Endo Iwate Medical University, Morioka, Japan Critical Care 2013, 17(Suppl 2):P37 (doi: 10.1186/cc11975) R Sato, Y Suzuki, M Sato, G Takahashi, M Kojika, Y Inoue, S Endo Iwate Medical University, Morioka, Japan Critical Care 2013, 17(Suppl 2):P37 (doi: 10.1186/cc11975) Methods We analyzed data from a large multinational database of patients with CAP (CAPO) [2] between 2001 and 2012. We performed logistic regression analysis and the area under the receiver operating characteristics (AUC) curve to study the association of these variables with the diagnosis of Legionella. Introduction As a method for the diagnosis of sepsis, we previously reported an ELISA method for measuring the serum levels of presepsin. That method, however, took approximately 2 hours to yield results.i Introduction As a method for the diagnosis of sepsis, we previously reported an ELISA method for measuring the serum levels of presepsin. That method, however, took approximately 2 hours to yield results. Methods To resolve this problem, we later developed a simplifi ed assay kit making use of immunochromatography (Point of Care test; POC test), and are currently evaluating the usefulness of this kit for diagnosis of sepsis and evaluation of its severity. In 21 septic patients with sepsis, we analyzed the serum levels of presepsin in relation to APACHE II and SOFA scores. pp y y Methods To resolve this problem, we later developed a simplifi ed assay kit making use of immunochromatography (Point of Care test; POC test), and are currently evaluating the usefulness of this kit for diagnosis of sepsis and evaluation of its severity. In 21 septic patients with sepsis, we analyzed the serum levels of presepsin in relation to APACHE II and SOFA scores. g g Results Data for 8,278 CAP patients were analysed; the infectious organism was known in 2,321 cases (28%), including a total of 101 patients with urinary antigen-confi rmed Legionnaires’ disease and 983 patients with confi rmed pneumococcal disease. All variables were predictors for Legionella with odds ratios ranging from 1.002 to 5.767. Combining the variables in a joint logistic regression model showed a high predictive accuracy with an AUC of 0.86. Results APACHE II and SOFA scores at baseline were 28.7  ±  7.5 and 10.8  ±  3.9, respectively, in these patients. Host genetic variants associated with community-acquired pneumonia LE Salnikova1, TV Smelaya1, VV Moroz1, A Golubev1, AV Rubanovich2 1V.A. Negovsky Research Institute of General Reanimatology, Moscow, Russia; 2N.I. Vavilov Institute of General Genetics, Russian Academy of Sciences, Moscow, Russia Critical Care 2013, 17(Suppl 2):P40 (doi: 10.1186/cc11978) Introduction Serum nitrite/nitrate (NOx) levels in the early phase of septic acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) have recently been reported to possibly play a key role in pathogenesis of ALI/ARDS. p g Methods NOx levels in the early phase of septic ALI/ARDS were measured by autoanalyzer (TCI-NOX 1000; Tokyo Kasei Kogyo Co., Ltd, Tokyo, Japan). Cytokine was measured by ELISA (Medogenix, Fleurus, Belgium). Introduction It has been shown that polymorphic variants at some host genes can modify risk of community-acquired pneumonia (CAP), including those critical for the host response to CAP – innate immune system, the lung’s defense against inhaled microorganisms, and inhibition of fi brinolysis and the renin–angiotensin system. The aim of the study was to analyze polymorphisms in genes potentially relevant to CAP pathogenesis mechanisms to reveal novel and confi rm reported genetic risk factors in the general Russian population. g Results Both NOx and TNFα levels were signifi cantly higher in the ARDS group than in the ALI group. A negative correlation was found between the PaO2/FIO2 (P/F) ratio and serum NOx levels. In addition, a positive correlation was found between the TNFα and serum NOx levels. The 30-day, 60-day and 90-day mortality rates were 8.7%, 15.2% and 19.6%, respectively, in the patients with ALI/ARDS. There were no diff erences in the P/F ratio, serum NOx levels or TNFα levels in the early phase of ALI/ARDS between the 30-day survival and death groups. On the other hand, the P/F ratio, serum NOx levels and TNFα levels in the early phase of ALI/ARDS were signifi cantly higher in the 60-day and 90-day death groups than in the corresponding survival groups. There were no signifi cant diff erences in the 90-day mortality rates between the ALI and ARDS groups.i Methods Patients with CAP (n = 334), volunteers without a previous history of CAP, control group A (n  = 141) and a second control group B (n  = 314) were included in the study. P38 P38 Relationship between serum nitrite/nitrate levels in the early phase of septic acute lung injury and prognosis M Sato, Y Suzuki, T Masuda, G Takahashi, M Kojika, Y Inoue, S Endo Iwate Medical University, Morioka, Japan Critical Care 2013, 17(Suppl 2):P38 (doi: 10.1186/cc11976) References 1. Fiumefreddo R, Zaborsky R, Haeuptle J, et al.: Clinical predictors for Legionella in patients presenting with community-acquired pneumonia to the emergency department. BMC Pulm Med 2009, 9:4. 2. Ramirez JA: Fostering international multicenter collaborative research: the CAPO Project. Int J Tuberc Lung Dis 2007, 11:1062-1065. Serum levels of presepsin refl ects the APACHE II and SOFA scores in patients with sepsis These were signifi cant correlations between the serum presepsin levels and the APACHE II score, and also between the serum presepsin levels and the SOFA score. Conclusion Furthermore, there was also a signifi cant correlation between the results of the POC test and the serum presepsin levels. These results indicate that measurement of the serum presepsin might be useful for evaluating the severity of sepsis. Conclusion This analysis validates the Legionella score in an inde- pendent sample and shows high diagnostic accuracy. Interventional trials with adapted antibiotic regimes for non-inferiority in a real live population are warranted. R f Compared values of presepsin (sCD14-ST) and procalcitonin as early markers of outcome in severe sepsis and septic shock: a preliminary report from the Albumin Italian Outcome Sepsis (ALBIOS) study Compared values of presepsin (sCD14-ST) and procalcitonin as early markers of outcome in severe sepsis and septic shock: a preliminary report from the Albumin Italian Outcome Sepsis (ALBIOS) study P Caironi1, S Masson2, E Spanuth3, R Thomae4, R Fumagalli5, A Pesenti5, M Romero6, G Tognoni6, R Latini2, L Gattinoni1 1Fondazione IRCCS Ca’ Granda – Ospedale Maggiore Policlinico, Università degli Studi di Milano, Milan, Italy; 2Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy; 3Diagnostic Engineering & Research GmbH, Heildelberg, Germany; 4Mitsubishi Chemical Europe GmbH, Munich, Germany; 5Ospedale San Gerardo, Monza, Italy; 6Consorzio Mario Negri Sud, Santa Maria Imbaro, Italy Critical Care 2013, 17(Suppl 2):P35 (doi: 10.1186/cc11973) , g , , 1Fondazione IRCCS Ca’ Granda – Ospedale Maggiore Policlinico, Università degli Studi di Milano, Milan, Italy; 2Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy; 3Diagnostic Engineering & Research GmbH, Heildelberg, Germany; 4Mitsubishi Chemical Europe GmbH, Munich, Germany; 5Ospedale San Gerardo, Monza, Italy; 6Consorzio Mario Negri Sud, Santa Maria Imbaro, Italy Results For the non-AKI patients, the median of presepsin in patients with nonsepsis (n = 78) and the sepsis group (n = 37) were 406 pg/ml (range: 86 to 4,374) and 1,065 pg/ml (range 86 to 9,960), respectively (P <0.0001). For the Risk patients, the median of presepsin in patients with nonsepsis (n = 25) and the sepsis group (n = 27) were 299 pg/ml (range: 71.2 to 3,361) and 831 pg/ml (range: 233 to 16,759), respectively (P <0.01). For the Injury patients, the median of presepsin in patients with nonsepsis (n = 12) and the sepsis group (n = 27) were 463 pg/ml (range: 122 to 1,197) and 1,451 pg/ml (range: 237 to 4,200), respectively (P <0.001). For the Failure patients, the median of presepsin in patients y Critical Care 2013, 17(Suppl 2):P35 (doi: 10.1186/cc11973) Introduction Sepsis results from complex interactions between infecting microorganisms and host responses, often leading to multiple organ failures and death. Over the years, its treatment has Introduction Sepsis results from complex interactions between infecting microorganisms and host responses, often leading to multiple organ failures and death. Over the years, its treatment has S14 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 P39 with nonsepsis (n = 14) and the sepsis group (n = 27) were 1,607 pg/ml (range: 454 to 8,516) and 1,523 pg/ml (range: 293 to 16,764), respectively (P = 0.175). The diagnostic accuracy of presepsin in patients with sepsis was determined by ROC analysis, the area under the curve was 0.789 for the non-AKI patient group, 0.735 for the Risk patient group, 0.855 for the Injury patient group and 0.593 for the Failure patient group. Conclusion In Failure and more progressed AKI patients, the diagnostic accuracy of the presepsin level was lower than the other groups. References with nonsepsis (n = 14) and the sepsis group (n = 27) were 1,607 pg/ml (range: 454 to 8,516) and 1,523 pg/ml (range: 293 to 16,764), respectively (P = 0.175). The diagnostic accuracy of presepsin in patients with sepsis was determined by ROC analysis, the area under the curve was 0.789 for the non-AKI patient group, 0.735 for the Risk patient group, 0.855 for the Injury patient group and 0.593 for the Failure patient group. Simple and fast prediction of Legionella sp. in community-acquired pneumonia: validation of a prediction rule S Haubitz1, F Hitz2, L Graedel2, T Wiemken3, P Peyrani Dicastelnuovo3, J Ramirez3, M Batschwaroff 1, C Fux1, B Mueller1, P Schütz1 1Kantonsspital Aarau, Switzerland; 2University of Basel, Switzerland; 3University of Louisville School of Medicine, Louisville, KY, USA Critical Care 2013, 17(Suppl 2):P39 (doi: 10.1186/cc11977) Conclusion In Failure and more progressed AKI patients, the diagnostic accuracy of the presepsin level was lower than the other groups. References 1. Endo S, et al.: J Infect Chemother 2012, 18:891-897. doi:10.1007/s10156-012-0435-2 2. Bellomo R, et al.: Crit Care 2004, 8:R204-R212. doi:10.1186/cc2872 1. Endo S, et al.: J Infect Chemother 2012, 18:891-897. doi:10.1007/s10156-012-0435-2 Introduction Ruling out Legionella sp. in patients presenting with community-acquired pneumonia (CAP) is important due to diff erences in treatment regimens. Yet antigen tests as well as blood cultures have low sensitivity and an important time delay, making empirical broad spectrum coverage necessary particularly in severe cases. Fiumefreddo and colleagues recently proposed a clinical score based on six clinical and laboratory variables (fever, cough, sodium, lactate-dehydrogenase, C-reactive protein, platelet count) which allowed assessing the likelihood of Legionella [1]. Yet these variables need validation in an independent patient cohort before implementation into clinical routine. 2. Bellomo R, et al.: Crit Care 2004, 8:R204-R212. doi:10.1186/cc2872 2. Bellomo R, et al.: Crit Care 2004, 8:R204-R212. doi:10.1186/cc2872 Eff ectiveness of early ureteric stenting for urosepsis associated with urinary tract calculi S Nishiguchi , Y Tokuda 1Shonan Kamakura General Hospital, Kamakura, Japan; 2Mito Kyodo General Hospital, Mito, Japan Critical Care 2013, 17(Suppl 2):P43 (doi: 10.1186/cc11981) S Nishiguchi , Y Tokuda 1Shonan Kamakura General Hospital, Kamakura, Japan; 2Mito Kyodo General Hospital, Mito, Japan Critical Care 2013, 17(Suppl 2):P43 (doi: 10.1186/cc11981) p Results A total of 536 (14%) of the 3,766 enrolled patients had SCAP. They were mostly male (66%) with median age 59 (29 to 82) years, median SAPS II 44 (21 to 80) and total SOFA score 8 (3 to 16). Thirty- seven per cent of the cases were microbiologically documented (St. pneumoniae  – 24%; Enterobacteriaceae  – 20%; infl uenza A (H1N1) virus – 18%) and 45% had septic shock. Antibiotic combination was used in 76% of the patients and 61% received a macrolide. Median hospital length of stay was 19 (3 to 70) days and hospital mortality was 35%. Comorbidities were present in 70% of the patients. The most frequent were: diabetes mellitus (21%), chronic respiratory failure (18%) and alcoholism (15%). Median Charlson’s comorbidity index (CCI) was 4 (0 to 13). In univariate analysis, the presence of at least one comorbidity (odds ratio (OR) 2.29; 95% CI 1.49 to 3.52), namely cancer (OR 3.80; 95% CI 2.14 to 6.74; P <0.001), chronic renal failure (OR 3.23; 95% CI 1.53 to 6.82; P = 0.001), immunosuppression (OR 2.12; 95% CI 1.15 to 3.92; P = 0.014) and neurologic disease (OR 1.87; 95% CI 1.10 to 3.17; P = 0.02), increased the chances of dying in the hospital. Median CCI was also signifi cantly higher in nonsurvivors (5 vs. 3; P <0.001; OR per point 1.10 (95% CI: 1.05 to 1.15)). The only independent risk factor for hospital mortality was the presence of at least one comorbidity (OR 2.09; 95% CI 1.13 to 3.85). Introduction Urosepsis associated with urinary tract calculi is a critical disease. Patients with this condition occasionally require drainage, mostly ureteric stent placement and these patients need longer hospitalization. However, indications for timely ureteric stenting for urosepsis associated with urinary tract calculi have not been clearly determined. The objective of the study was to evaluate whether earlier stent placement might lead to shorter length of stay (LOS) in hospitals. Outcome of severe community-acquired pneumonia: the impact of comorbidities Outcome of severe community-acquired pneumonia: the impact of comorbidities JM Pereira1, JA Paiva1, F Froes2, JP Baptista3, J Gonçalves-Pereira4 1Centro Hospitalar S. João, Porto, Portugal; 2Hospital Santa Maria-CHLN, Lisboa, Portugal; 3Hospitais da Universidade de Coimbra – CHUC, Coimbra, Portugal; 4Hospital São Francisco Xavier, Lisboa, Portugal Critical Care 2013, 17(Suppl 2):P41 (doi: 10.1186/cc11979) Conclusion In early surgery IE, it appears to be associated with mortality: age >70 years, previous heart disease, emergency surgery, antibiotics within 7 days before surgery, preoperative MOF, and high scores on scales of Pons and Ontario. The causal agent and echocardiography have no relation with worse prognosis. The general syndrome debut and the Streptococcus spp. etiology seem to have lower mortality. IE has pathologies with high .preoperative severity scores but these are not suffi cient to guide therapeutic decisions. Introduction Several comorbidities have been independently asso- ciated with both predisposition to community-acquired pneumonia and a worse outcome. The goal of this study was to evaluate the impact of comorbidities on the outcome of patients with severe community- acquired pneumonia (SCAP). Methods A prospective, multicentre, observational cohort study of all patients with SCAP consecutively admitted to 14 Portuguese ICUs during a 12-month period. Several comorbidities were evaluated: congestive heart failure, cancer, chronic renal failure, chronic respira- tory failure, chronic hepatic disease, alcoholism, diabetes mellitus, neurologic disease, immunosuppression, HIV infection. To evaluate the impact of comorbidities associated with hospital mortality in univariate analysis, a logistic regression analysis adjusted to other variables (clinical relevant or statistically signifi cant in univariate analysis) was performed. P42 P42 Prognostic scores and infective endocarditis P Fernandez Ugidos1, R Gomez Lopez1, P Vidal Cortes1, A Ceniceros Barros1, L Seoane Quiroga1, JM Lopez Perez2 1Complejo Hospitalario Universitario Ourense, Spain; 2Complejo Hospitalario Universitario A Coruña, Spain Critical Care 2013, 17(Suppl 2):P42 (doi: 10.1186/cc11980) Introduction Infective endocarditis (IE) is a high-mortality disease, especially in the early surgery subgroup. The aim of this study was try to identify prognostic factors of IE that will require surgery during the same admission of their diagnosis, including evaluation of surgical severity scores. Conclusion We have provided the fi rst experimental evidence for the associations of genes coding detoxifi cation enzymes with the risk of CAP. Our results also demonstrate that predisposition to CAP is strongly attributed to the eff ects of a number of genes with low penetrance and therefore imply that inter-locus interactions may be regarded as an important component of polygenic and multifactorial factors of susceptibility to CAP. Methods A retrospective study (5 years) of all patients admitted to a tertiary hospital in northern Spain with diagnosis of IE (modifi ed Duke criteria) who required early surgery. Demographic, clinical and microbiology data were collected. Chi-square and Student t tests. Signifi cance: P <0.05. SPSS17. gi Results We had 73 patients, 79.5% men, age 65  years. Eighty-two percent had positive blood cultures. Forty-one percent of cases required previous ICU admission. Surgery was urgent in 35%. Fifty-six per cent of patients had postoperative shock and 58% suff ered post- surgery ARF. Hospital mortality was 31.5%. Regarding prognostic scales: the mean EuroSCORE was 11.38 ± 3.93 points. No patient was placed in the low-risk group. Ninety-four per cent of cases were at high risk. The Parsonnet mean score was 27.7 ± 11.6. The mean Beth Israel Medical Center was 37.6 ± 10 points. The mean Ontario scale was 7.09 ± 2.7. The mean Surgical risk scale of Roques was 6.69. And the mean by Pons Scale was 24.02. Dead patients are older, with previous heart disease, require urgent surgery and have previous ICU stay; usually they have MOF and they had received less than 7 days of antibiotic treatment. The etiology did not worsen the prognosis. Outcome of severe community-acquired pneumonia: the impact of comorbidities JM Pereira1, JA Paiva1, F Froes2, JP Baptista3, J Gonçalves-Pereira4 1Centro Hospitalar S. P42 João, Porto, Portugal; 2Hospital Santa Maria-CHLN, Lisboa, Portugal; 3Hospitais da Universidade de Coimbra – CHUC, Coimbra, Portugal; 4Hospital São Francisco Xavier, Lisboa, Portugal Critical Care 2013, 17(Suppl 2):P41 (doi: 10.1186/cc11979) Host genetic variants associated with community-acquired pneumonia Using allele-specifi c tetraprimer PCR, all subjects were genotyped for 13 polymorphic variants in the genes of xenobiotic detoxifi cation CYP1A1 (rs2606345, rs4646903, rs1048943), GSTM1 (Ins/Del), GSTT1 (Ins/Del), ABCB1 (rs1045642); immune and infl ammation response IL-6 (rs1800795), TNFα (rs1800629), MBL2 (rs7096206), CCR5 (rs333), NOS3 (rs1799983), angiotensin-converting enzyme (ACE; rs4340), and occlusive vascular disease/hyperhomocysteinemia MTHFR (rs1801133). Conclusion Our fi ndings suggested that NOx may be involved in the pathogenesis of ALI/ARDS. S15 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Results Seven genes CYP1A1 rs2606345T/T, GSTM1 Ins/*, ABCB1 C/C, IL-6 C/C-G/G, NOS3 T/T-G/G, CCR5 Ins/Ins, and ACE Del/Del were associated with CAP. The highest eff ect was detected for the CYP1A1 rs2606345: in comparison with the control A, P  = 3.9×10–5, OR  = 2.40, 95% CI: 1.59 to 3.64; and in comparison with the control B, P = 1.4×10–5, OR = 2.0, 95% CI: 1.46 to 2.74. For the two genes CYP1A1 and GSTM1, associations remained signifi cant after correction for multiple comparisons. Multiple analysis by the number of all risk genotypes showed a highly signifi cant association with CAP (P = 2.4×10–7, OR = 3.03, 95% CI 1.98 to 4.64) with the threshold for three risk genotypes. Using the ROC analysis, the AUC value for multi-locus model was estimated as 68.38, which is rather high for genetic markers.i Yamamoto Y, et al.: BMC Urol 2012, 12:4. Yoshimura K, et al.: J Urol 2005, 173:458-462. Yamamoto Y, et al.: BMC Urol 2012, 12:4. Yoshim ra K et al J Urol 2005 173 458 462 2. Yoshimura K, et al.: J Urol 2005, 173:458-462. P46 Abdominal infection plays a role in the incidence of ventilator- associated pneumonia GB Bouroche1, S Ruckly2, B Misset3, JF Timsit4, F Philippart3 1Institut Gustave Roussy, Villejuif, France; 2Institut Albert Bonniot, La Tronche, France; 3Hôpital Saint Joseph, Paris, France; 4CHU Grenoble, France Critical Care 2013, 17(Suppl 2):P46 (doi: 10.1186/cc11984) g References 1. Clec’h C, Schwebel C, Français A: Does catheter-associated urinary tract infection increase mortality in critically ill patients. Infect Control Hosp Epidemiol 2007, 28:1367-1373. 1. Clec’h C, Schwebel C, Français A: Does catheter-associated urinary tract infection increase mortality in critically ill patients. Infect Control Hosp Epidemiol 2007, 28:1367-1373. p Methods To investigate, in humans, the potential involvement of previous intra-abdominal infection (IIA) in preventing or promoting VAP, we realized a prospective observational study using data from a multicenter database (OUTCOMEREA), including all patients admitted 2. Marx G, Reinhart K: Urosepsis: from the intensive care viewpoint. Int J Antimicrob Agents 2008, 31S:S79-S84. 2. Marx G, Reinhart K: Urosepsis: from the intensive care viewpoint. Int J Antimicrob Agents 2008, 31S:S79-S84. 2. Marx G, Reinhart K: Urosepsis: from the intensive care viewpoint. Int J Antimicrob Agents 2008, 31S:S79-S84. Figure 1 (abstract P46). Cumulative incidence of VAP. Eff ectiveness of early ureteric stenting for urosepsis associated with urinary tract calculi Methods For patients who required ureteric stent procedures for urosepsis associated with urinary tract calculi in our hospital from July 2008 to July 2012, we compared the LOS in our hospital between patients with earlier stenting and those without it. A linear regression model was used for multivariable-adjusted comparison. j Results In a total of 30 patients (mean age, 72; 13 males), the mean days from emergency room admission to ureteric stenting was 3.5 days (range, 1 to 14 days) and the overall mean LOS was 36 days (range, 8 to 102  days). The early-stenting group (mean LOS, 21  days) had signifi cantly shorter LOS than the delayed-stenting group (mean LOS, 50 days) with adjusted β coeffi cient of –26 days (95% CI, –46, –6). Conclusion In patients with urosepsis associated with urinary tract calculi, earlier stenting within 2 days of admission may reduce the hospital LOS. References Conclusion In SCAP, the presence of at least one comorbidity doubles the chances of dying in the hospital and is an independent risk factor for hospital mortality. Yamamoto Y, et al.: BMC Urol 2012, 12:4. Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 S16 (n = 13, 13.4%) and intraabdominal infections (n = 13, 13.4%). Patients with pneumonia were treated according to knowledge in a specifi c moment, thus diff erent antibiotics were used. The duration of antibiotic therapy was 20 ± 15.5 days. In nine episodes of pneumonia according to the CDC no germ was isolated in the cultures. Six of the episodes were polymicrobial infections. The most frequent microbes isolated were E. coli (n = 7, 22.5% of pneumonia cases), A. fumigatus (n = 7, 22.5%), S. aureus (n = 3, 9.68%), P. aeruginosa (n = 3, 9.68%), P. mirabilis, K. pneumoniae, E. cloacae, E. faecalis, C. glabrata, and S. marcescens (one case each, 3.22%). Pneumonia was suspected but not confi rmed in 75 patients. Despite this, antibiotic treatment was maintained for a media of 17.35 ± 7.01 days: 56 wide-spectrum treatments and 18 targeted therapy after knowing the antibiogram. The length of ICU stay was 38.4 ± 70.8 (3 to 264) days, of hospital stay was 66.2 ± 80.5 (3 to 304) days and of mechanical ventilation was 27.3 ± 50.2 (3 to 264) days. The mortality of patients with pneumonia was 32.3%. Abdominal infection plays a role in the incidence of ventilator- associated pneumonia GB Bouroche1, S Ruckly2, B Misset3, JF Timsit4, F Philippart3 1Institut Gustave Roussy, Villejuif, France; 2Institut Albert Bonniot, La Tronche, France; 3Hôpital Saint Joseph, Paris, France; 4CHU Grenoble, France Critical Care 2013, 17(Suppl 2):P46 (doi: 10.1186/cc11984) Introduction Despite many therapeutic interventions, ventilator- acquired pneumonias (VAP) are frequent in the ICU and are associated with major morbidity and mortality. Sepsis causes a time-dependent modifi cation of the infl ammatory response. This reprogramming could promote the occurrence of a secondary infection and worsen the prognosis. In animals, peritonitis is associated with an alteration of pulmonary immunity and an increasing mortality from secondary pneumonia. y Conclusion Urinary sepsis mortality is associated with late-onset and/ or inappropriate antibiotic use, as well as with immunodepression and advanced age. Introduction Analysis of mortality-related factors in urinary sepsis patients. Methods A retrospective descriptive study of urologic sepsis patients in the ICU from 2008 to 2010. Clinical, epidemiological and outcome variables were analysed. Quantitative variables are expressed as either mean and standard deviation or as median and interquartile range for asymmetric variables. Qualitative variables are expressed as percentages and absolute values. Mann–Whitney’s U test and Fisher’s exact test were applied (α error was 5% in both cases), as well as binary logistic regression for multivariate analysis. Results There was a total number of 44 patients (aged 59.39 ± 17.71; 63.8% females). APACHE II score upon admission was 18 ± 6. Out of these patients, 27.3% showed no underlying disorder and 18.2% (no = 8) showed chronic renal failure; 25% were immunodepressed patients; 31% underwent urinary instrumentation in the previous 15 days, yet only three of them had undergone permanent urine catheterization. Observed mortality was 25%, while sepsis-related mortality was 22.7%. The patients who died were, on average, older that those who survived (67.9 ±7 10.2 vs. 56.8 ± 18.7; P = 0.02). Besides, the former also reported greater delay in turning to the hospital after symptom onset (13.4 ± 6.6 vs. 6.2 ± 4.7 days; P = 0.0001). Immunodepressed patients presented higher mortality rate: OR 8.7 (95% CI 1.7 to 42.3), as well as those who underwent inappropriate initial antibiotic treatment: OR 10.8 (95% CI 2.1 to 54.7). No relation was observed between germ typology or resistance to β-lactam antibiotics and mortality. After adjustment of mortality due to APACHE II score upon admission, delay in the onset of appropriate antibiotic treatment was an independent predictor of mortality in our patients: OR 1.2, 95% CI (1.02 to 1.42). Conclusion Nosocomial pneumonia is the most frequent infection in our series. Despite when infection was not confi rmed, antibiotic therapy was maintained in suspect cases. We found a high incidence of aspergillosis. Limitations because of wide duration of this study should be considered. P46 P44 Outcomes in urinary sepsis Outcomes in urinary sepsis C Joya-Montosa, E Trujillo-Garcia, MJ Delgado-Amaya, E Curiel-Balsera Hospital Regional Carlos Haya, Málaga, Spain Critical Care 2013, 17(Suppl 2):P44 (doi: 10.1186/cc11982) Outcomes in urinary sepsis y p C Joya-Montosa, E Trujillo-Garcia, MJ Delgado-Amaya, E Curiel-Balsera Hospital Regional Carlos Haya, Málaga, Spain Critical Care 2013 17(Suppl 2):P44 (doi: 10 1186/cc11982) Introduction Analysis of mortality-related factors in urinary sepsis patients. Nosocomial pneumonia in the postoperative period after heart transplantation transplantation R Gómez López1, P Fernández Ugidos1, P Vidal Cortes1, M Bouza Vieiro2, J Miñiz2, S Fojon Polanco2, M Paniagua Martin2, R Marzoa Rivas2, E Barge Caballero2, M Crespo Leiro2 1Complexo Hospitalario Universitario de Ourense, Spain; 2Complexo Hospitalaro Universitario de A Coruña, Spain Critical Care 2013, 17(Suppl 2):P45 (doi: 10.1186/cc11983) p R Gómez López1, P Fernández Ugidos1, P Vidal Cortes1, M Bouza Vieiro2, J Miñiz2, S Fojon Polanco2, M Paniagua Martin2, R Marzoa Rivas2, E Barge Caballero2, M Crespo Leiro2 1Complexo Hospitalario Universitario de Ourense, Spain; 2Complexo Hospitalaro Universitario de A Coruña, Spain Critical Care 2013, 17(Suppl 2):P45 (doi: 10.1186/cc11983) Hospitalaro Universitario de A Coruña, Spain p p Critical Care 2013, 17(Suppl 2):P45 (doi: 10.1186/cc11983) Introduction Infections are a major complication during the postoperative period after heart transplantation (HT). In our hospital, nosocomial pneumonia is the most frequent infection in this period. The objective of this study is to determine the epidemiological and microbiological characteristics of this disease in our centre. Methods A descriptive retrospective study of all medical records of HT performed in a single institution from 1991 to 2009 followed until June 2010. Clinical and microbiological variables were considered. Centre for Diseases Control (CDC) criteria were used to defi ne nosocomial infections. Invasive aspergillosis was considered if there were criteria for probable aspergillosis according to IDSA criteria. Results In 594 HTs there were 97 infectious episodes in 75 patients (12.6%). Eighty-fi ve patients (14.3%) died during hospitalization. Infection is the second cause of mortality during the postoperative period (17.9% of dead patients). The most common locations of infections were pneumonia (n  = 31, 31.9% of infection episodes), bloodstream (n = 24, 24.7%), urinary tract (n = 14, 14.4%), surgical site Figure 1 (abstract P46). Cumulative incidence of VAP. S17 Critical Care 2013, Volume 17 Suppl 2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Figure 2 (abstract P46). Cumulative occurrence of death. that numbers of CVC, intubation and surgery, the use of muscle relaxant and steroid were independent risk factors for developing VAP. Ventilator days and ICU length of stay were longer in the VAP group (25 vs. 6 and 25 vs. 7 days, respectively). Lastly, the hospital mortality rate was signifi cantly higher in the VAP group (33% vs. 12%, P = 0.008). Nosocomial pneumonia in the postoperative period after heart transplantation Conclusion The incidence of VAP was 9.2% in the SICU of Siriraj Hospital, which was comparable with previous reports. Bundles of care to prevent VAP should include weaning from a ventilator. Muscle relaxant and steroid should be administered according to strong indication. Meticulous care of the airway should be implemented as protocol in order to prevent complications that can result in the development of VAP. Hospital-acquired bloodstream infection: Indian perspective g , FEHI, New Delhi, India FEHI, New Delhi, India Critical Care 2013, 17(Suppl 2):P48 (doi: 10.1186/cc11986) Figure 2 (abstract P46). Cumulative occurrence of death. Introduction This is a 1-year prospective study to determine the incidence, source and etiology of hospital-acquired bloodstream infection (HABSI) in the Indian context. The resistance pattern was also reviewed. Introduction This is a 1-year prospective study to determine the incidence, source and etiology of hospital-acquired bloodstream infection (HABSI) in the Indian context. The resistance pattern was also reviewed. to the ICU for severe sepsis or septic shock who required mechanical ventilation for at least 72 hours. Methods A single-centre prospective study in a 35-bed ICU. HABSI was defi ned according to current CDC guidelines. HCAP, catheter- associated UTI (CAUTI) and skin-related infections causing BSI was also defi ned according to recent guidelines and analysed. Results In total, 2,623 patients were included, of which 290 had an IIA. A total of 862 patients (33%) developed a VAP, 56 (19%) in the IIA group and 806 (34%) in the non-IIA group (P <0.01). VAP, after sepsis, occurred less frequently and later in patients with IIA. The occurrence of IIA, in comparison with another sepsis, is a protective factor against VAP (HR = 0.643 (0.478 to 0.863), P = 0.003). There is, however, no signifi cant diff erence between the groups in terms of ICU mortality (28% vs. 32%, P = 0.16). See Figures 1 and 2. i g g y Results Out of 332 positive samples, 90 samples (n = 45) were HABSI. The microbiological analysis showed 60% were Gram-negative, 6% were candida and 27% were Gram-positive. The commonest isolate was klebsiella and MRSA was commonest in Gram-positive. The source of HABSI showed CRBSI was the commonest cause at 69%, which correlates with international data. Ventilator-associated pneumonia and CAUTI caused 9.5% BSI respectively. The resistance pattern among Gram-negative bacteria showed multidrug-resistant (MDR) and extreme drug-resistant (XDR) isolates were highest. See Tables 1 and 2. g Conclusion In this study, the presence of an abdominal sepsis, in a context of severe sepsis or septic shock, was associated with a lower incidence of later VAP. These results have to be confi rmed in other studies, especially prospective. They open interesting new research directions. Table 1 (abstract P48). Source Source Total (%) CRBSI 69 HCAP 9.5 CAUTI 9.5 Devices 7 Skin 5 Table 2 (abstract P48). Reference 1. Werarak P, Kiratisin P, Thamlikitkul V: Hospital acquired pneumonia and ventilator associated pneumonia in adults at Siriraj hospital, etiology, clinical outcomes and impact of antimicrobial resistance. J Med Assoc Thai 2010, 93(Suppl 1):126-138. Hospital-acquired bloodstream infection: Indian perspective Resistance pattern Resistance Total (%) Non-MDR 15 MDR 44 XDR 33 PDR 8 Conclusion The incidence of HABSI is 27%. Of this, CRBSI cause 70% and Gram-negative bacteria were commonest with high resistance. This is in contrast to western data where Gram-positive infections are common. Our study highlights need for stringent guidelines for CRBSI prevention. References 1. Richard et al.: Crit Care Med 1999, 27:887-892. 2. Valles et al. : J Infect 2008, 56:27-34. P50 Analysis of risk factors for catheter-related bloodstream infection in a parenteral nutrition population P52 Results for introduction of a new hand hygiene program in the ICU P Vos, A Harts-Laurijsen, R Snoeren, R Ramnarain, JA Van Oers St Elisabeth Hospital, Tilburg, the Netherlands Critical Care 2013, 17(Suppl 2):P52 (doi: 10.1186/cc11990) Analysis of risk factors for catheter-related bloodstream infection in a parenteral nutrition population I Conrick-Martin1, M McGovern2, K Boner1, J Bourke1, E Fitzgerald1, R Hone1, M Lynch1, D Phelan1, C Walshe3 I Conrick-Martin1, M McGovern2, K Boner1, J Bourke1, E Fitzgerald1, R Hone1, M Lynch1, D Phelan1, C Walshe3 1Mater Misericordiae University Hospital, Dublin, Ireland; 2Harvard School of Public Health, Cambridge, MA, USA; 3Beaumont Hospital, Dublin, Ireland Critical Care 2013, 17(Suppl 2):P50 (doi: 10.1186/cc11988) 1Mater Misericordiae University Hospital, Dublin, Ireland; 2Harvard School of Public Health, Cambridge, MA, USA; 3Beaumont Hospital, Dublin, Ireland Critical Care 2013, 17(Suppl 2):P50 (doi: 10.1186/cc11988) 1Mater Misericordiae University Hospital, Dublin, Ireland; 2Harvard School of Public Health, Cambridge, MA, USA; 3Beaumont Hospital, Dublin, Ireland Critical Care 2013, 17(Suppl 2):P50 (doi: 10.1186/cc11988) Conclusion A low proinfl ammatory response is a key facilitating factor for the development of infection. Measuring serum TNFα levels before and after operations can thus predict the outcome. Introduction Catheter-related bloodstream infection (CRBSI) is a complication of central venous catheters (CVCs) with an attributable morbidity, mortality and cost [1]. We examined patient risk factors for CRBSI in an adult parenteral nutrition (PN) population. Incidence and risk factors for ventilator-associated pneumonia in Siriraj Hospital O Chaiwat, S Nakaviroj Siriraj Hospital Mahidol University, Bangkok, Thailand Critical Care 2013, 17(Suppl 2):P47 (doi: 10.1186/cc11985) Introduction Ventilator-associated pneumonia (VAP) is a serious illness with substantial morbidity and mortality and increases the cost of hospital care. Even when bundles of care to prevent VAP have been implemented, the incidence of VAP was not dramatically improved. This study aims to determine the incidence and risk factors of VAP in the SICU of Siriraj Hospital. Methods During a 1-year period, 228 patients admitted to the SICU were enrolled. All patients required ventilatory support longer than 48 hours. Data were collected by reviewing patient medical records including demographic data, onset of VAP, type of organisms, medication used, number of central venous catheters (CVC) used and blood transfusion. VAP outcomes were also reported. Results VAP occurred in 21 patients (9.21%) or 8.21 per 1,000 ventilator- days. The onset of VAP was late in the majority of patients. The most common organism was A. baumannii (66%), followed by P. aeuruginosa (19%). Compared with non-VAP groups, patients in the VAP group had higher APACHE II score (18 vs. 13, P <0.001), blood transfusion (95% vs. 75%, P = 0.04), numbers of CVC used (3 vs. 1, P <0.001), muscle relaxant used (43% vs. 3%, P <0.001) and steroid used (33% vs. 4%, P <0.001). The VAP group also had a signifi cantly higher number of intubation, reintubation and self-extubation. Multiple logistic regression showed Conclusion The incidence of HABSI is 27%. Of this, CRBSI cause 70% and Gram-negative bacteria were commonest with high resistance. This is in contrast to western data where Gram-positive infections are common. Our study highlights need for stringent guidelines for CRBSI prevention. S18 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 P49 Risk factors for catheter-related bloodstream infections in a surgical ICU A Kundakci, O Ozkalayci, P Zeyneloglu, H Arslan, A Pirat Baskent University Hospital, Ankara, Turkey Critical Care 2013, 17(Suppl 2):P49 (doi: 10.1186/cc11987) P49 Risk factors for catheter-related bloodstream infections in a surgical ICU A Kundakci, O Ozkalayci, P Zeyneloglu, H Arslan, A Pirat Baskent University Hospital, Ankara, Turkey Critical Care 2013, 17(Suppl 2):P49 (doi: 10.1186/cc11987) Results The study population was 1,961 patients in whom 3,213 CVCs were utilised over 19,511 CVC days. There were 256 CRBSI episodes in 216 patients. Median (IQR) patient age was 62 (23), and 58% were male. The incidence of CRBSI decreased signifi cantly (P <0.001) during the study period from 16% of patients in the period 1997 to 2003 to 7% in 2004 to 2010. The corresponding rate of CRBSI infection (per 1,000 CVC days) decreased from 18 to 10. There was a signifi cant decrease (P  <0.001) in numbers of CVCs inserted per patient (from 1.87 to 1.49). Each extra CVC PN day was associated with an increased risk of developing CRBSI of 3.4% (OR 1.034, P <0.05). The number of PN CVCs was associated with developing CRBSI (OR 1.218, P  <0.10). Patient factors signifi cantly associated with CRBSI included perioperative PN use (compared with medical patients) (OR 2.414, P <0.01), and male sex (OR 1.952, P <0.01). Introduction Preventing catheter-related bloodstream infections (CR-BSI) can reduce the duration of hospital stay, healthcare costs, and mortality rates. Identifying the risk factors and correction of modifi able factors should be one of the main objectives of infection control measures. The aim of this study is to determine the risk factors of CR-BSI in a cohort of surgical ICU patients admitted to Baskent University Hospital. p Methods Following Institutional Review Board approval, data for 876 patients admitted to the surgical ICU between January 2009 and July 2012 were reviewed retrospectively. After completing the review, 25 patients diagnosed with CR-BSI were compared with 66 appropriate matches who did not have CR-BSI. Demographical features, underlying diseases, APACHE II (Acute Physiology and Chronic Health Evaluation) and SOFA (Sequential Organ Failure Assessment) scores, length of stay, organ dysfunctions, laboratory values, use of vasopressors, mechanical ventilation, nutrition, antibiotics, transfusions, and features related to central venous catheterization were recorded. Patients who did not have a central venous catheter and were discharged within 2 days of ICU stay were excluded. Conclusion This prospective study demonstrated that perioperative PN use was associated with increased risk of CRBSI. The association between CRBSI and CVC PN days is consistent with the theory suggesting benefi t to limiting CVC duration and changing from PN to enteral nutrition as soon as appropriate. Can we predict the postoperative infections? 2 3 2 O’Grady NP: Guidelines for the prevention of intravascular catheter-related infections. Clin Infect Dis 2011, 52:e162-e193. 2. Bouza E: Catheter-related infections: diagnosis and intravascular treatment Clin Microbiol Infect 2002 8 265 274 2. Bouza E: Catheter-related infections: diagnosis and intravascular treatment. Clin Microbiol Infect 2002, 8:265-274. Results From 22 patients, eight deep wound and urine infections were found in 14 days and six urine and wound infections in 30 days after surgery, all survived. All patients were bacterially contaminated, as wound samples taken at the end of operation demonstrated. On day 0 the circulating TNFα values were lower in infected patients. TNF started to increase from day 3 to day 9, never reaching values of the uneventful healing group. Soluble TNF receptor I, IL-6, ACTH, and cortisol concentrations did not demonstrate any diff erence on day 0 but from day 1 started to increase transiently, reaching higher levels in septic patients.l Can we predict the postoperative infections? 2 3 2 y Results Out of 91 patients included in the fi nal analysis, 25 (27%) patients had CR-BSI. When compared with patients who did not have CR-BSI, those who did were older (P  = 0.029), required more blood product transfusions during the fi rst 3 days of ICU (P = 0.016), had a longer duration of catheter stay (P = 0.019), and were more frequently catheterized via the internal jugular vein (IJV) (P  = 0.022). A logistic regression model revealed that advanced age (OR: 1.037; 95% CI: 1.001 to 1.073; P = 0.042) was an independent risk factor for CR-BSI. Fourteen- day and 28-day mortality rates for CR-BSI were 12% (P = 0.749) and 28% (P = 0.406), respectively. p p p Mózes1, K Gornicsar2, A Grosz3, ZS Domján2, I Buzogány2 p p p T Mózes1, K Gornicsar2, A Grosz3, ZS Domján2, I Buzogány2 1Ministry of Defense Health Centre, Budapest, Hungary; 2Péterfy Hospital, Budapest, Hungary; 3University of Szeged, Hungary Critical Care 2013, 17(Suppl 2):P51 (doi: 10.1186/cc11989) T Mózes1, K Gornicsar2, A Grosz3, ZS Domján2, I Buzogány2 1Ministry of Defense Health Centre, Budapest, Hungary; 2Péterfy Hospital, Budapest, Hungary; 3University of Szeged, Hungary Critical Care 2013, 17(Suppl 2):P51 (doi: 10.1186/cc11989) Introduction Many patients develop infections following operations. Decreased immune competence has been demonstrated in acute neurological conditions. A strong cytokine-mediated anti- infl ammatory response was observed in stroke patients at infection, although infection due to the decreased proinfl ammatory mediators can be expected as well. To investigate this question the following experiment was performed. y Conclusion Although age, blood product transfusion, duration of catheter stay, and use of IJV were diff erent between patients who did and did not have CRBSI, advanced age was the only independent risk factor for CR-BSI. Early suspicion of CR-BSI by the other well-known risk factors has a substantial eff ect on the treatment of CR-BSI. References Methods Twenty-two urinary bladder cancer patients with radical cystectomy and lymphadenectomy were studied. Blood samples were taken on day 0 (before) and days 1, 3, 6, 9 and 14 after operation as well as on days 30, 60, 90 and 270 during follow-up. TNFα, soluble TNFα receptor I and IL-6 levels in sera were determined by HS ELISA and/or ELISA. Plasma ACTH and cortisol values were measured by RIA kits. 1. O’Grady NP: Guidelines for the prevention of intravascular catheter-related infections. Clin Infect Dis 2011, 52:e162-e193. 1. Reference 1. Blot SI, et al.: Clinical and economic outcomes in critically ill patients with nosocomial catheter-related bloodstream infection. Clin Infect Dis 2005; 41:1591-1598. Results for introduction of a new hand hygiene program in the ICU P Vos, A Harts-Laurijsen, R Snoeren, R Ramnarain, JA Van Oers St Elisabeth Hospital, Tilburg, the Netherlands Critical Care 2013, 17(Suppl 2):P52 (doi: 10.1186/cc11990) Methods The study was carried out in a 525-bed tertiary-referral teaching hospital over a 14-year study period (1997 to 2010). All in- patients referred for PN via CVCs were included. Prospectively collected data were recorded in a specifi c PN record. The CRBSI audit group met quarterly to review all sepsis episodes, assigning a diagnostic category (CRBSI or non-CRBSI). Patient risk factors for development of CRBSI were examined using a logistic regression model to take account of the dichotomous nature of the outcome. Odds ratios from a model incorporating demographic and clinical data were tested for statistical signifi cance. Introduction Although hand hygiene is known as a core element in prevention of healthcare-associated infections, compliance to its program is not high. With a new campaign we tried to enlarge awareness on hand hygiene. Introduction Although hand hygiene is known as a core element in prevention of healthcare-associated infections, compliance to its program is not high. With a new campaign we tried to enlarge awareness on hand hygiene. Methods We performed an experimental, before and after, study design on our 30-bed, mixed medical–surgical ICU. We conducted a baseline S19 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 evaluation during 3 days in May 2012 including direct observation of hand hygiene compliance by control nurses and hand cultures of 50 healthcare workers (HCW). Based on the WHO Guidelines on Hand Hygiene in Health Care [1], cleaning of hands with alcohol-based hand rubs (Sterillium) was prescribed before touching a patient and before aseptic procedures, after body fl uid exposure risk and after touching a patient and touching his/her surroundings. Promotion of the hand hygiene program consisted of lectures and web-based self-learning, posters located near points of care and verbal reminders by control nurses. New observations of hand hygiene by control nurses during 3 days and hand cultures of 50 healthcare providers were performed in September 2012. Consumption of alcohol-based hand rub (product volume use per patient-days) was used as a surrogate marker of hand hygiene over time. The diff erence in hand hygiene compliance during the two periods was examined using a chi-squared test. Diff erences in hand cultures were examined using a Student’s t test. Time trends in the consumption of alcohol-based hand rub were examined using linear correlation. P <0.05 was considered statistically signifi cant. Results for introduction of a new hand hygiene program in the ICU P Vos, A Harts-Laurijsen, R Snoeren, R Ramnarain, JA Van Oers St Elisabeth Hospital, Tilburg, the Netherlands Critical Care 2013, 17(Suppl 2):P52 (doi: 10.1186/cc11990) The study was approved by the institutional Ethics Review Board. from March through October 2012, and intervened to change behavior by providing monthly feedback to specifi c provider groups and services. We made use of the Unit Coordinator to measure compliance of all individuals in the ICU. Results Overall compliance by physicians was 82.1%, for nonphysicians was 84.8%. Feedback to physicians, individually and by service, dramatically increased hand hygiene compliance, defi ned as both on entry and exit from the patient room, over the study period. See Figure 1. Conclusion Physician behavior is responsive to monthly feedback that is specifi c to the individual or surgical service. Use of the Unit Coordinator was very eff ective at gathering a very large sample size in a short period of time. P54 Compliance with the implementation of an ICU cluster-randomized trial assessing the benefi ts and potential harms of universal glove and gowning D Kett1, DJ Morgan2, L Pineles2, MJ Zervos3, LS Munoz-Price1, AD Harris2, BUGG Investigators2 1University of Miami/Jackson Memorial Hospital, Miami, FL, USA; 2University of Maryland/VA Maryland Healthcare System, Baltimore, MD, USA; 3Henry Ford Health System, Detroit, MI, USA Critical Care 2013, 17(Suppl 2):P54 (doi: 10.1186/cc11992) Reference 1. Pittet D, et al.: Infect Control Hosp Epidemiol 2009, 30:611-622. Compliance with the implementation of an ICU cluster-randomized trial assessing the benefi ts and potential harms of universal glove and gowning Results During the survey, in May 158 opportunities to observe hand hygiene were presented and 286 in September. Overall compliance improved from 34.2% (54/158) to 51% (146/286), χ2 = 11.7 (P <0.001). In May, 50 HCW had a mean of 63.20 ± 39.37 colony-forming units (CFU) on their hands compared with 43.0 ± 40.19 CFU on the hands of 50 HCW in September (P = 0.024). We also observed an initial increased use of alcohol-based hand rubs from 21 ml per patient-day in May to a maximum 72 ml per patient-day in June, but a decline to 44 ml per patient-day in September, Pearson correlation coeffi cient = 0.31 (P = 0.61). D Kett1, DJ Morgan2, L Pineles2, MJ Zervos3, LS Munoz-Price1, AD Harris2, BUGG Investigators2 g 1University of Miami/Jackson Memorial Hospital, Miami, FL, USA; 2University of Maryland/VA Maryland Healthcare System, Baltimore, MD, USA; 3Henry Ford Health System, Detroit, MI, USA Critical Care 2013, 17(Suppl 2):P54 (doi: 10.1186/cc11992) Introduction The Benefi ts of Universal Glove and Gowning (BUGG) study is a cluster-randomized trial to evaluate the use of wearing gloves and gowns for all patient contact in the ICU. The primary outcome is VRE and MRSA acquisitions; secondary outcomes include frequency of healthcare worker visits, infection rates, hand hygiene compliance and adverse events. Conclusion Implementation of a new hand hygiene program at our ICU resulted in improved hand hygiene compliance and less CFU on the hands of HCW. There was no signifi cant increased use of alcohol-based hand rubs over time. The results indicate that constant awareness is vital for success. Methods We enrolled 20 ICUs in 15 states. ICUs collected nasal and perianal swabs on all patients at admission and discharge/transfer. After a 3-month baseline period, 10 units were randomized to the intervention arm and required to wear gloves and gowns for all patient contact. An intervention toolkit was created based on site feedback and compliance reports. Swab collection compliance was fed back and discussed during site conference calls on a weekly basis. Site coordinators monitored compliance with gloves and gowns, hand hygiene and frequency of HCW visits and reviewed patient charts for adverse events. Sinks as a correctable source of ESBL contamination for patients in the ICU p yf g p Methods Thirty-nine medical wards (MW) and 12 emergency depart- ments (ED) were enrolled. Every unit was asked to fi ll in a pre-agreed HOR Checklist focused on the main requirements suggested by the Guidelines. I Wolf1, P Bergervoet1, W Van der Zwet1, H Van den Oever1, P Savelkoul2, F Sebens1 Results Analysing the human resources available, we see that the bed- to-doctor ratio signifi cantly (P <0.01) increases from the day to the night shift: from 6 to 43 beds per doctor on the MW (median). Otherwise, the ED staff remains roughly the same: from 3.5 to 2.5 doctors on duty (median). The analysis of the organizational tools (Table 1) points out Introduction The incidence of patients carrying ESBL-positive bacteria in our ICU (12 in 780 admissions in 2011) was not considered problematic. However, routine cultures had identifi ed ESBL-negative patients who had become colonized with ESBL strains during their ICU stay. Self-disinfecting siphons, preventing bacterial growth by antibacterial coating and intermittent heating, and biofi lm formation by electromechanical vibration, were placed in all sinks in the ICU. The aim of the present study was to evaluate the eff ect of this intervention. Methods An intervention study in a 12-bed ICU. The intervention involved placement of 19 self-disinfecting siphons (Biorec). All patients with an expected ICU stay of 2 days or more between January 2011 and December 2012 were studied. Samples of throat, sputum and rectum were taken at admission and twice weekly, and cultured for ESBLs. Between June 2011 and October 2011, sinks in patient rooms were cultured regularly for ESBLs. After the intervention in April 2012, multiple repeat cultures were taken. Whenever the species and antibiogram of bacteria cultured from patients and sinks matched, they were typed by AFLP. P55 Fighting hospital sepsis l a low percentage of hospitals having: a Diagnostic and Therapeutic Protocol for sepsis management (8.3%), some Hospital Empirical Antibiotic Therapy Guidelines (0%) and an Infective Source Eradication Protocol (8.3%). Moreover, just 25% of hospitals involve an infectious diseases expert in every case of severe sepsis or septic shock. p y p p Conclusion We guess that the poor availability of HOR showed by the hospitals could have a role in the Guidelines implementation and in the patient’s outcome. Only a comparison between these results and data collected from a Clinical Checklist, focused on sepsis bundle compliance, and from a patient’s outcome summary could confi rm our hypothesis. This is the aim for our next part of the study. Introduction Sepsis accounts for a very high mortality. The Surviving Sepsis Campaign recommends a fi rst 6 hours resuscitative bundle to improve patient outcome. Despite this, the bundle is poorly performed because of several organizational and cultural barriers. In recognition of this, we guess that an Educational and Organizational Intervention out of the ICUs could impact on septic patient outcome. In order to test our hypothesis we carried out, in 12 hospitals, a pre-intervention survey of the human and organizational resources (HOR) available in the management of septic patients. The aim is to seek any barrier potentially aff ecting correct Guidelines implementation. 1. Dellinger RP, et al.: Intensive Care Med 2008, 34:17-60. Improving hand hygiene performance in a surgical ICU: a novel method for data collection to change physician behavior SA Nasraway Tufts Medical Center, Boston, MA, USA Critical Care 2013, 17(Suppl 2):P53 (doi: 10.1186/cc11991) Results During the 12-month study period, 100,210 swabs were collected. After the baseline period, we were able to achieve and maintain swab compliance rates between 85 and 97%. Monthly discharge compliance increased by 21% by the beginning of the intervention period (Figure 1). Observers found 86% compliance with universal glove and gowning over 1,242 30-minute observation periods (Figure 1). Ninety charts at each site were reviewed for adverse events. Conclusion Over a diverse group of US hospitals, we achieved high compliance with surveillance cultures and implementing universal gloving and gowning was achieved quickly with high compliance. Critical Care 2013, 17(Suppl 2):P53 (doi: 10.1186/cc11991) Introduction ICU-acquired infection is directly related to hospital mortality. Hand hygiene is an eff ective, low-cost intervention that can prevent the spread of bacterial pathogens, including multidrug- resistant organisms. Historical compliance with hand hygiene guidelines by physicians, nurses and other care providers is poor. Methods Present expectations by the Infection Control Committee are to ‘pump in, pump out’ of every room, using 63% isopropyl alcohol. We performed 17,622 observations of hand hygiene in the surgical ICU Figure 1 (abstract P54). Swab collection and gown/glove compliance. Figure 1 (abstract P53). Figure 1 (abstract P54). Swab collection and gown/glove compliance. Figure 1 (abstract P53). Figure 1 (abstract P54). Swab collection and gown/glove compliance. Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 S20 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 P55 Fighting hospital sepsis AR Calini, S Vesconi, R Fumagalli, S Marchesi, L Ghezzi, G Monti Ospedale Niguarda Ca’ Granda, Milan, Italy Critical Care 2013, 17(Suppl 2):P55 (doi: 10.1186/cc11993) P55 Fighting hospital sepsis AR Calini, S Vesconi, R Fumagalli, S Marchesi, L Ghezzi, G Monti Ospedale Niguarda Ca’ Granda, Milan, Italy Critical Care 2013, 17(Suppl 2):P55 (doi: 10.1186/cc11993) Clinical effi cacy of recombinant human soluble thrombomodulin in patients with septic disseminated intravascular coagulation Y Suzuki, R Sato, M Sato, S Endo Iwate Medical University, Morioka, Japan Critical Care 2013, 17(Suppl 2):P59 (doi: 10.1186/cc11997) Results All enalapril pigs received the maximum NE dose without reaching the target MAP. Enalapril resulted in lower MAP, higher CO, and transiently increased regional blood fl ows (Table  1). Fluid administration and urinary output were similar among groups. Liver mitochondrial respiration was reduced (State 3, State 4) in the enalapril group. One animal died in each ML and enalapril group. y p Critical Care 2013, 17(Suppl 2):P59 (doi: 10.1186/cc11997) Introduction Thrombomodulin is an endothelial cell cofactor and glycoprotein for thrombin-catalyzed activation of protein C. A recom- binant human soluble thrombomodulin (rhsTM) has been recently developed, and this new agent has a unique amino-terminal structure exhibiting anti-infl ammatory activity including sequestraction and cleavage of high-mobility group box 1(HMGB-1). Conclusion Enalapril enhances cardiac output and early regional blood fl ows in sepsis despite lower MAP, and reduces liver mitochondrial respiration, suggesting that RAS is involved in the hemodynamic and metabolic changes in sepsis. Methods In this study, 13 patients with septic disseminated intravascular coagulation (DIC) were treated with rhsTM, which is Recomodulin® Inj. 12800 (Asahi Kasei Pharma Co., Tokyo, Japan). Patients with septic DIC were treated with 130 to 380 U/kg/day.i Eff ects of intraperitoneal immunoglobulin therapy on behavior and cognitive functions in CLP-induced sepsis model in rats F Esen1, E Senturk1, P Ergin Ozcan1, G Orhun1, S Gümrü2, M Kaya1, N Orhan1, N Arıcan1, F Arıcıoglu2 1University of Istanbul, Turkey; 2Marmara University Faculty of Pharmacy, Istanbul, Turkey Critical Care 2013, 17(Suppl 2):P58 (doi: 10.1186/cc11996) Results There were signifi cant results for improvement of APACHE II score and DIC diagnostic criteria score for critically ill patients after treatment using rhsTM (P <0.01). Improvement for platelet count and D-dimer level were also observed in this study (P <0.05). Activation of antithrombin (AT) also was signifi cantly increased after treatment (P <0.05). Hospital mortality was 15.4% in this study. Critical Care 2013, 17(Suppl 2):P58 (doi: 10.1186/cc11996) Introduction The present study investigated the eff ects of a single dose of intraperitoneal (i.p.) IgG and IgGAM administration on various behavioral alterations in a cecal ligation perforation (CLP)-induced sepsis model in rats.i Conclusion The rhsTM might be one of most important endogenous regulators of coagulation, acting as the major inhibitor of thrombin as well as AT III. This new agent may play an important role in treatment for septic DIC. Sinks as a correctable source of ESBL contamination for patients in the ICU Table 1 (abstract P55) Availability Resource (%) Diagnostic and therapeutic protocol for septic patient management 8.3 Use of early warning score for diagnosis and management 8.3 Sepsis team 25 Microbiology laboratory: open 7 days a week 66.6 Lactate dosage: 24 hours a day availability 83.3 Central venous catheter insertion (CVC) available 24 hours a day 91.6 Hospital empirical antibiotic therapy guidelines 0 Infectious diseases team advice in any case of severe sepsis septic shock 25 Infective source eradication Infective source eradication protocols 8.3 Intervention radiology available 24 hours a day 50 Operating room available 24 hours a day 58.3 Specifi c infection management protocol (that is, CVC infection) 58.3 Imaging reporting service available 7 days a week 41.6 yp y Results Before intervention Multiple ESBL-forming strains were found in sinks of all patient rooms. Eighteen patients who were ESBL-negative on ICU admission became colonized with 11 diff erent ESBL strains, that were present in sinks of their admission rooms (Figure  1). Four contaminations were proven by AFLP-tying. One patient died of ESBL- positive E. cloacae pneumonia. After intervention All sinks were negative for ESBL strains. No further patients became ESBL colonized during the ICU stay. y Conclusion Wastewater sinks were the likely source of ESBL colonization for 18 ICU patients. After placing self-disinfecting siphons Figure 1 (abstract P56). Design and results of the intervention. Figure 1 (abstract P56). Design and results of the intervention. Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 S21 Table 1 (abstract P57). Hemodynamic variables, among times and groups there were no new ESBL colonizations in patients. This coincided with th di f ESBL t i f ll i k of immunoglobulin therapy on behavioral changes in CLP-induced i i t Table 1 (abstract P57). Clinical effi cacy of recombinant human soluble thrombomodulin in patients with septic disseminated intravascular coagulation Y Suzuki, R Sato, M Sato, S Endo Iwate Medical University, Morioka, Japan Critical Care 2013, 17(Suppl 2):P59 (doi: 10.1186/cc11997) Methods Female Wistar albino rats (200 to 250 g) were divided into fi ve groups (n = 8): a naive Control group, a Sham operated group receiving conventional antibiotic treatment, a CLP group receiving CLP procedure and conventional antibiotic treatment, and IgG and IgGAM groups which were also applied 1 g/kg, i.p. IgG and IGAM therapy 5 minutes after the CLP procedure. Ten, 30 and 60 days after the surgery, animals underwent three behavioral tasks: an open fi eld test to evaluate the locomotor activity, an elevated plus maze test to measure the level of anxiety, and a forced swim test to assess the possible depressive state. The results acquired from these tests were used to estimate the eff ect Sinks as a correctable source of ESBL contamination for patients in the ICU Immunoglobulin therapy prevented the occurrence of these behavioral changes. Especially, animals in the IgGAM group conserved the values quite near to those of the control group in measured parameters. Sinks as a correctable source of ESBL contamination for patients in the ICU Hemodynamic variables, among times and groups P BL R0h R12h R24h R36h R48h (time×group) MAP (ENL/MH/ML) 66 (9)/72 (15)/ 65 (11)/73 (10)/ 57 (4)/82 (6)/ 58 (8)/78 (6)/ 56 (12)/80 (5)/ 56 (11)/79 (9)/ 0.003 65 (9) 81 (16) 72 (13) 67 (12) 61 (6) 56 (13) CO (ENL/MH/ML) 106 (13)/89 (7)/ 92 (17)/67 (11)/ 169 (16)/95 (19)/ 187 (22)/129 (28)/ 161 (25)/139 (32)/ 157 (37)/140 (21)/ <0.0001 105 (13) 88 (22) 104 (13) 143 (31) 145 (13) 150 (18) Carotid fl ow (ENL/MH/ML) 6 (2)/7 (2)/7 (2) 5 (1)/4 (1)/5 (2) 10 (3)/5 (1)/6 (2) 10 (3)/8 (1)/9 (2) 8 (2)/8 (2)/8 (2) 8 (3)/8 (2)/8 (2) <0.0001 Femoral fl ow (ENL/MH/ML) 4 (1)/3 (1)/4 (2) 2 (1)/2 (0)/2 (1) 4 (1)/2 (1)/2 (0) 4 (1)/3 (1)/3 (1) 3 (1)/3 (1)/4 (1) 3 (2)/3 (1)/4 (1) 0.004 of immunoglobulin therapy on behavioral changes in CLP-induced sepsis in rats.ii Results In the open fi eld test, the CLP group showed a signifi cant decrease in total squares passed on days 10 and 30. Similarly, total numbers of rearing and grooming were dramatically decreased in the CLP group in comparison with control and sham groups (P <0.005). In the elevated plus maze test, the number of entries to open arms decreased in the CLP group. In the forced swim test, there was a tendency for increase in immobility time in the CLP group, although the data were statistically insignifi cant. All of these values which were indicating the importance of behavioral alterations were improved on day 60. Immunoglobulin therapy prevented the occurrence of these behavioral changes. Especially, animals in the IgGAM group conserved the values quite near to those of the control group in measured parameters. Results In the open fi eld test, the CLP group showed a signifi cant decrease in total squares passed on days 10 and 30. Similarly, total numbers of rearing and grooming were dramatically decreased in the CLP group in comparison with control and sham groups (P <0.005). In the elevated plus maze test, the number of entries to open arms decreased in the CLP group. In the forced swim test, there was a tendency for increase in immobility time in the CLP group, although the data were statistically insignifi cant. All of these values which were indicating the importance of behavioral alterations were improved on day 60. P57 Hemodynamic and mitochondrial eff ects of enalapril in experimental sepsis AJ Pereira, V Jeger, T Corrêa, M Vuda, S Djafarzadeh, J Takala, S Jakob University Hospital (Inselspital)/University of Bern, Switzerland Critical Care 2013, 17(Suppl 2):P57 (doi: 10.1186/cc11995) Introduction Sepsis may cause mitochondrial dysfunction. The renin–angiotensin system (RAS) activity is increased in sepsis, and can interfere with the mitochondrial function either directly or by modifying hemodynamics. We studied the eff ects of ACE inhibition by enalapril on hemodynamics and hepatic mitochondrial function in sepsis. Two septic groups without enalapril and with diff erent blood pressure targets served as controls. Conclusion Sepsis, even though it has been treated with conventional antibiotics, caused a negative eff ect on behavioral parameters. In this study, IgG and IgGAM treated animals in the presence of CLP did not show these behavioral changes. Therefore our results suggest that a single dose of i.p. IgG and IgGAM treatment, which was applied immediately after the sepsis procedure, prevents behavioral defects observed following sepsis. p g Methods Sepsis (fecal peritonitis) was induced in 24 anesthetized, mechanically ventilated pigs (40.1 ± 2.1 kg), divided into three groups (n = 8 for each): ENL (enalapril pretreatment for 7 days and at 0.005 to 0.02 mg/kg/hour during the study; MAP target 75 to 85 mmHg), ML (target MAP 50 to 60 mmHg), and MH (target MAP 75 to 85 mmHg). Hemodynamic support with fl uids, norepinephrine (NE; maximum dose 5,000  μg/hour), and antibiotics were started after 12  hours of peritonitis and continued for 48 hours. P59 59 Clinical effi cacy of recombinant human soluble thrombomodulin in patients with septic disseminated intravascular coagulation Y Suzuki, R Sato, M Sato, S Endo Iwate Medical University, Morioka, Japan Critical Care 2013, 17(Suppl 2):P59 (doi: 10.1186/cc11997) P61 A clinical randomized trial on the use of atorvastatin in patients with sepsis or septic shock: eff ects on endothelial function K Prado, C Ribeiro, T Furian, R Pinto Ribeiro, D Silvello, L Rohde, N Clausell, L Becker Hospital de Clinicas de Porto Alegre, Brazil Critical Care 2013, 17(Suppl 2):P61 (doi: 10.1186/cc11999) Results Forty-three patients (18 treated, 25 control) completed the study and were further analyzed. Incidence of organ dysfunction at enrollment (treatment vs. control) was: septic shock (94% vs. 100%, P = 0.42), acute respiratory distress syndrome (67% vs. 56%, P = 0.33), and renal failure (39% vs. 24%, P = 0.54). During 115 treatments no serious device-related adverse events occurred. On average, there were no changes in hematology and other blood parameters except for a modest reduction in platelet count (<10%) and albumin (<5%) with treatment. Hemoperfusion decreased IL-6 blood concentration signifi cantly (–49.1%, P  = 0.01), with similar reductions of MCP-1 (–49.5%, P = 0.002), IL-1ra (–36.5%, P = 0.001), and IL-8 (–30.2%, P = 0.002). The 28-day mortality (28% vs. 24% control, P = 0.84) and 60- day mortality (39% vs. 32% control, P = 0.75) did not diff er signifi cantly between the two studied groups.i Introduction Sepsis and septic shock are complex infl ammatory syndromes. Multiple cellular activation processes are involved, and many humoral cascades are triggered. Presumably, endothelial cells play a pivotal rule in the pathogenesis of sepsis, not only because they may infl uence the infl ammatory cascade but also because, upon interaction with excessive amounts of infl ammatory mediators, the function of these cells may become impaired. It is likely that a general dysfunction of the endothelium is a key event in the pathogenesis of sepsis [1]. HMG-CoA-reductase inhibitors have been shown to exhibit pronounced immunomodulatory eff ects independent of lipid lowering. Most of these benefi cial eff ects of statins appear to involve restoring or improving endothelial function [2]. We hypothesize that statins can improve endothelial dysfunction in septic patients. Conclusion In this fi rst clinical study of a novel sorbent hemoadsorption device in patients with severe sepsis and ALI, the device appeared to be safe and decreased the blood concentration of several cytokines. Further research is needed to study the eff ect of the device on the clinical outcome of septic patients. y Methods A double-blinded, placebo-controlled, randomized trial was undertaken. A multicenter randomized controlled study of an extracorporeal cytokine hemoadsorption device in septic patients D Schädler1, C Porzelius2, A Jörres3, G Marx4, A Meier-Hellmann5, C Putensen6, M Quintel7, C Spies8, C Engel2, N Weiler1, M Kuhlmann9 1University Medical Center Schleswig-Holstein, Campus Kiel, Germany; 2University of Leipzig, Germany; 3Charité University Hospital Campus Virchow- Klinikum, Berlin, Germany; 4RWTH University Hospital Aachen, Germany; 5HELIOS Klinikum, Erfurt, Germany; 6University of Bonn, Germany; 7University Hospital Göttingen, Germany; 8Campus Charité Mitte and Campus Charité Virchow-Klinikum, Charité-University Medicine Berlin, Germany; 9Vivantes Klinikum im Friedrichshain, Berlin, Germany i Results There were 85 patients (52 men, 33 women; age range 19 to 92 years (mean 69.0 ± 16.9)), and the mortality rate was 27.1% and APACHE II score was 20.1  ±  7.6. WBC, CRP, PT and SOFA score were signifi cantly improved after AT III concentrate therapy (13,176 ± 9,400 vs. 11,693 ± 7,089, P = 0.011, 15.9 vs. 13.3, P = 0.0015, 16.5 ± 4.4 vs. 15.9 ± 5.9, P = 0.0045, and 9.7 ± 3.8 vs. 8.6 ± 4.6, P = 0.01, respectively). Platelet was signifi cantly decreased (11.7 ± 9.7 vs. 10.6 ± 9.0, P = 0.028), while there were no signifi cant diff erences in fi brinogen, FDP and DIC score (387.3 ± 202.6 vs. 381.8 ± 163.2, P = 0.088, 36.9 ± 52.7 vs. 28.8 ± 39.9, P = 0.059, and 2.9 ± 1.6 vs. 2.6 ± 1.8, P = 0.25, respectively) after the therapy. One week after the therapy, platelet and DIC score were signifi cantly improved compared with before the therapy (15.6 ± 10.0, P = 0.0036 and 1.8 ± 1.9, P = 0.0041). , , y Critical Care 2013, 17(Suppl 2):P62 (doi: 10.1186/cc12000) Introduction A novel sorbent hemoadsorption device for cytokine removal (CytoSorbents, USA) was developed and successfully tested in animal models of sepsis. The experience in the clinical setting is still limited to case reports. In this fi rst clinical trial, we tested the hypothesis that treatment with sorbent hemoadsorption could safely and eff ectively reduce cytokines in septic patients with acute lung injury (ALI). Introduction A novel sorbent hemoadsorption device for cytokine removal (CytoSorbents, USA) was developed and successfully tested in animal models of sepsis. The experience in the clinical setting is still limited to case reports. In this fi rst clinical trial, we tested the hypothesis that treatment with sorbent hemoadsorption could safely and eff ectively reduce cytokines in septic patients with acute lung injury (ALI). References 1. Hack EC, et al.: The endothelium in sepsis: source of and a target for infl ammation. Crit Care Med 2001, 29:S21-S27.if 1. Hack EC, et al.: The endothelium in sepsis: source of and a target for infl ammation. Crit Care Med 2001, 29:S21-S27.if l p Methods From January 2009 to December 2011, adult septic patients, whom were given AT III concentrate in our medico-surgical ICU, were included in this study. AT III concentrate was administered 30 to 60 U/kg intravenously every 24 hours for 3 days in the patients with DIC status and AT III defi ciency. Between before and after the AT III concentrate therapy, WBC (/mm3), CRP (mg/dl), platelet (×104/μl), PT (seconds), fi brinogen (mg/dl), FDP (μg/ml), SOFA score and DIC score were evaluated. Values are expressed as mean ± SD. Data were analyzed by Wilcoxon signed-rank test. P <0.05 was considered signifi cant. 2. Davignon J: Benefi cial cardiovascular pleiotropic eff ects of statins. Circulation 2004, 109(Suppl III):III-39-III-43. 2. Davignon J: Benefi cial cardiovascular pleiotropic eff ects of statins. Circulation 2004, 109(Suppl III):III-39-III-43. P60 Antithrombin III concentrate therapy may have an effi cacy in sepsis N Saito, M Takeda, T Harada, R Moroi, M Namiki, A Yaguchi Tokyo Women’s Medical University, Tokyo, Japan Critical Care 2013, 17(Suppl 2):P60 (doi: 10.1186/cc11998) Introduction Antithrombin III (AT III) has been known to contribute to anti-infl ammatory response as well as its anticoagulation. Our previous S22 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Conclusion Our data showed no benefi t with the use of a potent statin acutely in patients with sepsis or septic shock with regards to improvement in endothelial function. study showed AT III defi ciency happened in the early stage of sepsis with no relation to DIC status. However, whether AT III concentrate is a benefi cial therapy or not for septic patients is controversial. Our hypothesis is that AT III concentrate may have effi cacy as anti- infl ammation for sepsis. study showed AT III defi ciency happened in the early stage of sepsis with no relation to DIC status. However, whether AT III concentrate is a benefi cial therapy or not for septic patients is controversial. Our hypothesis is that AT III concentrate may have effi cacy as anti- infl ammation for sepsis. P61 We enrolled adult patients within 24 hours of severe sepsis or septic shock diagnosis and randomized them to placebo or atorvastatin 80 mg/day for a short term. Endothelial dysfunction was assessed measuring plasmatic levels of IL-6, ET-1, VCAM-1 by ELISA and measuring fl ow-mediated vasodilatation of the brachial artery at basal, 24 and 72 hours after randomization. A multicenter randomized controlled study of an extracorporeal cytokine hemoadsorption device in septic patients Conclusion In the patients with septic DIC, WBC, CRP and SOFA score were immediately improved after the AT III concentrate therapy, while platelet and DIC score were improved later. AT III concentrate may also contribute to anti-infl ammatory for septic DIC with anticoagulation. Methods Ventilated patients fulfi lling the criteria for severe sepsis and ALI were enrolled in this multicenter randomized, controlled, open- label study comparing standard of care with or without hemoperfusion treatment. Primary endpoints were safety and IL-6 reduction. Treated patients underwent hemoperfusion at fl ow rates of ~200 to 300 ml/ minute for 6 hours per day for 7 consecutive days. The overall mean reduction in individual plasma cytokines for the control and treatment groups during the treatment period was calculated using a generalized linear model. P64 Methods Adult patients with septic shock who required renal replacement therapy (RRT), with a confi rmed endotoxemia, and suspected Gram-negative infection were consecutively added to the study within the fi rst 24  hours after diagnosis. All patients received full standard treatment for septic shock. Endotoxin elimination was performed using the membrane oXiris (Gambro, Sweden), a medical device for continued RRT with the unique feature of endotoxin adsorbtion. An endotoxin activity assay was used to monitor endotoxin elimination therapy at baseline (T0), 3  hours (T1), 12  hours (T2), 24 hours (T3), 48 hours (T4), and 72 hours (T5). Our key indicators were the improvement in hemodynamics and organ function, and decrease of endotoxin activity (EA) in blood. Continuous variables are presented as mean values with standard deviations. Combined use of pumpless extracorporeal lung assist system and continuous renal replacement therapy with citrate anticoagulation in polytrauma patients y HK Atalan, M Dumantepe, TB Denizalti, IA Tarhan, A Ozler Atasehir Memorial Hospital, Istanbul, Turkey Critical Care 2013, 17(Suppl 2):P64 (doi: 10.1186/cc12002) Introduction The usefulness of a pumpless extracorporeal lung assist system (pECLA) and continuous renal replacement therapy (CRRT) in critically ill patients has been demonstrated in previous studies [1,2]. The aim of this report was to examine combined use of pECLA and CRRT to improve carbon dioxide and infl ammatory mediator removal, which allows for lung protective ventilation strategies. Results High EA level at baseline (0.74 ± 0.14 endotoxin activity units (EAU)) signifi cantly decreased during RRT with oXiris membrane to 0.46 ± 0.02 (T1), 0.34 ± 0.01 (T2), 0.4 ± 0.02 (T3), 0.46 ± 0.04 (T4), 0.35 ± 0.07 (T5) EAU (P <0.05). MAP increased from baseline 72 ± 14 to 81 ± 18, 76 ± 6, 77 ± 7, 83 ± 13, 87 ± 10 mmHg (P <0.05), and the mean norepinephrine use decreased from 0.23 ± 0.04 to 0.19 ± 0.02, 0.11 ± 0.01, 0.09 ± 0.01, 0.04 ± 0.01, 0.0 μg/kg/minute (P <0.05) at T0, T1, T2, T3, T4, T5, respectively. The SOFA score had decreased from 14 ± 4 to 12 ± 2, 9 ± 3, 7 ± 3 points (P <0.05), and the procalcitonin level declined from 107 ± 123 to 45 ± 41, 29 ± 30, 17 ± 157 ± 1 ng/ml (P <0.05) at T0, T3, T4, T5. Methods In our 10 patients with ARDS due to polytrauma and sepsis, pECLA was established by insertion of cannulae to the femoral artery and vein. CRRT cannulae were introduced by venous line of the same vascular access (Figure 1). We preferred regional anticoagulation with trisodium citrate for both CRRT and ILA. Figure 1 (abstract P64). ILA and CRRT connected to the patient. Conclusion RRT with oXiris membrane resulted in the eff ective elimination of endotoxins from the blood. The therapy was associated with an increase in blood pressure, a reduction of vasopressor requirements, and an improvement of organ function. The application of the endotoxin activity assay was useful for bedside monitoring of endotoxemia in ICU patients. P63 Continuous renal replacement therapy with the adsorbent membrane oXiris in septic patients: a clinical experience F Turani, F Candidi, R Barchetta, E Grilli, A Belli, E Papi, A di Marzio, M Falco Aurelia Hospital/European Hospital, Rome, Italy Critical Care 2013, 17(Suppl 2):P63 (doi: 10.1186/cc12001) Results We studied 47 patients, 24 in the placebo group (mean age 52 ± 20 years, 29.1% male; APACHE II risk score 23.5 ± 7.3) and 23 in the statin group (mean age 49.5 ± 18 years, 53.4% male; APACHE II risk score 23 ± 6.9). The baseline characteristics of the placebo group were similar to statin patients as well as the mean length of stay in the ICU (8.6 ± 7.4 and 9.1 ± 8 days, respectively) and the time on vasopressors (49.3 ± 47.1 and 59 ± 91.1 hours, respectively). No signifi cant diff erence was observed on the temporal variation of biomarker levels (IL-6, VCAM-1, ET-1) between treatment and control groups. The intrahospital mortality rate was 26% in the statin group and 45% in the placebo group (P = 0.17). Introduction Renal failure is an important complication of sepsis and CRRT with adsorbing membranes may be useful in this clinical setting [1]. The aims of the study in septic/septic shock patients are to evaluate: the safety of a new hemofi lter membrane oXiris with adsorbing properties and anti-endotoxin activity; the renal and hemodynamic Introduction Renal failure is an important complication of sepsis and CRRT with adsorbing membranes may be useful in this clinical setting [1]. The aims of the study in septic/septic shock patients are to evaluate: the safety of a new hemofi lter membrane oXiris with adsorbing properties and anti-endotoxin activity; the renal and hemodynamic S23 Table 1 (abstract P63) Parameter Units T0 T1 Creatinine mg/dl 1.9 ± 0.1 1.18 ± 0.1* Diuresis ml/24 hours 1,284 ± 78 1,573 ± 98 Norepinephrine μg/kg/minute 0.17 ± 0.2 0.06 ± 0.1* IL-6 pg/ml 572 ± 78 278 ± 57* Procalcitonin ng/ml 35 ± 7 15 ± 2* Endotoxin Level/U 0.64 ± 0.2 0.49 ± 0.1 *P <0.05 vs. T0. Results Mean SAPS II and APACHE II scores were 55 and 23 respectively. Mean time on mechanical ventilation was 22 days. Mean ICU stay was 30 days for survivors and 38 days for nonsurvivors. 1. Marshall JC, Foster D, Vincent JL, Cook DJ, Cohen J, Dellinger RP, Opal S, Abraham E, Brett SJ, Smith T, Mehta S, Derzko A: Diagnostic and prognostic implications of endotoxemia in critical illness: results of the MEDIC Study. J Infect Dis 2004, 190:527-534. P66 Use of extracorporeal endotoxin elimination therapy for septic shock gi Results Table 1 presents the main results of this study. y Conclusion In septic/septic shock patients with renal failure, CRRT with a new treated heparin-coated membrane (oXiris; Gambro) is clinically feasible, and has a positive eff ect on renal function and hemodynamics. An adsorbing eff ect on proinfl ammatory mediators may have a role in these results. These data and the trend toward a decrease of endotoxin during the treatment warrant further investigation. Reference Introduction Endotoxin, a component of the outer membrane of Gram- negative bacteria, is considered an important factor in pathogenesis of septic shock [1]. The aim of our study was to determine whether endotoxin elimination treatment added to the standard treatment would improve organ function in patients with septic shock. 1. Rimmelé T, et al.: Nephrol Dial Transplant 2009, 24:421-427. P63 When compared with baseline values most relevant parameters were the improvement in tidal volumes, plateau pressures, PaCO2 levels and pH (Figure 2). Four patients survived while six patients died from sepsis-MOF.f Results Mean SAPS II and APACHE II scores were 55 and 23 respectively. Mean time on mechanical ventilation was 22 days. Mean ICU stay was 30 days for survivors and 38 days for nonsurvivors. When compared with baseline values most relevant parameters were the improvement in tidal volumes, plateau pressures, PaCO2 levels and pH (Figure 2). Four patients survived while six patients died from sepsis-MOF. Conclusion We concluded that pECLA can eff ectively address the impaired gas exchange in ARDS and CRRT is a safe procedure with potential therapeutic value for treating MOF. Citrate anticoagulation was well tolerated and fi lter life was appropriate. The use of the same vascular access for ILA and CRRT may minimize invasive procedures and related side eff ects. Conclusion We concluded that pECLA can eff ectively address the impaired gas exchange in ARDS and CRRT is a safe procedure with potential therapeutic value for treating MOF. Citrate anticoagulation was well tolerated and fi lter life was appropriate. The use of the same vascular access for ILA and CRRT may minimize invasive procedures and related side eff ects. References 1. Bein T, et al.: Int J Emerg Med 2010, 3:177-179. 2. Demetrios J, et al.: Kidney Int 2005, 67:2361-2367. 1. Bein T, et al.: Int J Emerg Med 2010, 3:177-179. response; and the changes of endotoxin and proinfl ammatory molecules. response; and the changes of endotoxin and proinfl ammatory molecules. Methods Forty septic/septic shock patients with renal failure were enrolled in the study. All patients had preoperative endotoxin >0.6 level/units (EAA Spectral D) and were submitted to high-volume hemodiafi ltration (50  ml/kg/hour, Prismafl ex; Gambro) with a new treated heparin-coated membrane (oXiris; Gambro). At T0 (pre- treatment) and T1 (24 hours) the main clinical and biochemical data were evaluated. All data are expressed as mean ± SD. One-way ANOVA test with Bonferroni correction was used to evaluate the data changes. P <0.05 was considered signifi cant. P65 Abstract withdrawn Reference 1. Marshall JC, Foster D, Vincent JL, Cook DJ, Cohen J, Dellinger RP, Opal S, Abraham E, Brett SJ, Smith T, Mehta S, Derzko A: Diagnostic and prognostic implications of endotoxemia in critical illness: results of the MEDIC Study. J Infect Dis 2004, 190:527-534. Figure 1 (abstract P64). ILA and CRRT connected to the patient. S24 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum com/supplements/17/S2 Figure 2 (abstract P64). Changes in tidal volume, plateau pressure, pH and PaCO2. CO Figure 2 (abstract P64). Changes in tidal volume, plateau pressure, pH and PaCO2. P67 (133; 286) to 132 (68; 155) mg/l, P = 0.015 and SOFA score from 7.0 (3,0; 8.0) to 6.0 (3,0; 7.0), P = 0.007. At 24 hours after Alteco LPS, signifi cantly decreased PCT from 8.7 (3.0; 25.9) to 4.8 (2.1; 10.0) ng/ml. The 28-day mortality rate was 14.2% (n = 2) in the PMX-F group and 31.3% (n = 10) in the Alteco LPS group. P67 Selective adsorption of lipopolysaccharide in the complex treatment of patients with severe sepsis S Rei, I Aleksandrova, V Kiselev, M Ilynskiy, G Berdnikov, L Marchenkova, G Bulava, N Borovkova Hospital Research Institute for Emergency Medicine named after N.V. Sklifosovsky, Moscow, Russia Critical Care 2013, 17(Suppl 2):P67 (doi: 10.1186/cc12005) Table 1 (abstract P67). Clinical characteristics of the groups Characteristic PMX-F Alteco LPS Sex M/F 9/5 17/15 Age (years) 41.6 ± 17.1 42.1 ± 13.5 Shock 6 (42.9%) 13 (40.6%) APACHE II 21 (17; 25) 21 (16; 24) SOFA 7 (3.0; 8) 7 (3.5; 11) Table 1 (abstract P67). Clinical characteristics of the groups Introduction Severe sepsis and septic shock remain the most serious problem of critical care medicine with a mortality rate of 30 to 55% [1]. Several studies have demonstrated positive eff ects of selective adsorption of LPS on blood pressure, PaO2/FiO2 ratio, endotoxin removal and mortality [2,3]. The purpose of the study was to evaluate the effi ciency of using the selective adsorption of LPS, Toraymyxin – PMX-F (Toray, Japan) and Alteco® LPS Adsorber (Alteco Medical AB, Sweden), in the complex treatment of patients with severe sepsis. Methods Forty-six patients with Gram-negative sepsis in the postoperative period were enrolled into the study. Toraymyxin – PMX-F was used in the PMX-F group (n = 14), while Alteco LPS adsorption was used in the Alteco LPS group (n = 32). P69 Results Survival is shown in Figure 1. Experiments in each animal were starting at 7:00 am. At 8:00 am, respective mean free cortisol of groups A, C and D was 1.81, 0.55 and 2.05 μg/dl; at 1:00 pm they were 0.92, 0.47 and 1.40 μg/dl. Reference The clinical characteristics are listed in Table 1. The SOFA score, PaO2/FiO2, procalcitonin (PCT), C-reactive protein (CRP), endotoxin activity assay (EAA) was noted before, 24 and 48 hours after the selective adsorption of LPS. Conclusion The use of the LPS-selective adsorption (particularly PMX-F) in patients with severe sepsis leads to improvement of systemic infl ammation and organ dysfunction. References 1. Vincent JL, et al.: Crit Care Med 2006, 34:344-353. Results At 48 hours after PMX-F, signifi cantly decreased PCT from 17.5 (5.0; 40.9) to 7.1 (4.8; 13.0) ng/ml, P = 0.028, decreased CRP from 180 2. Cruz DN, et al.: JAMA 2009, 301:2445-2452. 3. Yaroustovsky M et al.: Blood Purif 2009, 28:227-233. S25 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 or when vasopressor infusion is terminated whilst 11% after a fi xed duration. Withdrawal of CS also diff ered, in that 25% tapered/weaned steroids, 31% stopped it abruptly and 44% of CCPs would base their CS cessation pattern on the clinical context. Only 46% of CCPs believe that CS is benefi cial whereas 44% were unsure of the benefi ts in septic shock. Only 29 (36%) responders indicated that their critical care unit had a written protocol for CS in septic shock. References References 1. Dellinger et al.: Crit Care Med 2008, 36:296-327. 2. Bourne et al.: Anaesthesia 2003, 58:591-596. References 1. Dellinger et al.: Crit Care Med 2008, 36:296-327. 2. Bourne et al.: Anaesthesia 2003, 58:591-596. 1. Dellinger et al.: Crit Care Med 2008, 36:296-327. 2. Bourne et al.: Anaesthesia 2003, 58:591-596. Need for restoration of cortisol serum levels for successful antimicrobial therapy in experimental sepsis T Doulias1, A Pistiki2, P Christopoulos1, V Papaziogas1, E Giamarellos-Bourboulis2, I Koutelidakis1 1University of Thessaloniki, Medical School, Thessaloniki, Greece; 2University of Athens, Medical School, Athens, Greece Critical Care 2013, 17(Suppl 2):P70 (doi: 10.1186/cc12008) T Doulias1, A Pistiki2, P Christopoulos1, V Papaziogas1, E Giamarellos-Bourboulis2, I Koutelidakis1 1University of Thessaloniki, Medical School, Thessaloniki, Greece; 2University of Athens, Medical School, Athens, Greece Critical Care 2013, 17(Suppl 2):P70 (doi: 10.1186/cc12008) yg y y Results This study was terminated early as interim analysis identifi ed no signifi cant clinical benefi t. Baseline characteristics (age/APACHE IV score) were similar between two groups of patients. No signifi cant diff erence was noted between two groups with respect to change in total SOFA score (+1 vs. –5.5 for AT vs. ST, P = 0.382), vasopressor score (–29 vs. –46.6, P = 0.775), PaO2/FiO2 ratio (–26 vs. +163, P = 0.199), total urine output from 0 to 72 hours (3,850 ml vs. 4,570 ml, P = 0.355) and 28-day mortality (14.3% vs. 37.5%, P = 0.569). No signifi cant side eff ect was noted when using this new hemoadsorption device. Introduction It is postulated that clinical benefi t of low-dose hydrocortisone in septic shock is related to reversal of relative adrenal insuffi ciency [1]. This was proved in an animal model of sepsis. fi Methods Sixty-nine Wistar male rats were assigned to the following groups: A, sham-operation; B, sepsis; C, bilateral adrenalectomy and sepsis; D, bilateral adrenalectomy, sepsis and hydrocortisone treatment; E, bilateral adrenalectomy, sepsis and ertapenem treatment; and F, bilateral adrenalectomy, sepsis, hydrocortisone and ertapenem treatment. Sepsis was induced by the i.p. infusion of 1×106 cfu/ml of Escherichia coli after adrenalectomy. Hydrocortisone 10  mg/kg was infused i.v. bid starting 1  hour after bacterial challenge. Ertapenem 5  mg/kg was infused i.v. once daily starting 1  hour after bacterial challenge. Survival was recorded. In a separate set of experiments in 18 rats, animal sacrifi ce was performed to measure the free cortisol concentration. Conclusion This small study cannot identify any extra clinical benefi t on addition of Alteco endotoxin hemoadsorption to standard therapy in patients suff ering from intra-abdominal sepsis with shock due to Gram-negative bacterial infection. g Reference 1. Cruz DN, et al.: JAMA 2009, 301:2445-2452. 1. Cruz DN, et al.: JAMA 2009, 301:2445-2452. P68 Alteco endotoxin hemoadsorption in Gram-negative septic shock patients HP Shum, KC Chan, WW Yan Pamela Youde Nethersole Eastern Hospital, Hong Kong Critical Care 2013, 17(Suppl 2):P68 (doi: 10.1186/cc12006) Introduction Septic shock is a common cause of mortality and morbidity in the ICU. Endotoxin hemoadsorption using a Polymyxin B fi ber column can improve patient outcome [1]. This study investigated the therapeutic eff ect of a new endotoxin hemoadsorption device in Gram-negative septic shock patients. Conclusion The perceptions, usage and cessation of CS in septic shock vary but do appear to have shifted in the last decade. A UK survey in 2003 identifi ed that only 60% of ICUs used CS for septic shock and over 22% perform a short synacthen test [2]. It appears that many intensivists are using CS for septic shock, despite confl icting outcome data. We all strive to practice evidence-based medicine but until we have a robust, reliable and methodical randomised control trial that attempts to resolve the CS debate, practice will remain diverse on this subject, as refl ected by our survey. g p p Methods An open, controlled, prospective, randomized, single-centre trial conducted between February 2010 and June 2012. Patients with septic shock due to intra-abdominal sepsis were recruited and randomized to either standard therapy (ST, n = 8) or standard therapy plus two 2 hourly sessions of Alteco endotoxin hemoperfusion (AT, n = 7). Standard therapy included infective source control, appropriate early antibiotic, low-dose steroid, early continuous renal replacement therapy in the presence of acute kidney injury (RIFLE Injury class or more), hemodynamic optimization and lung-protected ventilatory support. Primary outcome was change in organ dysfunction at 48 hours measured by Sequential Organ Failure Assessment (SOFA) scores. Secondary outcomes were change in vasopressor requirement (measured by vasopressor score), PaO2/FiO2 (fraction of inspired oxygen) ratio, total urine output at 72 hours and 28-day mortality.i Effi cacy and utility of preemptive anti-Gr(+) coverage in selected critically ill patients Effi cacy and utility of preemptive anti-Gr(+) coverage in selected critically ill patients VT Todorova, SM Milanov, G Georgiev, M Milanov Emergency Hospital ‘Pirogov’, Sofi a, Bulgaria Critical Care 2013, 17(Suppl 2):P73 (doi: 10.1186/cc12011) critically ill patients VT Todorova, SM Milanov, G Georgiev, M Milanov Emergency Hospital ‘Pirogov’, Sofi a, Bulgaria Critical Care 2013, 17(Suppl 2):P73 (doi: 10.1186/cc12011) Introduction Based on the results of our previous studies [1] we have identifi ed clinical risk factors for the emergence of Gr(+) infections in our ICU and we have developed a new algorithm for combating them. The choice of the particular antibiotic drug is guided by additional risk factors for severity of illness and data on the infectious focus. The response to therapy and its duration are also stated. The aim of the current study was to evaluate the effi cacy and safety of this preemptive approach. Results In total, 383 patients were admitted with septic shock, of whom 253 (66%) were included; 231(92%) had appropriate antibiotic therapy, of whom 52 (22.5%) died at 7 days. Average time to initiation of appropriate antibiotic therapy was 9  ±  23 hours. By bivariate analysis, body mass index (BMI) <20, SAPS II ≥56, SOFA score ≥11 and bacteremia were signifi cantly associated with 7-day mortality. Urinary tract infection (UTI) was a protective factor. Age, sex, comorbidities (particularly immunosuppression), Knaus score, nosocomial infection and type of germ had no infl uence on 7-day mortality. By multivariate logistic regression, BMI <20 (OR = 4.87, 95% CI = 1.36 to 17.43, P = 0.01) and SOFA score ≥11 (OR = 7.99, 95% CI = 3.11 to 20.5, P <0.001) were the only factors signifi cantly associated with 7-day mortality. UTI was a signifi cant protective factor (OR = 0.30, 95% CI = 0.10 to 0.88, P = 0.03). Time to initiation of appropriate antibiotherapy was not associated with 7-day mortality (OR = 0.99, 95% CI = 0.99 to 1.00, P = 0.48). pp Methods A randomized prospective controlled trial was carried out from September 2010 to September 2012. Patients were submitted to block randomization and stratifi ed on the basis of their initial SAPS II exp score. Antibiotic therapy was started on the day of inclusion in the treatment group and only with proven Gr(+) pathogen in the control group. Steroid therapy in septic shock: survey of practice amongst UK critical care physicians G Rajendran, K Dasari, A Dhrampal j , , p Norfolk & Norwich University Hospital, Norwich, UK Critical Care 2013, 17(Suppl 2):P69 (doi: 10.1186/cc12007) Figure 1 (abstract P70). Survival of rats. Introduction Corticosteroid (CS) therapy in sepsis remains contro- versial and was fi rst introduced in sepsis management for its anti- infl ammatory property. CS has found a role in septic shock amelioration with inconsistent outcomes. The Surviving Sepsis Campaign (SSC) includes CS as a Level 2C recommendation in septic shock [1]. Adapting and practicing SSC guidelines vary between critical care units. Accordingly, a survey was conducted to elucidate the usage of CS for septic shock by UK critical care physicians (CCPs). p y p y Methods Following approval by the UK Intensive Care Society (ICS), the survey was publicised on the ICS website and its newsletter. y Results A total of 81 intensivists responded to this online survey. Seventy-four (92.5%) CCPs prescribed CS only if the septic shock is poorly responsive to fl uid resuscitation and vasopressor therapy. Six (7.5%) initiated CS at the same time as vasopressor therapy. None initiated CS for patients with severe sepsis. No CS other than hydrocortisone is being used. The most commonly used intravenous regimen is 50 mg 6 hourly (65%) followed by 50 mg 8 hourly (11%). Only 10% of CCPs would prescribe it by infusion. Less commonly used regimens were 100 mg 8 hourly (6%) and 100 mg 6 hourly (5%). Only 5% would consider adding fl udrocortisone. Prior to initiating CS, 5% of CCPs would perform a short synacthen test, while 94% would not. The majority (89%) of CCPs would stop CS after resolution of shock state Figure 1 (abstract P70). Survival of rats. S26 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 cases during the outbreak. Completed questionnaires describing 27 patients, 11 nonsurvivors and 16 survivors, were returned.i cases during the outbreak. Completed questionnaires describing 27 patients, 11 nonsurvivors and 16 survivors, were returned.i Conclusion Even when eff ective antimicrobial treatment is adminis- tered, administration of hydrocortisone at a regimen restoring normal secretion is mandatory for survival. Milanov S, et al.: Gram positive nosocomial infections in a general ICU – emerging new clues. Crit Care 2011, 15(Suppl 1):P224. Steroid therapy in septic shock: survey of practice amongst UK critical care physicians p Results In preliminary analysis of categorical data, a signifi cantly (Fisher exact test) greater proportion of patients with compared with without the following fi ndings did not survive; history of alcohol use (P  = 0.05); the presence of lethargy (P  = 0.01), confusion (P  = 0.03), nausea (P  = 0.04), abdominal pain (P  = 0.02), or the need for vasopressors (P = 0.002), oxygen, mechanical ventilation, or steroids (all P = 0.004) at presentation; and excessive bleeding at surgery (P = 0.01). Initial analysis of continuous data demonstrated that, compared with survivors at presentation, nonsurvivors had signifi cantly (one-way ANOVA) increased respiratory rate, percent neutrophils on complete blood count, hemoglobin, INR, C-reactive protein, and bilirubin and signifi cantly decreased temperature, systolic blood pressure, platelets, sodium, albumin, calcium (corrected for albumin), base excess and bicarbonate (all P ≤0.05).f 1. Annane D, et al.: JAMA 2002, 288:862-871. Effi cacy and utility of preemptive anti-Gr(+) coverage in selected critically ill patients Initial data were gathered on demographics, diagnosis, proven risk factors for sepsis-related mortality, severity of infl ammatory response, ventilator-associated pneumonia and organ dysfunction. Dynamics of SIRS, CPIS and SOFA scores, subsequent infectious isolates, ventilator-free days, length of ICU stay and outcome were followed for each patient. y y Conclusion Prognosis at 7 days of patients with septic shock is largely related to the number of failing organs. The majority received appropriate antibiotic therapy although time to initiation is longer than recommended. Further eff orts are warranted to reduce mortality in patients with septic shock. p Results A total of 170 patients were enrolled. No statistically signifi cant diff erences in their basal characteristics were found. The subsequent score values, length of ICU stay and the number of ventilator-free days were also comparable between groups. The majority of Gr(+) pathogens were isolated between 6 and 10 days of inclusion. No diff erences were found regarding the concomitant Gr(–) fl ora and the related antibiotic therapy. The new organ dysfunction severity was similar in both groups (P = 0.37). The in-hospital mortality was 26.2% in the treatment group versus 18.6% in the control group (P  = 0.56). Signifi cant diff erences between the Kaplan–Meier estimates of survival were also not found (log-rank test P = 0.81). No major adverse reactions were observed. Results A total of 170 patients were enrolled. No statistically signifi cant diff erences in their basal characteristics were found. The subsequent score values, length of ICU stay and the number of ventilator-free days were also comparable between groups. The majority of Gr(+) pathogens were isolated between 6 and 10 days of inclusion. No diff erences were found regarding the concomitant Gr(–) fl ora and the related antibiotic therapy. The new organ dysfunction severity was similar in both groups (P = 0.37). The in-hospital mortality was 26.2% in the treatment group versus 18.6% in the control group (P  = 0.56). Signifi cant diff erences between the Kaplan–Meier estimates of survival were also not found (log-rank test P = 0.81). No major adverse reactions were observed. Conclusion The implementation of this new policy failed to reduce the degree of organ dysfunction severity and was not associated with signifi cant survival benefi t. Moreover, even though it did not reach statistical signifi cance, a second peak of Gr(+) isolates was observed as a possible complication of the preemptive therapy. P71 Impact of time to initiation of appropriate antibiotic therapy on early mortality of patients with septic shock JP Quenot1, C Binquet2, S Vinault2, A Pavon1 1University Hospital Dijon, France; 2CHU de Dijon, Centre d’Investigation Clinique – Epidémiologie Clinique, Dijon, France Critical Care 2013, 17(Suppl 2):P71 (doi: 10.1186/cc12009) P71 Impact of time to initiation of appropriate antibiotic therapy on early mortality of patients with septic shock JP Quenot1, C Binquet2, S Vinault2, A Pavon1 1University Hospital Dijon, France; 2CHU de Dijon, Centre d’Investigation Clinique – Epidémiologie Clinique, Dijon, France Critical Care 2013, 17(Suppl 2):P71 (doi: 10.1186/cc12009) Impact of time to initiation of appropriate antibiotic therapy on early mortality of patients with septic shock Impact of time to initiation of appropriate antibiotic therapy on early mortality of patients with septic shock JP Quenot1, C Binquet2, S Vinault2, A Pavon1 Introduction The impact of appropriate antibiotic therapy on prognosis of patients with septic shock is well established. However, the prognostic infl uence of time to initiation of antibiotics remains debated. We evaluated the eff ect on 7-day mortality of time to initiation of appropriate antibiotic therapy in patients hospitalised in critical care for septic shock. Conclusion The implications of the apparent diff erences noted between nonsurvivors and survivors in this survey of cases from the fi rst known outbreak of injectional anthrax require further study. However, these diff erences might inform the design of research during future outbreaks or of methods to identify patients most in need of anthrax-specifi c therapies such as toxin-directed antibodies. Methods Secondary analysis from the EPISS cohort. We included only patients admitted to the University Hospital Dijon. Septic shock was defi ned as initiation of vasopressors in a patient with suspected or documented infection with at least one criterion of hypoperfusion (metabolic acidosis and/or renal insuffi ciency and/or hepatic dys func- tion). We excluded patients with no available bacteriological data. Anti- biotherapy was considered appropriate if at least one of the antibiotics prescribed was active against the germ identifi ed. Bivariate and multivariate logistic regression analysis was used to assess the impact of time to initiation of appropriate antibiotherapy on 7-day mortality. Effi cacy and utility of preemptive anti-Gr(+) coverage in selected critically ill patients Whether this approach could lead to vancomycin MIC creep or there could still be a niche for it later in the course of treatment and/or in nontrauma patients remains to be further explored. P72 P74 Gram-negative resistance and need for ICU among urinary tract infections in the United States M Zilberberg1, A Shorr2 1EviMed Research Group, LLC, Goshen, MA, USA; 2Washington Hospital Center Washington, DC, USA Critical Care 2013, 17(Suppl 2):P74 (doi: 10.1186/cc12012) t e p e a e ce o ect o t e t e u t d ug es sta t ( ) o CPE. We defi ned MDR-PA as any PA isolate resistant to ≥3 drug classes. Enterobacteriaceae were classifi ed as CPE if resistant to both a third- generation cephalosporin and a carbapenem. We evaluated specimens from respiratory, bloodstream, urinary tract (UTI) and complicated intra-abdominal infections. Introduction Urinary tract infection (UTI) can lead to both hospitali- za tion and severe sepsis. We theorized that UTI due to Gram- negative (GN) multidrug-resistant P. aeruginosa (MDR-PA), extended- spectrum β-lactamase (ESBL), E. coli (EC) and Klebsiella sp. (KP), and carbapenemase-producing Enterobacteriaceae (CPE) would be frequently isolated in the ICU. Results We identifi ed 327,912 PA (60,695 (18.5%) MDR-PA) and 279,600 Enterobacteriaceae (2,558 (0.9%) CPE) specimens. More than one- quarter (26.1%) of all PA were recovered from ICU patients as compared with 17.9% of all Enterobacteriaceae specimens. Of those specimens originating in the ICU, MDR-PA represented 21.9% of all PA organisms, while CPE represented 1.6% of all Enterobacteriaceae. Pneumonia and UTI accounted for 92.4% of all PA and 84.0% of all Enterobacteriaceae specimens. The proportion of both MDR-PA and CPE was highest in pneumonia (22.0% and 1.6%, respectively) and lowest in UTI (13.7% and 0.6%, respectively). Over the time frame of the study, CPEs emerged and stabilized at approximately 2.8% of all Enterobacteriaceae, while MDR-PA increased slightly from 16.0% of all PA in 2000 to 17.8% in 2009. Conclusion Although CPE organisms have emerged as an important pathogen, MDR-PA remains an order of magnitude more prevalent in the United States. Pneumonia patients and those in the ICU are at an increased risk for both MDR-PA and CPE infections compared with those outside the ICU. y Methods We analyzed a large US-based microbiology database, Eurofi ns TSN, between the years 2000 and 2009. We determined the proportion of isolates caused by MDR-PA, ESBL-EC, ESBL-KP, and CPE relative to their susceptible counterparts. We defi ned MDR-PA as any isolate resistant to ≥3 drug classes. ESBL organisms were defi ned as E. coli and K. pneumoniae resistant to a third-generation cephalosporin. Prevalence of multidrug resistance, extensive drug resistance and pandrug resistance among multiple Gram-negative isolates: experience in a tertiary-care hospital ICU in North India S Dewan, T Sahoo, N Chandra, A Varma Fortis Escorts Heart Institute, New Delhi, India Critical Care 2013, 17(Suppl 2):P76 (doi: 10.1186/cc12014) Prevalence of multidrug resistance, extensive drug resistance and pandrug resistance among multiple Gram-negative isolates: experience in a tertiary-care hospital ICU in North India S Dewan, T Sahoo, N Chandra, A Varma Fortis Escorts Heart Institute, New Delhi, India Critical Care 2013, 17(Suppl 2):P76 (doi: 10.1186/cc12014) Prevalence of multidrug resistance, extensive drug resistance and pandrug resistance among multiple Gram-negative isolates: experience in a tertiary-care hospital ICU in North India g y g Conclusion The microbiology of GN UTI hospitalizations has shifted over the last decade and greater antimicrobial resistance is evident. The prevalence of MDR-PA, ESBL-EC, ESBL-KP, and CPE is higher in the ICU than in other hospital locations. Introduction Antibiotic resistance is a major worldwide problem in the ICU [1]. The situation in developing countries like India is particularly serious. Since the presence of drug-resistant bacteria in the environment is a threat for public health, up-to-date information on local pathogens and the drug sensitivity pattern is very crucial to treat patients. This study was carried out to evaluate the prevalence of multidrug resistance (MDR), extensive drug resistance (XDR) and pandrug resistance (PDR) among multiple Gram-negative isolates in a medical–surgical ICU in a tertiary care hospital in North India. P75 Multidrug resistance among P. aeruginosa and Enterobacteriaceae in US hospitals, 2000 to 2009 M Zilberberg1, A Shorr2 1EviMed Research Group, LLC, Goshen, MA, USA; 2Washington Hospital Center, Washington, DC, USA Critical Care 2013, 17(Suppl 2):P75 (doi: 10.1186/cc12013) Introduction Gram-negative resistance remains a major challenge in the care of critically ill patients. Traditionally, P. aeruginosa (PA) has represented the most concerning pathogen. However, carbapenemase- producing Enterobacteriaceae (CPE) have emerged as a challenge, but h id i l f hi h i l d d Figure 1 (abstract P74). Figure 1 (abstract P74). Figure 1 (abstract P74). Methods We conducted a prospective observational study. All data were analysed using descriptive statistics. All Gram-negative culture isolates over a period of 13 months (October 2011 to October 2012) were included in this study. Isolation and identifi cation were performed using the bact alert system and VITEK2, respectively. Sensitivities were determined by Kirby Bauer disc diff usion and broth dilution using VITEK2-AST cards and interpreted according to Clinical and Laboratory Standards Institute criteria. P72 Enterobacteriaceae were considered CPE if resistant to both a third- generation cephalosporin and a carbapenem. We further examined the evolution of the frequency of resistance among GN UTIs over time. Results We identifi ed 115,201 PA (13.7% MDR-PA), 359,090 EC (5.6% ESBL), 97,419 KP (12.9% ESBL), and 176,110 Enterobacteriaceae (0.6% CPE) UTI specimens. The prevalence of resistance rose for each organism of interest from 2000 through 2009: MDR-PA, 11.6 to 12.3%; ESBL-EC, 3.3 to 8.0%; ESBL-KP, 9.1 to 18.6%; CPE 0 to 2.3%. For each organism the proportion of resistant pathogens was consistently higher among ICU specimens than among specimens from other hospital locations, reaching nearly 20% for MDR-PA (Figure 1). P72 Comparison of survivors and nonsurvivors in 27 confi rmed injectional anthrax cases from the 2009 outbreak in Scotland M Booth1, L Donaldson1, C Xizhong2, S Junfeng2, P Eichacker2 1Glasgow Royal Infi rmary, Glasgow, UK; 2National Institutes for Health, Bethesda, MD, USA Critical Care 2013, 17(Suppl 2):P72 (doi: 10.1186/cc12010) Critical Care 2013, 17(Suppl 2):P72 (doi: 10.1186/cc12010) ( g ) j Conclusion The implementation of this new policy failed to reduce the degree of organ dysfunction severity and was not associated with signifi cant survival benefi t. Moreover, even though it did not reach statistical signifi cance, a second peak of Gr(+) isolates was observed as a possible complication of the preemptive therapy. Whether this approach could lead to vancomycin MIC creep or there could still be a niche for it later in the course of treatment and/or in nontrauma patients remains to be further explored. Introduction From December 2009 to December 2010, 47 patients in Scotland presented with confi rmed anthrax infection manifested by soft tissue disease related to heroin injection. These cases represent the fi rst known outbreak of a recently recognized form of anthrax, termed injectional anthrax, which appears to be associated with a high mortality rate (28% in confi rmed cases from the UK outbreak). While epidemiologic data from this outbreak have been published, no report has systematically described fi ndings in patients at presentation or compared these fi ndings in nonsurvivors and survivors. p Reference Methods To better describe injectional anthrax, we developed a questionnaire and sent it to clinicians who had cared for confi rmed Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 S27 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 P74 Methods We analyzed a large US-based microbiology database, Eurofi ns TSN, between the years 2000 and 2009. We aimed to describe the prevalence of infection with either multidrug-resistant (MDR) PA or CPE. We defi ned MDR-PA as any PA isolate resistant to ≥3 drug classes. Enterobacteriaceae were classifi ed as CPE if resistant to both a third- generation cephalosporin and a carbapenem. We evaluated specimens from respiratory, bloodstream, urinary tract (UTI) and complicated intra-abdominal infections. Risk factors associated with Acinetobacter baumannii septicemia and its mortality rates in critically ill patients Risk factors associated with Acinetobacter baumannii septicemia and its mortality rates in critically ill patients K Kontopoulou, K Tsepanis, I Sgouropoulos, A Triantafyllidou, D Socratous, P Tassioudis, E Chasou, E Antypa, F Renta, E Antoniadou, K Mandraveli General Hospital G. Gennimatas, Thessaloniki, Greece Critical Care 2013, 17(Suppl 2):P77 (doi: 10.1186/cc12015) Results Sixty-three patients were admitted. The mean length of stay (MLS) was 3  ±  3 days, and the mean age was 25  ±  14 (13 patients <15  years). Patients were hospitalized for combat-related trauma (74%), noncombat-related trauma, medical pathologies (10%), and postoperative care (8%). They were Afghans (92%) or westerners (8%). Swabs were not realized for eight patients. Forty-three percent revealed an ESBLB colonization: Escherichia coli (22 patients), Klebsiella pneumoniae (one patient), Acinetobacter baumanii (one patient). No patients were colonized with MRSA. Ten patients (16%) were directly admitted to the ICU, 12 (19%) had been hospitalized before admission, 39 (62%) were transferred after resuscitative and stabilization care in a level 2 unit. For the two last categories, the MLS (for previous hospitalization) was respectively 14 ± 28 days and 8 ± 6 hours. Among patients transferred after care in a level 2 unit, MLS was no diff erent between colonized and noncolonized patients: 8 ± 7 versus 9 ± 6 hours (P = 0.5, Mann–Whitney test). y y p K Kontopoulou, K Tsepanis, I Sgouropoulos, A Triantafyllidou, D Socratous, P Tassioudis, E Chasou, E Antypa, F Renta, E Antoniadou, K Mandraveli General Hospital G. Gennimatas, Thessaloniki, Greece Critical Care 2013, 17(Suppl 2):P77 (doi: 10.1186/cc12015) K Kontopoulou, K Tsepanis, I Sgouropoulos, A Triantafyllidou, D Socratous, P Tassioudis, E Chasou, E Antypa, F Renta, E Antoniadou, K Mandraveli General Hospital G. Gennimatas, Thessaloniki, Greece Critical Care 2013, 17(Suppl 2):P77 (doi: 10.1186/cc12015) Introduction Acinetobacter baumannii (A. baum) is a leading cause of septicemia of patients hospitalized in the ICU with high mortality rates. The aim of our study is to investigate the risk factors associated with A. baum bacteremia and its mortality rates. y Methods A total of 937 patients (457 women and 480 men, median age 59) admitted to the ICU during the period 1 January 2009 to 30 September 2012 were enrolled in our retrospective study. Blood cultures were obtained from all patients. The identifi cation and the antimicrobial susceptibility testing were performed by the automated system VITEK2 (Bio Merieux, France). Prevalence of multidrug resistance, extensive drug resistance and pandrug resistance among multiple Gram-negative isolates: experience in a tertiary-care hospital ICU in North India S Dewan, T Sahoo, N Chandra, A Varma Fortis Escorts Heart Institute, New Delhi, India Critical Care 2013, 17(Suppl 2):P76 (doi: 10.1186/cc12014) For the purpose of this study, we used MDR to denote isolates resistant to representatives three or more classes of antimicrobial agents, XDR as those resistant to all but one or two classes and PDR as those resistant to all classes of antimicrobial agents available [2,3]. Results Out of a total 2,796 culture specimens sent over 13 months, 250 isolates were Gram-negative (8.9%). Among these 250 (n) Gram- negative isolates, 195 (78%) were extended-spectrum β-lactamase (ESBL) producers and the remaining 55 (22%) were non-ESBL producers. Among the ESBL producers, PDR, XDR and MDR isolates were 14 (5.6%), 113 (45.2%) and 68 (27.2%), respectively (Figure  1). Among the XDR-positive organisms, seven (6.1%) organisms were New Delhi metallo-β-lactamase-1 (NDM-1) producers and fi ve (4.4%) organisms were NDM-2 producers. Among ESBL-positive isolates, the most predominant isolate was Klebsiella pneumoniae (29.7%) followed by Acinetobacter aeruginosa (22.5%) and Escherichia coli (20.5%) (Figure 2). Among non-ESBL-positive isolates, the most predominant isolate was Escherichia coli (34.5%) followed by Klebsiella pneumoniae (21.8%) and Pseudomonas aeruginosa (14.5%) (Figure 2). P75 Multidrug resistance among P. aeruginosa and Enterobacteriaceae in US hospitals, 2000 to 2009 M Zilberberg1, A Shorr2 1EviMed Research Group, LLC, Goshen, MA, USA; 2Washington Hospital Center, Washington, DC, USA Critical Care 2013, 17(Suppl 2):P75 (doi: 10.1186/cc12013) Multidrug resistance among P. aeruginosa and Enterobacteriaceae in US hospitals, 2000 to 2009 Introduction Gram-negative resistance remains a major challenge in the care of critically ill patients. Traditionally, P. aeruginosa (PA) has represented the most concerning pathogen. However, carbapenemase- producing Enterobacteriaceae (CPE) have emerged as a challenge, but the epidemiology of this pathogen is poorly understood. Conclusion ESBL producers were the most frequently isolated Gram- negative bacterial isolates in this tertiary-care hospital in north India. Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 S28 pp http://ccforum.com/supplements/17/S2 Figure 1 (abstract P76). Prevalence of drug-resistant Gram-negative isolates. Figure 2 (abstract P76). Prevalence of Gram-negative isolates. P  <0.05 from the previous model and those mentioned at recent medical literature as signifi cantly related with A. baum septicemia and its mortality. Figure 1 (abstract P76). Prevalence of drug-resistant Gram-negative isolates. y Results A total of 101 patients (10.78%) developed bloodstream infection caused by A. baum and the mortality rate due to A. baum septicemia was estimated as 49.5% (50/101). Whitman TJ: Infection control challenges related to war wound infections in the ICU setting. J Trauma 2007, 62:S53. Prevalence of multidrug resistance, extensive drug resistance and pandrug resistance among multiple Gram-negative isolates: experience in a tertiary-care hospital ICU in North India S Dewan, T Sahoo, N Chandra, A Varma Fortis Escorts Heart Institute, New Delhi, India Critical Care 2013, 17(Suppl 2):P76 (doi: 10.1186/cc12014) Multiple regression analysis revealed adjusted mortality >55% (Exp(B) = 2.01, P = 0.013), MIS (Exp(B) = 1.97, P = 0.017), ABL (Exp(B) = 2.34, P = 0.009), LS >14 days (Exp(B) = 1.34, P = 0.034), MV (Exp(B) = 2.67, P = 0.005) and INV.PR (Exp(B) = 3.27, P = 0.001) as independent risk factors associated with A. baum septicemia. p Conclusion The fact that the probability of A. baum bacteremia increases in immunocompromised patients refl ects the opportunistic characteristic of these infections. MV accelerates respiratory A. baum colonization, which is a risk factor for AB bacteremia. Recent INV. PR increased the incidence of A. baum bacteremia and this result is probably related to the severe status in patients with central venous catheter. The administration of carbapenems inhibits the growth of other more susceptible bacteria, allowing the growth of multidrug- resistant A. baum. Figure 1 (abstract P76). Prevalence of drug-resistant Gram-negative isolates. Figure 2 (abstract P76). Prevalence of Gram-negative isolates. P78 Prevalence of colonization by multiresistant bacteria on admission to the ICU in the French military hospital in Kabul, Afghanistan JV Schaal, P Pasquier, H Delacour, A Salvadori, A Jarrassier, JR Renner, SM Mérat Military Teaching Hospital Bégin, Saint-Mandé, France Critical Care 2013, 17(Suppl 2):P78 (doi: 10.1186/cc12016) Prevalence of colonization by multiresistant bacteria on admission to the ICU in the French military hospital in Kabul, Afghanistan Prevalence of colonization by multiresistant bacteria on admission to the ICU in the French military hospital in Kabul, Afghanistan JV Schaal, P Pasquier, H Delacour, A Salvadori, A Jarrassier, JR Renner, SM Mérat Military Teaching Hospital Bégin, Saint-Mandé, France Critical Care 2013, 17(Suppl 2):P78 (doi: 10.1186/cc12016) Military Teaching Hospital Bégin, Saint-Mandé, France Military Teaching Hospital Bégin, Saint-Mandé, France Critical Care 2013, 17(Suppl 2):P78 (doi: 10.1186/cc12016) Figure 2 (abstract P76). Prevalence of Gram-negative isolates. Introduction The French military hospital at the Kaboul International Airport (KaIA) base provides surgical care for International Force and Afghan National Army soldiers, and also local patients. The development of multiresistant bacteria (MRB) nosocomial infections has raised a major problem complicating the care of combat casualties [1]. The aim of this study is to assess the prevalence of MRB carriage on admission to the ICU in this combat support hospital. Introduction The French military hospital at the Kaboul International Airport (KaIA) base provides surgical care for International Force and Afghan National Army soldiers, and also local patients. The development of multiresistant bacteria (MRB) nosocomial infections has raised a major problem complicating the care of combat casualties [1]. The aim of this study is to assess the prevalence of MRB carriage on admission to the ICU in this combat support hospital. Among ESBL producers, XDR organisms were most frequent, followed by MDR and PDR organisms. Few of the XDR isolates were NDM producers, which have propensity to spread to other bacteria. In view of signifi cant prevalence of multidrug resistance amongst Gram-negative organisms in the ICU, regular surveillance of antibiotic susceptibility patterns plays a crucial role for setting orders to guide the clinician in choosing empirical or directed therapy of infected patients. References Methods We used a prospective observation study on patients admitted to the French military ICU in KaIA over 3 months (July to September 2012). All hospitalized patients were assessed for the presence of colonization with MRB: nasal and rectal swabs were performed to identify, respectively, methicillin-resistant Staphylococcus aureus (MRSA) and extended-spectrum β-lactamases bacteria (ESBLB). The following data were recorded for each patient on admission: demographic characteristics, bacteriological results, length of stay, type of previous hospitalization. References 1. Radji M, et al.:Asian Pac J Trop Biomed 2011, 1:39-42. 2. Falagas ME, et al.: Clin Infect Dis 2008, 46:1121-1122. 3. Souli M, et al.: Euro Surveill 2008, 13:30-40. 1. Radji M, et al.:Asian Pac J Trop Biomed 2011, 1:39-42. 2. Falagas ME, et al.: Clin Infect Dis 2008, 46:1121-1122. 3. Souli M, et al.: Euro Surveill 2008, 13:30-40. References 1. Wu HS, et al.: Clin Microbiol Infect 2012, 18:E373-E376 2. Huang ST, et al.: J Microbiol Immunol Infect 2012, 45:356-362. Risk factors associated with Acinetobacter baumannii septicemia and its mortality rates in critically ill patients Data collected included underlying diseases, malignancies and immune suppression (MIS), prognostic factors (APACHE score, adjusted mortality), age, sex, length of ICU stay (LS), recent administration of broad-spectrum β-lactam antibiotics (especially carbapenems; ABL), mechanical ventila- tion (MV), implementation of invasive procedures (central venous catheter and urine catheter; INV.PR) and outcome. At fi rst a univariate statistical model was used with signifi cance level set at P = 0.05. For the multivariate statistical analysis model we used all variables with Conclusion In this study, prevalence of colonization with ESBLB at admission is very high, suggesting a high prevalence of MDR colonization in the local population in Afghanistan. It remains important to intensify the prevention policy against MRB cross-transmission in the deployed ICU. p y Reference p y Reference Whitman TJ: Infection control challenges related to war wound infections in the ICU setting. J Trauma 2007, 62:S53. Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 S29 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 (63 patients: 30 received SD and 33 HD), the clinical cure rate and microbiological eradication percentage were higher when TGC was used at higher doses (57.6% vs. 33.3%; P = 0.08 and 57.1% vs. 30.4%; P = 0.1). Table 1 shows multivariate analysis of clinical cure predictors in the VAP subgroup. (63 patients: 30 received SD and 33 HD), the clinical cure rate and microbiological eradication percentage were higher when TGC was used at higher doses (57.6% vs. 33.3%; P = 0.08 and 57.1% vs. 30.4%; P = 0.1). Table 1 shows multivariate analysis of clinical cure predictors in the VAP subgroup. P79 Extended-spectrum β-lactamase Providencia stuartii in a general ICU P Myrianthefs1, E Evodia2, G Fildissis1, G Baltopoulos1 1Athens University, Athens, Greece; 2Iatriko Kentro Athinon, Athens, Greece Critical Care 2013, 17(Suppl 2):P79 (doi: 10.1186/cc12017) Table 1 (abstract P80). Predictors of clinical cure in patients with VAP (multivariate analysis) OR (95% CI) P value HD TGC 5.93 (1.38 to 25.6) 0.02 IIAT 0.23 (0.06 to 0.85) 0.03 SOFA score 0.62 (0.45 to 0.86) <0.01 Table 1 (abstract P80). Predictors of clinical cure in patients with VAP (multivariate analysis) Introduction Providencia stuartii, a member of the family of Entero- bacteriacea, is a Gram-negative pathogen causing colonization and opportunistic infections in ICU patients. pp p Methods We retrospectively recorded the characteristics of P. stuartii infections in our ICU in a total period of 1 year (six-bed general ICU). Methods We retrospectively recorded the characteristics of P. stuartii infections in our ICU in a total period of 1 year (six-bed general ICU). Results A total of 116 patients (80 males, 68.9%) were hospitalized in our ICU of mean age 58.5 ± 1.8, mean ICU stay: 23.2 ± 3.3 days, APACHE II: 19.3 ± 0.7, SAPS II: 45.7 ± 1.6, SOFA: 7.9 ± 0.4 and mortality: 18.9%. Admission diagnosis was multiple trauma (29.3%), emergency surgery (37.1%), and medical (33.6%). Of them 21 (18.1%) developed P. Reference 1. Tasina E, et al.: Effi cacy and safety of tigecycline for the treatment of infectious diseases: a meta-analysis. Lancet Infect Dis 2011, 11:834-844. P81 p y Reference stuartii infection that was related to multiple trauma (P = 0.0289), length of ICU stay 69.8 ± 12.6 (median 51.0) versus 12.6 ± 1.7 (median 6.0) (P <0.0001) and illness severity APACHE II (21.7 ± 1.3 vs. 17.5 ± 0.8; P = 0.0056), SAPS II (54.9 ± 2.9 vs. 43.6 ± 1.8; P = 0.0296) and SOFA (10.6 ± 0.5 vs. 7.2 ± 0.4; P <0.0001). There was no statistically signifi cant diff erence regarding sex, age or mortality (P  = 0.3789). Mean day of fi rst isolation was 31.1 ± 2.1 (median 28.0). The number of isolations per site were – blood: 11 (median day: 25.0), tracheal aspirates: 9 (median: 28.5), catheter tip: 15 (median: 31.0), urine: 6 (median: 32.0), wound: 12 (median: 25.0), biological fl uids: 4 (median: 29.5), other catheters: 1 (median: 28.0), middle ear: 2 (median: 32.5), and nose: 1 (median: 25.0). Six patients had only one site isolation and the remaining 15 had multiple sites of P. stuartii isolation. We totally recorded 63 isolates of which 82.5% were second-generation and third-generation cephalosporin-resistant, 80.3% aztreonam-resistant and 81% carbapenem-resistant strains expressing an extended-spectrum β-lactamase phenotype. All patients had previously received colistin or meropenem or tygecycline for a median period of 18.5, 15 and 10.5 days, respectively. Conclusion In critically ill patients, HD TGC use seems to be safe and, combined with other active antibiotics, may increase the rate of MDR germ VAP clinical success. IIAT and the severity degree of patients’ clinical condition still remain major determinants of VAP treatment failure. In vitro effi ciency of Amikacin Inhale, a novel drug-device delivery system y N Kadrichu1, S Boc1, K Corkery1, P Challoner2 1Novartis Pharmaceuticals, San Carlos, CA, USA; 2Nektar Therapeutics, San Francisco, CA, USA Critical Care 2013, 17(Suppl 2):P81 (doi: 10.1186/cc12019) Introduction Amikacin Inhale (NKTR-061, BAY41-6551) is a drug- device combination in clinical development for adjunctive treatment of intubated and mechanically ventilated patients with Gram-negative pneumonia. The product uses a proprietary vibrating mesh nebulizer system (PDDS Clinical) with amikacin sulfate formulated for inhalation (3.2 ml of 125 mg/ml amikacin solution) for a 10-day twice-daily course of therapy. It is designed for use with two delivery systems: one system for intubated patients (On-vent; Figure 1), and a second Handheld (HH) system for patients who are extubated before completing the course of therapy (Figure 2). We investigated in vitro the amikacin lung dose delivered by PDDS Clinical. Conclusion P. stuartii infection/colonization may develop in critically ill patients, especially those with multiple trauma and prolonged ICU stay, and maybe isolated in blood after the 25th day of ICU stay. Also, infections due to extended-spectrum β-lactamase-producing multidrug-resistant P. stuartii are an emerging problem. P80 High-dose tigecycline use in severe infections G De Pascale1, L Montini1, T Spanu2, V Bernini1, A Occhionero1, DL Grieco1, M Biancone1, P De Santis1, ES Tanzarella1, SL Cutuli1, MA Pennisi1, MA Antonelli1 1Sacro Cuore Catholic University, Rome, Italy; 2Institute of Microbiology, Sacro Cuore Catholic University, Rome, Italy Critical Care 2013, 17(Suppl 2):P80 (doi: 10.1186/cc12018) P80 High-dose tigecycline use in severe infections G D P l 1 L M i i1 T S 2 V B i i1 A O Methods An estimated lung dose (ELD) for On-vent setting was measured in vitro after collecting aerosolized amikacin from a fi lter at the end of an endotracheal tube during ventilation. The ELD for the HH device was calculated from the fi ne particle fraction (FPF <5 μm) post- mouthpiece, multiplied by the in vitro delivered dose post-mouthpiece. FPF <5 μm refl ects lung deposition observed during phase 2 clinical trials [1]. Eighty-one nebulizers with volume median diameter (VMD) Introduction The aim of this study is to describe the clinical and epidemiological profi le of ICU patients receiving tigecycline (TGC) and to evaluate the potential benefi ts of TGC higher doses. Figure 1 (abstract P81). On-vent system with brown blinder shell for clinical trials. i Methods All patients admitted to our ICU between 1 June 2009 and 31 May 2012 who received TGC were evaluated. Cases were excluded when infections were not microbiologically confi rmed.i i Results Over the study period, 100 patients fulfi lled the inclusion criteria: 54 in the SD group (50 mg every 12 hours) and 46 in the HD group (100 mg every 12 hours). The SD group and the HD group were not signifi cantly diff erent in terms of age, severity of disease, duration of TGC therapy, rate of concomitant other active antibiotic use and of inadequate empirical antimicrobial therapy (IIAT) (P  = NS). MDR A. baumannii and K. pneumoniae were the main pathogens isolated. The percentage of germs other than A. baumannii and K. pneumoniae was higher in the SD TGC group (P <0.01). Otherwise infections due to less susceptible germs (TGC MIC value ≥1 μg/ml) were mainly treated with TGC higher doses (P <0.01). No signifi cant diff erences were found in terms of ICU mortality (P  = 0.8). The rate of abnormal laboratory measures during TGC treatment was similar between the two groups (P = NS). No patients required TGC discontinuation or dose reduction because of suspected adverse events. In the VAP subpopulation Figure 1 (abstract P81). On-vent system with brown blinder shell for clinical trials. Critical Care 2013, Volume 17 Suppl 2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 S30 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 parameters infl uencing the relation between amikacin dose and peak concentration. Figure 2 (abstract P81). Handheld system with brown blinder shell for clinical trials. Cost implication of antibiotic-associated diarrhoea and the fi nancial impact of probiotic use in its prevention p p p C Ebm, M Cecconi, C Moran, A Rhodes, T Rahman C Ebm, M Cecconi, C Moran, A Rhodes, St Georges Healthcare Trust, London, UK St Georges Healthcare Trust, London, UK Reference g Critical Care 2013, 17(Suppl 2):P83 (doi: 10.1186/cc12021) 1. Fink J, et al.: High in vivo amikacin lung deposition after NKTR-061 dosing correlates with in vitro aerosol characterization. Am J Respir Crit Care Med 2008, 177:A530. Introduction Antibiotic-associated diarrhoea (AAD) occurs in as many as 30% of patients receiving antibiotics, often leading to increased morbidity, prolonged in-hospital stay and additional healthcare resource utilisation. Age, antibiotics and prolonged postoperative ward and ICU stay have been suggested to be independent risk factors. In such patient populations, probiotics may be used to prevent antibiotic- associated diarrhoea, yet they are not routinely recommended as a component of perioperative care. The aim of this study was to model the long-term costs associated with AAD and to assess the eff ectiveness of probiotics as a preventive strategy. P80 High-dose tigecycline use in severe infections G D P l 1 L M i i1 T S 2 V B i i1 A O Methods In this retrospective study we looked at 47 patients (128 peak levels) between 2003 and 2012. Multivariate linear regression analysis was done for several parameters: administered dose calculated with total body weight, ideal body weight, adjusted body weight, type of intensive care patient, BMI, daily fl uid balance, SOFA score and APACHE score, and patient characteristics were analyzed. Results A linear correlation between dose and amikacin peak level was confi rmed (Figure 1). A total 54.69% of all amikacin administrations did not result in a therapeutic peak level. The multivariate linear regression analysis showed the best linear correlation with adjusted body weight and SOFA score. The comparison of variables between four patient groups, based on the deviation between measured peak level and predicted peak level (according the linear correlation), showed new variables that may infl uence peak level. yl Conclusion This confi rms that low doses (<18 mg/kg) of amikacin in intensive care patients seldom result in a therapeutic peak level. The proposed loading dose of 25 mg/kg is good for reaching a therapeutic level, although 29.6% remains subtherapeutic. Due to the linear correlation, more therapeutic levels may be reached with higher doses (25 to 30 mg/kg). New variables need further investigation to explain the high variability in achieved peak level. Figure 2 (abstract P81). Handheld system with brown blinder shell for clinical trials. of 4.4 ± 0.5 μm and output rates of 0.23 ± 0.10 ml/minute were tested for each system. Delivered dose data were fi t to the independent variables (that is, VMD and output rates) using a least-squares fi t with 95% confi dence limits. P83 y y Conclusion The results support the use of the PDDS Clinical with either system to administer aerosolized amikacin with high effi ciency and no dose adjustment is required when switching from the On-vent to the HH system for extubated patients. References References 1. Delattre IK, et al.: Ther Drug Monit 2010, 32:749-756. 2. Taccone FS, et al.: Crit Care 2010, 14:R53. 3. Gálvez R, et al.: Int J Antimicrobial Agents 2011, 38:146-151. 4. Avent ML, et al.: Internal Med J 2011, 41:441-449. 5. Fernández de Gatta MM, et al.: J Clin Pharm Ther 1996, 21:417-421. 1. Delattre IK, et al.: Ther Drug Monit 2010, 32:749-756. 2. Taccone FS, et al.: Crit Care 2010, 14:R53. 3. Gálvez R, et al.: Int J Antimicrobial Agents 2011, 38:146-151. 4. Avent ML, et al.: Internal Med J 2011, 41:441-449. 5. Fernández de Gatta MM, et al.: J Clin Pharm Ther 1996, 21:417-421. 1. Delattre IK, et al.: Ther Drug Monit 2010, 32:749-756. 2. Taccone FS, et al.: Crit Care 2010, 14:R53. 2. Taccone FS, et al.: Crit Care 2010, 14:R53. 3. Gálvez R, et al.: Int J Antimicrobial Agents 2011, 38:146-151. i Results Total percentage recoveries for On-vent and HH test runs were between 85% and 115% of the nominal dose. The mean ELDs were 50 ± 9% (On-vent) and 49 ± 11% (HH) of the nominal dose. Nebulizers with longer dosing times and lower VMDs had higher ELD values for both delivery systems. 4. Avent ML, et al.: Internal Med J 2011, 41:441-449. 4. Avent ML, et al.: Internal Med J 2011, 41:441-449. 5 Fernández de Gatta MM et al : J Clin Pharm Ther 1996 21:417 42 5. Fernández de Gatta MM, et al.: J Clin Pharm Ther 1996, 21:417-42 Empirical versus preemptive antimycotic therapy in terms of outcome benefi t l d l Figure 1 (abstract P85). Cologne antifungal strategy. Figure 1 (abstract P85). Cologne antifungal strategy. Introduction Novel treatment strategies for invasive candidiasis (IC) are constantly emerging. Nevertheless, diffi culties in diagnosis pose a challenge on their reliability, effi cacy and safety. We have previously developed and approbated in our ICU an algorithm for empirical antimycotic therapy, combining the most signifi cant risk factors for IC with three major clinical criteria for persistent nonbacterial sepsis [1]. On the other hand, preemptive therapy, based on identifi cation of mycotic antigens and/or anti-mycotic antibodies in serum, is regarded as more reliable, even though it is known for its low sensitivity. The aim of the current study was to compare and evaluate the possible outcome benefi t of our protocol implementation versus detection of galactomanan in patient’s serum as a trigger for antimycotic treatment initiation. Results Mean age was 67.4  years with 64% male patients. Most patients had combined CABG and valve procedure (n  = 20), other groups were HTX and LTX (n = 4), assist therapy (n = 4), TAVI (n = 3) and other procedures. Mean predicted mortality using the logarithmic CASUS score at the onset of IFI was 59%. C. albicans was isolated in 73%, C. glabrata in 21%. Length of antifungal treatment using micafungin in 30 cases was 14 ± 5 days. Eradication of yeast was successful in 79% but mortality of all patients remains high at 36.8% but was lower than predicted in the CASUS score. Mortality was not yeast related. Methods A randomized prospective controlled trial was carried out from September 2010 to September 2012. After the implication of the inclusion and exclusion criteria, patients were submitted to block randomization and stratifi ed on the basis of their initial SAPS II exp score. Antimycotic therapy was started on the day of inclusion in the control group and only with positive galactomanan serum test in the preemptive therapy group. Initial data were gathered on demographics, proven risk factors for IC-related mortality, severity of infl ammatory response and organ dysfunction. Dynamics of SIRS and SOFA values, Candida colonization index, ventilator-free days, length of ICU stay and outcome were followed for each patient.i p y y Conclusion Our described treatment approach shows encouraging results for the treatment of IFI especially in high-risk cardiothoracic patients. Results A total of 106 patients were enrolled. Empirical versus preemptive antimycotic therapy in terms of outcome benefi t l d l No statistically signifi cant diff erences in their basal characteristics were found. The subsequent SIRS and SOFA scores showed fi rm dynamics in the control group, although the new organ dysfunction severity was insignifi cantly lower. The length of ICU stay and the number of ventilator-free days were comparable. The in-hospital mortality was 47.1% in the preemptive therapy group versus 31.3% in the control group (P = 0.94). A total of seven adverse reactions were observed among treated patients, yet not associated with higher mortality risk. Analysis of parameters in critically ill patients infl uencing amikacin peak levels p M Hollevoet1, SJ Toye2, J De Waele1 p IM Hollevoet1, SJ Toye2, J De Waele1 Introduction Recent studies demonstrate that a loading dose of 25  mg/kg (total body weight) of amikacin in septic patients is required to reach a suffi cient peak concentration. This study examines Methods We developed a simulation model to determine clinical costs and outcomes attributable to AAD. To assess the cost-eff ectiveness of probiotics, as part of a perioperative regime, we constructed a decision Figure 1 (abstract P82). Correlation between dose of amikacin and peak concentration. Figure 1 (abstract P82). Correlation between dose of amikacin and peak concentration. S31 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 tree. The model observes long-term costs and outcomes of probiotics as compared with conventional therapy, from a societal perspective. Input parameters, extracted from meta-analysis, clinical trials and national databases, include incidence numbers, costs and quality- adjusted health states for the remaining life (QALYs). Outcomes assessed were overall costs attributable to ADD and the cost-eff ectiveness of probiotics, described as costs/QALY. References . Eggiman et al.: Invasive Candida infections in the ICU. Mycoses 20 1. Eggiman et al.: Invasive Candida infections in the ICU. Mycoses 2012, 55(Suppl):65-72. 55(Suppl):65-72. 2. Andes et al.: Impact of treatment strategy on outcomes in patients with candidemia and other forms of invasive candidiasis: a patient-level quantitative review of randomized trials. Clin Infect Dis 2012, 54:1110-1122. P86 Conclusion The choice of empirical versus preemptive therapy led to earlier and more stable reduction in the degree of organ dysfunction severity. It showed to be at least not inferior if not equal; in terms of survival benefi t and expediency of treatment. Moreover, galactomanan detection fails to guide the choice of the individual antimycotic, based on the expected Candida spp. Impact of echinocandins as fi rst-choice therapy in cardiothoracic high-risk patients with candidemia Impact of echinocandins as fi rst-choice therapy in cardiothoracic high-risk patients with candidemia g p G Langebartels, Y Choi, J Catteleans, T Wahlers University of Cologne, Germany Critical Care 2013, 17(Suppl 2):P85 (doi: 10.1186/cc12023) g p G Langebartels, Y Choi, J Catteleans, T Wahlers University of Cologne, Germany Critical Care 2013, 17(Suppl 2):P85 (doi: 10.1186/cc12023) Introduction Invasive candidemia is a major cause of increased mortality among ICU patients. Antifungal agents like liposomale amphotericin B and azoles could not accomplish the claim to be fi rst choice in the treatment of invasive fungal infection (IFI) because of side eff ects and eff ectiveness. Especially, cardiothoracic surgery patients as a group of high-risk patients are in a focus for new strategies and agents. A new class of antimycotic agents, the echinocandins, with a low profi le of side eff ects, low interactive potential and high eff ectiveness in the treatment of candidemia, is a powerful option in the treatment of IFI. We report our single-center experience with a modifi ed clinical treatment approach based on clinical score of Leon and using echinocandins as fi rst-line therapy for proven and suspected fungal infection. Results Our results indicate an estimated incremental lifetime cost of £13,272.53 per ADD patient, largely driven by increased ICU length of stay and readmission rates. The addition of probiotics to the standard perioperative regime is associated with a small survival benefi t of 1.2 months, yet a cost reduction of £917.3/ADD patient. The main cost was increased duration of ICU stay and readmissions, which contribute to 85% of total expenses. p Conclusion AAD is associated with a signifi cant increase in costs from a societal perspective. The provision of probiotics can achieve substantial cost savings and can be recommended as a cost-eff ective regime in the perioperative setting. Preventing ADD off ers a potentially signifi cant reduction of in-hospital costs and resource expenditures. g Methods From May 2011 to October 2012, 2,844 patients were treated on our cardiothoracic ICU. We evaluated 37 cardiothoracic postoperative patients with proven or suspected IFI or prophylaxis (Figure 1). The records were evaluated for cardiothoracic procedures, microbiological and yeast date, cardiothoracic surgery score (CASUS), ICU and clinical data. P84 Empirical versus preemptive antimycotic therapy in terms of outcome benefi t S Milanov, VT Todorova, G Georgiev, M Milanov Emergency Hospital ‘Pirogov’, Sofi a, Bulgaria Critical Care 2013, 17(Suppl 2):P84 (doi: 10.1186/cc12022) Cost-eff ectiveness analysis of anidulafungin in the treatment of candidaemia g Results A total of 2,426 patients were admitted to Heart of England NHS Foundation Trusts ICUs during the study period. Of these, 31 patients were identifi ed as having an IFI (1.3%). Baseline demographic data were similar between groups. Patients with IFI had signifi cantly longer ICU length of stay (19 days vs. 5 days, P <0.0001) and required more days of advanced organ support (12 days vs. 3 days, P <0.0001). A trend towards higher hospital mortality (41% vs. 27%, P = 0.08) was observed. Candida albicans was the predominant organism cultured (64%), followed by Candida glabrata (23%), with other Candida species accounting for the remaining 13% of IFIs. Sites of IFIs were blood (45%), intra-abdominal (39%), and pleural (16%). Most patients (52%) who acquired an IFI had had intra-abdominal surgery prior to ICU admission. The majority of patients (71%) were treated with echinocandins, whilst of the nine patients who were initially treated with fl ucanazole, six (67%) required therapy escalation to an echinocandin. G Auzinger1, G Playford2, C Graham3, H Narula3, C Charbonneau4, D Weinstein4, M Kantecki4, H Schlamm5, M Ruhnke6 1King’s College Hospital, London, UK; 2Princess Alexandra Hospital, Brisbane, Australia; 3RTI Health Solutions, Durham, NC, USA; 4Pfi zer International Operations, Paris, France; 5Pfi zer Inc., New York, NY, USA; 6Charité University Medicine, Berlin, Germany Critical Care 2013, 17(Suppl 2):P87 (doi: 10.1186/cc12025) G Auzinger1, G Playford2, C Graham3, H Narula3, C Charbonnea D Weinstein4, M Kantecki4, H Schlamm5, M Ruhnke6 1King’s College Hospital, London, UK; 2Princess Alexandra Hospital, Brisbane, Australia; 3RTI Health Solutions, Durham, NC, USA; 4Pfi zer International Operations, Paris, France; 5Pfi zer Inc., New York, NY, USA; 6Charité University Medicine, Berlin, Germany y Critical Care 2013, 17(Suppl 2):P87 (doi: 10.1186/cc12025) Critical Care 2013, 17(Suppl 2):P87 (doi: 10.1186/cc12025) Introduction Echinocandins are recommended fi rst-line treatment for candidaemia [1]. A cost-eff ectiveness model developed from a UK perspective examined costs and outcomes of antifungal treatment for candidaemia and other forms of invasive candidiasis based on European clinical guidelines [1]. Methods Costs and treatment outcomes with the echinocandin anidula fungin were compared with caspofungin, micafungin, fl uconazole, voriconazole and amphotericin B. The model included non-neutropenic patients aged ≥16 years with confi rmed candidaemia/ another form of invasive candidiasis receiving intravenous fi rst-line treatment [2]. Fungal ventricular assist device infections occur in colonized patients and are associated with high mortality rate Fungal colonization (FC), infection and outcome during VAD support n FC Mortality No IFI 24 12 9 IFI 10 9 8 P value 0.029 0.024 Conclusion In our center, we observed a high incidence of IFI in IC patients with VAD that was associated with a mortality rate of 80% Screening of fungal colonization appears to be very important durin the ICU stay for VAD patients. Trials are needed for investigating th use, the drug choice and the timing of antifungal prophylaxis for suc high-risk patients. Reference 1. Aslam S, et al.: Clin Infect Dis 2010, 50:664-671. P87 were all-cause 6-week mortality, cost of treatment-related adverse events (AEs) and other medical resource use costs. Life-years were calculated using a published model [4]. Antifungal agent-related AEs were taken from the product label/literature. Resource use was derived from the literature and discussion with clinical experts. Drug acquisition/ administration costs were taken from standard UK costing sources. with fungi [1]. The relationship between colonization and invasive fungal infection (IFI) in severely ill ICU patients with a VAD support is not described. This study analyzes the incidence and outcome of fungal infection and colonization in VAD patients in bridge to transplantation or in destination therapy. py Methods We conducted a retrospective review of all VAD implantations in our surgical ICU between 2007 and 2012. The incidence of fungal colonization, antifungal prophylaxis, bacterial sepsis and the mortality of IFI versus no IFI patients were compared. Results First-line anidulafungin for treatment of candidaemia was cost-eff ective per life-year gained versus fl uconazole (incremental cost-eff ectiveness ratio £813). Anidulafungin was cost saving versus caspofungin and micafungin in terms of life-years gained due to lower ICU costs and a higher rate of survival combined with a higher probability of clinical success.f Results In the study period, 34 patients with severe heart failure or cardiogenic shock were selected for a VAD implantation (nine in destination therapy). The overall mortality rate was 50% during mechanical assistance. Confi rmed (n = 8) and highly suspected (n = 2) IFI occurred during the ICU stay in 29% of patients who were treated with echinocandins, voriconazole and/or liposomal amphotericin B. The isolated fungi were: six Candida albicans, two parapsilosis, one glabrata and one invasive pulmonary aspergillosis. Antifungal prophylaxis with fl uconazole was administered to 18% of patients at mean for 5 days mainly in the more recent implantations. Allou N, et al.: Curr Infect Dis Rep 2011, 13:426-432. Zaragosa R, et al.: Adv Sepsis 2008, 6:90-98. Fungal ventricular assist device infections occur in colonized patients and are associated with high mortality rate In the no IFI population, 54% (n = 13) had a systemic or VAD bacterial sepsis with a mortality rate about 54%. The mortality without any sepsis was reduced to 18%. Fungal colonization was signifi cantly more present (90% vs. 50%) before IFI in VAD patients. The mortality rate was dramatically higher with IFI (80% vs. 38%) in accordance with the literature [1]. See Table 1. y Conclusion Anidulafungin was cost-eff ective compared with fl uconazole for treatment of candidaemia and was cost saving versus other echinocandins in the UK. European guidelines recommend echinocandins as fi rst-line treatments for candidaemia [1]; this model indicates that anidulafungin marries clinical eff ectiveness and cost-eff ectiveness. f References 1. Cornely OA: Clin Microbiol Infect 2012, 18:19-37. 2. Reboli AC, et al.: N Engl J Med 2007, 356:2472-2482. 3. Mills EJ, et al.: Ann Clin Microbiol Antimicrob 2009, 8:23. 4. Sidhu MK, et al.: Curr Med Res Opin 2009, 25:2049-2059 2. Reboli AC, et al.: N Engl J Med 2007, 356:2472 2482. 3. Mills EJ, et al.: Ann Clin Microbiol Antimicrob 2009, 8:23. 4. Sidhu MK, et al.: Curr Med Res Opin 2009, 25:2049-2059 Table 1 (abstract P86). Fungal colonization (FC), infection and outcome during VAD support Table 1 (abstract P86). Fungal colonization (FC), infection and outcome during VAD support n FC Mortality No IFI 24 12 9 IFI 10 9 8 P value 0.029 0.024 Reference 1. Aslam S, et al.: Clin Infect Dis 2010, 50:664-671. Cost-eff ectiveness analysis of anidulafungin in the treatment of candidaemia Patients were categorised as a clinical success or failure (patients with persistent/breakthrough infection); frequency data for each outcome were taken from a mixed-treatment comparison [3]. Successfully treated patients switched to oral therapy. Clinical failures switched to a diff erent antifungal class. It was assumed that second-line treatment duration was equivalent to that of fi rst-line treatment and only two lines of therapy were required to treat infection. Other inputs Conclusion The results of our study are consistent with other published data, in that whilst IFI prevalence is low, they are associated with increased morbidity in critically ill patients. This study has led to a change in hospital policy regarding antifungal use in the ICU, with echinocandins being fi rst-line in the pre-emptive treatment of IFI. We keenly await the results of the FIRE study, which will provide important insights to identifi cation of patients at risk of IFIs and optimal drug therapy. Prevalence and impact of invasive fungal infections in intensive care JM Patel K Couper T Melody R O’Brien D Parekh Introduction Invasive fungal infections (IFI) aff ect 1% of ICU patients and are increasing in incidence. IFIs are associated with a poor prognosis, which is further complicated by diffi culties in identifi cation of fungal organisms by traditional culture methods and the emergence of Candida species resistant to triazole therapy [1,2]. This study aimed to assess the prevalence of IFIs, the organisms responsible and outcomes of patients aff ected. Conclusion In our center, we observed a high incidence of IFI in ICU patients with VAD that was associated with a mortality rate of 80%. Screening of fungal colonization appears to be very important during the ICU stay for VAD patients. Trials are needed for investigating the use, the drug choice and the timing of antifungal prophylaxis for such high-risk patients. R f f Methods Patients admitted to the Heart of England NHS Foundation Trust ICUs who acquired an IFI were identifi ed through the hospital Fungal Infection Risk Evaluation (FIRE) study database. All ICU patients admitted between October 2009 and March 2011 were used as a comparative cohort. Data collected included: baseline demographics, length of stay, ICU and hospital mortality, and nature of IFI. Data were analysed using Student’s t test for continuous data and Fischer’s exact test for categorical data. Fungal ventricular assist device infections occur in colonized patients and are associated with high mortality rate P Gaudard, J Eliet, N Zeroual, G Culas, P Colson CHRU Montpellier, France Critical Care 2013, 17(Suppl 2):P86 (doi: 10.1186/cc12024) Referencefi Introduction Infection is a common complication of the ventricular assist device (VAD) and is associated with poor outcome especially 1. S Milanov, et al.: Effi cacy and utility of a protocol for pre-emptive antimycotic therapy. Crit Care 2010, 14(Suppl 1):P70. 1. S Milanov, et al.: Effi cacy and utility of a protocol for pre-emptive antimycotic therapy. Crit Care 2010, 14(Suppl 1):P70. Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 S32 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 with fungi [1]. The relationship between colonization and invasiv fungal infection (IFI) in severely ill ICU patients with a VAD support not described. This study analyzes the incidence and outcome of fung infection and colonization in VAD patients in bridge to transplantatio or in destination therapy. Methods We conducted a retrospective review of all VAD implantation in our surgical ICU between 2007 and 2012. The incidence of fung colonization, antifungal prophylaxis, bacterial sepsis and the mortali of IFI versus no IFI patients were compared. Results In the study period, 34 patients with severe heart failu or cardiogenic shock were selected for a VAD implantation (nin in destination therapy). The overall mortality rate was 50% durin mechanical assistance. Confi rmed (n = 8) and highly suspected (n = IFI occurred during the ICU stay in 29% of patients who were treate with echinocandins, voriconazole and/or liposomal amphotericin B. Th isolated fungi were: six Candida albicans, two parapsilosis, one glabra and one invasive pulmonary aspergillosis. Antifungal prophylaxis wi fl uconazole was administered to 18% of patients at mean for 5 da mainly in the more recent implantations. In the no IFI populatio 54% (n = 13) had a systemic or VAD bacterial sepsis with a mortali rate about 54%. The mortality without any sepsis was reduced to 18% Fungal colonization was signifi cantly more present (90% vs. 50% before IFI in VAD patients. The mortality rate was dramatically high with IFI (80% vs. 38%) in accordance with the literature [1]. See Table  Table 1 (abstract P86). References Allou N, et al.: Curr Infect Dis Rep 2011, 13:426-432. Zaragosa R, et al.: Adv Sepsis 2008, 6:90-98. S33 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 P89 Beliefs and actual practice of oxygen therapy in the ICU HJ Helmerhorst1, MJ Schultz2, PH Van der Voort3, E De Jonge1, DJ Van Westerloo1 1Leiden University Medical Center, Leiden, the Netherlands; 2Academic Medical Center, Amsterdam, the Netherlands; 3Onze Lieve Vrouwe Gasthuis, Amsterdam, the Netherlands Critical Care 2013, 17(Suppl 2):P89 (doi: 10.1186/cc12027) P90 FiO2/PEEP index: a simple tool for opitimizing ventilator settings D Trasy, M Nemeth, K Kiss, Z Till, Z Molnar University of Szeged, Hungary Critical Care 2013, 17(Suppl 2):P90 (doi: 10.1186/cc12028) Introduction During mechanical ventilation, oxygenation can be infl uenced by adjusting FiO2 and positive end-expiratory pressure (PEEP). There have been recommendations for how the FiO2 and PEEP should be set [1]. However, in a recent audit we found that the compliance of doctors of these recommendations is very low [2]. Therefore we invented a simple parameter called the FiO2/PEEP index (FPi) of which the physiologic value is ≤7 (that is, FiO2 = 21%/PEEP = 3 cmH2O), which corresponds to the ARDSNet trial’s minimum FiO2/ PEEP settings: 35%/5 cmH2O [2]. The aim of this case–control study was to investigate the impact of an FPi ≤7 targeted protocol on clinical practice. Introduction The aim of this study was to compare self-reported beliefs with actual clinical practice of oxygen therapy in the ICU. Hyperoxia is frequently encountered in ventilated patients and prolonged exposure has repeatedly been shown to induce lung injury and (systemic) toxicity. y Methods An online questionnaire for ICU clinicians was conducted to investigate beliefs and motives regarding oxygen therapy for critically ill patients. Furthermore, arterial blood gas (ABG) samples and corresponding ventilator settings were retrieved to retrospectively assess objective oxygenation between 1 April 2011 and 31 March 2012 in the ICUs of three teaching hospitals in the Netherlands. p Methods A prospective observational study in 2010 and 2012. Every mechanically ventilated patient was recruited. Demographics, outcome data and Lung Injury Score (LIS) were recorded after admission. Respiratory settings, oxygenation and ventilation parameters were recorded after mechanical ventilation was commenced and the fi rst arterial blood gas sample was taken (T0). Measurements were repeated in 24 hours (T1). Data are presented as mean ± SD, paired-sample and independent-sample t test and chi-square tests were used for statistics. References Results In 2010, 75 patients, and in 2012, 130 patients were included. There was no diff erence in demographics, disease severity, but LIS was higher in 2012: 1.34 ± 1.13 versus 0.84 ± 0.98, P = 0.001. There was no signifi cant diff erence in FPi between the two groups at T0: 10.91 ± 4.25 versus 10.26 ± 5.01 (2012 vs. 2010, respectively). At T24 the FPi was signifi cantly lower in 2012 as compared with 2010: 7.28 ± 2.58 versus 8.17 ± 3.3, P = 0.001; which was due to the higher PEEP applied: 7.08 ± 2.87 versus 6.63 ± 2.91, P = 0.014. Although in 2012 signifi cantly more patients, 112 (86%), were ventilated with FiO2 ≥50% at T0 as compared with 2010, 44 (58%) (P  = 0.001), by T24 signifi cantly less patients received FiO2 ≥50%, 46 (35%) vs. 34 (45%) (P = 0.011). There was no signifi cant diff erence between the two groups regarding FiO2, PaO2 and PaCO2 at T0 and T24.i Results Analyzable questionnaire responses were received from 200 ICU physicians and nurses. The majority of respondents believed that oxygen-induced lung injury is a concern, although barotrauma and volutrauma are generally considered to impose a greater risk in mechanical ventilation. Frequently allowed minimal saturation ranges in the questionnaire were 85 to 95% and 7 to 10 kPa (Figure 1). Self- reported FiO2 adjustment in hypothetical patient cases with variable saturation levels was moderately impacted by the underlying clinical condition. To study actual clinical practice, a total of 107,888 ABG samples with corresponding ventilator settings, covering 5,565 patient admissions, were retrieved. Analysis showed a median (IQR) PaO2 of 11.7 kPa (9.9 to 14.3), median FiO2 was 0.4 (0.4 to 0.5), median PEEP was 5 (5 to 8). A total 63.5% of all PaO2 registries were higher than previously suggested oxygenation goals (7.3 to 10.7 kPa) [1]. In 56.8% of cases with PaO2 higher than the target range, neither FiO2 nor PEEP levels had been lowered when the next ABG sample was taken. Figure 1 (abstract P89). Conclusion Most clinicians acknowledge the detrimental eff ects of prolonged exposure to hyperoxia in the ICU and report a low tolerance for high saturation levels. 1. Brower RG, et al.: N Engl J Med 2004, 351:327-336. References However, the self-reported intention for conservative oxygen therapy is not consistently expressed in our objective data of actual clinical practice and a large proportion of patients was exposed to high and potentially toxic oxygen levels. Reference 1. Brower RG, et al.: N Engl J Med 2004, 351:327-336. Figure 1 (abstract P89). 2 2 Conclusion Implementing an FPi ≤7-based algorithm signifi cantly reduced the FiO2 and increased the PEEP applied in mechanically ventilated within the fi rst 24 hours. Whether this has any impact on earlier weaning due to reaching the weaning criteria of FiO2 sooner, and as a result shortening the duration of mechanical ventilation, has to be investigated in the future. g References References 1. ARDSNet: N Engl J Med 2000, 342:1301. 2. Kiss K, et al.: Intensive Care Med 2011, 37(Suppl 1):S195. 1. ARDSNet: N Engl J Med 2000, 342:1301. 1. ARDSNet: N Engl J Med 2000, 342:1301. 2. Kiss K, et al.: Intensive Care Med 2011, 37(Suppl 1):S195. g , 2. Kiss K, et al.: Intensive Care Med 2011, 37(Suppl 1):S195. 2. Kiss K, et al.: Intensive Care Med 2011, 37(Suppl 1):S195. P92 9 Relation between PaO2/FiO2 ratio, SpO2/FiO2 ratio, oxygenation index and ventilation ratio in critically ill patients T Aslanidis, A Myrou, E Chytas, E Anastasiou, E Geka, E Efthimiou, V Ourailoglou, I Soultati, S Primikiri, M Giannakou-Peftoulidou A.H.E.P.A. University Hospital, Thessaloniki, Greece Critical Care 2013, 17(Suppl 2):P92 (doi: 10.1186/cc12030) Relation between PaO2/FiO2 ratio, SpO2/FiO2 ratio, oxygenation index and ventilation ratio in critically ill patients y A Batchinsky1, C Necsoiu1, T Langer2, V Vecchi2, W Baker1, J Salinas1, L Cancio1 1US Army Institute of Surgical Research, Fort Sam Houston, TX, USA; 2University of Milan, Italy Critical Care 2013, 17(Suppl 2):P93 (doi: 10.1186/cc12031) A Batchinsky1, C Necsoiu1, T Langer2, V Vecchi2, W Baker1, J Salinas1, L Cancio1 1US Army Institute of Surgical Research, Fort Sam Houston, TX, USA; 2University of Milan, Italy Critical Care 2013, 17(Suppl 2):P93 (doi: 10.1186/cc12031) Introduction Respiratory complexity (RC) as assessed by sample entropy (SampEn) is lower in patients failing spontaneous breathing trials. We evaluated the role of RC in monitoring subjects with severe acute respiratory distress syndrome (ARDS) treated with the Cardiohelp (Maquet, Rastatt, Germany) extracorporeal life-support system (ECLS). We hypothesized that RC is reduced during apnea. Introduction Many authors have proposed less invasive ways to measure oxygenation in patients with ALI. The aim of the present study is to compare the already popular oxygenation index (OI) and PaO2/ FiO2 ratio (PFr) with other recently proposed indices such as SpO2/FiO2 ratio (SFr) and ventilation ratio (VR) in a mixed ICU population. Methods During a 6-month prospective observational study carried out in a polyvalent 10-bed adult ICU, ABGs were obtained from 145 patients. Two independent measurements were taken from each patient under the same mode of ventilation (SIMVPSV). PFr, SFr, VR and OI were calculated. Demographic data (APACHE II score, age, sex, yp g p Methods Six sheep sedated with midazolam were connected to the Cardiohelp via a 23F Avalon catheter. Blood fl ow was ~2  l/minute and FiO2 was 0.5. Sheep were on CPAP of 8 cmH2O with FiO2 of 1 via tracheostomy. P91 Use of high-fl ow nasal canulae: eff ect on alveolar pressure and its limitation limitation H Hayami, K Mizutani, M Shioda, S Takaki, H Maejima, K Ueno, Y Yamaguchi, T Kariya, T Gotoh Yokohama City University Hospital, Yokohama, Japan Critical Care 2013, 17(Suppl 2):P91 (doi: 10.1186/cc12029) Figure 1 (abstract P89). Introduction High-fl ow nasal canulae (HFNC) deliver high-fl ow humidifi ed gas at up to 60 l/minute. There are two types of respiratory circuit to generate mix gas fl ow, Blender type (typeB) and Venturi type (typeV). The therapy is well established in the pediatric population and HFNC use has been described in the adult population. It has been reported that HFNC provide higher FIO2 compared with low-fl ow canulae, and also create mild positive pharyngeal airway pressure, but the eff ect on alveolar pressure is unknown. We aimed to investigate the eff ect of HFNC on alveolar pressure, by measuring intratracheal pressure in patients with a cricothyrotomy catheter (CTC). At the same time, we measured the actual gas fl ow rate (AGFR) by fl owmeter and compared it with assumed fl ow. Conclusion Most clinicians acknowledge the detrimental eff ects of prolonged exposure to hyperoxia in the ICU and report a low tolerance for high saturation levels. However, the self-reported intention for conservative oxygen therapy is not consistently expressed in our objective data of actual clinical practice and a large proportion of patients was exposed to high and potentially toxic oxygen levels. Reference l Methods Seven patients with a CTC were participated. A tube was connected to the CTC and the tube was then connected to a pressure transducer to measure intratracheal pressure. The HFNC (Optifl ow S34 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 diagnosis, body weight) were also recorded. Kolmogorov–Smirnov test for normality was calculated followed by bivariate nonparametric analysis (statistical signifi cance: P <0.05). system) were applied with the humidifi er to optimize humidication. TypeB was used in three patients and typeV in four patients. The fl ow was started at 10  l/minute. This fl ow rate was titrated upwards to a maximum of 60 l/minute (10, 25, 30, 40, 50, 60 l/minute) and the AGFR was measured. Intratracheal pressure tracing was done over 1 minute. Airway pressure measurement was repeated and the maximal expiratory pressure was measured in mmHg. y gi Results A total of 290 measurements were included for further analysis. References References 1. Rice TW, et al.: Chest 2007, 132:410-417. 2. Sinha P, et al.: Br J Anaesth 2009, 102:692-697. 1. Rice TW, et al.: Chest 2007, 132:410-417. 1. Rice TW, et al.: Chest 2007, 132:410-417. 1. Rice TW, et al.: Chest 2007, 132:410-417. 2. Sinha P, et al.: Br J Anaesth 2009, 102:692-697. Conclusion HFNC are eff ective in providing higher expiratory pressure. It is important to know the fl ow rate is lower than expected when the Venturi type is used. 1. Rice TW, et al.: Chest 2007, 132:410-417. 2. Sinha P, et al.: Br J Anaesth 2009, 102:692-697. *Signifi cant diff erence versus Baseline. #Signifi cant diff erence between Preinjury and Postinjury apnea. P91 Use of high-fl ow nasal canulae: eff ect on alveolar pressure and its limitation Mean ± SD values for age and APACHE II score were 61.2 ± 16 years and 15.4 ± 1.8, respectively, while median ± SD values for PFr are 257 ± 135, for SFr 211  ±  58, for OI 4.8  ±  4.46 and for VR 1.19  ±  0.31. Relations between PFr, SFr and OI are displayed in Table 1. p y p g Results The AGFR in the respiratory circuit was almost same in typeB, but there was obvious decrease in the AGFR in typeV (7.1  ±  1.0, 17.7 ± 0.8, 21.9 ± 0.9, 29.9 ± 3.6, 36.9 ± 2.7, 45.0 ± 5.5 l/minute at assumed fl ow, 10, 25, 30, 40, 50, 60 l/minute, respectively). HFNC signifi cantly increased maximal expiratory pressure in both groups, 1.5  ±  2.1, 2.0 ± 1.0, 3.0 ± 2.8, 4.5 ± 3.5 mmHg for typeV and 2.5 ± 0.7, 5.8 ± 2.4, 6.0 ± 2.8, 8.0 ± 2.8 mmHg (maximum 10 mmHg) for typeB, when AGFR was set at 30, 40, 50, 60 l/minute. Higher AGFRs were found to result in larger increase in maximum expiratory pressure. The data indicate that HFNC are associated with an increase in intratracheal expiratory pressure. Because it was diffi cult to determine end-expiratory pressure, we chose maximal expiratory pressure for a substitute. The reason why AGFR in typeV was lower than assumed fl ow may be the resistance generated by NC. The larger increase in expiratory pressure in our study than previously reported may be due to the eff ect of high respiratory resistance of Japanese who have relatively small airway structure compared with western people.f Table 1 (abstract P92). Correlations (Spearman ρ) between VR, PFr, SFr and OI Table 1 (abstract P92). Correlations (Spearman ρ) between VR, PFr, SFr and OI PFr SFr OI Table 1 (abstract P92). Correlations (Spearman ρ) between VR, PFr, SFr and OI PFr SFr OI VR –0.326* –0.325* 0.373* PFr – 0.759* –0.939* SFr – – –0.773* *P <0.01. Conclusion Our study identifi ed a strong relation between PFr, SFr and OI but not VR. Thus, these markers may be used interchangeably as bedside indices of oxygenation in critically ill patients. Yet larger studies are needed to come to a safer conclusion. R f Acute respiratory distress syndrome – the Berlin defi nition: impact on an ICU of a university hospital Acute respiratory distress syndrome – the Berlin defi nition: impact on an ICU of a university hospital A Agrifoglio, J Lopez, J Figueira, M Hernández, L Fernández, M Irazabal, S Yus, M Jiménez Hospital Universitario La Paz, Madrid, Spain Critical Care 2013, 17(Suppl 2):P94 (doi: 10.1186/cc12032) y A Agrifoglio, J Lopez, J Figueira, M Hernández, L Fernández, M Irazabal, S Yus, M Jiménez Hospital Universitario La Paz, Madrid, Spain Critical Care 2013, 17(Suppl 2):P94 (doi: 10.1186/cc12032) g Results A weaning-induced pulmonary edema was diagnosed in 12 instances (PAOP signifi cantly increased from 15.6 ± 0.6 to 25.8 ± 0.9 in these cases). EVLWI, BNP, plasma protein and hemoglobin concentrations signifi cantly increased in these instances (28.3 ± 5.7%, 20.2 ± 7.8%, 9.6 ± 0.8% and 9.3 ± 1.3%, respectively) while they did not signifi cantly changed in cases without weaning-induced pulmonary edema. The increase of EVLWI ≥8.5% (+1.5  ml/kg), an increase in BNP ≥6.7% (+23 pg/ml), an increase in plasma protein concentration ≥5% and in hemoglobin concentration ≥5% exhibited good areas under the ROC curves to predict weaning-induced pulmonary edema (0.97 ± 0.03, 0.80 ± 0.11, 1.0 ± 0.00 and 0.92 ± 0.05, respectively). These areas under the ROC curves were not statistically diff erent. The baseline values of EVLWI, BNP, plasma protein and hemoglobin concentrations did not predict weaning-induced pulmonary edema. Introduction The current defi nition of ARDS was established in 1994 by the AECC. In 2011 emerged the Berlin defi nition. The aim of this study was to fi nd a new approach from the impact of ARDS in an ICU and to analyze APACHE II score, days of mechanical ventilation (MV), duration of ARDS and mortality. y Methods We analyzed the database of our ICU collected prospectively from October 2008 to January 2011 and regarded all patients undergoing MV for 48 hours or more and who met the AECC criteria. All patients were ventilated according to the ARDSNet protocol. p g p Results During this period 1,900 patients were admitted, 607 were under MV for at least 48 hours and 104 had ARDS criteria. Moderate ARDS 60.6%: 60% male, median age 53 years, APACHE II mean 22.49 (95% CI = 20.71 to 24.27), mean duration MV 23.84 (95% CI = 18.08 to 29.60), ARDS duration 12.87 (95% CI = 9.83 to 15.90), average stay 29.22, mortality 52.4%. P92 Reference ICU admission have a longer duration of ARDS, which means more days on MV and therefore increased ICU stay. Reference of 0.1 ml/kg oleic acid. Apneas (defi ned as >60% reduction in minute ventilation) developed both before (Preinjury) and after ARDS (Postinjury). Heart rate (HR), mean arterial pressure (MAP), respiratory rate (RR) and tidal volume (TV) were recorded, and the PaO2:FiO2 ratio (PFR) calculated corresponding to sections of respiratory pressure waveforms containing 200 breaths during Baseline, during Preinjury and Postinjury apnea periods. Similar periods were selected before and after apneas. Inter-breath interval (IBI) and its deviation, SampEn, percentage of forbidden words (FW), bit per-word entropy (BPWEn), symbol distribution entropy (DisNEn), and stationarity (StatAv) were calculated using software. Statistics by one-way ANOVA with adjustment for multiple comparisons. 1. Ranieri M, et al.: JAMA 2012, 307:2526-2533. P95 Extravascular lung water, B-type natriuretic peptide and blood volume contraction for diagnosing weaning-induced pulmonary edema M Dres, JL Teboul, N Anguel, C Richard, X Monnet Service de réanimation médicale, Hôpital de Bicêtre, Le Kremlin Bicêtre, France Critical Care 2013, 17(Suppl 2):P95 (doi: 10.1186/cc12033) Extravascular lung water, B-type natriuretic peptide and blood volume contraction for diagnosing weaning-induced pulmonary edema M Dres, JL Teboul, N Anguel, C Richard, X Monnet Service de réanimation médicale, Hôpital de Bicêtre, Le Kremlin Bicêtre, France Critical Care 2013, 17(Suppl 2):P95 (doi: 10.1186/cc12033) M Dres, JL Teboul, N Anguel, C Richard, X Monnet Service de réanimation médicale, Hôpital de Bicêtre, Le Kremlin Bicêtre, France Critical Care 2013, 17(Suppl 2):P95 (doi: 10.1186/cc12033) Results See Table  1. SampEn was associated with PFR (r2  = 0.31, P <0.001). Introduction We compared three diff erent methods for diagnosing pulmonary edema induced by weaning from mechanical ventilation: the increase in extravascular lung water index (EVLWI), the increase in B-type natriuretic peptide (BNP) and blood volume contraction, refl ected by increases in plasma protein and in hemoglobin concen- trations, all observed during a spontaneous breathing trial (SBT).fi Conclusion Respiratory complexity during apnea and ECLS remains unchanged in a healthy state but decreases after ARDS. It has a signifi cant association with PFR and may serve as an index of injury severity. P92 After ~6 hours of ECLS, ARDS was induced by injection Table 1 (abstract P93) Preinjury Preinjury Preinjury Postinjury Postinjury Postinjury BL pre-apnea apnea post-apnea pre-apnea apnea post-apnea RR (breaths/minute) 22 ± 2 22 ± 3 17 ± 2 23 ± 4 24 ± 7 5 ± 1* 25 ± 7 TV (ml) 388 ± 31 217 ± 32 145 ± 14 205 ± 21 211 ± 51 116 ± 30* 229 ± 40 HR (beats/minute) 156 ± 15 153 ± 21 152 ± 21 166 ± 21 162 ± 22 165 ± 21 151 ± 16 MAP (mmHg) 102 ± 4.12 102 ± 6.13 103 ± 6.91 104 ± 6.08 106 ± 3.76 108 ± 3.86 111 ± 5.69 PFR (unitless) 435 ± 24.80 417 ± 48.06 354 ± 49.52 429 ± 37.87 139 ± 50.90* 92 ± 30.43*# 91 ± 27.25* IBI (ms) 2,765.1 ± 230.5 3,149.8 ± 378.0 5,151.7 ± 467.7 3,404.7 ± 663.0 2,253.2 ± 427.0 10,334.0 ± 1,739.2*# 2,162.0 ± 387.6 IBI Dev (ms) 771.8 ± 117.49 1,630.0 ± 230.64 4,505.7 ± 785.34 1,598.3 ± 292.45 1,404.8 ± 445.04 25,065.0 ± 11,480.37*# 844.6 ± 236.65 SampEn (unitless) 1.7 ± 0.12 1.6 ± 0.16 1.3 ± 0.13 1.6 ± 0.11 1.2 ± 0.14 0.5 ± 0.25*# 1.3 ± 0.20 FW (%) 29.9 ± 4.60 41.5 ± 7.43 41.8 ± 10.15 34.5 ± 6.10 44.0 ± 8.80 62.2 ± 4.16* 33.6 ± 5.26 BPwEn (unitless) 4.9 ± 0.18 4.3 ± 0.27 4.1 ± 0.37 4.6 ± 0.22 4.1 ± 0.42 2.7 ± 0.38*# 4.4 ± 0.24 DisNEn (unitless) 0.8 ± 0.03 0.7 ± 0.04 0.7 ± 0.06 0.8 ± 0.05 0.7 ± 0.07 0.4 ± 0.06*# 0.7 ± 0.04 StatAv (unitless) 0.42 ± 0.06 0.37 ± 0.09 0.37 ± 0.04 0.41 ± 0.10 0.31 ± 0.05 0.51 ± 0.07 0.42 ± 0.08 *Signifi cant diff erence versus Baseline. #Signifi cant diff erence between Preinjury and Postinjury apnea. S35 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 ICU admission have a longer duration of ARDS, which means more days on MV and therefore increased ICU stay. Reference 1. Ranieri M, et al.: JAMA 2012, 307:2526-2533. ICU admission have a longer duration of ARDS, which means more days on MV and therefore increased ICU stay. P94 Methods We included 12 diffi cult-to-wean patients (22 recordings). Before and at the end of a SBT (T tube), we measured pulmonary occlusion arterial pressure (PAOP), EVLWI (PiCCO device), BNP, plasma protein and hemoglobin concentrations. Weaning-induced pulmonary edema was confi rmed if a clinical intolerance to SBT was associated with an increase of PAOP >18 mmHg at the end of SBT. Acute respiratory distress syndrome – the Berlin defi nition: impact on an ICU of a university hospital Severe ARDS 39.4%: 63% male, median age 45 years, APACHE II mean score 23.19 (95% CI = 20.62 to 25.75), mean duration MV 18.52 (95% CI = 13.01 to 24.03), ARDS duration 12.15 (95% CI = 8.72 to 15.58), average stay 22.65, mortality 48.8% with no statistically signifi cant diff erence with moderate ARDS (P = 0.72). Leading cause of death in both groups: multiorgan dysfunction (60%). See Tables 1 and 2. Conclusion In this new approach of our ARDS patients we found no statistically signifi cant diff erences in relation to the variables of interest to analyze in the two groups. Patients with severe ARDS who survive Conclusion The increases in EVLWI, in plasma protein and hemoglobin concentration and in BNP are valuable alternatives to the pulmonary artery catheter for diagnosing weaning-induced pulmonary edema. P96 P96 Airway pressures and S36 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 the other hand, auto open lung management off ers a distinctive result of ventilation. A signifi cant improvement of oxygenation and lung compliance was observed within a few breaths after the recruitment maneuvers. Both subjects were ventilated at the same tidal volume of 6 ml/kg and the comparative results of automatic ventilation settings and BGA are provided in Table 1 for every 2 hours. Conclusion The auto open lung management concept gave much better gas exchange than the auto ARDSNet protocol. These preliminary results showed a necessity to evaluate the two diff erent ventilation strategies. Therefore, further experiments with pig models will be implemented in the near future to obtain results with statistical signifi cance and to ensure the safety of automation in a mechanical ventilation system. the other hand, auto open lung management off ers a distinctive result of ventilation. A signifi cant improvement of oxygenation and lung compliance was observed within a few breaths after the recruitment maneuvers. Both subjects were ventilated at the same tidal volume of 6 ml/kg and the comparative results of automatic ventilation settings and BGA are provided in Table 1 for every 2 hours. ventilatory mechanics were measured twice daily. Data are presented as mean ± 95% CI. *P > 0.05. the other hand, auto open lung management off ers a distinctive result of ventilation. A signifi cant improvement of oxygenation and lung compliance was observed within a few breaths after the recruitment maneuvers. Both subjects were ventilated at the same tidal volume of 6 ml/kg and the comparative results of automatic ventilation settings and BGA are provided in Table 1 for every 2 hours. Results A total of 166 patients with mean age 55 (52 to 58) years, LIS of 2.27 (2.16 to 2.37) and MODS of 3.13 (2.9 to 3.3) were enrolled and stratifi ed by LISL (65.8%) and LISH (34.2%). Mortality, and incidence of pneumothorax and atelectasis were 21% (14.9 to 28.2), 1.6% (0.8 to 3.1) and 6.4 (3.2 to 11.2). Duration of MV was 6 days (4.83 to 7.16). Pneumothorax in LISL and LISH was 1% (0 to 5.2) versus 2.4% (0.7 to 6.1)*, and mortality 11.4% (3.8 to 24.6) versus 25% (9.8 to 46.7)*. P97 Comparison of a fully automatic ARDSNet protocol and a feedback-controlled open lung management concept A Pomprapa1, D Schwaiberger2, B Lachmann2, S Leonhardt1 1RWTH Aachen University, Aachen, Germany; 2Charite Berlin, Germany Critical Care 2013, 17(Suppl 2):P97 (doi: 10.1186/cc12035) Introduction Intellivent-ASV has been developed to provide fully closed loop mechanical ventilation using a ventilation controller keep- ing EtCO2 and SpO2 within expert-based ranges. Ventilation of ARDS patients focuses on delivering adequate oxygenation and allowing elimination of CO2 while protecting the lung. The objectives were to compare Intellivent-ASV with conventional ventilation on safety and effi cacy, and to compare the number of manual adjustments between the two ventilatory modalities. Introduction The aim of this study is to compare two ventilation strategies, the ARDSNet protocol and open lung management, using computer control for 6 hours. The standard therapy for patients with ARDS does typically apply a mechanical ventilator to support breathing. The cost of therapy is high and it requires much attention from physicians to adjust the proper ventilation settings in a timely manner. A closed-loop ventilation concept has therefore been developed and tested with two induced ARDS pigs. y Methods A randomized, controlled study including all consecutive patients receiving mechanical ventilation for at least 48 hours. Patients were randomly ventilated either with Intellivent-ASV or conventional ventilation, with a S1 (Hamilton, Bonaduz, Switzerland). Parameters were adjusted by the clinician in charge of the patient. Ventilatory and oxygenation parameters were recorded cycle by cycle during 48 hours and blood gases were performed every 6 hours. g Methods The hardware system is composed of a ventilator (Servo 300), a spectrophotometry (CEVOX), a capnography device (CO2SMO+), an electrical impedance tomography device (GOE MF II) and a patient monitor (Sirecust). The software is developed with Labview 7.1. With approval from the ethical committee, two 27 kg pigs were exposed to surfactant depletion with a warm saline washout to induce ARDS (PaO2/ FiO2 <200 mmHg). One pig model was ventilated with an automatic ARDSNet protocol and another was automatically ventilated with open lung management. Blood gas analysis (BGA) was carried out every half an hour.i Results Twenty-four patients with ARDS were included, 10 female, 14 male, median age 58 (46 to 63) years, APACHE II score 22 (17 to 29), PaO2/FiO2 at inclusion 136 (107 to 154). Eleven were ventilated in the conventional group and 13 in the Intellivent-ASV group. P96 Pressure support ventilation with minimal sedation as the main ventilatory mode in critically ill patients with lung injury: eff ect on mortality and incidence of complications A Gomez1, A Leon1, G Fernandez1, G Montenegro1, H Gomez2 1Clinica Palermo, Bogota, Colombia; 2University of Pittsburgh, PA, USA Critical Care 2013, 17(Suppl 2):P96 (doi: 10.1186/cc12034) Pressure support ventilation with minimal sedation as the main ventilatory mode in critically ill patients with lung injury: eff ect on mortality and incidence of complications A Gomez1, A Leon1, G Fernandez1, G Montenegro1, H Gomez2 1Clinica Palermo, Bogota, Colombia; 2University of Pittsburgh, PA, USA Critical Care 2013, 17(Suppl 2):P96 (doi: 10.1186/cc12034) Table 1 (abstract P94). Subgroup analysis: moderate ARDS Variable Survivors Nonsurvivors P value APACHE II 20.06 ± 6.96 24.83 ± 6.17 0.006 MV (days) 25.63 ± 29.13 22.21 ± 15.43 0.557 ARDS (days) 10.29 ± 9.07 15.20 ± 12.92 0.107 ICU stay 34.33 ± 33.26 24.57 ± 16.91 0.142 Table 2 (abstract P94). Subgroup analysis: severe ARDS Variable Survivors Nonsurvivors P value APACHE II 20.82 ± 8.89 25.31 ± 5.63 0.761 MV (days) 26.95 ± 18.03 10.10 ± 11.64 0.001 ARDS (days) 15.8 ± 11.60 8.5 ± 8.6 0.031 ICU stay 33.52 ± 18.25 11.25 ± 11.80 0.001 Table 1 (abstract P94). Subgroup analysis: moderate ARDS Introduction The primary aim of this study is to assess the impact of pressure support ventilation (PSV) on the rate of pneumothorax and mortality in critically ill patients with lung injury. The secondary aim is to evaluate pressure–volume (P–V) relationships. Spontaneous modes of ventilation have been associated with lower rates of atelectasis, less muscle atrophy, better airfl ow distribution and importantly lower sedation requirements, which relates to lower mortality. Accordingly, we hypothesized that the use of PSV in patients with moderate/severe lung injury would have rates of pneumothorax and mortality within the standard of care. We further hypothesized that given its spontaneous nature, set pressures (PEEP and PS) but not tidal volume (Vt) would be related to airway pressures. Methods All adult patients admitted to two surgical/medical ICUs subjected to invasive mechanical ventilation (MV) were enrolled. Patients were stratifi ed by Lung Injury Score (LIS) in two groups: <2.5 (LISL); ≥2.5 (LISH). Exclusion criteria included pneumothorax on admission, use of other ventilatory strategies, and inability to trigger ventilation. Patients were ventilated with PSV, and treated only with pro re nata haldol, morphine and clozapine. P96 Mean parameters were: Vt 10.3 ml/kg (10.1 to 10.6); PEEP 10.6 (9.9 to 11); PS 16.6 (15.9 to 17.1); plateau pressure (Ppl) 25.7 (25.1 to 26.2). Ppl was >26.2 only in 2.5%. PEEP and PS (P <0.0001), and MODS were associated with Pplt, but not Vt* or LIS. Only lower Vt was associated with barotrauma (OR = 0.996, P = 0.02). p y Conclusion The auto open lung management concept gave much better gas exchange than the auto ARDSNet protocol. These preliminary results showed a necessity to evaluate the two diff erent ventilation strategies. Therefore, further experiments with pig models will be implemented in the near future to obtain results with statistical signifi cance and to ensure the safety of automation in a mechanical ventilation system. Conclusion We demonstrate that PSV in minimally sedated patients with severe lung injury is safe as it is associated with low incidence of barotrauma, atelectasis and mortality, and with Ppl and duration of MV within standard of care. We also demonstrate in PSV that P–V relationships may diff er and that in this setting higher Vt may not be deleterious. P98 Comparison of an entirely automated ventilation mode, Intellivent-ASV, with conventional ventilation in ARDS patients: a 48-hour study E Bialais1, L Vignaux2, X Wittebole1, D Novotni2, J Meyer2, M Wysocki2, T Sottiaux3, G Reychler1, J Roeseler1, P Laterre1, P Hantson1 1Cliniques Universitaires Saint Luc, Brusells, Belgium; 2Hamilton Medical AG, Bonaduz, Switzerland; 3Clinique Notre-Dame de Grâce, Gosselies, Belgium Critical Care 2013, 17(Suppl 2):P98 (doi: 10.1186/cc120236) P97 The study was stopped for one patient from the Intellivent-ASV group because of a pneumothorax not caused by ventilation. The delivered Vt was slightly higher during Intellivent-ASV (7.9 (7.5 to 8.5) vs. 7.2 (6.8 to 7.8) ml/kg, P = 0.013). The time spent by the various parameters in the suboptimal zone (safety) is the same for the two ventilation modes. The time spent in the optimal zone (effi cacy) is the same for the two ventilation modes, Results Artifi cial ventilation using the auto ARDSNet protocol success- fully stabilized oxygenation, minimized plateau pressure (<30 cmH2O), and controlled the pH value for acidosis and alkalosis management. On Table 1 (abstract P97). Comparative results between auto ARDSNet protocol and auto open lung management Before auto ventilation After 2 hours After 4 hours After 6 hours Auto ARDSNet Auto OLM Auto ARDSNet Auto OLM Auto ARDSNet Auto OLM Auto ARDSNet Auto OLM FiO2 1.0 1.0 0.40 0.25 0.40 0.25 0.50 0.25 RR (bpm) 22 40 23 38 23 39 23 35 I:E ratio 1:2 1:1 1:2 1:1 1:2 1:1 1:2 1:1 VT (ml/kg) 6 6 6 6 6 6 6 6 Pplat /PIP (mmHg) 22 22 18 20.6 15.8 17.7 14.6 15.4 PEEP (cmH2O) 5 10 8 15 2 13 10 11 pH 7.34 7.30 7.32 7.52 7.37 7.60 7.38 7.51 pO2/FiO2 (mmHg) 59 59 180 380.8 155 387.2 122 404 pCO2 (mmHg) 52 64 56 39.6 58 31.5 54 41 OLM, open lung management. Table 1 (abstract P97). Comparative results between auto ARDSNet protocol and auto open lung management . Comparative results between auto ARDSNet protocol and auto open lung management S37 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 expect for SpO2 (78% (75 to 89) with Intellivent-ASV vs. 44% (41 to 58), P  = 0.001). Intellivent-ASV required less manual adjustments during 48 hours (2 (0 to 7) vs. 17 (10 to 27), P <0.001). The total number of adjustments during 48 hours, including automatic regulation, was higher in the Intellivent-ASV group (379 (274 to 493) vs. 17 (10 to 27), P <0.001). Methods This observational study in ICU mechanically ventilated septic patients with intra-abdominal surgical infections was carried out at the V.A. Negovsky Research Institute of General Reanimatology in 2010 to 2012. ARDS was diagnosed and staged according to the research institute criteria [1] and the American–European criteria. Diff erence in pulmonary permeability between indirect and direct acute respiratory distress syndrome assessed by the transpulmonary thermodilution technique K Morisawa1, Y Taira1, M Yanai1, Y Takamatu1, S Kushimoto2, S Fujitani3 1St Marianna University, Kawasaki-Shi, Japan; 2Tohoku University Graduate School of Medicine, Sendai-shi, Japan; 3Tokyo Bay Urayasu/Ichikawa Medical Center, Urayasu-shi, Japan Critical Care 2013, 17(Suppl 2):P99 (doi: 10.1186/cc12037) Introduction ARDS is characterized by increased pulmonary capillary permeability secondary to diff use alveolar infl ammation and injury. Common risk factors can be classifi ed into two groups: extrapulmonary causes (indirect etiologies: ARDSexp) or pulmonary causes (direct etiologies: ARDSp). There are few quantitative methods to distinguish between the diff erences in these two ARDS categories. Conclusion Plasma SPD level ≤253.0 ng/ml is a sensitive and specifi c biomarker of the early stage of ARDS in septic patients. Reference f g Methods A subanalysis of the trial by the PiCCO Pulmonary Edema Study (prospective, observational, multi-institutional study) in 23 ICUs of academic tertiary referral hospitals in Japan. All consecutive adult patients requiring mechanical ventilation with the diagnosis of ARDS were monitored by the transpulmonary thermodilution technique system (PiCCO; Pulsion Medical Systems, Munich, Germany) for 3 days: day 0, day 1 and day 2. The pulmonary vascular permeability index (PVPI), extravascular lung water index and intrathoracic blood volume index were measured concurrently. Three experts retrospectively determined the pathophysiological mechanism causing ARDS by patient history, clinical presentation, chest computed tomography and radiography. Patients were classifi ed into two groups: patients with ARDS triggered by ARDSexp and ARDSp. 1. Kuzovlev A, et al.: Semin Cardiothorac Vasc Anesth 2010, 14:231-24 P101 Intravenous perfl uorocarbons for prevention of ventilator-associated ARDS A Scultetus1, A Haque1, F Arnaud1, G McNamee2, C Auker1, R McCarron1, P McKay2, R Mahon1 1Naval Medical Research Center, Silver Spring, MD, USA; 2Uniformed Services University of the Health Sciences, Bethesda, MD, USA Critical Care 2013, 17(Suppl 2):P101 (doi: 10.1186/cc12039) A Scultetus1, A Haque1, F Arnaud1, G McNamee2, C Auker1, R McCarron1, P McKay2, R Mahon1 Results During the study period from March 2009 to August 2011, a total of 173 patients were assessed including 56 ARDSexp patients and 117 ARDSp patients, with the most common cause of ARDSexp secondary to sepsis (71%) and of ARDSp pneumonia (80%). The measure ment of PVPI was signifi cantly elevated in the ARDSp group on all days. There were no signifi cant diff erences in mortality at 28 days, mechanical ventilation days, and hospital length-of-stay between the two groups, while the ARDSexp group seemed to be associated with prolonged mechanical ventilation days and hospital length-of-stay.f Introduction Emulsifi ed perfl uorocarbons (PFC) are synthetic hydro- carbons that can carry 50 times more oxygen than human plasma. Their properties may be advantageous in applications requiring preservation of tissue viability in oxygen-deprived states [1,2], making them a potential candidate for combat and civilian prehospital resuscitation. Our hypothesis is that an intravenous dose of PFC increases vital organ tissue oxygenation, improves survival after hemorrhagic shock (HS) and may reduce or prevent the development of ventilator-associated ARDS. Here we report data from the fi rst part (HS only) of a multiphase swine study to study the benefi ts of PFC in treating HS and preventing ARDS. This initial study was designed to ensure safe use of PFC in traumatized animals. Conclusion This study suggests the existence of several diff erences in pathogenetic pathways and the degree of pulmonary permeability between patients with ARDSexp and ARDSp. We therefore believe that using the PVPI may provide us with timely quantitative information to make clinical decisions. Methods Anesthetized Yorkshire swine were hemorrhaged 55% of their estimated blood volume (Time 0 (T0)) over 15 minutes. At T15 minutes, pigs received a bolus of the PFC Oxycyte or 10% hydroxyethyl starch (HES). At T60 animals underwent continuous hemorrhage (0.5 cm3/kg/ minute) until death. Time to death and physiological parameters were primary endpoints. P97 Plasma concentrations of SPD and CCP were measured on ARDS diagnosis (day 0) and days 3, 5, and 7 by immunoenzyme essay (Bio Vendor, USA). Data were statistically analyzed by STATISTICA 7.0, ANOVA method, and presented as mean ± σ, ng/ml. P <0.05 was considered statistically signifi cant. Areas under the receiver operating curves (ROC) were calculated. Conclusion Over 48 hours, Intellivent-ASV allowed safe ventilation for ARDS patients, with better oxygenation and an identical ventilation effi cacy when compared with conventional ventilation, with less manual adjustments. Results Fifty-fi ve patients (out of 250 screened) were enrolled according to the inclusion/exclusion criteria. Patients were assigned into groups: ARDS (n = 30; subgroups – ARDS stage 1 (n = 15), stage 2 (n = 15)) and noARDS (n = 25). In the ARDS group SPD was higher at all points than in the noARDS group (day 0 – 352.5 ± 287.8 vs. 141.0 ± 103.4; day 3 – 278.6 ± 204.8 vs. 151.8 ± 125.9; day 5 – 339.9 ± 331.4 vs. 162.3 ± 138.8; day 7 – 339.9 ± 300.1 vs. 169.8 ± 154.5; P <0.05). SPD levels were lower at ARDS stage 1 in comparison with stage 2 (day 0 – 154.6 ± 125.2 vs. 451.8 ± 299.2; day 3 – 129.3 ± 74.9 vs. 362.9 ± 202.0; day 5 – 167.8 ± 120.4 vs. 428.5  ±  372.4; day 7  – 186.2  ±  127.1 vs. 415.5  ±  337.0; P <0.05). Plasma SPD had a good diagnostic capacity for stage 1 ARDS: SPD on day 0 ≤253.0 ng/ml yielded a sensitivity of 90% and specifi city of 74% (AUC = 0.83; 95% CI = 0.631 to 0.951; P = 0.0001). No such diff erences for CCP were detected.i P103 End-expiratory esophageal pressure versus lower infl ection point in acute lung injury Results In the multivariable model, age, lung compliance, serum lactate and serum ACE levels were signifi cantly associated with both 28-day and 90-day mortality. No signifi cant correlation was found between serum and BALF ACE levels (Spearman’s ρ = 0.054; P = 0.66). Serum ACE concentrations were signifi cantly higher (P  = 0.046) in patients with D/D genotype versus the two other groups combined (I/D and I/I genotypes). A meta-analysis of six studies (including ours) provided evidence that the D allele is signifi cantly associated with increased mortality in ALI/ARDS patients, yielding a per-allele odds ratio of 1.76 (95% CI: 1.19 to 2.59). See Figure 1 and Table 1.f Introduction No recommendations available concerning protocols of static PV loop and esophageal pressure measurements use set positive end-expiratory pressure (PEEP). The aim of the study was evaluation of the signifi cance of the lower infl ection point (LIP) and esophageal pressure monitoring for PEEP adjustment in ALI and ARDS. Conclusion Serum ACE levels appear to be aff ected by the I/D poly- morphism and are correlated with prognosis in patients with ALI/ARDS, indicating that further investigation of the clinical signifi cance of the ACE in ARDS might be of value. Methods A prospective study performed in one general ICU. We include 72 patients who received mechanical ventilation before evaluation and met ARDS criteria by AECC (1994) – acute onset, PaO2/FiO2 lower than 250 Torr, bilateral infi ltrates on chest X-ray. Exclusion criteria were age <15 years and pregnancy. We drew a static pressure–volume loop with sustained infl ation 40×30 (PV loop) for all patients using a low- fl ow technique (Hamilton G5) and measured the esophageal pressure (Avea) in 36 of 72 patients. After that PEEP was set according to zero end-expiratory transpulmonary pressure. We compare PV loop data with esophageal pressure measurements.l Figure 1 (abstract P102). Figure 1 (abstract P102). Results The low infl ection point median was 8 (95% CI = 5 to 10.5) mbar, which does not correspond to the empirically set optimal PEEP of 13 (95% CI = 12 to 15) mbar (P <0.001, Wilcoxon test). End-expiratory esophageal pressure (EEEP) median was 14 (95% CI = 12 to 18) mbar, the correlation between LIP and EEEP was poor (ρ = 0.279, P = 0.049). We fi nd signifi cant correlation between static compliance and EEEP (ρ = –0.421, P = 0.005). P100 P102 Eff ect of angiotensin-converting enzyme gene I/D polymorphism and its expression on clinical outcome in acute respiratory distress syndrome I Tsangaris, A Tsantes, P Kopterides, G Tsaknis, S Kokkori, I Karampela, D Konstantonis, S Karabi, E Vrigkou, A Pappas, S Orfanos, A Armaganidis University Hospital ‘Attikon’, University of Athens Medical School, Athens, Greece Critical Care 2013, 17(Suppl 2):P102 (doi: 10.1186/cc12040) Introduction The role of the D allele of the angiotensin-converting enzyme (ACE) gene I/D polymorphism in the clinical outcomes of patients with acute lung injury and acute respiratory distress syndrome (ALI/ARDS) remains controversial. We assessed simultaneously the eff ect of the ACE I/D polymorphisms as well as the serum and BALF ACE levels on prognosis of ARDS patients. Methods We recruited 69 mechanically ventilated ALI/ARDS patients. ACE activity levels in serum and BALF were assessed by chemical th d P ti t t d f ACE I/D l hi Ti Table 1 (abstract P102). Characteristics of the study population (n = 69) Age (years) 64.4 ± 17.9; 69 (28 to 89) Sex (males, %) 43 (62.3%) APACHE II score 22.1 ± 6.2; 21 (14 to 35) SOFA score 8.4 ± 3.2; 8 (4 to 16) Lung compliance (ml/cmH2O) 29.4 ± 7.2; 30 (17 to 40) PaO2/FiO2 139, 1 ± 47.1; 140 (72 to 223) PEEP 8.1 ± 2.9; 8 (5 to 15) Lung Injury Score 2.5 ± 0.6; 2.5 (1.75 to 3.50) Blood lactate (mmol/l) 1.7 ± 1.7; 1.3 (0.5 to 5.2) Septic status 39 (56.5%) Serum ACE (U/l) 16.5 ± 10.8; 13.6 (4.8 to 39.8) BALF (U/l) 2.3 ± 1.4; 2.0 (0.3 to 4.7) ACE I/D polymorphism: D/D 27 (39.1%) I/D 28 (40.6%) I/I 14 (20.3%) 28-day mortality 34/69 (49.3%) 90-day mortality 43/69 (62.3%) Ventilator-free days 5.1 ± 7.8; (0 to 23) Days w/o cardiovascular failure 14.0 ± 10.2; 16 (0 to 27) Days w/o renal failure 15.4 ± 10.6; 16 (0 to 28) Table 1 (abstract P102). 0 Eff ect of angiotensin-converting enzyme gene I/D polymorphism and its expression on clinical outcome in acute respiratory distress syndrome Eff ect of angiotensin-converting enzyme gene I/D polymorphism and its expression on clinical outcome in acute respiratory distress syndrome y I Tsangaris, A Tsantes, P Kopterides, G Tsaknis, S Kokkori, I Karampela, D Konstantonis, S Karabi, E Vrigkou, A Pappas, S Orfanos, A Armaganidis University Hospital ‘Attikon’, University of Athens Medical School, Athens, Greece Critical Care 2013, 17(Suppl 2):P102 (doi: 10.1186/cc12040) Introduction The role of the D allele of the angiotensin-converting enzyme (ACE) gene I/D polymorphism in the clinical outcomes of patients with acute lung injury and acute respiratory distress syndrome (ALI/ARDS) remains controversial. We assessed simultaneously the eff ect of the ACE I/D polymorphisms as well as the serum and BALF ACE levels on prognosis of ARDS patients. p g p Methods We recruited 69 mechanically ventilated ALI/ARDS patients. ACE activity levels in serum and BALF were assessed by chemical methods. Patients were genotyped for ACE I/D polymorphisms. Time- to-event analysis evaluated the variables associated with the 28-day and 90-day mortality. P100 Characteristics of the study population (n = 69) Age (years) 64.4 ± 17.9; 69 (28 to 89) Sex (males, %) 43 (62.3%) APACHE II score 22.1 ± 6.2; 21 (14 to 35) SOFA score 8.4 ± 3.2; 8 (4 to 16) Lung compliance (ml/cmH2O) 29.4 ± 7.2; 30 (17 to 40) PaO2/FiO2 139, 1 ± 47.1; 140 (72 to 223) PEEP 8.1 ± 2.9; 8 (5 to 15) Lung Injury Score 2.5 ± 0.6; 2.5 (1.75 to 3.50) Blood lactate (mmol/l) 1.7 ± 1.7; 1.3 (0.5 to 5.2) Septic status 39 (56.5%) Serum ACE (U/l) 16.5 ± 10.8; 13.6 (4.8 to 39.8) BALF (U/l) 2.3 ± 1.4; 2.0 (0.3 to 4.7) ACE I/D polymorphism: D/D 27 (39.1%) I/D 28 (40.6%) I/I 14 (20.3%) 28-day mortality 34/69 (49.3%) 90-day mortality 43/69 (62.3%) Ventilator-free days 5.1 ± 7.8; (0 to 23) Days w/o cardiovascular failure 14.0 ± 10.2; 16 (0 to 27) Days w/o renal failure 15.4 ± 10.6; 16 (0 to 28) Table 1 (abstract P102). Characteristics of the study population (n = 69) on these data we will continue to the next phase of this project and test PFC in the prevention of ARDS alone, and in combination with HS. References on these data we will continue to the next phase of this project and test PFC in the prevention of ARDS alone, and in combination with HS. References 1. Keipert PE, et al.: Enhanced oxygen delivery by perfl ubron emulsion during acute hemodilution. Artif Cells Blood Substit Immobil Biotechnol 1994, 22:1161-1167.l 2. Habler O, et al.: IV perfl ubron emulsion versus autologous transfusion in severe normovolemic anemia: eff ects on left ventricular perfusion and function. Res Exp Med (Berl) 1998, 197:301-318. 1. Keipert PE, et al.: Enhanced oxygen delivery by perfl ubron emulsion during acute hemodilution. Artif Cells Blood Substit Immobil Biotechnol 1994, 22:1161-1167. P100 Biomarkers for early stage of acute respiratory distress syndrome in septic patients: surfactant protein D and Clara cell protein A Kuzovlev1, V Moroz1, A Goloubev1, S Polovnikov2 1V.A. Negovsky Scientifi c Research Insitute of General Reanimatology RAMS, Moscow, Russia; 2N.N. Burdenko Main Military Hospital, Moscow, Russia Critical Care 2013, 17(Suppl 2):P100 (doi: 10.1186/cc12038) Results Average survival time after onset of second hemorrhage was 60 minutes in PFC-treated animals versus 65 minutes in the HES group. Mean arterial pressure (MAP) was similar between T0 and T60, thereafter PFC-treated animals had lower MAP, mean pulmonary artery pressure (MPAP), heart rate and cardiac output (P >0.05).if Conclusion There was no signifi cant diff erence in survival time, MAP and MPAP in the PFC group compared with HES control. These data suggest that it is safe to administer in this HS model. Regulatory approval of hemoglobin-based oxygen carriers has been halted due to possible side eff ects related to vasoconstriction. In this model, and with this class of oxygen-carrying drugs (PFCs), we did not observe evidence of vasoconstriction. Using PFCs did not result in a survival advantage here; however, there was also no observation of adverse events. Based Introduction The injury of alveolar epithelium and endothelium is the basis of pathogenesis for the early stage of acute respiratory distress syndrome (ARDS). A prompt detection of this injury will provide us with a possibility for early ARDS diagnosis and treatment. Currently no biomarkers of alveolar epithelial injury are clinically available. The purpose of our study was to investigate the role of surfactant protein D (SPD) and Clara cell protein (CCP) as biomarkers of early ARDS. S38 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 on these data we will continue to the next phase of this project and test PFC in the prevention of ARDS alone, and in combination with HS. References 1. Keipert PE, et al.: Enhanced oxygen delivery by perfl ubron emulsion during acute hemodilution. Artif Cells Blood Substit Immobil Biotechnol 1994, 22:1161-1167. 2. Habler O, et al.: IV perfl ubron emulsion versus autologous transfusion in severe normovolemic anemia: eff ects on left ventricular perfusion and function. Res Exp Med (Berl) 1998, 197:301-318. P103 P103 End-expiratory esophageal pressure versus lower infl ection point in acute lung injury A Yaroshetskiy1, D Protsenko1, E Larin2, O Ignatenko1, B Gelfand1 1Russian National Research Medical University, Moscow, Russia; 2City Hospital #7, Moscow, Russia Critical Care 2013, 17(Suppl 2):P103 (doi: 10.1186/cc12041) 2. Habler O, et al.: IV perfl ubron emulsion versus autologous transfusion in severe normovolemic anemia: eff ects on left ventricular perfusion and function. Res Exp Med (Berl) 1998, 197:301-318. Quantifi cation of stress raisers in ARDS Quantifi cation of stress raisers in ARDS M Cressoni1, M Amini1, P Cadringher2, C Chiurazzi1, D Febres1, E Gallazzi1, A Marino1, M Brioni1, F Menga1, I Cigada1, E Carlesso1, D Chiumello2, L Gattinoni2 1Università degli Studi di Milano, Milan, Italy; 2Fondazione IRCCS Ca’ Granda- Ospedale Maggiore Policlinico, Milan, Italy Critical Care 2013, 17(Suppl 2):P105 (doi: 10.1186/cc12043) Results We included in our study the CT scans of 89 patients (24 mild, 55 moderate and 10 severe ARDS, age 58 ± 17 years, BMI 25.6 ± 5.4, PaO2/FiO2 165  ±  66, PaCO2 42  ±  9, 60 discharged alive from ICU (67%)). At PEEP 5 cmH2O 39 ± 16% of the lung parenchyma was not infl ated, 33 ± 13% poorly infl ated, 28 ± 15% well infl ated and 0 ± 1% overinfl ated. The median lung recruitability between 5 and 15 cmH2O PEEP was 6% (3 to 10%) of lung parenchyma (71 g (26 to 182 g)). Mean EELV at PEEP 5 cmH2O was 1,233 ± 709 while it increased to 1,784 ± 848 at PEEP 15 cmH2O (ΔEELV = 551 ± 407 ml). More than one-half (52% (31 to 75%)) of the increase in end-expiratory lung volume (EELV) was due to an increase in infl ation of the already well-aerated tissue; 38% (23 to 43%) was due to the infl ation of poorly aerated tissue, while only 4% (1 to 8%) to the recruitment of lung tissue. The CLsp of the well, poorly and recruited tissue was 0.07 (0.031 to 0.13), 0.03 (0.01 to 0.06) and 0.02 (0.01 to 0.04) ml/cmH2O/g, respectively (P <0.0001). Introduction Ventilator-induced lung injury (VILI) is a well-known side eff ect of mechanical ventilation. The pressures and volumes needed to induce VILI in healthy animals are far greater than pressure and volumes applied in clinical practice [1]. A possible explanation may be the presence of local pressure multipliers (stress raisers). Methods We retrospectively analyzed CT scans of 147 patients with ARDS and CT scans of 100 healthy subjects. A homogeneous lung would have the same gas/tissue ratio in all its regions. If a lung region expands less than the neighbour regions these will be more strained to vicariate the non/less expanding region. Recruited lung tissue does not resume normal mechanical properties g Results Patients were 65 ± 13 years old, ventilation days were 9 ± 10 and PaO2/FiO2 was 194 ± 49 mmHg at PEEP 8 ± 2 cmH2O. Introduction of Sigh improved PaO2/FiO2 (P  <0.001) and ΔEELVgl (P  <0.001). The increase in EELV was diff use across all lung regions as both ΔEELVnondep and ΔEELVdep changed (P <0.01 and P = 0.06). minute ventilation, arterial blood pH and hemodynamics were not signifi cantly aff ected by introduction of Sigh, while mean airway pressure increased (P <0.001), albeit only at the highest Sigh rate and by a limited extent (~2 cmH2O). Patients with mean ΔEELVgl over the three Sigh phases >200  ml (that is, Sigh-responders) had signifi cantly lower PaO2/ FiO2 values at baseline (that is, at Sigh rate  = 0/minute) than Sigh- nonresponders (163 ± 25 mmHg vs. 222 ± 39 mmHg, P <0.01). p p M Cressoni1, C Chiurazzi1, M Amini1, D Febres1, E Gallazzi1, E Carlesso1, P Cadringher1, T Langer1, D Chiumello1, L Gattinoni2 1Università degli Studi di Milano, Milan, Italy; 2Fondazione IRCCS Ca’ Granda – Ospedale Maggiore Policlinico, Milan, Italy Critical Care 2013, 17(Suppl 2):P106 (doi: 10.1186/cc12044) Introduction Positive end-expiratory pressure (PEEP) is commonly used in patients with ARDS to prevent end-expiratory collapse. The increase in gas volume and/or change in mechanical properties of the lung are used at the bedside to estimate lung recruitment and individualize PEEP selection. Methods Retrospective analysis of patients who underwent whole- lung CT scan at 5 and 15 cmH2O PEEP. Every chest CT was divided both into 10 apex-base levels of equal height and into 10 sterno-vertebral levels, yielding 100 units per scanned lung (200 units per patient); each unit was classifi ed according to its average CT number (<–100 not infl ated, –100 to –500 poorly infl ated, –500 to –900 well infl ated and –900 overinfl ated). Lung regions defi ned as not infl ated at PEEP 5 cmH2O and infl ated (poorly, well or over) at PEEP 15 cmH2O were classifi ed as recruited. Moreover, a surrogate specifi c lung compliance (CLsp) was determined on the expiratory limb of the pressure–volume curve (15 to 5  cmH2O PEEP at CT) and was defi ned as Δgas/cmH2O PEEP/gram of tissue. Gattinoni L, et al.: Int Care Med 2005. 31:776-784. Lung recruitment induced by Sigh in hypoxemic intubated critically ill patients p TM Mauri1, G Bellani1, A Coppadoro1, P Tagliabue2, A Barletta1, V Meroni2, M Teggia Droghi1, N Patroniti1, A Pesenti1 1University of Milan-Bicocca, Monza, Italy; 2San Gerardo Hospital, Monza, Italy Critical Care 2013, 17(Suppl 2):P104 (doi: 10.1186/cc12042) of the stress raisers the fraction of lung volume above this threshold. Results The extent of stress raisers increased with the severity of ARDS (14 ± 5, 18 ± 8, 23 ± 1% of lung parenchyma in mild, moderate and severe ARDS, P <0.0001). The extent of stress raisers correlated with the dead space fraction (r2  = 0.34, P <0.001), with the fraction of poorly aerated tissue (r2 = 0.36, P <0.0001) and also has a negative correlation with the fraction of well infl ated tissue (r2 = 0.47, P <0.0001). The response to PEEP, passing from 5 to 45 cmH2O is minimal (average decrease of stress raiser extent 6 ± 5%) and inter-individual variability is great (in 11 patients, stress raisers increased passing from PEEP 5 to PEEP 45). Stress raisers turn out to be greater in nonsurvivor patients than in survivor patients (17 ± 7 vs. 20 ± 9% of lung volume, P = 0.03). Introduction In intubated critically ill patients, cyclic short recruitment manoeuvres (Sigh) introduced during pressure support ventilation (PSV) improve oxygenation, probably by increasing end-expiratory lung volume (EELV). We assessed Sigh eff ects on regional distribution of EELV by electrical impedance tomography (EIT): a bedside, radiation- free lung imaging technique. Moreover, we investigated baseline characteristics correlated with response to Sigh. Methods We enrolled 20 intubated critically ill patients undergoing PSV with PaO2/FiO2 ≤300 mmHg and PEEP ≥5 cmH2O. We applied on each patient’s thorax a 16-electrode belt connected to an EIT monitor (PulmoVista 500®; Dräger Medical GmbH, Lübeck, Germany). Sigh was introduced as 35 cmH2O continuous positive airway pressure phase lasting 3 to 4 seconds at diff erent rates (0, 0.5, 1, 2 per minute, random order) for 20 minutes. From raw EIT data, we calculated global changes in EELV (ΔEELVgl), calculated as changes in end-expiratory lung impedance calibrated versus tidal volume to estimate changes in ΔEELV and considering 0 Sigh/minute as baseline; and ΔEELV of nondependent and dependent lung regions (ΔEELVnondep and ΔEELVdep). Together, we collected ventilation parameters, hemodynamics and arterial blood gases. Reference 1. Protti A: Am J Respir Crit Care Med 2011, 183:1354-1362. 1. Protti A: Am J Respir Crit Care Med 2011, 183:1354-1362. Recruited lung tissue does not resume normal mechanical properties p g g Conclusion Introduction of Sigh in intubated critically ill patients undergoing PSV improves gas exchange by inducing alveolar recruitment across all lung regions with minimal increase of airway pressure. Patients with more severe gas exchange impairment present the highest degree of potentially recruitable lung. Quantifi cation of stress raisers in ARDS We measured the stress raisers by computing the ratio between the gas fraction of the region of interest and the neighbouring regions: if the infl ation would be the same (homogeneity), the ratio will be equal to one; if the infl ation of the surrounding regions would be greater than the region of interest (that is, more strained), the ratio between the two will be greater than one 2 Conclusion Most of the PEEP-related EELV increase is due to an increased infl ation of already well aerated lung tissue. In agreement with the baby lung theory [1], the well-aerated tissue showed near to normal mechanical properties. Lung recruitment induced by Sigh in hypoxemic intubated critically ill patients P106 Recruited lung tissue does not resume normal mechanical properties M Cressoni1, C Chiurazzi1, M Amini1, D Febres1, E Gallazzi1, E Carlesso1, P Cadringher1, T Langer1, D Chiumello1, L Gattinoni2 1Università degli Studi di Milano, Milan, Italy; 2Fondazione IRCCS Ca’ Granda – Ospedale Maggiore Policlinico, Milan, Italy Critical Care 2013, 17(Suppl 2):P106 (doi: 10.1186/cc12044) P103 End-expiratory esophageal pressure versus lower infl ection point in acute lung injury Sustained infl ation did not lead to improved oxygenation (P >0.05). PEEP adjustment by EEEP led to an increase in PaO2/FiO2 – median 107 mmHg (95% CI = 18 to 147, P <0.001). EEEP was similar to empirically set PEEP (P >0.05). Conclusion LIP has poor correlation with EEEP. PEEP adjustment by esophageal pressure was close to empirically set PEEP and can improve oxygenation. S39 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 P104 and was taken as a measure of stress raiser. We considered pathological stress raisers as the regions showing infl ation ratio greater than the 95th percentile of the control group (1.61) and defi ned as the extent P104 Lung recruitment induced by Sigh in hypoxemic intubated critically ill patients TM Mauri1, G Bellani1, A Coppadoro1, P Tagliabue2, A Barletta1, V Meroni2, M Teggia Droghi1, N Patroniti1, A Pesenti1 1University of Milan-Bicocca, Monza, Italy; 2San Gerardo Hospital, Monza, Italy Critical Care 2013, 17(Suppl 2):P104 (doi: 10.1186/cc12042) Reference Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 S40 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 P107 Intraoperative management of hypoxemia with recruitment maneuvers: are the benefi ts worth the costs? V Grosomanidis, B Fyntanidou, K Karakoulas, K Kotzampasi, E Oloktsidou, C Nouris, C Skourtis Aristotle Medical School, Thessaloniki, Greece Critical Care 2013, 17(Suppl 2):P107 (doi: 10.1186/cc12045) P107 and without inotropic support were enrolled. A transthoracic echo- cardiography was performed to assess diastolic function: E/A, isovolumic relaxation time (IVRT), deceleration time (DT), E/e’ were assessed. Eight patients (fi ve with poor relaxation pattern, three with decreased left ventricle compliance) were identifi ed in diastolic dysfunction group (Group 1) and eight were included in the normal diastolic function group (Group 2). A recruitment manoeuvre was performed by applying a PEEP of 40 cmH2O for 30 seconds. Haemodynamic parameters of cardiac output (CO), stroke volume (SV), heart rate (HR), pulse pressure variation (PPV), and cardiac cycle effi ciency (CCE) were continuously recorded during the manoeuvre using the MostCare pulse contour method.ii and without inotropic support were enrolled. A transthoracic echo- cardiography was performed to assess diastolic function: E/A, isovolumic relaxation time (IVRT), deceleration time (DT), E/e’ were assessed. Eight patients (fi ve with poor relaxation pattern, three with decreased left ventricle compliance) were identifi ed in diastolic dysfunction group (Group 1) and eight were included in the normal diastolic function group (Group 2). A recruitment manoeuvre was performed by applying a PEEP of 40 cmH2O for 30 seconds. Haemodynamic parameters of cardiac output (CO), stroke volume (SV), heart rate (HR), pulse pressure variation (PPV), and cardiac cycle effi ciency (CCE) were continuously recorded during the manoeuvre using the MostCare pulse contour method. Introduction Several strategies can be applied for the prevention and management of anesthesia-related hypoxemia. The aim of our study was to investigate the cardiovascular eff ects of a specifi c recruitment maneuver (RM) used for the management of intraoperative hypoxemia. Methods Forty-nine patients (29 male/20 female) of mean age 57.4 ± 9.9 years, mean weight 80.2 ± 13.6 kg and ASA-PS classifi cation I to IV (I: 2, II: 27, III: 16, IV: 6), undergoing general surgery procedures, who developed intraoperative hypoxemia (PaO2/FiO2 <200), were enrolled in our study. For the management of hypoxemia a total of 67 RMs have been applied, which consisted of a manual increase of the airway pressure to 40 cmH2O. Reference This rise has been maintained for 10 seconds and was followed by an increase of positive end-expiratory pressure from 5 to 10 cmH2O. Before RM application an oesophageal Doppler monitor probe was inserted into the patients for measuring stroke volume (SV), cardiac output (CO), peak velocity (PV) and fl ow corrected time (FTc). Standard monitoring also included ECG, IBP, ETCO2 and SpO2. Heart rate (HR), systemic arterial pressure (SAP), SV, CO, PV and FTc were recorded directly before, during, right after and 5 minutes after RM application (T1 to T4). Kolmogorov–Smirnoff was used to test normal distribution of data and ANOVA was used for the statistical analysis. P <0.05 was considered statistically signifi cant.i Results No signifi cant haemodynamic changes happened in the fi rst part of the recruitment (t0 to t15 seconds). A signifi cant decrease in CO (P <0.05), SV (P <0.01), systolic pressure (P <0.04) and PPV (P <0.04) occurred in Group 1 during the remaining part of the manoeuvre. No signifi cant decrease in the same haemodynamic parameters were noted when only E/A was used to discriminate the diastolic dysfunction. See Table 1. Table 1 (abstract P108). CO diff erence associated with diastolic dysfunction parameters E/A <1 E/A >1 P value CO 15 to 30 3.9 4.8 0.20 E/e’ <1 E/e’ >1 CO 15 to 30 3.92 5.6 0.02 Table 1 (abstract P108). CO diff erence associated with diastolic dysfunction parameters y gi Results HR, SAP, SV, CO, PV and FTc showed a statistically signifi cant decrease during and right after RM compared with the baseline values but they were gradually restored to control values after RM discontinuation (Table 1). Conclusion The recruitment manoeuvre can compromise left diastolic fi lling by increasing the transpulmonary pressure. In patients with diastolic dysfunction a signifi cant decrease in SV can be observed during the recruitment manoeuvre with no compensatory mechanisms evoked determining a signifi cant decrease in CO. The E/A ratio is not able to discriminate between the two groups, confi rming the power of tissue Doppler imaging to recognize the correct diastolic pattern. Reference Table 1 (abstract P107) T1 T2 T3 T3 HR (beats/minute) 69.8±7.1 60.2±10.6* 67.1±7.4*^ 68.4±6.9^ SAP mean (mmHg) 89.8±7.7 53.4±5.7* 81.5±7.7*^ 87.6±6.7^ SV (ml) 82.2±21.2 22.5±15.5* 75.4±21.7*^ 79.6±6.4^ CO (l/minute) 5.75±1.7 1.4±0.9* 5.1±1.7*^ 5.5±1.5^ PV (cm/second) 72.6±17.1 26±15.9* 68±16.8*^ 70.7±15.9^ FTc (seconds) 0.4±0.06 0.19±0.08* 0.36±0.06*^ 0.36±0.06^ *P <0.05 versus T1. ^P <0.05 versus T2. 1. Reference Gernoth C, et al.: Respiratory and haemodynamic changes during decremental open lung positive end-expiratory pressure titration in patients with acute respiratory distress syndrome. Crit Care 2009, 13:R59. P109 Alveolar Recruitment for ARDS Trial: preliminary results AB Cavalcanti1, EA Suzumura1, M Abreu1, GF Ribeiro1, A Kodama1, F Moreira1, HP Guimarães1, E Romano1, MB Amato2, O Berwanger1, CR Carvalho2, ART Investigators1 1Hospital do Coração – HCor, São Paulo, Brazil; 2Universidade de São Paulo, Brazil Critical Care 2013, 17(Suppl 2):P109 (doi: 10.1186/cc12047) Conclusion According to our results, RM application causes a profound impairment of the cardiovascular system. This can be attributed to the increase of the airway pressures, which results in preload decrease and afterload increase of the right ventricle and cardiac contractility attenuation. Nevertheless, these eff ects are transient and reversible and RM application is a safe technique for the management of intraoperative hypoxemia, provided that adequate cardiac preload is ensured and the anesthesiologist is alert to discontinue the RM if it exceeds cardiovascular reserves. Critical Care 2013, 17(Suppl 2):P109 (doi: 10.1186/cc12047 Introduction The aim of the ongoing Alveolar Recruitment for ARDS Trial (ART) is to evaluate whether a maximum stepwise alveolar recruit- ment maneuver (MSARM) followed by ventilation at optimal PEEP may decrease 28-day mortality in patients with moderate to severe ARDS compared with ventilation with the ARDSNet strategy (NCT01374022). Here we report the results of a planned a priori preliminary analysis involving 100 patients to assess feasibility, physiologic variables and safety outcomes. P108 P108 Haemodynamic changes during alveolar recruitment manoeuvre in patients with diastolic dysfunction A Rubino, E Marini, B Ferro, M Collareta, F Forfori, F Guarracino, F Giunta Azienda Ospedaliero Universitaria Pisana, Pisa, Italy Critical Care 2013, 17(Suppl 2):P108 (doi: 10.1186/cc12046) y Methods ART is an event-driven multicenter randomized controlled trial planned to last until 520 deaths within 28  days are observed. Patients assigned to the experimental group receive a MSARM achieving PEEP of 45 cmH2O and plateau pressure of 60 cmH2O plus PEEP titrated according to the static compliance of the respiratory system (ART strategy). The target tidal volume is 4 to 6 ml/kg predicted body weight (PBW) and plateau pressure ≤30 cmH2O in both groups. Results We randomized 101 patients between November 2011 and October 2012 in 51 centers. Considering 124 active sites in July 2013 and the current recruitment rate, we will fi nish enrollment by November 2014. MSARM was complete in 34/47 (72%) of patients allocated to the P112f Eff ect of protective ventilation on organ-specifi c cytokine production in an experimental postoperative sepsis model J Sperber1, M Lipcsey2, A Larsson3, A Larsson2, J Sjölin3, M Castegren1 1Centre for Clinical Research Sörmland, Uppsala University, Uppsala, Sweden; 2Surgical Sciences, Uppsala University, Uppsala, Sweden; 3Medical Sciences, Uppsala University, Uppsala, Sweden Critical Care 2013, 17(Suppl 2):P112 (doi: 10.1186/cc12050) Methods We prospectively evaluated 270 patients immediately after cardiac surgery, presenting hypoxemia and PaO2/FiO2 <250. Patients were randomized to an intensive alveolar recruitment maneuver (ARM) or a moderate ARM strategy. Intensive ARM group: recruitment with an inspiratory pressure amplitude of 15 cmH2O and PEEP of 30 cmH2O, followed by ventilation with PEEP  = 13  cmH2O, during 4  hours of protective mechanical ventilation with VT = 6 ml/kg/pbw. Moderate ARM group: recruitment with opening pressures of 20 cmH2O in the airways, followed by ventilation with PEEP = 8 cmH2O, during 4 hours of protective mechanical ventilation with VT = 6 ml/kg/pbw. The primary outcome was a composite endpoint of severe pulmonary complications in the postoperative period defi ned as intra-hospital death, need for mechanical ventilation for more than 48 hours after surgery, pulmonary infection or after reintubation within 28 days after randomization. The secondary outcome was the incidence of nonpulmonary complications as postoperative myocardial ischemia, acute renal failure (RIFLE-R), respiratory mechanics and blood gas analysis after ARM, ICU length of stay, hospital length of stay and 30-day mortality. Introduction Low tidal volume (VT) ventilation in intensive care patients without lung injury attenuates the systemic infl ammatory response [1]. The contribution of the specifi c organ infl ammatory responses to the systemic picture remains to be elucidated. We investigated the eff ect of low VT ventilation compared with medium high VT on hepatic, splanchnic and cerebral cytokine responses in an experimental large animal postoperative sepsis model. Methods Twenty pigs, group Protective Ventilation (PV), were ventilated with low VT (6 ml/kg) and PEEP 10 cmH2O while 10 pigs, group Control (C), were ventilated with a VT of 10 ml/kg and PEEP 5 cmH2O. Catheters were introduced into an artery, the jugular bulb, the hepatic vein and the portal vein. Laparotomy for 2 hours simulated a surgical procedure after which baseline ensued and a continuous endotoxin infusion was started at 0.25 μg/kg/hour for 5 hours. Diff erences were analyzed with ANOVA for repeated measures. 1. Amato MB, et al.: N Engl J Med 1998, 338:347-354. P110 0 Intensive alveolar recruitment protocol reduces pulmonary complications and intensive care permanence after cardiac surgery A Leme1, L Hajjar1, E Nozawa1, C Hashizume1, J Almeida1, J Fukushima1, J Auler Jr1, R Santiago1, R Ianotti1, M Amato2, E Osawa1, M Feltrim1, F Galas1 1Heart Institute, São Paulo, Brazil; 2Faculdade de Medicina da Universidade de São Paulo, Brazil Critical Care 2013, 17(Suppl 2):P110 (doi: 10.1186/cc12048) Conclusion The reliability of pressure measurements and also of compliance estimation via the tested catheters is high. Only in two catheters was the fi lling volume a critical point for a precise measurement of pressure or for estimation of compliance. Immediately after unpacking, adhesion of the balloon material might prevent reliable pressure measurement, therefore before the fi rst measurement overfi lling of the balloon and retention of the excess gas seems strongly recommended. Introduction Cardiac surgical procedures are associated with a high incidence of postoperative complications, increasing costs and mortality. The purpose of this study is to evaluate prospectively the impact of two protective mechanical ventilation strategies, both using low-tidal volume ventilation (6 ml/kg/ibw) after cardiac surgery. Haemodynamic changes during alveolar recruitment manoeuvre in patients with diastolic dysfunction Introduction To investigate the haemodynamic eff ects of an alveolar recruitment manoeuvre in intubated patients with diastolic dysfunction compared with patients with normal diastolic function in a mixed ICU. Methods Between October 2011 and February 2012, 16 mechanically ventilated patients admitted to the ICU with normal systolic function 2 Results We randomized 101 patients between November 2011 and October 2012 in 51 centers. Considering 124 active sites in July 2013 and the current recruitment rate, we will fi nish enrollment by November 2014. MSARM was complete in 34/47 (72%) of patients allocated to the S41 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 ART group. The main reason for MSARM interruption was hypotension (8/9 (90%)). Mean titrated PEEP in the ART group was 16.1 ± 3.5 versus 12.9 ± 3.4 cmH2O in ARDSNet (P <0.001). One hour after randomization, tidal volume was similar between the ART and ARDSNet groups (5.1 ± 0.8 vs. 5.3 ± 0.7 ml/kg PBW, respectively; P = 0.08). Mean values remained below 6 ml/kg PBW up to day 3. Few patients had plateau pressure >30  cmH2O 1  hour after randomization in the ART and ARDSNet groups (3/43 (7.0%) vs. 4/47 (8.5%), respectively; P = 1.00) and on subsequent days. PaO2/FiO2 was signifi cant higher in the ART group (179.5 ± 84.5 vs. 143.3 ± 46.8, P = 0.01) and increased over time up to 7 days after randomization (272.3 ± 136.5 vs. 192.6 ± 72.3, P = 0.003). The ART strategy did not increase the risk of barotrauma (relative risk (RR) = 0.78, 95% CI = 0.19 to 3.30) in the fi rst 7 days after randomization or the need to initiate or increase vasopressors or mean arterial pressure <65 mmHg (RR = 1.14, 95% CI = 0.65 to 2.02, P = 0.67) 1 hour after randomization. However, the ART strategy increased the risk for severe acidosis (pH <7.10) 1 hour after randomization (RR = 3.20, 95% CI = 1.12 to 9.20, P = 0.03). Reliability of pressure measurements via balloon catheters is high: an evaluation of six esophageal catheters Reliability of pressure measurements via balloon catheters is high: an evaluation of six esophageal catheters p g S Walterspacher, L Isaak, HJ Kabitz, J Guttmann, S Schumann University Medical Center Freiburg, Germany Critical Care 2013, 17(Suppl 2):P111 (doi: 10.1186/cc12049) y g y Critical Care 2013, 17(Suppl 2):P111 (doi: 10.1186/cc12049) Introduction Reliable measurement of esophageal pressure is a prerequisite for analysis of respiratory system mechanics in spontaneous breathing patients. For that purpose, various types of balloon catheters exist that diff er in material, size and shape. In physical models we studied the quality of pressure measurement via six diff erent balloon catheters, three of them containing a second balloon for measurement of gastric pressure. g p Methods Nine balloons of six esophageal catheters were investigated in three conditions: measurement of balloon pressure during initial infl ations immediately after unpacking; measurement of static pressures at diff erent fi lling volumes; and compliance estimation in physical models (27, 54, 90  ml/cmH2O) at diff erent levels of superimposed pressure. Conclusion ART is feasible. The incidence of adverse events was similar between groups except for severe acidosis 1 hour after randomization. Hence we adjusted the study protocol, increasing the respiratory rate (from 10 to 15/minute) during MSARM. Results During the initial infl ation most catheters showed pressure artifacts resulting from material adhesion. Those artifacts disappeared during following infl ations. Static pressure measurements could be performed with an error below 1 cmH2O if the balloon was fi lled appropriately. Overfi lling of the balloon resulted in larger errors only in two catheters. Compliance estimations resulted in errors below 1 ml/ cmH2O. Superimposed pressure had no relevant eff ect on compliance estimation. P112f y y y y Results The intensive ARM group compared with the moderate ARM group had lower incidence of the primary outcome, mainly due to the reduced rate of pulmonary infection (2.3% vs. 10.1%, P = 0.009). Moreover, the intensive ARM group presented higher lung compliance (68 ± 19 vs. 51 ± 17 ml/cmH2O, P <0.001) and PaO2/FiO2 ratio (360 ± 68 vs. 240  ±  74, P  <0.001) after intervention when compared with the moderate group. Also, the intensive ARM presented a lower length of ICU stay (3 days vs. 4 days, P = 0.027) than the moderate ARM. There are no diff erences regarding severe nonpulmonary complications and 30- day mortality between groups. Results TNFα levels were higher in the hepatic vein than in the artery, the jugular bulb and the portal vein. IL-6 levels were higher in the artery and the jugular bulb compared with the portal and hepatic veins. IL-10 levels were higher in the portal vein compared with the jugular bulb and hepatic vein. The organ-specifi c IL-10 concentrations were all higher than the arterial concentration. Comparison between the ventilation groups showed that TNFα, IL-6 and IL-10 in the hepatic vein were higher in group C compared with group PV at the end of the experiment. Peak concentrations of TNFα and IL-6 in the portal vein were higher in group C compared with group PV. Conclusion An intensive ARM strategy reduces postoperative pulmo- nary complications, reduces hypoxemia, increases lung compliances and decreases the length of ICU stay after cardiac surgery. Reference S42 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Methods Mechanically ventilated patients (PEEP 10  cmH2O and TV 7 ml/kg – Baseline) were randomized to two groups: in the PEEP 15 group, PEEP was increased from 10 to 15 cmH2O; while in the PEEP 5 group, PEEP was decreased from 10 to 5 cmH2O. Arterial gas analyses were performed in both groups after 5, 15, 30 and 60 minutes from the change of PEEP. Conclusion In this experiment TNFα was mainly generated in the liver while the results point to signifi cant nonhepatic IL-6 and IL-10 production. Ventilation with low VT and medium-high PEEP attenuated hepatic and splanchnic cytokine production compared with medium- high VT and lower PEEP. Reference 1. N Engl J Med 2006, 354:1775-1786. 1. N Engl J Med 2006, 354:1775-1786. 1. N Engl J Med 2006, 354:1775-1786. P112f Reference g Results We enrolled 44 ARDS patients: 23 in the PEEP 15 group and 21 in the PEEP 5 group. At Baseline, PaO2/FiO2 (P/F) and PaCO2 were similar in both groups (P/F 169.5 ± 78.8 vs. 165.4 ± 80.6; PaCO2 45.6 ± 8.5 vs. 41.7  ±  5.0  mmHg, PEEP 15 vs. PEEP 5, respectively). In the PEEP 15 group, P/F signifi cantly continuously increased over time. In PEEP 5, P/F signifi cantly decreased after 5 minutes and remained stable over time. In the PEEP 15 group, PaCO2 did not change within 60 minutes after PEEP increase. When PEEP was reduced (PEEP 5) PaCO2 remained stable for the fi rst two steps, while at 30 and 60 minutes PaCO2 was signifi cantly higher than at Baseline (Figures 1 and 2). 1. Determann et al.: Crit Care 2010, 14:R1. doi:10.1186/cc8230 P114 Comparison between the standard and low-dose chest CT scans on the lung quantitative analysis in critically ill patients. S Froio1, D Chiumello1, I Cigada2, M Brioni2, S Coppola1, F Menga2, L Gattinoni2 1Fondazione IRCCS Ca’ Granda – Ospedale Maggiore Policlinico, Milan, Italy; 2Università degli Studi di Milano, Milan, Italy Critical Care 2013, 17(Suppl 2):P114 (doi: 10.1186/cc12052) Comparison between the standard and low-dose chest CT scans on the lung quantitative analysis in critically ill patients. S Froio1, D Chiumello1, I Cigada2, M Brioni2, S Coppola1, F Menga2, L Gattinoni2 Comparison between the standard and low-dose chest CT scans on the lung quantitative analysis in critically ill patients. S Froio1, D Chiumello1, I Cigada2, M Brioni2, S Coppola1, F Menga2, L Gattinoni2 Figure 2 (abstract P113). Two-way ANOVA RM. P <0.05, #versus 5 minutes. Figure 1 (abstract P113). Two-way ANOVA RM. P <0.05, *versus Time 0, #versus 5 minutes, °versus 15 minutes. Comparison between the standard and low do the lung quantitative analysis in critically ill pat S Froio1, D Chiumello1, I Cigada2, M Brioni2, S Coppo L Gattinoni2 1Fondazione IRCCS Ca’ Granda – Ospedale Maggiore P 2Università degli Studi di Milano, Milan, Italy Critical Care 2013, 17(Suppl 2):P114 (doi: 10.1186/cc Introduction Quantitative analysis of a lung C the reference method to study lung recruitab ventilatory strategy in ARDS patients. However radiation exposure, especially when CT analysis is the evolution of ARDS. The aim of this study was t of a lower radiation dose on lung CT quantitati with analysis computed by standard CT. Methods Sedated and paralyzed patients und scans: a Standard CT (120 kV, 110 mAs, pitch 1.2 Care Dose Technology) and a CT performed by a (Lowdose: 120  kV, 60  mAs). CTs were perform inspiratory or expiratory hold, for diff erent valu Each CT image was manually delineated exclud and mediastinal structures. We analyzed Stand images with dedicated software to quantify lung regions with diff erent degrees of infl atio data computed by Standard and by Lowdose s according to Bland–Altmann analysis. Figure 2 (abstract P113). Two-way ANOVA RM. P <0.05, #versus 5 minutes. Figure 1 (abstract P113). Two-way ANOVA RM. P <0.05, *versus Time 0, #versus 5 minutes, °versus 15 minutes. Figure 1 (abstract P114). Total tissue weight. Figure 1 (abstract P113). Two-way ANOVA RM. P <0.05, *versus Time 0, #versus 5 minutes, °versus 15 minutes. Time required for gas exchange equilibration after a change of positive end-expiratory pressure in acute respiratory distress syndrome y S Coppola1, D Chiumello1, F Menga2, M Brioni2, I Cigada2, S Froio1 1Fondazione IRCCS Ca’ Granda – Ospedale Maggiore Policlinico, Milan, Italy; 2Università degli Studi di Milano, Milan, Italy Critical Care 2013, 17(Suppl 2):P113 (doi: 10.1186/cc12051) i y Conclusion Our data indicate that it is important in critically ill patients to allow suffi cient time for the full eff ect of PEEP increase on oxygenation and to prevent excessive delay when P/F decrease occurs following the application of a lower level of PEEP. Introduction Positive end-expiratory pressure (PEEP) is fundamental to prevent lung collapse in ARDS patients. A common method to titrate PEEP is to perform a PEEP test, recording the variation of cardio- respiratory parameters after a PEEP change [1]. The aim of this study was to evaluate the time-course changes of gas exchanges following a PEEP test. P116 P116 Feasibility and eff ectiveness of prone position in morbidly obese ARDS patients: a case–control clinical study A De Jong, N Molinari, M Sebbane, A Prades, S Jaber Montpellier University Hospital, Montpellier, France Critical Care 2013, 17(Suppl 2):P116 (doi: 10.1186/cc12054) Introduction Obese patients are at risk of developing atelectasis and acute respiratory distress syndrome (ARDS) [1]. The prone position (PP) may reduce atelectasis, and improves oxygenation and outcome in severe hypoxemic patients in ARDS [2], but little is known about its eff ect in obese ARDS patients. Results We enrolled 13 patients admitted to our ICU. In the Bland– Altman analysis of the lung total tissue and not infl ated tissue, the bias and agreement bands for Standard CT scan and Lowdose CT quantitative analysis were –16.97 g (–77.43 to +43.49 g) and –9.65 g (–116.81 to 77.51 g), respectively (Figures 1 and 2). Results We enrolled 13 patients admitted to our ICU. In the Bland– Altman analysis of the lung total tissue and not infl ated tissue, the bias and agreement bands for Standard CT scan and Lowdose CT quantitative analysis were –16.97 g (–77.43 to +43.49 g) and –9.65 g (–116.81 to 77.51 g), respectively (Figures 1 and 2). f Methods Morbidly obese patients (body mass index (BMI) ≥35 kg/m2) in PP with ARDS (PaO2/FiO2 ratio ≤200 mmHg) were matched to nonobese (BMI <30  kg/m2) ARDS patients in a case–control clinical study. The primary endpoints were safety and complications of PP; the second endpoints were the eff ect on oxygenation (PaO2/FiO2 ratio at the end of PP), length of mechanical ventilation and ICU stay, nosocomial infections and mortality. g p y g Conclusion Lung CT quantitative analysis computed by Lowdose CT scans could be a useful tool for monitoring and ventilatory management of ARDS patients. y Results Between January 2005 and December 2009, 149 patients were admitted for ARDS. Thirty-three obese patients were matched with 33 nonobese patients. Median PP duration was 9 (6 to 11) hours in obese patients and 8 (7 to 12) hours in nonobese patients (P = 0.28). We collected 51 complications, of which 25 in obese patients and 26 in nonobese patients. The number of patients with at least one complication was similar across groups (n = 10, 30%). P114 1Fondazione IRCCS Ca’ Granda – Ospedale Maggiore Policlinico, Milan, Italy; 2Università degli Studi di Milano, Milan, Italy Critical Care 2013, 17(Suppl 2):P114 (doi: 10.1186/cc12052) Introduction Quantitative analysis of a lung CT scan is considered the reference method to study lung recruitability to optimize the ventilatory strategy in ARDS patients. However, CT implies a risk of radiation exposure, especially when CT analysis is necessary to monitor the evolution of ARDS. The aim of this study was to evaluate the impact of a lower radiation dose on lung CT quantitative analysis compared with analysis computed by standard CT. y p y Methods Sedated and paralyzed patients underwent two chest CT scans: a Standard CT (120 kV, 110 mAs, pitch 1.2, collimation 0.6 mm; Care Dose Technology) and a CT performed by a lower radiation dose (Lowdose: 120  kV, 60  mAs). CTs were performed during the same inspiratory or expiratory hold, for diff erent values of airway pressure. Each CT image was manually delineated excluding pleural eff usions and mediastinal structures. We analyzed Standard and Lowdose CT images with dedicated software to quantify the tissue weight of lung regions with diff erent degrees of infl ation. Lung quantitative data computed by Standard and by Lowdose scans were compared according to Bland–Altmann analysis. Figure 1 (abstract P113). Two-way ANOVA RM. P <0.05, *versus Time 0, #versus 5 minutes, °versus 15 minutes. Figure 1 (abstract P113). Two-way ANOVA RM. P <0.05, *versus Time 0, #versus 5 minutes, °versus 15 minutes. Figure 1 (abstract P113). Two-way ANOVA RM. P <0.05, *versus Time 0, #versus 5 minutes, °versus 15 minutes. Figure 2 (abstract P113). Two-way ANOVA RM. P <0.05, #versus 5 minutes according to Bland Altmann analysis. Figure 2 (abstract P113). Two-way ANOVA RM. P <0.05, #versus 5 minutes. Figure 1 (abstract P114). Total tissue weight. Figure 1 (abstract P114). Total tissue weight. Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 S43 Critical Care 2013, Volume 17 Suppl 2 , pp http://ccforum.com/supplements/17/S2 therapy was higher in APRV than the CMV group (72% vs. 49%, respectively, P <0.0001). The mortality rate was signifi cantly higher in CMV (n = 46, 51%) as compared with APRV (n = 11, 29%) (P = 0.022). Conclusion The use of APRV in septic shock patients restores hemodynamic stability earlier than the CMV mode. There was a signifi cant improvement in ICU survival using APRV over CMV. P114 Early initiation of APRV in ventilated septic shock patients was associated with a decrease in ICU mortality. Figure 2 (abstract P114). Not infl ated tissue weight. Figure 2 (abstract P114). Not infl ated tissue weight. P116 The PaO2/FiO2 ratio (Figure 1) increased signifi cantly more in obese patients (from 118 ± 43 to 222 ± 84 mmHg) than in nonobese patients (from 113 ± 43 mmHg to 174 ± 80 mmHg, P = 0.03). Length of mechanical ventilation, ICU stay and nosocomial infections did not diff er signifi cantly, but mortality at 90 days was signifi cantly lower in obese patients (27 vs. 48%, P <0.05). 1. N Engl J Med 2006, 354:1775-1786. Benefi cial eff ects of intrapulmonary percussive ventilation in patients with respiratory insuffi ciency in the ICU I Blum, R Janssen-Dean, A Overdijk van, B Speelberg St Anna Hospital Geldrop, Geldrop, the Netherlands Critical Care 2013, 17(Suppl 2):P119 (doi: 10.1186/cc12057) I Blum, R Janssen-Dean, A Overdijk van, B Speelberg St Anna Hospital Geldrop, Geldrop, the Netherlands Critical Care 2013, 17(Suppl 2):P119 (doi: 10.1186/cc12057) l Methods A case–control study was conducted following ethics approval. A case was defi ned as a ventilated pregnant/postpartum woman reported to the Australian and New Zealand INFINITE H1N1 09 study from 1 June 2009 to 31 August 2009. Controls were ventilated nonpregnant women (15 to 49 years) reported to the INFINITE H1N1 09 study during the same time frame. Data were entered into SPSS and analysed using nonparametric statistics; two-tailed P <0.05 was considered signifi cant. Introduction Intrapulmonary percussive ventilation (IPV) is a therapy that is used to clear endobronchial secretions. The IPV ventilator was designed and developed by FM Bird in 1979. The ventilator consists of a phasitron that delivers rapid, high-fl ow, mini bursts of oxygen, mixed with air. The potential aims of this mechanism are pulmonary recruitment, improved mucus clearance with a direct high-frequency oscillatory eff ect. We investigated whether IPV has a positive eff ect on ventilatory values in adult patients on the ICU. i Results We studied 36 index cases and 38 controls. Index cases were more likely to have a single arterial blood gas (ABG) taken prior to intubation (P  <0.05). Similar reasons were given for the trigger to intubate (high respiratory rate, low PaO2, increased work of breathing) apart from a high PaCO2, which was a trigger in the control group only (P <0.05). There were no diff erences in the pre-intubation and post- intubation ABGs apart from a lower PaCO2 (P <0.05) and lower HCO3 (P  <0.05) in cases, and cases presented with a lower haemoglobin (P  <0.05). There were six diffi cult intubations documented with no diff erences between groups. Initial ventilator settings including mode, tidal volume, minute volume and respiratory rate demonstrated no diff erences. Both groups showed increases in PaO2 and PaCO2, and a decrease in pH from the pre-intubation to post-intubation ABGs (P <0.05).f y p Methods All patients presenting during a 4-month period in 2011 with respiratory insuffi ciency on our mixed adult ICU were included in this study. Patients were monitored before, directly after and 15 minutes after therapy with IPV using a Bird Intrapulmonary Ventilator Model IPV-2C. Preliminary experience with airway pressure release ventilation on hemodynamics in patients with septic shock in a medical/surgical ICU A Hussain, H Lababidi, A Mir, A AlHamoud, A Al Oheli, A Al Enezi King Fahd Medical City, Riyadh, Saudi Arabia Critical Care 2013, 17(Suppl 2):P115 (doi: 10.1186/cc12053) Introduction Airway pressure release ventilation (APRV) allows spontaneous breathing throughout the ventilation cycle. It increases venous return and cardiac index, which will signifi cantly improve organ perfusion. This is important in septic shock patients to prevent extrathoracic organ system failure secondary to poor perfusion. Benefi ts of APRV with cardiovascular changes are noticed in patients with acute lung injury and acute respiratory distress syndrome. It is not well established whether applying APRV will improve the survival outcome for septic shock patients. The primary outcome is whether the use of APRV in septic shock patients restores hemodynamic stability earlier than the CMV mode. The secondary hypothesis is whether the use of APRV in septic shock patients improves their survival in the ICU. Methods After Institutional Review Board approval, we retrospectively analyzed the clinical data of 129 septic shock patients who received ventilator support between January and December 2011 at a tertiary care hospital. The Cox proportional hazards model was used in adjusting potential confounding factors. The nonparametric Wilcoxon rank sum test was used to assess signifi cant outcome diff erences between groups. Time to event/survival data will be analyzed using Kaplan–Meier methods. These analyses were accomplished using SAS, version 9.3. Figure 1 (abstract P116). Individual variations of PaO2/FiO2 ratio between supine and prone positions in obese and nonobese patients. Results Among the 187 patients, 58 were excluded as per the exclusion criteria: incomplete data (n  = 28), do not resuscitate (n  = 16), ICU readmission (n = 12) and head injury (n = 4). Finally, 129 patients were included, from these 91 received CMV and 38 received APRV. At the beginning of the study, there were no diff erences between the groups in relation to hemodynamic parameters. Reversal of shock achieved in less than 72 hours was statistically signifi cant between the groups (APRV, n = 16 (42%) and CMV, n = 8 (9%), P = 0.0101). The proportion of patients recovering from septic shock after initiation of ventilator Figure 1 (abstract P116). Individual variations of PaO2/FiO2 ratio between supine and prone positions in obese and nonobese patients. S44 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 years. Provision of mechanical ventilation to pregnant/postpartum women with H1N1 infl uenza: a case–control study l WE Pollock1, R Bellomo2, S Webb3, I Seppelt4, A Davies5, E Sullivan6, S Morrison7, B Howe7 1Mercy Hospital for Women, Heidelburg, Australia; 2Austin Health, Heidelberg, Australia; 3Royal Perth Hospital, Perth, Australia; 4Nepean Hospital, Penrith, Australia; 5Alfred Hospital, Prahran, Australia; 6University of New South Wales, Randwick, Australia; 7Monash University, Prahran, Australia Critical Care 2013, 17(Suppl 2):P117 (doi: 10.1186/cc12055) Conclusion In this study, inhalation injury is common in burn patients. They had higher severity scores (APACHE II, ABSI) and higher mortality. These patients had a higher need for mechanical ventilation and lower PaO2/FiO2 ratio, but there was no signifi cant increase in ARDS or respiratory sepsis. Introduction During the infl uenza pandemic of 2009, clinicians delivered mechanical ventilation to pregnant women with little evidence to guide practice. The objective of this study was to compare the provision of mechanical ventilation to pregnant/postpartum women and a nonpregnant matched control group admitted to the ICU with H1N1 infl uenza. P120 P120 High-frequency ventilation for acute traumatic and nontraumatic lung injury R Varutti1, R Bigai1, M Fiorillo1, D Tomasello1, W Mercante1, G Trillò2 1Azienda Ospedaliera Santa Maria degli Angeli, Pordenone, Italy; 2Udine University Hospital, Udine, Italy Critical Care 2013, 17(Suppl 2):P120 (doi: 10.1186/cc12058) Preliminary experience with airway pressure release ventilation on hemodynamics in patients with septic shock in a medical/surgical ICU The mean total burn surface area (TBSA) was 28% (± 25). Forty- fi ve patients (53.5%) had TBSA >20%. These patients had higher severity scores: ABSI 8 (± 2.8) versus 6 (± 2.2) (P = 0.0001), APACHE II 13 (± 6.9) versus 7 (± 6.7) (P = 0.0001). They also had a higher need for mechanical ventilation (88.1% vs. 34.9%, P = 0.001) and shock (59.5% vs. 27.0%, P = 0.001). The PaO2/FiO2 ratio at admission with and without inhalation was 245 versus 359 (P = 0.003), and at 72 hours was 207 versus 306 (P  = 0.002). There were no signifi cant diff erences in the appearance of sepsis, ARDS and renal failure. The length of stay with and without inhalation was 25.2 (1 to 95) days versus 22.5 (1 to 92) days (P = 0.4). The inhalation injury group showed a signifi cantly higher mortality (9.0% vs. 28.9%, P = 0.001). Conclusion PP seems safe in obese patients and may improve oxygenation more than in nonobese patients. Obese patients could be a subgroup of ARDS patients who may benefi t most from PP. References 1. Gong MN, et al.: Thorax 2010, 65:44-50. 2. Charron C, et al.: Intensive Care Med 2011, 37:785-790. References 1. Gong MN, et al.: Thorax 2010, 65:44-50. 2. Charron C, et al.: Intensive Care Med 2011, 37:785-790. 1. Gong MN, et al.: Thorax 2010, 65:44-50. 2. Charron C, et al.: Intensive Care Med 2011, 37:785-790. Benefi cial eff ects of intrapulmonary percussive ventilation in patients with respiratory insuffi ciency in the ICU All patients received IPV for a period of 20 minutes consisting of two cycles of 10 minutes. Peripheral oxygen saturation (SpO2), tidal volume (Vt), respiratory rate, end-tidal CO2 (ET-CO2), dynamic lung compliance (C-dyn) and work of breathing (WOB) were monitored at the diff erent time points. Paired Student t tests were performed in order to compare the values immediately before IPV, with directly after therapy and 15 minutes later. P <0.05 was considered signifi cant.i yi Results Eighty-three patients were examined. SpO2 improved signifi - cantly from 93.7 ± 3.7 before IPV to 95.7 ± 2.8 after IPV (P <0.001) and 15 minutes later to 95.2 ± 2.8 (P <0.001). Vt improved from 418 ± 111 before IPV to 476  ±  102 directly after (P  <0.001) and to 480  ±  131 15 minutes later (P <0.01). Respiratory rate improved from 24 ± 6 to 23 ± 6 only after 15 minutes signifi cantly (P <0.01). WOB and C-dyn did not change. ET-CO2 decreased from 34.9 ± 14.8 to 33.3 ± 13.3 (P <0.05) directly after IPV and to 32.1 ± 12.8 (P <0.01) 15 minutes later.if Conclusion There were physiological diff erences between the two groups with pregnant/postpartum women showing lower PaCO2 and HCO3. However, initial ventilator support was not signifi cantly diff erent for pregnant/postpartum women compared with controls. Reference p p Critical Care 2013, 17(Suppl 2):P118 (doi: 10.1186/cc12056) 1. Salim A, Martin M: High-frequency percussive ventilation. Crit Care Med 2005, 33:S241-S245. Introduction The objective is to analyze the epidemiology and mortality of critical burn patients with inhalation injury. Introduction The objective is to analyze the epidemiology and mortality of critical burn patients with inhalation injury. Methods A prospective, observational and descriptive study was carried out in all patients admitted to an ICU from October 2008 to June 2011. Inhalation injury was defi ned with two or more of the following criteria: history of injury in an enclosed space, facial burns with singed nasal hair, carbonaceus sputum or stridor. If they were intubated it was diagnosed by bronchoscopy. Demographic data, length of stay, ABSI, APACHE II, duration of mechanical ventilation, hospital course and mortality data were collected. Data are presented as number and percentage or as median and interquartile range and were analyzed with the Fisher exact test and Mann–Whitney test. P119i Benefi cial eff ects of intrapulmonary percussive ventilation in patients with respiratory insuffi ciency in the ICU I Blum, R Janssen-Dean, A Overdijk van, B Speelberg St Anna Hospital Geldrop, Geldrop, the Netherlands Critical Care 2013, 17(Suppl 2):P119 (doi: 10.1186/cc12057) Inhalation injury in critical burn patients L C h f M S h E H L F á d Conclusion In this study we demonstrated a benefi cial eff ect of IPV on oxygen saturation, tidal volume and end-tidal CO2. IPV has this eff ect in addition to the mobilization of bronchial secretions. Reference P120 High-frequency ventilation for acute traumatic and nontraumatic lung injury high-frequency oscillatory ventilation (HFOV) was considered mostly a rescue therapy. py Methods Fifteen adult patients admitted to our ICU for acute traumatic and nontraumatic lung injury were submitted to HFOV when conventional mechanical ventilation failed. Acknowledgement This research has been cofi nanced by the European Union and Greek national funds through the operating programme ‘Education and Lifelong Learning’ – Research Funding Programme Heracleitus II. Results Clinical and demographic data are shown in Table 1. Figure 1 shows the trend of gas parameters during the recovery. At baseline PaO2 was 94  ±  28  mmHg; after 6  hours of HFOV: 135  ±  41  mmHg, P <0.01. At baseline PaO2/FiO2 was 182 ± 97 mmHg; after 6 hours of HFOV: 264 ± 101 mmHg, P <0.01. The benefi ts are maintained when returned to conventional ventilation. Reference 1. Zeravik J, et al.: Acta Anaesthesiol Scand 1989, 33(Suppl 90):149-152. Figure 1 (abstract P121). Scatter plot and fi t line of ELWI versus dPaO2/FiO2. Conclusion HFOV may therefore be anticipated to improve end- organ perfusion and gas exchange; it should be considered in severe traumatic and nontraumatic respiratory failure [1]. 1. BMJ 2010, 340:c2327. 1. BMJ 2010, 340:c2327. P120 High-frequency ventilation for acute traumatic and nontraumatic lung injury R Varutti1, R Bigai1, M Fiorillo1, D Tomasello1, W Mercante1, G Trillò2 1Azienda Ospedaliera Santa Maria degli Angeli, Pordenone, Italy; 2Udine University Hospital, Udine, Italy C i i l C 2013 17(S l 2) P120 (d i 10 1186/ 12058) y p y Critical Care 2013, 17(Suppl 2):P120 (doi: 10.1186/cc12058) Introduction ARDS is commonly observed in trauma patients. In some instances the severity of the clinical presentation is such that all conventional ventilatory support mode fails. In this setting, Results Of the 362 patients admitted, 84 (23.2%) had inhalation injury. Seventy-six percent were male and the average age was 52 (± 17.5) S45 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Figure 1 (abstract P120). Mean PaO2, PaCO2, PaO2/FiO2 at baseline and during HFOV (P, percussionator; C, conventional). *P <0.05 versus baseline, †P <0.01 versus baseline. Figure 1 (abstract P120). Mean PaO2, PaCO2, PaO2/FiO2 at baseline and during HFOV (P, percussionator; C, conventional). *P <0.05 versus baseline, †P <0.01 versus baseline. Figure 1 (abstract P120). Mean PaO2, PaCO2, PaO2/FiO2 at baseline and during HFOV (P, percussionator; C, conventional). *P <0.05 v versus baseline. Table 1 (abstract P120). Clinical and demographic data Mean Age (years) 64 Male/female 11/4 Dead 4 Trauma 3 SAPS II 45/40 APACHE 23/8 Table 1 (abstract P120). Clinical and demographic data fl ow were 3.5 Hz, 85 to 95 cmH2O, and 40 l/minute, respectively; a 2.5 to 3.5 cmH2O tracheal tube cuff leak was used. HFO mean airway pressure (mPaw) exceeded preceding conventional ventilation (CV)-mPaw by 7 to 13 cmH2O. PaO2/FiO2, lung mechanics, and hemodynamics were documented during lung-protective CV (baseline) and 1 hour following the initiation of HFO-TGI ventilation. PULSION PICCOplus v7.0 was used for hemodynamic measurements including the ELWI.i y g Results Oxygenation (PaO2/FiO2) improved signifi cantly with HFO- TGI compared with CMV (125.5  ±  54.7 vs. 195.6  ±  108.7, P  <0.001). Changes in PaO2/FiO2 were positively correlated with ELWI at baseline (Spearman’s ρ = 0.56, P = 0.016). See Figure 1. There were no signifi cant changes in patients’ fl uid balance and hemodynamics including the ELWI. Conclusion Estimation of the ELWI can help to predict the oxygenation response of ARDS patients considered for HFO-TGI ventilation. The possibility that HFO-TGI exerts an eff ect on pulmonary oedema needs further investigation.i high-frequency oscillatory ventilation (HFOV) was considered mostly a rescue therapy. P121 Improvement in oxygenation with high-frequency oscillatory ventilation combined with tracheal gas insuffl ation is correlated to the extravascular lung water index CS Vrettou, S Malachias, SG Zakynthinos, SD Mentzelopoulos Evaggelismos General Hospital, Athens, Greece Critical Care 2013, 17(Suppl 2):P121 (doi: 10.1186/cc12059) Physiologic eff ects and regional ventilation of high-frequency positive-pressure ventilation using a conventional ventilator in a severe ARDS animal model associated with an inspiratory pause or recruitment maneuvers RL Cordioli1, M Park2, M Amato3, S Gomes3, E Leite2, L Azevedo2 1Hopitaux Universitaire de Geneve, Geneva, Switzerland; 2Hospital Sirio Libanes IEP, São Paulo, Brazil; 3Hospital das Clinicas LIM 09, São Paulo, Brazil Critical Care 2013, 17(Suppl 2):P122 (doi: 10.1186/cc12060) RL Cordioli1, M Park2, M Amato3, S Gomes3, E Leite2, L Azevedo2 1Hopitaux Universitaire de Geneve, Geneva, Switzerland; 2Hospital Sirio Libanes IEP, São Paulo, Brazil; 3Hospital das Clinicas LIM 09, São Paulo, Brazil Critical Care 2013, 17(Suppl 2):P122 (doi: 10.1186/cc12060) i g Results Data displayed by VDR4® overestimated pulmonary pressures by more than 10%, but were reliable for other parameters. During ascent, an off set appeared for all respiratory parameters: Vt increased by 59% and PmEI by 53% between 0 and 8,000  ft. During descent, the off set was reversely directed with a 39% decrease in Vt and a 28% decrease in PmEE between 8,000 and 0 ft. Modifying working pressure adequately corrected PmEI and PmEE, but not Vt. In all cases, manually correcting VDR4® parameters to their 0  ft level also corrected these off sets. Multivariate analysis further established that, adjusting for other parameters, Vt, PmEI and PmEE did practically not depend on altitude. Oxygen consumption of the respirator was high, 25 l/minute at 0 ft, and stable with altitude. It was reduced with percussive rate and with FiO2. Introduction Protective mechanical ventilation (MV) in ARDS is based on reduced stretch of pulmonary tissue, sometimes resulting in severe hypoventilation that can be avoided when using high respiratory rate. High-frequency positive-pressure ventilation (HFPPV) has not been fully explored, especially when associated with other strategies aiming to avoid hypercapnia. y Methods We induced ARDS in eight pigs by lung lavage with saline plus 3 hours of injurious MV with low PEEP and high driving pressure (DP). We then performed a recruitment maneuver (RM) followed by PEEP titration using the amount of alveolar collapse in electrical impedance tomography (EIT). Then stabilization during 1 hours with tidal volume (VT) at 6 ml/kg, respiratory rate (RR) 35 breaths/minute and PEEP selected with the PEEP-FiO2 table (ARMA study), which was kept constant during two steps of HFPPV with a RR 60: one without an inspiratory pause (HFPPV-60), and one with a pause of 30% of inspiratory time (HFPPV-60 w/P30%). In another HFPPV step, we used PEEP titrated with EIT after RM (HFPPV-60 w/RM). Recruitable volume is comparable in acute respiratory distress syndrome and in healthy lungs CA Stahl1, K Moeller2, D Steinmann1, D Henzler3, S Lundin4, O Stenqvist4 1University Medical Center Freiburg, Germany; 2Biomedical Engineering, Villingen-Schwenningen, Germany; 3Klinikum Herford, Germany; 4Sahlgrenska University Hospital, Gothenburg, Sweden Critical Care 2013, 17(Suppl 2):P124 (doi: 10.1186/cc12062) Table 1 (abstract P122). Physiological variables Variable VT = 6, stabilization HFPPV-60 HFPPV-60 w/RM DP (cmH2O) 16 ± 2 16 ± 3 12 ± 2* P/F ratio 95 ± 13 149 ± 60 246 ± 99* *Tukey’s post-hoc analysis, P < 0.05 versus others. Table 1 (abstract P122). Physiological variables Introduction The application of PEEP is commonly used in acute respiratory distress syndrome (ARDS) and has been shown to improve oxygenation. To identify patients that most benefi t from the application of PEEP, the discrimination of recruiters and nonrecruiters has been postulated by Gattinoni and colleagues [1]. Recently, Dellamonica and colleagues [2] presented a method to predict alveolar recruitment. We hypothesised that the amount of recruitable volume allows the discrimination between ARDS patients and patients with healthy lungs (HL). Conclusion HFPPV with a conventional mechanical ventilator is able to maintain stable PaCO2 in clinically acceptable values, allowing reductions in VT. HFPPV-60 w/RM and PEEP titration using EIT allowed further physiologic benefi ts in a severe ARDS model. ( ) Methods We recalculated the recruited volume (RV) in 25 patients with ARDS [3] according to the method proposed by Dellamonica and colleagues during an incremental PEEP manoeuvre (PEEP increased until the plateau pressure reached 45 cmH2O). RV was calculated as the change in end-expiratory lung volume minus total respiratory system compliance times the PEEP change (RV  = ΔEELV  – CTOT×ΔPEEP). For comparison, 12 patients with HL undergoing elective surgery in general anaesthesia were measured using the same protocol. Results Both ARDS and HL patients exhibited typical P–V curves and stepwise recruitment (Figure 1). By raising PEEP from 0 to 12 cmH2O, ARDS patients recruited 331 ± 195 ml (mean ± SD) and HL patients 435 ± 43 ml. There was a strong correlation (R2 = 0.88) of the total RV with the end-inspiratory volume at a plateau pressure of 45 cmH2O in both groups; that is, recruitment was found to the same extent in both groups (Figure 2). Methods We recalculated the recruited volume (RV) in 25 patients with ARDS [3] according to the method proposed by Dellamonica and colleagues during an incremental PEEP manoeuvre (PEEP increased until the plateau pressure reached 45 cmH2O). g References g References 1. Velmahos GC, et al.: Chest 1999, 116:440-446. 2. Chung KK, et al.: Crit Care Med 2010, 38:1970-1977. References 1. Velmahos GC, et al.: Chest 1999, 116:440-446. 2. Chung KK, et al.: Crit Care Med 2010, 38:1970-1977. 2 Results HFPPV allowed reduction in PaCO2 levels: 81 (77, 94) versus 60 (58, 61), 59 (58, 60), 60 (58, 61)  mmHg, besides using lower VT: 5.2 (5.0, 5.9), 5.1 (4.5, 6.0), 4.7 (4.2, 5.7) and 4.8 (4.5, 5.6) ml/kg during stabilization, HFPPV-60, HFPPV-60 w/P30% and HFPPV-60 w/RM, respectively. It had no signifi cant diff erent results comparing HFPPV-60 with and without an inspiratory pause. HFPPV-60 w/RM allowed a better alveolar homogenization and improvement in oxygenation, shunt, dead space and DP compared with the other steps. See Table 1. P124 Recruitable volume is comparable in acute respiratory distress syndrome and in healthy lungs CA Stahl1, K Moeller2, D Steinmann1, D Henzler3, S Lundin4, O Stenqvist4 1University Medical Center Freiburg, Germany; 2Biomedical Engineering, Villingen-Schwenningen, Germany; 3Klinikum Herford, Germany; 4Sahlgrenska University Hospital, Gothenburg, Sweden Critical Care 2013, 17(Suppl 2):P124 (doi: 10.1186/cc12062) Recruitable volume is comparable in acute respiratory distress syndrome and in healthy lungs RV was calculated as the change in end-expiratory lung volume minus total respiratory system compliance times the PEEP change (RV  = ΔEELV  – CTOT×ΔPEEP). For comparison, 12 patients with HL undergoing elective surgery in general anaesthesia were measured using the same protocol. Physiologic eff ects and regional ventilation of high-frequency positive-pressure ventilation using a conventional ventilator in a severe ARDS animal model associated with an inspiratory pause or recruitment maneuvers During each HFPPV step, VT was set to reach a PaCO2 of 60  ±  3  mmHg. Distribution of regional ventilation was analyzed using EIT. Equilibrium was considered if PaCO2 was stable (<5% of variation) for >30 minutes. 2 Conclusion HFPV can be safely used at altitude, provided that VDR4®- displayed parameters are used to manually adjust settings in order to avoid exposing patients to volutrauma or barotrauma during ascent, and to major hypoventilation and alveolar collapse during descent. The high oxygen consumption is currently the main limit to its use for long- range aeromedical evacuations. Improvement in oxygenation with high-frequency oscillatory ventilation combined with tracheal gas insuffl ation is correlated to the extravascular lung water index HFPPV better alveolar homogenization and improvem shunt, dead space and DP compared with the ot Table 1 (abstract P122). Physiological variables Variable VT = 6, stabilization HFPPV-6 DP (cmH2O) 16 ± 2 16 ± 3 P/F ratio 95 ± 13 149 ± 60 *Tukey’s post-hoc analysis, P < 0.05 versus others. Conclusion HFPPV with a conventional mechan to maintain stable PaCO2 in clinically accept reductions in VT. HFPPV-60 w/RM and PEEP titra further physiologic benefi ts in a severe ARDS mo P123 High-frequency percussive ventilation at altitu hypobaric chamber with a mechanical test lung AC Cirodde1, S Montmerle2, ND Donat1, CB Bourhill L Bargues1, T Leclerc1 1Military Hospital Percy, Clamart, France; 2IRBA, Brétig Critical Care 2013, 17(Suppl 2):P123 (doi: 10.1186/c Introduction High-frequency percussive ventilat technique for most severe acute lung injury/acu syndrome (ARDS) patients [1], especially with respiratory burns [2]. This study aimed at ch delivered by Percussionnaire VDR4® and at evalua interferes with HFPV, in order to assess its usabili Methods Using a mechanical test lung mimicki 17 ml/cmH2O) with two resistance levels (5 and and ventilated with VDR4® in a hypobaric cham between 0 and 5,000 and then 0 and 8,000 ft were P122 VDR4® parameters were modifi ed one at a time at each altitude. Besides these parameters (cross-measured with standalone hardware), oxygen consumption of the respirator and three calculated parameters were studied: low-frequency tidal volume (Vt, integrated from instantaneous fl ows measured with a Fleisch pneumotachograph), end-inspiratory (PmEI) and end-expiratory (PmEE) mean pressures. PmEI and PmEE in HFPV refl ect plateau pressure and positive end-expiratory pressure in conventional ventilation. The correction of altitude-induced off set with the modifi cation of working pressure was also tested. Physiologic eff ects and regional ventilation of high-frequency positive-pressure ventilation using a conventional ventilator in a severe ARDS animal model associated with an inspiratory pause or recruitment maneuvers RL Cordioli1, M Park2, M Amato3, S Gomes3, E Leite2, L Azevedo2 1Hopitaux Universitaire de Geneve, Geneva, Switzerland; 2Hospital Sirio Libanes IEP, São Paulo, Brazil; 3Hospital das Clinicas LIM 09, São Paulo, Brazil Critical Care 2013, 17(Suppl 2):P122 (doi: 10.1186/cc12060) Physiologic eff ects and regional ventilation of high-frequency positive-pressure ventilation using a conventional ventilator in a severe ARDS animal model associated with an inspiratory pause or recruitment maneuvers Improvement in oxygenation with high-frequency oscillatory ventilation combined with tracheal gas insuffl ation is correlated to the extravascular lung water index Introduction High-frequency oscillatory ventilation combined with tracheal gas insuffl ation (HFO-TGI) can signifi cantly improve oxygenation in patients with ARDS. It has been demonstrated that oxygenation in patients with ARDS has a better response to HFO when extravascular lung water is >15 ml/kg body weight (BW). Our aim is to examine whether the extravascular lung water index (ELWI) correlates with changes in PaO2/FiO2 in patients ventilated with HFO-TGI. Figure 1 (abstract P121). Scatter plot and fi t line of ELWI versus dPaO2/FiO2. 2 2 Methods Data from 18 sessions of HFO-TGI in six patients were included in the analysis. HFO frequency, oscillatory pressure amplitude, and bias Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 S46 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 P122 Physiologic eff ects and regional ventilation of h positive-pressure ventilation using a conventio severe ARDS animal model associated with an recruitment maneuvers RL Cordioli1, M Park2, M Amato3, S Gomes3, E Leite2, 1Hopitaux Universitaire de Geneve, Geneva, Switzerlan Libanes IEP, São Paulo, Brazil; 3Hospital das Clinicas LIM Critical Care 2013, 17(Suppl 2):P122 (doi: 10.1186/c Introduction Protective mechanical ventilation on reduced stretch of pulmonary tissue, sometim hypoventilation that can be avoided when using High-frequency positive-pressure ventilation (H fully explored, especially when associated with o to avoid hypercapnia. Methods We induced ARDS in eight pigs by lu plus 3 hours of injurious MV with low PEEP and (DP). We then performed a recruitment man by PEEP titration using the amount of alveolar impedance tomography (EIT). Then stabilization tidal volume (VT) at 6 ml/kg, respiratory rate (R and PEEP selected with the PEEP-FiO2 table (AR kept constant during two steps of HFPPV with an inspiratory pause (HFPPV-60), and one wit inspiratory time (HFPPV-60 w/P30%). In another PEEP titrated with EIT after RM (HFPPV-60 w/RM step, VT was set to reach a PaCO2 of 60  ±  3  m regional ventilation was analyzed using EIT. Equil if PaCO2 was stable (<5% of variation) for >30 mi Results HFPPV allowed reduction in PaCO2 lev 60 (58, 61), 59 (58, 60), 60 (58, 61)  mmHg, be 5.2 (5.0, 5.9), 5.1 (4.5, 6.0), 4.7 (4.2, 5.7) and 4.8 ( stabilization, HFPPV-60, HFPPV-60 w/P30% a respectively. It had no signifi cant diff erent results with and without an inspiratory pause. P123 We compared incidence rates of nosocomial bacteremia using the Hospitals in Europe Link for Infection Control through Surveillance (HELICS) between general ICU and VV-ECMO patients and used multiple logistic regression analysis to control for possible confounders. Figure 2 (abstract P124). p Results During the study period 1,146 patients were admitted and 16 received VV-ECMO. The incidence of bloodstream infection in patients with ECMO was 19/1,000 exposure-days versus 4,9/1,000 exposure- days in general ICU patients (incidence rate ratio of 3.82; 95% CI: 2.0 to 7.3; P <0.001). Bacteremia was mostly due to Gram-negative agents (65%). The patients with bloodstream infections under ECMO (n  = 10) had a nonsignifi cant younger age (P = 0.08) and a lower SAPS II score (P = 0.03) compared with non-ECMO patients (n = 25). VV-ECMO patients had a signifi cantly higher risk of primary bacteremia than non- ECMO patients (P = 0.04). Patients with bloodstream infections in the VV-ECMO group had a lower crude mortality rate (OR: 0.1, P = 0.04), not confi rmed in the adjusted analysis. There were no crude or adjusted diff erences in the time to bacteremia or infections due to multiple drug-resistant microorganisms (OR: 0.26; P = 0.085) between groups. g g g p Conclusion This study suggests that VV-ECMO patients have a signifi cantly higher risk for primary nosocomial bloodstream infection. A larger study is needed to confi rm such fi ndings and to assess the need for specifi c intervention, namely routine antibiotic prophylaxis. Referencesfi Figure 2 (abstract P124). 1. Peek GJ, et al.: Effi cacy and economic assessment of conventional ventilatory support versus extracorporeal membrane oxygenation for severe adult respiratory failure (CESAR): a multicentre randomised controlled trial. Lancet 2009, 374:1351-1363. 1. Peek GJ, et al.: Effi cacy and economic assessment of conventional ventilatory support versus extracorporeal membrane oxygenation for severe adult respiratory failure (CESAR): a multicentre randomised controlled trial. Lancet 2009, 374:1351-1363. P123 P123 High-frequency percussive ventilation at altitude: study in a hypobaric chamber with a mechanical test lung AC Cirodde1, S Montmerle2, ND Donat1, CB Bourhillon2, P Jault1, L Bargues1, T Leclerc1 1Military Hospital Percy, Clamart, France; 2IRBA, Brétigny, France Critical Care 2013, 17(Suppl 2):P123 (doi: 10.1186/cc12061) Results Both ARDS and HL patients exhibited typical P–V curves and stepwise recruitment (Figure 1). By raising PEEP from 0 to 12 cmH2O, ARDS patients recruited 331 ± 195 ml (mean ± SD) and HL patients 435 ± 43 ml. There was a strong correlation (R2 = 0.88) of the total RV with the end-inspiratory volume at a plateau pressure of 45 cmH2O in both groups; that is, recruitment was found to the same extent in both groups (Figure 2). Introduction High-frequency percussive ventilation (HFPV) is a rescue technique for most severe acute lung injury/acute respiratory distress syndrome (ARDS) patients [1], especially with smoke inhalation or respiratory burns [2]. This study aimed at characterizing HFPV as delivered by Percussionnaire VDR4® and at evaluating how hypobarism interferes with HFPV, in order to assess its usability at altitude. Conclusion The relative contribution of RV to lung volume gain is similar in ARDS and in patients with healthy lungs. Our results question the relevance of recruitability as defi ned by Dellamonica and colleagues as a typical phenomenon of ARDS, but support the baby lung concept, as the recruited volume was closely related to the size of the lung. Methods Using a mechanical test lung mimicking ARDS (compliance 17 ml/cmH2O) with two resistance levels (5 and 15 cmH2O/l/second) and ventilated with VDR4® in a hypobaric chamber, ascents/descents between 0 and 5,000 and then 0 and 8,000 ft were performed. Adjustable Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum com/supplements/17/S2 S47 http://ccforum.com/supplements/17/S2 References 1. Gattinoni et al.: N Engl J Med 2006, 354:1775. 2. Dellamonica et al.: Intensive Care Med 2011, 37:1595. 3. Stahl et al.: Crit Care Med 2006, 34:2090. P125 Nosocomial bloodstream infection and venovenous extracorporeal membrane oxygenation: a retrospective cohort study B Serra de Oliveira, S Mendes Fernandes, C França Hospital de Santa Maria CHLN, Lisboa, Portugal Critical Care 2013, 17(Suppl 2):P125 (doi: 10.1186/cc12063) Methods A retrospective cohort study from patients admitted to our ICU between April 2009 and June 2012. P123 We compared incidence rates of nosocomial bacteremia using the Hospitals in Europe Link for Infection Control through Surveillance (HELICS) between general ICU and VV-ECMO patients and used multiple logistic regression analysis to control for possible confounders. Results During the study period 1,146 patients were admitted and 16 received VV-ECMO. The incidence of bloodstream infection in patients with ECMO was 19/1,000 exposure-days versus 4,9/1,000 exposure- days in general ICU patients (incidence rate ratio of 3.82; 95% CI: 2.0 to 7.3; P <0.001). Bacteremia was mostly due to Gram-negative agents (65%). The patients with bloodstream infections under ECMO (n  = 10) had a nonsignifi cant younger age (P = 0.08) and a lower SAPS II score (P = 0.03) compared with non-ECMO patients (n = 25). VV-ECMO patients had a signifi cantly higher risk of primary bacteremia than non- ECMO patients (P = 0.04). Patients with bloodstream infections in the VV-ECMO group had a lower crude mortality rate (OR: 0.1, P = 0.04), not confi rmed in the adjusted analysis. There were no crude or adjusted diff erences in the time to bacteremia or infections due to multiple drug-resistant microorganisms (OR: 0.26; P = 0.085) between groups. Conclusion This study suggests that VV-ECMO patients have a signifi cantly higher risk for primary nosocomial bloodstream infection. A larger study is needed to confi rm such fi ndings and to assess the need for specifi c intervention, namely routine antibiotic prophylaxis. References 1. Peek GJ, et al.: Effi cacy and economic assessment of conventional ventilatory support versus extracorporeal membrane oxygenation for severe adult respiratory failure (CESAR): a multicentre randomised controlled trial. Lancet 2009, 374:1351-1363. 2. Conrick-Martin I, et al.: Nosocomial infections in a cohort of extracorporeal life support patients. Crit Care Resusc 2012, 14:198-201. P126 Monitoring anticoagulation during extracorporeal membrane oxygenation in patients with acute respiratory failure M Panigada1, C Mietto2, F Pagan2, L Bogno2, V Berto2, L Gattinoni2 1F d i IRCCS C ’ G d O d l M i P li li i Mil It l Figure 1 (abstract P124). Figure 2 (abstract P124). Figure 1 (abstract P124). Figure 1 (abstract P124). Figure 2 (abstract P124). Methods A retrospective cohort study from patients admitted to our ICU between April 2009 and June 2012. References 1. Gattinoni et al.: N Engl J Med 2006, 354:1775. 2. Dellamonica et al.: Intensive Care Med 2011, 37:1595. 3. Stahl et al.: Crit Care Med 2006, 34:2090. 2. Conrick-Martin I, et al.: Nosocomial infections in a cohort of extracorporeal life support patients. Crit Care Resusc 2012, 14:198-201. 2. Conrick-Martin I, et al.: Nosocomial infections in a cohort of extracorporeal life support patients. Crit Care Resusc 2012, 14:198-201. P125 Nosocomial bloodstream infection and venovenous extracorporeal membrane oxygenation: a retrospective cohort study B Serra de Oliveira, S Mendes Fernandes, C França Hospital de Santa Maria CHLN, Lisboa, Portugal Critical Care 2013, 17(Suppl 2):P125 (doi: 10.1186/cc12063) P127 Extracorporeal membrane oxygenation: the MOTOR of cytokine production? p Y Hara1, O Nishida1, T Nakamura1, S Uchiyama1, J Shibata1, C Yamashita1, M Yumoto1, Y Shimomura1, N Kuriyama1, N Yasuoka1, M Ito1, K Kawata1, S Hayakawa1, S Yamada2, T Miyasho3, K Moriyama1 1Fujita Health University School of Medicine, Toyoake, Japan; 2Shino Corporation, Tokyo, Japan; 3Rakuno Gakuen University, Ebetsu, Japan Critical Care 2013, 17(Suppl 2):P127 (doi: 10.1186/cc12065) g Results In all patients RR decreased with the increase of extracorporeal CO2 removal and a negative correlation was found between RR and ECCO2 Cl/total VCO2 (r2 = 0.42, P <0.01). In all patients we were able to obtain a reduction of RR below 15 (28 ± 4 vs. 8 ± 4, RR at low gas fl ow vs. RR at maximal gas fl ow, P <0.001). The selected maximal gas fl ow was variable between diff erent patients (6.7 ± 2 l/minute), corresponding to diff erent levels of ECCO2 Cl/total VCO2 (83 ± 17%, range 53 to 100%) and RR response (8 ± 4, range 5 to 14). Introduction The usefulness of extracorporeal membrane oxygenation (ECMO) is being rediscovered in the wake of the pandemic of H1N1 infl uenza. However, it has been reported that patients who received ECMO often developed virus-associated hemophagocytic syndrome (VAHS), compared with those without ECMO support. Although there is ample evidence that extensive cytokine activation is a key factor in VAHS, ECMO itself could be a potential trigger to exacerbate the pathology by amplifying cytokine activation. In this study, we investigated whether mediators such as cytokines may be produced by ECMO. Conclusion In patients with COPD exacerbation, who failed non- invasive ventilation, VV-ECMO allows one to maintain spontaneous breathing. Titration of extracorporeal CO2 removal leads to control RR. This approach could interrupt the vicious circle of dynamic hyperinfl ation and allow the defl ation of lung parenchyma. Figure 1 (abstract P128). y Methods Patients with severe respiratory failure who were placed on ECMO were enrolled between June and July 2012. This study was approved by the ethics committee. Blood specimens were drawn from the blood circuit at the inlet of the centrifugal pump (before) and outlet of the hollow fi ber oxygenator (after) at a frequency of three to four times per day. Blood IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12(p70), IL-13, IL-17, G-CSF, GM-CSF, IFNγ, MCP-1, MIP-1β, and TNFα were measured globally using a multiplex cytokine bead array system (Bio-Plex; Bio-Rad, Tokyo, Japan). Eff ect of extracorporeal CO2 removal on respiratory rate in spontaneously breathing patients with chronic obstructive pulmonary disease exacerbation Table 1 (abstract P126). Comparison between aPTT and K-TEG R Table 1 (abstract P126). Comparison between aPTT and K-TEG R aPTT range (ratio) K-TEG R range Low Therapeutic High (minutes) (<1.5) (1.5 to 2) (>2) Low (<20) 13 (9%) 8 (5%) 0 21 (14%) Therapeutic (20 to 90) 10 (7%) 25 (17%) 11 (7%) 46 (30%) High (>90) 15 (10%) 55 (36%) 15 (10%) 85 (56%) 38 (25%) 88 (58%) 26 (17%) 152 total samples Conclusion Anticoagulation was excessive in more than one-half of the samples according to TEG monitoring, while negligible based on aPTT. Reference 1. Oliver WC: Semin Cardiothorac Vasc Anesth 2009, 13:154-175. P127 Extracorporeal membrane oxygenation: the MOTOR of cytokine production? Y Hara1, O Nishida1, T Nakamura1, S Uchiyama1, J Shibata1, C Yamashita1, M Yumoto1, Y Shimomura1, N Kuriyama1, N Yasuoka1, M Ito1, K Kawata1, S Hayakawa1, S Yamada2, T Miyasho3, K Moriyama1 1Fujita Health University School of Medicine, Toyoake, Japan; 2Shino Corporation, Tokyo, Japan; 3Rakuno Gakuen University, Ebetsu, Japan Critical Care 2013 17(Suppl 2):P127 (doi: 10 1186/cc12065) Table 1 (abstract P126). Comparison between aPTT and K-TEG R aPTT range (ratio) Table 1 (abstract P126). Comparison between aPTT and K-TEG R PTT ( ti ) Table 1 (abstract P126). Comparison between aPTT and K-TEG R aPTT range (ratio) K-TEG R range Low Therapeutic High (minutes) (<1.5) (1.5 to 2) (>2) Low (<20) 13 (9%) 8 (5%) 0 21 (14%) Therapeutic (20 to 90) 10 (7%) 25 (17%) 11 (7%) 46 (30%) High (>90) 15 (10%) 55 (36%) 15 (10%) 85 (56%) 38 (25%) 88 (58%) 26 (17%) 152 total samples Introduction During severe exacerbation of chronic obstructive pulmonary disease (COPD) tachypnea, as a consequence of respiratory acidosis, and airfl ow limitation, due to small airway obstruction, lead to lung hyperinfl ation, respiratory distress and gas exchange impairment. Invasive mechanical ventilation could worsen lung hyperinfl ation and produce a vicious circle. We investigated whether increasing extracorporeal carbon dioxide removal (ECCO2 Cl) could reduce the respiratory rate (RR), so prolonging time for lung emptying and allowing resolution of hyperinfl ation. g yl Methods Six patients with COPD exacerbation with respiratory acidosis (PaCO2 83  ±  27  mmHg, pH 7.19  ±  0.1) and tachypnea (RR 39  ±  5) despite maximal non-invasive ventilation underwent venovenous extracorporeal membrane oxygenation (VV-ECMO). All patients were awake and spontaneously breathing an adequate air–oxygen mixture to correct hypoxemia (PaO2 72 ± 27 mmHg). Monitoring anticoagulation during extracorporeal membrane oxygenation in patients with acute respiratory failure yg p B Serra de Oliveira, S Mendes Fernandes, C França Hospital de Santa Maria CHLN, Lisboa, Portugal p , , g Critical Care 2013, 17(Suppl 2):P125 (doi: 10.1186/cc12063) M Panigada1, C Mietto2, F Pagan2, L Bogno2, V Berto2, L Gattinoni2 1Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy; 2IRCCS Ca’ Granda Maggiore Policlinico Hospital Foundation, Milan, Italy Critical Care 2013, 17(Suppl 2):P126 (doi: 10.1186/cc12064) Introduction Venovenous extracorporeal membrane oxygenation (VV-ECMO) for respiratory failure in the ICU is used in a variety of clinical situations and has been demonstrated to signifi cantly improve survival without disability in adult respiratory distress syndrome [1]. ECMO has been presented as a risk factor for bloodstream infection although recently published data do not support this view or the use of antibiotic prophylaxis [2]. We aimed to examine VV-ECMO as a risk factor for nosocomial bloodstream infection. Introduction aPTT is a common tool for anticoagulation monitor ing during extracorporeal membrane oxygenation (ECMO). Thrombo- elasto graphy (TEG) is another available option in this setting. Methods A prospective observational study on 12 consecutive patients during venovenous ECMO. Anticoagulation was provided g p y ( ) p g Methods A prospective observational study on 12 consecutive patients during venovenous ECMO. Anticoagulation was provided S48 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 0.22; IL-8, P = 0.43; IL-10, P = 0.84; MCP-1, P = 0.10; and MIP-1β, P = 0.65; Wilcoxon signed-rank test). with unfractioned heparin titrated to an aPTT ratio target of 1.5 to 2. Kaolin-activated TEG (K-TEG) was contemporarily measured but did not guide heparin infusion. Baseline K-TEG reaction time (R) >20 minutes is accepted for anticoagulation but when it exceeds 90  minutes anticoagulation may be too great [1]. with unfractioned heparin titrated to an aPTT ratio target of 1.5 to 2. Kaolin-activated TEG (K-TEG) was contemporarily measured but did not guide heparin infusion. Baseline K-TEG reaction time (R) >20 minutes is accepted for anticoagulation but when it exceeds 90  minutes anticoagulation may be too great [1]. g Conclusion The use of ECMO in patients with severe respiratory failure did not induce systemic infl ammatory changes. These observations are preliminary, but may nevertheless have important implications for the future management of patients with severe infections. g y g Results Mean ECMO duration was 9 ± 4 days. Eff ect of extracorporeal CO2 removal on respiratory rate in spontaneously breathing patients with chronic obstructive pulmonary disease exacerbation While keeping the blood fl ow stable (2.9 ± 0.5 l/minute), we changed the gas fl ow of the artifi cial lung to modify the extracorporeal CO2 clearance as a percentage of total patient CO2 production (% ECCO2 Cl/total VCO2) and we observed the variations of RR. We recorded RR at three levels of gas fl ow in each patient (Figure 1). Conclusion Anticoagulation was excessive in more than one-half of the samples according to TEG monitoring, while negligible based on aPTT. Reference Reference 1. Oliver WC: Semin Cardiothorac Vasc Anesth 2009, 13:154-175. Monitoring anticoagulation during extracorporeal membrane oxygenation in patients with acute respiratory failure A total of 152 K-TEGs were collected. Comparison between aPTT and K-TEG R is reported in Table 1. Four patients (33%) had hemorrhagic complications. Neither aPTT nor K-TEG R were signifi cantly diff erent in patients with hemorrhagic events compared with patients without hemorrhagic events but the latter received a signifi cantly lower total heparin dose (P = 0.0097). P128f P128f Eff ect of extracorporeal CO2 removal on respiratory rate in spontaneously breathing patients with chronic obstructive pulmonary disease exacerbation E Spinelli, S Crotti, L Zacchetti, N Bottino, V Berto, R Russo, M Chierichetti, A Protti, L Gattinoni Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy Critical Care 2013, 17(Suppl 2):P128 (doi: 10.1186/cc12066) P131 Feasibility of minimally invasive stimulation of the phrenic nerves for supporting ventilation in fully anesthetized swine J Bijwadia1, M Karamanoglu2 1Regions Hospital, St Paul, MN, USA; 2Respithera, Bloomington, MN, USA Critical Care 2013, 17(Suppl 2):P131 (doi: 10.1186/cc12069) ECCO2 removal with a phosphorylcholine-coated membrane oxygenator in diffi cult respiratory weaning patients Table 1 (abstract P129) p g Results There was a close correlation between the measured build-up of end-expiratory lung volume during a PEEP-step manoeuvre and ΔPEEP/EL where EL was conventionally determined using oesophageal pressure measurements (see Figure 1).fi Conclusion Esophageal pressure measurements are diffi cult to perform [3] and rarely used in routine clinical practice. Our fi ndings indicate that a change in PEEP together with measurements of the resulting change in end-expiratory volume by spirometry in the ventilator could be used to determine lung elastance separately, the relation between lung and chest wall elastance as well as the transpulmonary pressure. References Conclusion ECCO2 removal with a phosphorylcholine-coated mem- brane oxygenator is clinically safe, avoids clotting of the oxygenator circuit and allows adequate CO2 removal. Reference 1. Stenqvist O, et al.: Acta Anaesthesiol Scand 2012, 56:738-747. 2. Baydur A: Chest 2002, 121:324-326. 3. Hedenstierna G: Minerva Anestesiol 2012, 78:959-966. 1. Stenqvist O, et al.: Acta Anaesthesiol Scand 2012, 56:738-747. 2. Baydur A: Chest 2002, 121:324-326. 2. Baydur A: Chest 2002, 121:324 326. 3. Hedenstierna G: Minerva Anestesiol 2012, 78:959-966. 1. Livigni S, et al.: Crit Care 2006, 10:R151. P127 Extracorporeal membrane oxygenation: the MOTOR of cytokine production? One-way ANOVA was used to compare the changes of parameters. P <0.05 was considered signifi cant. Results Table 1 presents the main results. The CO2 removal by mem- brane oxygenator ranged from 56 to 37  ml/minute. All patients survived to the treatment and 7/10 were weaned from the ventilator at the end of ECCO2 removal. Only one oxygenator was used for every patient without clotting of the circuit or any major bleeding problem. Table 1 (abstract P129) Day 0 Day 2 Day 4 pH 7.24 ± 0.06 7.38 ± 0.1 7.41 ± 0.07* PaCO2 (mmHg) 70 ± 5 57 ± 8 52 ± 3* PP (cmH2O) 48 ± 4 28 ± 4* 25 ± 4 VCO2 (ml/minute) 220 ± 15 201 ± 14 180 ± 13* *P <0.05 versus T0. Conclusion ECCO2 removal with a phosphorylcholine-coated mem- brane oxygenator is clinically safe, avoids clotting of the oxygenator circuit and allows adequate CO2 removal. Reference 1. Livigni S, et al.: Crit Care 2006, 10:R151. P130 Lung elastance can be determined without esophageal pressure measurements C Grivans, S Lundin, O Stenqvist Sahlgrenska University Hospital, Gothenburg, Sweden Critical Care 2013, 17(Suppl 2):P130 (doi: 10.1186/cc12068) Introduction We have previously shown, in an ex vivo porcine model, that lung elastance calculated as the PEEP change divided by lung volume increase (ΔPEEP/ΔEELV) is closely correlated to conventionally measured lung elastance using oesophageal pressure [1]. In this study we hypothesize that the successive change in lung volume during a PEEP-step manoeuvre could be predicted from ΔPEEP and lung l h b f h d l d h P129 ECCO2 removal with a phosphorylcholine-coated membrane oxygenator in diffi cult respiratory weaning patients F Turani, S Martini, A Marinelli, M Falco, R Barchetta, F Candidi, F Gargano 1Aurelia Hospital/European Hospital, Rome, Italy Critical Care 2013, 17(Suppl 2):P129 (doi: 10.1186/cc12067) Figure 1 (abstract P130). Correlation between spirometrically measured and calculated increase in EELV. ECCO2 removal with a phosphorylcholine-coated membrane oxygenator in diffi cult respiratory weaning patients Introduction ECCO2 removal may be a useful support in patients with diffi cult respiratory weaning. The aim of this study is to evaluate a new phosphorylcholine-coated EECO2 removal system with no thrombogenic activity to assess the clinical safety of the system, the changes of main cardiorespiratory indices and CO2 removal by the system. y Methods Ten patients were enrolled. Before starting with ECCO2 removal all patients were ventilated with TV <6 ml/kg, peak pressure >35 cmH2O and pH <7.25. ECCO2 removal was initiated using a modifi ed continuous venovenous hemofi ltration system with a membrane oxygenator (ABYLCAP; Bellco, Mirandola, Italy; membrane surface area: 0.67  m2, blood fl ow 280 to 350  ml/minute, phosphorylcholine coated). Femoral vein cannulation with a double-lumen catheter was used to connect the patients to the extracorporeal system. Heparin was infused to maintain ACT <190 to 240 seconds. All patients had ECCO2 for 4 days. Every 12 hours the pH, PaCO2, peak pressure and PaO2 were evaluated. VCO2 was determined by indirect calorimetry using a gas analyser equipped on an Engstrom Carestation Ventilator. During the ECCO2 removal the patients were ventilated with TV ≤6 ml/kg and peak pressure <30 cmH2O. All data are expressed as mean ± SD. One-way ANOVA was used to compare the changes of parameters. P <0.05 was considered signifi cant. Figure 1 (abstract P130). Correlation between spirometrically measured and calculated increase in EELV. i Results Table 1 presents the main results. The CO2 removal by mem- brane oxygenator ranged from 56 to 37  ml/minute. All patients survived to the treatment and 7/10 were weaned from the ventilator at the end of ECCO2 removal. Only one oxygenator was used for every patient without clotting of the circuit or any major bleeding problem. pressure minus tidal variation in oesophageal pressure divided by tidal volume. Position of the oesophageal catheter was verifi ed according to Baydur [2]. The measured change in end-expiratory lung volume during the PEEP-step manoeuvre using spirometry was compared with the end-expiratory lung volume change calculated from EL and stepwise changes in PEEP as ΔPEEP/EL. Table 1 (abstract P129) Day 0 Day 2 Day 4 pH 7.24 ± 0.06 7.38 ± 0.1 7.41 ± 0.07* PaCO2 (mmHg) 70 ± 5 57 ± 8 52 ± 3* PP (cmH2O) 48 ± 4 28 ± 4* 25 ± 4 VCO2 (ml/minute) 220 ± 15 201 ± 14 180 ± 13* *P <0.05 versus T0. P127 Extracorporeal membrane oxygenation: the MOTOR of cytokine production? HMGB1 was measured using an ELISA kit (Shino-Test, Tokyo, Japan). Results Two patients with interstitial pneumonia were studied. The ECMO system consisted of a Rotafl ow Centrifugal Pump (Maquet Japan, Tokyo, Japan), a Biocube TNC coating 6000 (NIPRO, Osaka, Japan), and a percutaneous cardiopulmonary support system (Capiox EBS; Terumo, Tokyo, Japan). The blood fl ow rate was 2.0 ± 4.0 l/minute. A total of 34 blood sets were collected. In most cases, blood levels of IL-1β, IL-2, IL- 4, IL-5, IL-12(p70), IL-13, IL-17, GM-CSF, IFNγ, and TNFα were below the detection limit and did not increase during ECMO. The other mediators were detected at the inlet (before), but no signifi cant increase was observed at the outlet (after) (HMGB1, P = 0.33; IL-6, P = 0.12; IL-7, P = Figure 1 (abstract P128). S49 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 P129 ECCO2 removal with a phosphorylcholine-coated membrane oxygenator in diffi cult respiratory weaning patients F Turani, S Martini, A Marinelli, M Falco, R Barchetta, F Candidi, F Gargano 1Aurelia Hospital/European Hospital, Rome, Italy Critical Care 2013, 17(Suppl 2):P129 (doi: 10.1186/cc12067) Introduction ECCO2 removal may be a useful support in patients with diffi cult respiratory weaning. The aim of this study is to evaluate a new phosphorylcholine-coated EECO2 removal system with no thrombogenic activity to assess the clinical safety of the system, the changes of main cardiorespiratory indices and CO2 removal by the system. Methods Ten patients were enrolled. Before starting with ECCO2 removal all patients were ventilated with TV <6 ml/kg, peak pressure >35 cmH2O and pH <7.25. ECCO2 removal was initiated using a modifi ed continuous venovenous hemofi ltration system with a membrane oxygenator (ABYLCAP; Bellco, Mirandola, Italy; membrane surface area: 0.67  m2, blood fl ow 280 to 350  ml/minute, phosphorylcholine coated). Femoral vein cannulation with a double-lumen catheter was used to connect the patients to the extracorporeal system. Heparin was infused to maintain ACT <190 to 240 seconds. All patients had ECCO2 for 4 days. Every 12 hours the pH, PaCO2, peak pressure and PaO2 were evaluated. VCO2 was determined by indirect calorimetry using a gas analyser equipped on an Engstrom Carestation Ventilator. During the ECCO2 removal the patients were ventilated with TV ≤6 ml/kg and peak pressure <30 cmH2O. All data are expressed as mean ± SD. P134 Novel biomarkers for prediction of mortality after acute exacerbation of chronic obstructive pulmonary disease H Michalopoulou, H Michalopoulou, P Stamatis, F Kattis, D Stamatis Metaxa Hospital, Athens, Greece Critical Care 2013, 17(Suppl 2):P134 (doi: 10.1186/cc12072) Results Ninety patients were included (77% male, 23% female). Mean age 69  years (±  11.88). Fifty-fi ve percent were smokers, 52% had arterial hypertension, 39% received long-term oxygenotherapy, 18% received antibiotics in the 7  days before, 18% corticosteroids, and 14% were on long-term NIV support at home. An emergency medical ambulance was immediately sent for 86% of patients. Ninety-two percent had normal consciousness (Glasgow Coma Scale 15), 78% had bronchospasm, 71% had signs of respiratory struggle, and 12% were unable to speak. The mean respiratory rate was 31.4 cycles/ minute (±  8.18), the average cardiac pulse was 103.6  beats/minute (± 23.14). Nasal EtCO2 44.92 mmHg (± 16.38), pulse oximetry with air was 83.48% (± 12.09), and the average fl ow rate of oxygen delivered was 5.69 l/minute (± 2.93). None of the patients had fever. Eighty-fi ve percent were supported on spontaneous ventilation, 22% received prehospital non-invasive ventilation, they all showed signs of severity and 3% need tracheal intubation. Seventy-fi ve percent of patients received β2-agonist and anticholinergic nebulization, 45% intravenous corticosteroids. Seventy-one percent were admitted to the emergency room, 29% to the ICU. Introduction Retrospective studies suggest that cardiac troponin levels are often elevated in patients with acute exacerbation of chronic obstructive pulmonary disease (AECOPD) indicating a poor survival. Novel high-sensitivity cardiac troponin (hs-cTnT) assays have better analytical precision than standard troponin (cTnT) assays. We elaborated a prospective cohort study to investigate the prognostic value of this novel biomarker in patients with AECOPD. Methods Fifty-six patients (mean age 64 years, 68% male) with the fi nal diagnosis of AECOPD were enrolled. Those who were diagnosed with acute coronary syndromes were excluded. We measured cardiac troponin T with a standard fourth-generation assay and a high- sensitivity assay. Clinical, electrocardiographic and echocardiographic data were collected at admission and the primary prognostic endpoint was death during 30 days of follow-up. Results Mean hs-cTnT levels at admission were 34  ng/l. During the follow-up period seven patients (12%) died. Thirty-eight percent of patients had hs-cTnT above the range of 14 ng/l. Lung elastance can be determined without esophageal pressure measurements C Grivans, S Lundin, O Stenqvist Sahlgrenska University Hospital, Gothenburg, Sweden Sahlgrenska University Hospital, Gothenburg, Sweden g y g Critical Care 2013, 17(Suppl 2):P130 (doi: 10.1186/cc12068) Critical Care 2013, 17(Suppl 2):P130 (doi: 10.1186/cc12068) Introduction Long-term use of mechanical ventilators may lead to ventilator-induced diaphragmatic dysfunction (VIDD) and increase the duration of weaning from MV [1]. It was hypothesized that stimulating the diaphragm during MV may prevent VIDD and may lead to early weaning [2]. In this study, the feasibility of generating coordinated contraction of both diaphragms was investigated using a novel transvenous diaphragmatic pacing system. Introduction We have previously shown, in an ex vivo porcine model, that lung elastance calculated as the PEEP change divided by lung volume increase (ΔPEEP/ΔEELV) is closely correlated to conventionally measured lung elastance using oesophageal pressure [1]. In this study we hypothesize that the successive change in lung volume during a PEEP-step manoeuvre could be predicted from ΔPEEP and lung elastance as ΔPEEP/EL. The objective of the study was to validate this hypothesis in patients with acute respiratory failure (ARF). Methods Two juvenile pigs were anesthetized with propofol (150 to 250 μg/kg/minute) and ventilated (VENT) with an assist control mode MV (Nellcor Puritan Bennett 840). Using fl uoroscopy, a novel multipolar neurostimulation catheter (Inspirx RL PICC53; Respithera, Bloomington, MN, USA) was threaded into the left internal jugular vein and advanced to the junction of right atrium and the superior vena cava using a Methods Thirteen patients with ARF were studied during an incremental PEEP trial, 0–4–8–12–16 cmH2O. ΔEELV was determined as the change in expiratory tidal volume following each PEEP step. Conventional calculation of lung elastance was obtained from tidal variation in airway S50 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 modifi ed Seldinger technique. The successful capture of the right and left phrenic nerves was confi rmed by fl uoroscopic visualization. Peak airway pressures (PAWP) and blood gases were determined after 10 minutes MV (MV), MV and stimulation applied together (MV+STIM) and stimulation only (STIM). modifi ed Seldinger technique. The successful capture of the right and left phrenic nerves was confi rmed by fl uoroscopic visualization. Peak airway pressures (PAWP) and blood gases were determined after 10 minutes MV (MV), MV and stimulation applied together (MV+STIM) and stimulation only (STIM). P133 N-terminal pro-brain natriuretic peptide as a prognostic marker in patients with exacerbation of chronic obstructive pulmonary disease Table 1 (abstract P131). Ventilation parameters MV MV+STIM STIM PAWP (cmH2O) 20 16 9 PO2 (mmHg) 89 101 95 PCO2 (mmHg) 32 26 26 Table 1 (abstract P131). Ventilation parameters Table 1 (abstract P131). Ventilation parameters P Stamatis, H Michalopoulou, D Stamatis Metaxa Hospital, Athens, Greece Critical Care 2013, 17(Suppl 2):P133 (doi: 10.1186/cc12071) P Stamatis, H Michalopoulou, D Stamatis Metaxa Hospital, Athens, Greece Critical Care 2013, 17(Suppl 2):P133 (doi: 10.1186/cc12071) Introduction Plasma N-terminal pro-brain natriuretic peptide (NT-pro- BNP) levels are elevated in patients with pulmonary disease especially in those with concomitant right ventricular dysfunction. The aim of the present study was to investigate the use of plasma NT-pro-BNP levels as a prognostic tool in patients with exacerbation of chronic obstructive pulmonary disease (COPD). Conclusion It was possible to capture and stimulate both phrenic nerves using a minimally invasive approach to support respiration and sustain blood gases at physiological levels. This development could help wean MV-dependent ICU patients earlier. Further long-term studies are needed to assess the full potential of this novel system. References Methods A retrospective medical records analysis of all patients hospitalized between June 2009 and June 2012 with the fi nal diagnosis of acute exacerbation of COPD who had undergone NT-pro-BNP measurements at admission followed by echocardiogram. 1. Vassilakopoulos T, Petrof BJ: Am J Respir Crit Care Med 2004, 169:336-341. 2. Sassoon CS, Zhu E, Caiozzo VJ: Am J Respir Crit Care Med 2004, 170:626-632. 1. Vassilakopoulos T, Petrof BJ: Am J Respir Crit Care Med 2004, 169:336-341. 2. Sassoon CS, Zhu E, Caiozzo VJ: Am J Respir Crit Care Med 2004, 170:626-632. 1. Vassilakopoulos T, Petrof BJ: Am J Respir Crit Care Med 2004, 169:336-341. y Results Seventy-two patients (mean age 69 years, 58% male) with COPD exacerbation but without clinical or echocardiography-oriented evidence of acute cardiac disease were enrolled. The mean forced expiratory volume in 1 second (FEV 1)/forced vital capacity ratio was 44.2% and the mean FEV 1 was 0.82 l. Median NT-pro-BNP levels at admission were 424.4 pg/ml and the tertile limits were 262.1 and 930 pg/ml. NT-pro-BNP levels signifi cantly predicted 30-day mortality (OR: 7.2, 95% CI: 3.9 to 15.2; P <0.001), (OR: 25, 95% CI: 16.3 to 37.3; P  <0.001) and (OR: 44, 95% CI: 44.5 to 76.2) for each tertile. These associations persisted after adjusting for arterial CO2 pressure, body mass index, age, gender and systolic pulmonary artery pressure. References References 1. Antoine G: The epidemiology and outcome of medical emergency team call patients treated with non invasive ventilation. Resuscitation 2011, 82:1218-1223. 2. Schmidbauer W: Early prehospital use of non invasive ventilation improves acute respiratory failure in acute exacerbation of chronic obstructive pulmonary disease. Emerg Med J 2011, 28:626-627. 1. Antoine G: The epidemiology and outcome of medical emergency team call patients treated with non invasive ventilation. Resuscitation 2011, 82:1218-1223. 2. Schmidbauer W: Early prehospital use of non invasive ventilation improves acute respiratory failure in acute exacerbation of chronic obstructive pulmonary disease. Emerg Med J 2011, 28:626-627. y Results No animal–ventilator dyssynchrony during stimulation (MV+STIM) was noted while peak airway pressures were reduced. During STIM there was no discernible paradoxical movement of the diaphragm. In addition, PCO2 and PO2 confi rmed that adequate ventilation and oxygenation can be provided by the system, while PAWP could be reduced (Table 1). Prehospital management of COPD patients in respiratory failure and short-term outcome G Campagne, J Cuny, P Gosselin, P Goldstein, N Assez, E Wiel Lille University Hospital Center, Lille Cedex, France y p Critical Care 2013, 17(Suppl 2):P132 (doi: 10.1186/cc12070) Introduction Respiratory failure in COPD patients is a frequent call in French emergency dispatching centers. We have evaluated the prehospital management of COPD patients and severity signs, and analyse outcome in the emergency department or ICU. Conclusion Among patients with acute exacerbation of chronic pulmonary disease, NT-pro-BNP could be a useful marker for severity and poor prognosis. Methods We conducted an observational, descriptive, retrospective, single-center study during a 4-month period. All COPD patients with respiratory failure and prehospital care were included. Diff erent data were recorded. P134 Prognostic accuracy of hs-cTnT for death was signifi cantly higher, with area under the ROC curve (AUC) of 0.83 (95% CI: 0.72 to 0.86), than that of cTnT (AUC: 0.63, 95% CI: 0.56 to 0.74; P <0.001). After adjustment for age, gender, creatinine levels, heart rate, left ventricular ejection fraction, arterial O2 pressure and systolic pulmonary artery pressure, hs-cTnT above the Conclusion Most of the patients had signs of severity and bronchospasm. The absence of fever and antibiotics allows us to think that the cause of decompensation is not pneumonia. Although most of them were hypoxic and hypercapnic, they seem to be good candidates for NIV support in prehospital care. Very few studies report the use of NIV in cases of COPD respiratory failure in the fi rst care delivered at home. Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 S51 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 equal groups: group I (cases, 25 patients) were subjected to additional intervention, early FOB during the fi rst 6 to 12 hours from admission; while group II (control, 25 patients) received the conventional treatment and NIPPV only. Outcome parameters measured were changes in ABG data, duration of NIPPV, rate of its success, ICU stay and mortality as well as the safety of FOB and possible complications.if 99th percentile was associated with a hazard ratio for death of 3.2 (95% CI: 1.5 to 6.7). Conclusion In AECOPD, novel biomarkers such as hs-cTnT appear to be positively associated with COPD severity and could be a determinant of mortality. 99th percentile was associated with a hazard ratio for death of 3.2 (95% CI: 1.5 to 6.7). Conclusion In AECOPD, novel biomarkers such as hs-cTnT appear to be positively associated with COPD severity and could be a determinant of mortality. y Results No signifi cant diff erence was detected between the two groups regarding the baseline characteristics. No serious complications happened from FOB, oxygen desaturation occurred in 4/25 patients (16%), tachycardia in 2/25 patients (8%). In group I, 23 patients (92%) were successfully weaned from NIPPV versus 16 patients (64%) in group II (P = 0.037). Total duration of NIPPV was 28.52 hours in group I versus 56.25 hours in group II (P = 0.001). Length of ICU stay was 4.84 days in group I versus 8.68 days in group II (P = 0.001). SMART-COP score for patients admitted with community-acquired pneumonia (CAP) to an ICU in a district general hospital: a smarter way of identifying patients with severe CAP? SMART-COP score for patients admitted with community-acquired pneumonia (CAP) to an ICU in a district general hospital: a smarter way of identifying patients with severe CAP? y y g p M Pachucki1, F Kelly2, A Padkin2 M Pachucki1, F Kelly2, A Padkin2 y 1Frenchay Hospital, Bristol, UK; 2RUH, Bath, UK Results Thirty-four patients were enrolled. Median age was 66 (54 to 71) years; 65% of the patients were male; median APACHE IV score on admission was 76 (61 to 96). Median ICU stay was 8 (5 to 17) days, with an overall hospital mortality of 12%. Thirteen patients (38%) tested positive for a respiratory virus. The most frequently found virus was infl uenza (37%), followed by RSV (15%), rhinovirus (15%), MPV (15%), corona virus (9%) and parainfl uenza virus (9%). Two of the patients (6%) had a bacterial–viral co-infection (blood culture and PCR positive for Streptococcus pneumonia and rhinovirus in one patient and for Haemophilus infl uenza and infl uenza virus in the second patient). Length of stay in the ICU was signifi cantly longer in PCR-negative patients, in comparison with PCR-positive patients (11 (6 to 20) vs. 7 (2 to 8) days, respectively, P <0.05).l y p Critical Care 2013, 17(Suppl 2):P137 (doi: 10.1186/cc12075) Introduction British Thoracic Society guidelines on community- acquired pneumonia (CAP) advocate ICU referral for patients with CURB65 score of 4 and 5. A recently developed scoring system, SMART- COP, designed to identify patients at need of intensive respiratory or vasopressor support (IRVS), has been validated in a variety of settings. It predicts the need for ICU admission (defi ned as need for IRVS) with greater accuracy than CURB65, but is not used routinely in our UK institution. Methods We retrospectively analysed critical care admissions of patients with a diagnosis of CAP in a UK district general hospital – ICNARC-coded diagnoses of pneumonia (bacterial, viral, no organisms isolated) over a 7-month period (August 2011 to January 2012). We ascertained the CURB65 and SMART-COP scores on referral to the ICU and matched them in relation to the need for IRVS, length of inotropic and ventilatory support and ICU length of stay. Conclusion Respiratory viruses, and particularly infl uenza virus, are frequently found in adult patients with respiratory failure admitted to the ICU. P136 Role of bronchoscopy during non-invasive ventilation in hypercapnic respiratory failure W Rady, A Abouelela, A Aly, W Youssef Alexandria University, Alexandria, Egypt Critical Care 2013, 17(Suppl 2):P136 (doi: 10.1186/cc12074) References 1. Carrat F, et al.: Intensive Care Med 2006, 2:156-159. 2. Choi SH, et al.: Am J Respir Crit Care Med 2012, 186:325-332. 3. Miggins M, et al.: PLoS One 2011, 6:18890. 1. Carrat F, et al.: Intensive Care Med 2006, 2:156-159. 2. Choi SH, et al.: Am J Respir Crit Care Med 2012, 186:325-332. 3. Miggins M, et al.: PLoS One 2011, 6:18890. Results Our search revealed 28 potential matches. Five patients were excluded (not CAP) and the notes for seven patients were not available for analysis. We analysed the notes of 16 patients matching our criteria. In this small sample, there was a strong association between increasing SMART-COP score and the need for IRVS (correlation coeffi cient r  = 0.96). There was also a strong correlation with longer inotropic support (r  = 0.85) and longer ventilatory support (r  = 0.96) with increasing SMART-COP scores but a weaker correlation with length of ICU stay (r = 0.49). Moreover, none of the patients admitted to the ICU had CURB65 score higher than 3 at the time of ICU referral. P134 Only one patient died in group I versus three patients in group II (P = 0.609). High incidence of respiratory viruses in critically ill adult patients with respiratory failure Introduction Laboratory testing for viral infections is not routinely performed in adult patients admitted to the ICU. However, reports indicate that viruses may contribute to signifi cant morbidity and mortality and that viral–bacterial co-infection is associated with poor outcome in this particular patient population. The aim of the study was to document the incidence of respiratory virus infections in critically ill adult patients admitted to the ICU for acute respiratory failure. g p p g p Conclusion The early application of FOB during NIPPV in patients with ARF due to COPD exacerbation was shown to be safe. Signifi cant improvement in the outcome of patients who underwent FOB was noticed in terms of improved ABG data, shorter duration of NIPPV, higher percentage of success and shorter ICU stay while no signifi cant diff erence was detected in mortality. References 1. Scala R, et al.: Crit Care 2010, 14:R80. y Methods The prospective, observational study took place during two consecutive winter seasons in a mixed 16-bed ICU at the Medical Center Leeuwarden, the Netherlands. Adult patients admitted to the ICU, suspected of respiratory failure due to community-acquired pneumonia, were included. After informed consent brushed nasopharyngeal swab (Copan®) samples were taken within 24 hours after admission and analyzed by PCR assays for the presence of respiratory syncytial virus (RSV), infl uenza virus, metapneumovirus (MPV), parainfl uenza virus, adenovirus, rhinovirus and coronavirus. Data are presented as median (IQR). 1. Scala R, et al.: Crit Care 2010, 14:R80. 2. Ambrosino N, et al.: Eur Respir J 2008, 31:874-886. 2. Ambrosino N, et al.: Eur Respir J 2008, 31:874-886. 2. Ambrosino N, et al.: Eur Respir J 2008, 31:874-886. 3. Heunks LM, et al.: Intensive Care Med 2010, 36:143-147. p 3. Heunks LM, et al.: Intensive Care Med 2010, 36:143-147. 3. Heunks LM, et al.: Intensive Care Med 2010, 36:143-147. Role of bronchoscopy during non-invasive ventilation in hypercapnic respiratory failure W Rady, A Abouelela, A Aly, W Youssef W Rady, A Abouelela, A Aly, W Youssef Alexandria University, Alexandria, Egypt Critical Care 2013, 17(Suppl 2):P136 (doi: 10.1186/cc12074) Conclusion In our small sample, higher SMART-COP score was associated with increased likelihood of IRVS. This suggests that a further study with a larger sample size should be performed to investigate whether SMART-COP is an improvement on CURB65 in predicting the need for IRVS in UK intensive care patients. Introduction Non-invasive positive-pressure ventilation (NIPPV) is the fi rst-line treatment for hypercapnic acute respiratory failure (ARF) secondary to COPD exacerbation in selected patients. Limited data exist supporting the use of fi beroptic bronchoscopy (FOB) during this clinical setting. The aim of this study is to assess the role of FOB during NIPPV in patients with decompensated COPD acute exacerbation. P135 High incidence of respiratory viruses in critically ill adult patients with respiratory failure M Sietses1, TE Faber1, L Bont2, H Buter1, EC Boerma1 1Medical Center Leeuwarden, the Netherlands; 2University Medical Center Utrecht, the Netherlands Critical Care 2013, 17(Suppl 2):P135 (doi: 10.1186/cc12073) Ventilation distribution measured with electrical impedance tomography at varying levels of assist during pressure support versus neurally adjusted ventilatory assist in ICU patients MS Van Mourik1, P Blankman1, D Hasan2, D Gommers1 1Erasmus Medical Center, Rotterdam, the Netherlands; 2Maasstad, Rotterdam, the Netherlands Critical Care 2013, 17(Suppl 2):P140 (doi: 10.1186/cc12078) 1Erasmus Medical Center, Rotterdam, the Netherlands; 2Maasstad, Rotterdam, the Netherlands y y Results The APACHE scores in the S group and NS group were 27 ± 10 and 23 ± 4, Sequential Organ Failure Assessment scores were 8 ± 4 and 7 ± 3, respectively. These scores showed no signifi cant diff erence between the groups. Procalcitonin (PCT) in the S and NS groups was 20.7  ±  21.7 and 45.0  ±  47.7  ng/ml, respectively, and there was no signifi cant diff erence between the groups. PCT declined signifi cantly in both groups. PaO2/FiO2 of the NS group was signifi cantly higher than the S group on ICU admission and 4 days after admission, but no signifi cant diff erence on 7 days after ICU admission. IL-6 of the NS group declined signifi cantly after ICU admission, and the S group also tended to decline. Introduction Electrical impedance tomography (EIT) is a non-invasive and nonradiating imaging technique, which can be used to visualize ventilation distribution of the lungs and could distinguish between the dependent (dorsal) and nondependent (ventral) parts. Introduction Electrical impedance tomography (EIT) is a non-invasive and nonradiating imaging technique, which can be used to visualize ventilation distribution of the lungs and could distinguish between the dependent (dorsal) and nondependent (ventral) parts. Methods The aim of this study was to observe ventilation distribution between dependent and nondependent lung regions, for the individual patient, during three diff erent levels of support during pressure support (PS) and neurally adjusted ventilatory assist (NAVA) ventilation. Ten mechanically ventilated patients in the ICU were included. y p Results The ratio for dependent/nondependent distribution of ventilation is signifi cantly higher at lower support levels compared with higher support levels in both PS and NAVA. However, during NAVA there was signifi cantly less impedance loss between the diff erent levels of assist compared with PS. Tidal volumes decreased when decreasing assist levels during PS whereas not during NAVA ventilation. The electrical activity of the diaphragm decreased in both PS and NAVA with higher levels of assist. Three patients showed an increase in dependent tidal impedance variation (TIV) after lowering the assist level from 15 to 10 cmH2O. This increase in TIV did not occur during NAVA ventilation. 3. Robins-Browne K, et al.: The SMART-COP score performs well for pneumonia risk stratifi cation in Australia’s Tropical Northern Territory: a prospective cohort study. Trop Med Int Health 2012, 17:914-919. 4. Fang et al.: Application and comparison of scoring indices to predict outcomes in patients with healthcare associated pneumonia. Crit Care 2011, 15:R32. P138f P138 Eff ects of steroid therapies to severe streptococcal pneumoniae that required mechanical ventilation T Nagura, T Ikeda, K Ikeda, T Ueno, S Suda Tokyo Medical University, Hachioji Medical Center, Tokyo, Japan Critical Care 2013, 17(Suppl 2):P138 (doi: 10.1186/cc12076) Introduction Streptococcal pneumonia remains the most common cause of community-acquired pneumonia (CAP), bacterial meningitis and bacteremia. Severe pneumonia caused by streptococcal pneu- monia frequently exists in the emergency room or ICU. We performed this study to evaluate the eff ect of steroid therapy for severe strepto- coccal pneumonia patients with mechanical ventilation retrospectively. Methods We enrolled 13 adults of streptococcal pneumonia patients who required mechanical ventilation. Seven of 13 patients (S group) were administered with steroid (hydrocortisone 200 to 300 mg/day), and the remaining six patients received no steroid therapy (NS group). As the conventional therapies, mechanical ventilation was commenced when a patient’s PaO2/FiO2 showed less than 200 or they clinically complained of being short of breath. All patients received appropriate fl uid therapies, vasoactive agents and blood transfusion according to the protocol of early goal-directed therapy in the Surviving Sepsis Campaign Guidelines 2008, and also were treated with antibiotics, immunoglobulins (5 g/day for 3 days) and sivelestat sodium hydrate (4.8 mg/kg/day for 7 days). Conclusion The presence of diabetes mellitus and immunosuppression, higher APACHE II scores, longer mechanical ventilation duration, and presence of open wounds were predictors of VAP. Reference 1. Guidelines for the management of adults with hospital-acquired, ventilator-associated, and healthcare-associated pneumonia. Am J Respir Crit Care Med 2005, 171:388-416. Ventilation distribution measured with electrical impedance tomography at varying levels of assist during pressure support versus neurally adjusted ventilatory assist in ICU patients MS Van Mourik1, P Blankman1, D Hasan2, D Gommers1 1Erasmus Medical Center, Rotterdam, the Netherlands; 2Maasstad, Rotterdam, the Netherlands Critical Care 2013, 17(Suppl 2):P140 (doi: 10.1186/cc12078) Conclusion Steroid therapy for severe streptococcal pneumonia patients with mechanical ventilation may have a potential to maintain oxygenation of the lung, but no signifi cant eff ects on changes of infl ammatory markers (IL-6, CRP). P139 Risk factors for ventilator-associated pneumonia in a surgical ICU A Kundakci, O Ozkalaycı, P Zeyneloglu, H Arslan, A Pirat Baskent University Hospital, Ankara, Turkey Critical Care 2013, 17(Suppl 2):P139 (doi: 10.1186/cc12077) 2 Conclusion There is more ventilation in the dependent part of the lung, compared with the nondependent part, at lower levels of assist. This could indicate that at higher support levels the contribution of the diaphragm is reduced. During NAVA ventilation, there is an autoregulation in which the patient is adjusting his tidal ventilation to maintain homogeneous ventilation distribution. Introduction Ventilator-associated pneumonia (VAP) is the most common nosocomial infection in ICU patients who require mechanical ventilation support. The aim of this study was to determine predictors for the development of VAP in surgical ICU patients admitted to Baskent University Hospital. y Methods Following Institutional Review Board approval we performed this retrospective study, including 876 patients admitted to the surgical ICU between January 2009 and July 2012. After completing a review of patient data, 45 patients diagnosed with VAP were compared with 26 appropriate matches who were not. Patients’ demographical features (age, sex, body weight), underlying diseases, etiology for ICU admission, APACHE II and Sequential Organ Failure Assessment scores, duration of hospitalization, organ dysfunctions, fl uid balances, laboratory values, use of vasopressors, mechanical ventilation, nutrition, antibiotics, transfusions, features related to central venous catheterization, urinary catheterization, and intubation were the recorded parameters. Patients P140 Ventilation distribution measured with electrical impedance tomography at varying levels of assist during pressure support versus neurally adjusted ventilatory assist in ICU patients MS Van Mourik1, P Blankman1, D Hasan2, D Gommers1 1Erasmus Medical Center, Rotterdam, the Netherlands; 2Maasstad, Rotterdam, the Netherlands Critical Care 2013, 17(Suppl 2):P140 (doi: 10.1186/cc12078) References 1. Lim WS, et al.: BTS guidelines for the management of community acquired pneumonia in adults: update 2009. Thorax 2009, 64(Suppl 3):iii1-iii55. doi:10.1136/thx.2009.121434 Methods This study is a randomized prospective case–control pilot study carried out on 50 patients admitted to critical care units at Alexandria University Hospital, Egypt suff ering from hypercapnic ARF secondary to COPD exacerbation with Kelly Matthay Score from 2 to 4. All patients received NIPPV. Patients were divided randomly into two doi:10.1136/thx.2009.121434 2. Charles PG, et al.: SMART-COP: a tool for predicting the need for intensive respiratory or vasopressor support in community-acquired pneumonia. J Clin Infect Dis 2008, 47:375-384. 2. Charles PG, et al.: SMART-COP: a tool for predicting the need for intensive respiratory or vasopressor support in community-acquired pneumonia. J Clin Infect Dis 2008, 47:375-384. Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 S52 who were not intubated and were discharged or died within 2 days of ICU admission were excluded. Results Out of 71 patients who were included in the fi nal analysis, 45 patients (63%) had VAP. Comparing with the control group, patients who developed VAP were more likely to have diabetes mellitus and immunosuppression (P  = 0.020 and P  = 0.014, respectively). These patients also had higher APACHE II scores (P  = 0.020) and a longer duration of mechanical ventilation (P <0.001), and more frequently had an open wound (P  = 0.001). Following regression analysis, presence of diabetes mellitus (OR: 12.048; 95% CI: 1.157 to 125.000; P = 0.037), immunosuppression (OR: 16.949, 95% CI: 2.463 to 111.111; P = 0.004), and open wound (OR: 5.714; 95% CI: 1.017 to 37.258; P = 0.048), higher APACHE II scores (OR: 1.132; 95% CI: 1.022 to 1.254; P = 0.018), and prolonged duration of mechanical ventilation (OR: 1.084; 95% CI: 1.002 to 1.171; P = 0.043) were determined as risk factors for VAP. Fourteen- day and 28-day mortality rates for VAP were 19% and 29%, respectively (P = 0.760 and P = 1.000, respectively). References 1. Marik PE, et al.: J Intensive Care Med 2012. [Epub ahead of print] 2. Piper AJ, et al.: Am J Respir Crit Care Med 2011, 183:292-298. 1. Marik PE, et al.: J Intensive Care Med 2012. [Epub ahead of print] 2. Piper AJ, et al.: Am J Respir Crit Care Med 2011, 183:292-298. 1. Marik PE, et al.: J Intensive Care Med 2012. [Epub ahead of print] 2. Piper AJ, et al.: Am J Respir Crit Care Med 2011, 183:292-298. Non-invasive ventilation in patients with a clinical diagnosis of pneumonia: a clinical audit Results Three hundred and fi fty-nine subjects were analyzed. Table 1 shows the time on BiPAP per visit. Children whose weight was >97 percentile revealed trends towards longer treatment times on BiPAP compared with the other two groups. The moderate subjects who weighed >97 percentile had statistically signifi cant longer treatment periods (P <0.006) when compared with the <90 percentile moderate group. Initial BiPAP settings are listed in Table 2. When controlling for age, higher BiPAP settings correlated with increasing weight. There were no weight-based trends for admissions to the PICU or overall hospital lengths of stay. No serious complications were noted.if Introduction The benefi ts of non-invasive ventilation (NIV) in patients with type 2 respiratory failure secondary to exacerbations of COPD are widely acknowledged [1], but its effi cacy in respiratory failure of other aetiologies is less clear. We describe use of NIV (bilevel and/ or continuous positive airway pressure modes) in the subgroup of patients with a clinical diagnosis of pneumonia in a 35-bed adult critical care unit (ACCU). Methods A retrospective review of data recorded prospectively on an electronic clinical information management system at University College Hospital (UCH), London, UK. Patients requiring NIV acutely at UCH are managed on the ACCU. Electronic records reviewed for patients admitted from 1 November 2010 to 2 December 2011 with clinical diagnosis of pneumonia, and in whom NIV was recorded on ≥6 occasions on the electronic chart. We concede some patients on NIV <6 hours may have been missed. We excluded patients in whom NIV was the ceiling of therapy and those using domiciliary NIV. The following data were collected: baseline patient demographics, comorbidities, therapy received in the ACCU, respiratory physiological variables, length of stay/unit outcome. Descriptive statistics of the cohort and variables associated on univariate analysis with requirement for intubation were generated using MedCalc Version 12.3.0.0 (MedCalc Software, Mariakerke, Belgium).i Conclusion This is the fi rst study to analyze the weight eff ect on BiPAP application in children with status asthmaticus. Subjects who weighed more trended greater mean time on BiPAP and initial BiPAP settings. Weight did aff ect PICU admissions or overall length of hospital stay. Analysis of bilevel positive airway pressure therapy in children based on weight Subjects were divided into moderate and severe exacerbations and then further subdivided into the following growth curve-based weight subgroups: <90 percentile, 90 to 97 percentile and >97 percentile. Subjects received standard asthma therapies in addition to BiPAP. Data were obtained at the bedside by the respiratory therapist or collected retrospectively by study investigators. Data were stored and analyzed using a RedCap database.i in status asthmaticus. Our purpose was to analyze BiPAP use and outcomes for children with status asthmaticus and obesity in our PED. Methods Patients placed on BiPAP in the PED for status asthmaticus from 1 January 2010 to 31 August 2012 were included in the analysis. Subjects were divided into moderate and severe exacerbations and then further subdivided into the following growth curve-based weight subgroups: <90 percentile, 90 to 97 percentile and >97 percentile. Subjects received standard asthma therapies in addition to BiPAP. Data were obtained at the bedside by the respiratory therapist or collected retrospectively by study investigators. Data were stored and analyzed using a RedCap database.i Analysis of bilevel positive airway pressure therapy in children based on weight Analysis of bilevel positive airway pressure therapy in children based on weight A Williams, T Abramo Vanderbilt University Medical Center, Nashville, TN, USA Critical Care 2013, 17(Suppl 2):P141 (doi: 10.1186/cc12079) A Williams, T Abramo Vanderbilt University Medical Center, Nashville, TN, USA Critical Care 2013, 17(Suppl 2):P141 (doi: 10.1186/cc12079) Introduction Obesity as a comorbidity adds a challenge when treating children presenting to the PED in status asthmaticus. Bilevel positive airway pressure (BiPAP) is an accepted treatment modality for children S53 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Table 1 (abstract P141). Time on BiPAP by weight percentile and severity of asthma exacerbation (hours) Moderate exacerbation Severe exacerbation Table 1 (abstract P141). Time on BiPAP by weight percentile and severity of asthma exacerbation (hours) Methods A monocentric prospective observational study including 48 patients with MOHS and treated by NIV for ARF over a 2-year period. Results NIV failed to reverse ARF in only seven patients. NIV failure was associated with a sixfold increase in in-hospital mortality (85.7% vs. 14.6%; P <0.001). Factors associated with NIV failure and need for endotracheal intubation included pneumonia (n = 5, 71.4% vs. n = 8, 19.5%; P = 0.01), higher SOFA (11 vs. 5; P <0.001) and SAPS 2 (69 vs. 39; P  =  0.001) score at admission. The only factor associated with successful response to NIV was idiopathic decompensated MOHS (n = 19, 100% vs. n = 0, 0%; P = 0.03). In these patients, pH (7.23 vs. 7.28 vs. 7.44, respectively, at H0, H2, H24; P <0.05) and PCO2 (74.5 vs. 68.5 vs. 57 mmHg, respectively, at H0, H2, H24; P <0.05) dramatically improved with NIV.fi Conclusion NIV provides a very effi cient therapy to reverse hypercapnic ARF in morbidly obese patients. Multiple organ failure and pneumonia are the main factors associated with NIV failure in patients with MOHS. Conclusion NIV provides a very effi cient therapy to reverse hypercapnic ARF in morbidly obese patients. Multiple organ failure and pneumonia are the main factors associated with NIV failure in patients with MOHS. References in status asthmaticus. Our purpose was to analyze BiPAP use and outcomes for children with status asthmaticus and obesity in our PED. Methods Patients placed on BiPAP in the PED for status asthmaticus from 1 January 2010 to 31 August 2012 were included in the analysis. Non-invasive ventilation in patients with a clinical diagnosis of pneumonia: a clinical audit CJ Wright1, J Hornsby2, DA Barr3, SR Moonesinghe2 1Glasgow Royal Infi rmary, Glasgow, UK; 2University College Hospital, London, UK; 3Brownlee Institute, Glasgow, UK Critical Care 2013, 17(Suppl 2):P143 (doi: 10.1186/cc12081) Non-invasive ventilation in patients with a clinical diagnosis of pneumonia: a clinical audit CJ Wright1, J Hornsby2, DA Barr3, SR Moonesinghe2 1Glasgow Royal Infi rmary, Glasgow, UK; 2University College Hospital, London, UK; 3Brownlee Institute, Glasgow, UK Critical Care 2013, 17(Suppl 2):P143 (doi: 10.1186/cc12081) P142 Determinants of non-invasive ventilation success or failure in morbidly obese patients in acute respiratory failure M Lemyze, P Taufour, J Mallat, O Nigeon, D Thevenin Schaff ner Hospital, Lens, France Critical Care 2013, 17(Suppl 2):P142 (doi: 10.1186/cc12080) Introduction Malignant obesity hypoventilation syndrome (MOHS) refers to the association between morbid obesity (body mass index >40  kg/m2), daytime hypercapnia (PCO2 >45  mmHg) after other respiratory or neuromuscular causes of alveolar hypoventilation have been excluded, and multiple organ disorders (diabetes mellitus, arterial hypertension, metabolic syndrome, obstructive sleep apnea syndrome) [1]. Acute respiratory failure (ARF) is a common life-threatening complication of MHOS. Non-invasive ventilation (NIV) provides the cornerstone of the therapeutic management of decompensated MOHS [2]. We aimed to identify the determinants of NIV success or failure in this indication. g Results Fifty-two patients fulfi lled the inclusion criteria, 29 (55.8%) were male, median age was 62.4 (IQR 48.8 to 71.6), median unit stay was 8 days (IQR 4.5 to 16.5), mean duration of NIV use was 2 days (IQR 1 to 3), 27 (51.9%) required intubation of whom 48% died. Total mortality was 13 (25%) – all intubated. Patients with recorded comorbidities were more likely to be intubated than those without (OR = 4.3, P = 0.0362); pH was signifi cantly lower in those requiring intubation at all recorded time points; and mean FiO2 at 4 to 6 hours was signifi cantly higher in those requiring intubation(0.72 vs. 0.56, P = 0.032). There was a trend S54 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 toward patients with COPD requiring intubation more often (OR = 8.4, P = 0.0711). are specialized and relatively invasive. Visualizing diaphragm thickening during inspiration by ultrasound may permit non-invasive monitoring. We explored the feasibility, reliability and physiological signifi cance of diaphragm thickening on ultrasound. Conclusion NIV was successful in 48.1% of patients with pneumonia, the remainder requiring intubation. Given the high mortality in those patients who failed NIV we believe its use in a ward setting is hazardous. We conclude that NIV may be useful in some individuals with pneumonia, but they should be managed in the ACCU setting. Further work is required to identify those patients at risk of deterioration on NIV given the possible excess mortality. Methods Five healthy subjects participated. Analysis of management of non-invasive ventilation support in prehospital care for COPD patients and short-term outcome J Cuny, G Campagne, P Gosselin, P Goldstein, N Assez, E Wiel Lille University Hospital Center, Lille Cedex, France Critical Care 2013, 17(Suppl 2):P144 (doi: 10.1186/cc12082) Analysis of management of non-invasive ventilation support in prehospital care for COPD patients and short-term outcome J Cuny, G Campagne, P Gosselin, P Goldstein, N Assez, E Wiel Lille University Hospital Center, Lille Cedex, France Critical Care 2013, 17(Suppl 2):P144 (doi: 10.1186/cc12082) y Results Diaphragm thickening in the zone of apposition was readily visualized by ultrasound in all fi ve subjects. Mean end-expiratory diaphragm thickness was 2.1  mm (SD  =  0.3  mm). During tidal breathing, the diaphragm thickened by a mean of 35% (SD = 31%). The Bland–Altman coeffi cient of reproducibility was 0.5 mm; approximately 50% of measurement variability arose from caliper positioning on the ultrasound machine; diaphragm thickness measurements changed as the probe was placed in diff erent intercostal interspaces. Diaphragm inspiratory thickening increased signifi cantly with increasing inspiratory eff ort but also varied with lung volume independent of eff ort. At inspiratory volumes below 40% of inspiratory capacity, lung volume change contributed minimally to diaphragm thickening. Introduction Benefi ts of the use of NIV in emergency departments are well established. Training and available staff , and choice of respiratory machines are essential criteria for success. Methods We conducted an observational, descriptive, retrospective, single-center study in a 4-month period. COPD patients with respiratory failure who received prehospital NIV were included. We compared two groups: COPD patients with NIV, and COPD patients without NIV. g p p p Results Forty-two patients were included, mean age 68.86 years (± 11.98), 57.14% smokers, 64.28% arterial hypertension, 100% long- term oxygenotherapy, 23.80% antibiotics in the 7 days before, 28.57% corticosteroids. A total of 88.09% had bronchospasm, 78.26% had struggle signs, 28.57% were unable to speak, 14.28% of patients were sweating. The mean respiratory rate was 30.5 cycles/minute (± 7.17), mean pulse rate was 105.76 (± 25.34). Nasal EtCO2 was 47.75 mmHg (±  16.53), pulse oxymetry in air was 85% (±  10.94), oxygen fl ow rate was 5.45 l/minute (±  2.42), temperature was 37.14°C (±  8.15). Twenty patients received NIV. A total of 61.90% were admitted to the emergency department, 35.71% to the ICU, and one patient was left at home. One patient received tracheal intubation in the hosting service. Mortality in the fi rst month was 13.04%. Analysis of management of non-invasive ventilation support in prehospital care for COPD patients and short-term outcome J Cuny, G Campagne, P Gosselin, P Goldstein, N Assez, E Wiel Lille University Hospital Center, Lille Cedex, France Critical Care 2013, 17(Suppl 2):P144 (doi: 10.1186/cc12082) A signifi cant diff erence (P  <0.05) was found for: sweats (30%/0), respiratory rate (34  ±  8.23/27  ±  6.11), nasal EtCO2 (55.0  ±  24.4/40.50  ±  9.03), pulse oxymetry in air (80%  ±  8.63/90%  ±  13.25), pulse oxymetry with oxygen (89.4% ± 4.24/87.90% ± 2.55), β2-mimetic and anti-cholinergic nebulization (60%  ±  0.5/90%  ±  0.29), emergency room admission (35%  ±  0.35/86%  ±  0.48), ICU admission (60%  ±  0.5/13%  ±  0.35), arterial blood gases on arrival in the host service (PaCO2 76.6 ± 18.66/43.93 ± 11.78). No diff erence in mortality at 1 month (2/3). Conclusion Non-invasive ventilation has improved the management and prognosis of COPD patients admitted to the emergency room. Very few studies concern the prehospital management. NIV seems to show an eff ect on prehospital care, especially in patients with signs of severity, hypercapnia, and without fever. Oxygenation and hypercapnia seem to be improved. Also fewer patients are admitted to the ICU. Bronchospasm does not seem to be an obstacle. R f y Conclusion Visualizing diaphragm thickening in the zone of appo- sition by ultrasound provides a feasible non-invasive technique for monitoring diaphragm activation in healthy subjects. Diaphragm thickening primarily refl ects muscular eff ort rather than altered muscle conformation induced by changes in lung volume, especially at lower inspiratory volumes. Reference 1. Brochard L, et al.: Noninvasive ventilation for acute exacerbations of chronic obstructive pulmonary disease. N Engl J Med 1995, 333:817-822. Retrospective review of utilisation and outcomes of diaphragmatic EMG monitoring and neurally adjusted ventilatory assist in a central London teaching hospital over a 3-year periodi Retrospective review of utilisation and outcomes of diaphragmatic EMG monitoring and neurally adjusted ventilatory assist in a central London teaching hospital over a 3-year period A Skorko, D Hadfi eld, A Vercueil, C Bell, A Feehan, K Peters, P Hopkins King’s Health Partners AHSC, London, UK Critical Care 2013, 17(Suppl 2):P146 (doi: 10.1186/cc12084) Introduction The theoretical advantages of monitoring the electrical activity of the diaphragm (EAdi) and neural triggering of support breaths (NAVA-Maquet) have not yet been shown to translate into signifi cant clinical benefi t [1]. Here we assess the eff ect of EAdi monitoring, in patients at risk of prolonged weaning, on outcomes. Figure 1 (abstract P146). Figure 1 (abstract P146). 1. Schmidbauer W, et al.: Early pre hospital use of NIV. Emerg Med J 2011, 28:626-627. P145 P145 A window into diaphragm kinetics: feasibility, precision, and physiological meaning of ultrasound measurements of diaphragm thickness EC Goligher1, F Laghi2, ND Ferguson1 1University of Toronto, Canada; 2Loyola University, Chicago, IL, USA Critical Care 2013, 17(Suppl 2):P145 (doi: 10.1186/cc12083) P142 We monitored inspiratory fl ow, volume, esophageal and gastric pressures, and diaphragm electrical activity (by esophageal and surface electromyography) while subjects performed a series of inspiratory maneuvers: tidal breathing, threshold-loaded breathing, a Muller maneuver, and inspiration to various lung volumes above functional residual capacity. At the end of each inspiratory eff ort, subjects were instructed to close the glottis and relax the respiratory muscles (so as to maintain lung volume while eliminating diaphragm activation). Sonographic images of diaphragm thickening during these maneuvers were obtained using M-mode with a 13 MHz linear array probe placed in the right ninth, 10th, or 11th intercostal space between the middle and anterior axillary lines. Risk of weaning failure with PAOP ≥15 versus PAOP ≥18 Y Nassar, A Abdelbary Risk of weaning failure with PAOP ≥15 versus PAOP ≥18 Y Nassar, A Abdelbary Cairo University, Giza, Egypt Critical Care 2013, 17(Suppl 2):P148 (doi: 10.1186/cc12086) Cairo University, Giza, Egypt Critical Care 2013, 17(Suppl 2):P148 (doi: 10.1186/cc12086) Introduction Despite 18 mmHg being recognized as a classical cutoff value, there is no defi nite value of PAOP above which cardiogenic oedema develops. We aimed to assess the risks of weaning failure with PAOP ≥15 and with PAOP ≥18. Results In total, 493/2,684 (18.3%) patients had heart–lung risk factors for prolonged weaning. One hundred and four patients received EAdi monitoring. Ventilated days were signifi cantly reduced (Figure  1) in the EAdi monitored group (median 9) versus the nonmonitored group (median 12); P = 0.024 (Mann–Whitney U test). ICU mortality was not signifi cantly diff erent and there was no correlation between time spent in NAVA and days ventilated.i Methods A prospective enrollment of 30 adult medical ICU patients on invasive mechanical ventilation who were fulfi lling the criteria for initiation of weaning from mechanical ventilation according to the Sixth International Conference Statement of Intensive Care Medicine. We followed the two-step weaning strategy, which involves assessment regarding readiness for weaning followed by a spontaneous breathing trial (SBT) as a diagnostic test to determine the likelihood of successful extubation. Invasive right heart catheterization to measure PAOP, and non-invasive transthoraxic echo and tissue Doppler parameters E/E’, E/A, DT, IVRT were measured before initiation and after termination of the SBT. Conclusion EAdi catheter insertion was associated with a signifi cant reduction in time spent on MV. A prospective RCT will be needed to confi rm benefi t and explore mechanisms. Reference 1. Brander L, et al.: Chest 2009, 135:695-703. Results E/E’ was signifi cantly correlated with PAOP (r = 503, P <0.001). A cutoff value of septal E/E’ >11.3 predicted PAOP elevation ≥18 mmHg with a sensitivity of 90.9% and specifi city of 87.8%, while a value of septal E/E’ >10.9 predicted PAOP elevation ≥15 mmHg with a sensitivity of 59.1% and specifi city of 87.8%. The incidence of weaning failure was 23%, and 72% of patients who failed to be weaned exhibited signifi cantly higher PAOP during the trial. Patients with PAOP ≥18 at the end of the SBT mostly (72.4%) failed weaning while a minority (27.6%) successfully weaned. 9 Impact of continuous positive airway pressure on the treatment of acute exacerbation of asthma: a randomised controlled trial Y Sutherasan, S Kiatboonsri, P Theerawit, C Kiatboonsri, S Trakulsrichai Ramathibothi Hospital, Bangkok, Thailand Critical Care 2013, 17(Suppl 2):P149 (doi: 10.1186/cc12087) Introduction Despite the extensive use of continuous positive airway pressure (CPAP) in various respiratory failure conditions, its role in acute asthmatic attack is uncertain [1-3]. This study aims at exploring the effi cacy of CPAP when use in addition to the conventional treatment of acute asthma exacerbation in the emergency department (ED). Results Forty-two patients were evaluated (42% male), mean age was 73 years (SD ± 15) and APACHE II score was 18.77 (median 20). Based upon evaluation of the fi ndings of BLUE protocol, a positive correlation was found in 83.78% of the sample. For the diagnosis of pulmonary edema (n = 15), sensitivity was 85% and specifi city was 86%, while for the diagnosis of pneumonia (n = 17), sensitivity was 88% and specifi city was 90%. In this sample there were only four cases of reversible airway obstruction and one case of pulmonary embolism. Five patients were excluded of the analysis because they had rare diagnoses. g y Methods During May to December 2009, acute asthma patients attending ED were randomly assigned to CPAP and control groups. In addition to the conventional treatment, CPAP ventilation of 8 cmH2O was applied to patients in the CPAP group. Data collections in both groups included peak expiratory fl ow rates (PEFR, %predicted) at each 15-minute interval (T0, T15, …), the number of short-acting bronchodilators (SABA) used, ED length of stay, relapse rate and admission rate. The primary outcome is the improvement in peak expiratory fl ow rate during a short period stay in the ED. Results There were 86 patients enrolled, with 43 patients in each study group. PEFR in the CPAP group showed no diff erence from the control group at T0 (52.7  ±  20.33 vs. 47.2  ±  16.64, P  =  NS) but was signifi cantly better at T15 (65.58 ± 21.64 vs. 56.49 ± 18.53%, P <0.05). Such improvement was consistently observed for all subsequent PEFR measurement, along with the fewer SABA used and relapse rate, but the fi gures did not meet statistical diff erence. Methods During May to December 2009, acute asthma patients attending ED were randomly assigned to CPAP and control groups. In addition to the conventional treatment, CPAP ventilation of 8 cmH2O was applied to patients in the CPAP group. 1. Lichtenstein DA, Mezière GA: Relevance of lung ultrasound in the diagnosis of acute respiratory failure: the BLUE protocol. Chest 2008, 134:117-125. 1. Brander L, et al.: Chest 2009, 135:695-703. 1. Lichtenstein DA, Mezière GA: Relevance of lung ultrasound in the diagnosis of acute respiratory failure: the BLUE protocol Chest 2008 134:117 125 Risk of weaning failure with PAOP ≥15 versus PAOP ≥18 Y Nassar, A Abdelbary The majority (91.3%) of the patients with PAOP  <18 had a successful weaning outcome and 8.7% failed weaning outcome. PAOP ≥18 signifi cantly negatively correlated with weaning success (P = 0.001, r = –0.627). On the other hand, PAOP ≥15 did not show a signifi cant correlation (P >0.05, r = –0.274). The PAOP ≥18 weaning failure risk estimate was 8.2, with 95% CI 0.004 to 0.3. The PAOP ≥15 weaning failure risk estimate was 1.83, with a signifi cant 95% CI of 0.913 to 3.648.i P147 Validation of a lung ultrasound protocol in acute respiratory failure: preliminary results RV Cremonese1, AC Tabajara Raupp1, JM Stormovski de Andrade1, RS Townsend1, FS Neres1, AS Machado1, MC Prestes1, JG Maccari2, FL Dexheimer Neto1 1Ernesto Dornelles, Porto Alegre, Brazil; 2Hospital Moinhos de Vento, Porto Alegre, Brazil Critical Care 2013, 17(Suppl 2):P147 (doi: 10.1186/cc12085) P147 Validation of a lung ultrasound protocol in acute respiratory failure: preliminary results RV Cremonese1, AC Tabajara Raupp1, JM Stormovski de Andrade1, RS Townsend1, FS Neres1, AS Machado1, MC Prestes1, JG Maccari2, FL Dexheimer Neto1 1Ernesto Dornelles, Porto Alegre, Brazil; 2Hospital Moinhos de Vento, Porto Alegre, Brazil Critical Care 2013, 17(Suppl 2):P147 (doi: 10.1186/cc12085) Critical Care 2013, 17(Suppl 2):P147 (doi: 10.1186/cc12085) Introduction Patients with acute respiratory failure demand dynamic evaluation and interventions. Lung ultrasound is a bedside technique, very promising in this setting. Previously, Lichtenstein and colleagues proposed an algorithm approach with a diagnostic accuracy of 90.5%, named Bedside Lung Ultrasound in Emergency (BLUE protocol). However, this approach has never been validated in other populations or ultrasound operators. The aim of this study was to evaluate the diagnostic accuracy of the lung ultrasound algorithm (BLUE protocol) in ICU patients admitted with acute respiratory failure. Conclusion E/E’ was signifi cantly correlated with PAOP (r  =  503, P <0.001). E/E’ > 11.3 predicted PAOP elevation ≥18 mmHg (sensitivity 90.9% and specifi city 87.8%), while E/E’ >10.9 predicted PAOP elevation ≥15 mmHg (sensitivity 59.1% and specifi city 87.8%). Weaning failure risk is greater (8.2 vs. 1.8) with PAOP ≥18 versus PAOP ≥15, respectively. P149 y Methods A prospective study, single-center consecutive case series evaluating critically ill adult patients in acute respiratory failure, admitted to a 23-bed general ICU between October 2011 and November 2012. The ultrasound examination was performed by an ICU physician until 20 minutes after admission, without interfering with usual care, and the ultrasound operator was blinded to the medical history of the patient. Three items were assessed: artifacts (horizontal A lines or vertical B lines indicating interstitial syndrome), lung sliding, alveolar consolidation and/or pleural eff usion. BLUE protocol results were compared with clinical diagnosis by the medical assistant team at the end of the ICU stay. P149 Impact of continuous positive airway pressure on the treatment of acute exacerbation of asthma: a randomised controlled trial Y Sutherasan, S Kiatboonsri, P Theerawit, C Kiatboonsri, S Trakulsrichai Ramathibothi Hospital, Bangkok, Thailand Critical Care 2013, 17(Suppl 2):P149 (doi: 10.1186/cc12087) A window into diaphragm kinetics: feasibility, precision, and physiological meaning of ultrasound measurements of diaphragm thickness EC Goligher1, F Laghi2, ND Ferguson1 g , g , g 1University of Toronto, Canada; 2Loyola University, Chicago, IL, USA Critical Care 2013, 17(Suppl 2):P145 (doi: 10.1186/cc12083) Introduction Inadequate respiratory muscle activity has been linked to ventilator-induced diaphragm dysfunction. Current techniques for monitoring respiratory muscle activity during mechanical ventilation S55 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 P148 Methods Institutional ethics approval was obtained. The Medtrack clinical information system was searched to identify patients who received MV for >48 hours and who had signifi cant chronic pulmonary disease or left/right ventricular impairment between April 2009 and March 2012. Age, APACHE II score, ventilated days, time in NAVA and outcome were compared between groups who had or had not received EAdi monitoring. P150 Novel method to confi rm tracheal intubation based on airway pressures: validation in patients with lung disease A Stemerdink1, KG Monsieurs2, F Luijmes1, W Dieperink1, JG Zijlstra1, AF Kalmar1 1University Medical Center Groningen, the Netherlands; 2Antwerp University Hospital, Antwerp, Belgium Critical Care 2013, 17(Suppl 2):P150 (doi: 10.1186/cc12088) P150 Novel method to confi rm tracheal intubation based on airway pressures: validation in patients with lung disease A Stemerdink1, KG Monsieurs2, F Luijmes1, W Dieperink1, JG Zijlstra1, AF Kalmar1 1University Medical Center Groningen, the Netherlands; 2Antwerp University Hospital, Antwerp, Belgium Critical Care 2013, 17(Suppl 2):P150 (doi: 10.1186/cc12088) P150 Novel method to confi rm tracheal intubation based on airway pressures: validation in patients with lung disease A Stemerdink1, KG Monsieurs2, F Luijmes1, W Dieperink1, JG Zijlstra1, AF Kalmar1 1University Medical Center Groningen, the Netherlands; 2Antwerp University Hospital, Antwerp, Belgium Critical Care 2013, 17(Suppl 2):P150 (doi: 10.1186/cc12088) Results A total of 470 BC were carried out in 240 patients admitted to the ICU (68% were male and 32% were female). All of the procedures were performed in mechanically ventilated patients. The mean patient age was 61 ± 14 years, with an APACHE II score at admission of 20 ± 7. The main cause for ICU admission was respiratory failure (27%). The most common comorbidities were smoking, chronic obstructive pulmonary disease, cardiovascular risk factors and atrial fi brillation. The main indications for performing the BC were percutaneous tracheostomy endoscopically guided (28%), resolution of atelectasis (27%), aspiration of secretions (24%) and hemoptysis (10%). Bronchial brushing was performed in 435 cases (93%) obtaining microbiological growth in 64% of them, mainly Pseudomonas aeruginosa and Acinetobacter baumanii. Bronchoalveolar lavage was performed in 62 cases (13%) obtaining microbiological growth in 61% of them, mainly cytomegalovirus and Stenotrophomona maltophilia. Citology was performed in 60 cases (13%); 7% showed the presence of microorganism and 13% revealed precancerous/cancerous tissue changes. Introduction Emergency endotracheal intubation results in accidental oesophageal intubation in up to 17% of patients often with disastrous consequences. We have previously published a highly specifi c and sensitive novel method to detect endotracheal intubation based on diff erences in ventilation pressure waveforms in the oesophagus and in the trachea in patients with healthy lungs [1]. A detection algorithm, based on diff erences in compliance/elasticity between the lung and the oesophagus, generated a D-value indicating tracheal intubation if D >0.5 and oesophageal intubation if D <0.5. The aim of the current study was to validate the algorithm in patients with lung disease. p g Conclusion BC can be performed at the bedside, avoiding transfers outside the ICU that may cause clinical worsening. Most frequent indications were bronchoscopy-guided percutaneous tracheostomy, resolution of atelectasis, aspiration of secretions and hemoptysis. P150 Novel method to confi rm tracheal intubation based on airway pressures: validation in patients with lung disease A Stemerdink1, KG Monsieurs2, F Luijmes1, W Dieperink1, JG Zijlstra1, AF Kalmar1 1University Medical Center Groningen, the Netherlands; 2Antwerp University Hospital, Antwerp, Belgium Critical Care 2013, 17(Suppl 2):P150 (doi: 10.1186/cc12088) Microbiological results obtained revealed the presence of aggressive pathogens. BC in ICU provides valuable diagnostic information and has therapeutic utility. y g p g Methods After obtaining institutional approval, 20 intubated and ventilated ICU patients were included. Inclusion criteria were controlled mechanical ventilation and at least mild to moderate lung injury according to a Murray lung injury score >0.1. A connecting piece was placed between the endotracheal tube and the ventilation bag. This piece comprised a thin air-fi lled catheter inserted through the tube lumen at 1 cm from the distal end, and a second catheter located at the proximal end of the tube. We performed three consecutive manual bag ventilations while recording the pressure curves through both catheters. For each ventilation, a D-value was calculated. y References References 1. Shivaram U, et al.: Eff ects of continuous positive airway pressure in acute asthma. Respiration 1987, 52:157-162. 2. Meduri GU, et al.: Noninvasive positive pressure ventilation in status asthmaticus. Chest 1996, 110:767-774. 3. Lin HC, et al.: Eff ect of nasal continuous positive airway pressure on methacholine-induced bronchoconstriction. Respir Med 1995, 89:121-128. 1. Shivaram U, et al.: Eff ects of continuous positive airway pressure in acute asthma. Respiration 1987, 52:157-162. 2. Meduri GU, et al.: Noninvasive positive pressure ventilation in status asthmaticus. Chest 1996, 110:767-774. 3. Lin HC, et al.: Eff ect of nasal continuous positive airway pressure on methacholine-induced bronchoconstriction. Respir Med 1995, 89:121-128. 1. Shivaram U, et al.: Eff ects of continuous positive airway pressure in acute asthma. Respiration 1987, 52:157-162. 2. Meduri GU, et al.: Noninvasive positive pressure ventilation in status asthmaticus. Chest 1996, 110:767-774. 3. Lin HC, et al.: Eff ect of nasal continuous positive airway pressure on methacholine-induced bronchoconstriction. Respir Med 1995, 89:121-128. 1. Shivaram U, et al.: Eff ects of continuous positive airway pressure in acute asthma. Respiration 1987, 52:157-162. Introduction Bronchoscopy (BC) is a frequently used diagnostic and therapeutic tool in our ICU. We planned to review the patients and conditions in which the procedure was performed. The objective was to determine indications, diagnostic and therapeutic yield. 2. Meduri GU, et al.: Noninvasive positive pressure ventilation in status asthmaticus. Chest 1996, 110:767-774. 2. Meduri GU, et al.: Noninvasive positive pressure ventilation in status asthmaticus. Chest 1996, 110:767-774. 3. Lin HC, et al.: Eff ect of nasal continuous positive airway pressure on methacholine-induced bronchoconstriction. Respir Med 1995, 89:121-128. 3. Lin HC, et al.: Eff ect of nasal continuous positive airway pressure on methacholine-induced bronchoconstriction. Respir Med 1995, 89:121-128. 3. Lin HC, et al.: Eff ect of nasal continuous positive airway pressure on methacholine-induced bronchoconstriction. Respir Med 1995, 89:121-128. Methods A retrospective, single-center observational study was carried out. All BC performed in a 20-bed polyvalent ICU of a tertiary university hospital over a period of 30 months (from January 2010 to June 2012) were included. The main variables collected were demographic data and clinical data including comorbidities, endobronchial techniques and results. References 1. Lucena CM, et al.: Med Intensiva 2012, 36:389-395. 2. Estella A, et al.: Med Intensiva 2012, 36:396-401. 3. Shennib H, et al.: Chest Surg Clin N Am 1996, 6:349-361. 4. Turner JS, et al.: Crit Care Med 1994, 22:259-264. 1. Lucena CM, et al.: Med Intensiva 2012, 36:389-395. Results Mean age (SD) of the patients was 60 (16)  years, 60% were male. The mean (SD) Murray score was 1.4 (0.6). Pathologies included pulmonary oedema, pneumonia, atelectasis and traumatic lung injury. All 60 D-values are represented in Figure  1. The median (IQR, range) D-value was 38 (16 to 74, 0.8 to 1,272). Our algorithm therefore confi rmed a high sensitivity to detect correct endotracheal intubation also in patients with lung disease. Under the hypothesis that oesophageal compliance does not increase signifi cantly in patients with lung disease, the specifi city of our algorithm will not be aff ected. 9 Impact of continuous positive airway pressure on the treatment of acute exacerbation of asthma: a randomised controlled trial Y Sutherasan, S Kiatboonsri, P Theerawit, C Kiatboonsri, S Trakulsrichai Ramathibothi Hospital, Bangkok, Thailand Critical Care 2013, 17(Suppl 2):P149 (doi: 10.1186/cc12087) Data collections in both groups included peak expiratory fl ow rates (PEFR, %predicted) at each 15-minute interval (T0, T15, …), the number of short-acting bronchodilators (SABA) used, ED length of stay, relapse rate and admission rate. The primary outcome is the improvement in peak expiratory fl ow rate during a short period stay in the ED. Conclusion Preliminary results demonstrate diagnostic accuracy of lung ultrasound examination performed on the admission of patients with acute respiratory failure similar to the original publication. Lung ultrasound, as proposed in the BLUE protocol, has good accuracy, and seems reproducible and useful in this group of patients. f p yl g p y Results There were 86 patients enrolled, with 43 patients in each study group. PEFR in the CPAP group showed no diff erence from the control group at T0 (52.7  ±  20.33 vs. 47.2  ±  16.64, P  =  NS) but was signifi cantly better at T15 (65.58 ± 21.64 vs. 56.49 ± 18.53%, P <0.05). Such improvement was consistently observed for all subsequent PEFR measurement, along with the fewer SABA used and relapse rate, but the fi gures did not meet statistical diff erence. 1. Lichtenstein DA, Mezière GA: Relevance of lung ultrasound in the diagnosis of acute respiratory failure: the BLUE protocol. Chest 2008, 134:117-125. Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 S56 http://ccforum.com/supplements/17/S2 Conclusion The addition of CPAP to conventional acute asthma treatment in the ED could accelerate PEFR improvement with a trend showing fewer SABA needed, lower relapse rate and shorter ED length of stay. P151 Main indications, diagnostic and therapeutic yield of bronchoscopy in the ICU J Iglesias, B Gonzalez, O Rajas La Princesa Hospital, Madrid, Spain Critical Care 2013, 17(Suppl 2):P151 (doi: 10.1186/cc12089) P152 P152 Benchtop study comparing leakages across cuff s of three endotracheal tubes SM Lam, CW Lau, WW Yan Pamela Youde Nethersole Eastern Hospital, Hong Kong Critical Care 2013, 17(Suppl 2):P152 (doi: 10.1186/cc12090) Conclusion The algorithm to detect correct endotracheal intubation performed excellent in patients with lung disease. Reference Introduction The aim was to compare two novel endotracheal tubes (ETT), Mallinckrodt TaperGuard (TG, tapered polyvinyl chloride (PVC) cuff ) and KimVent Microcuff (MC, cylindrical polyurethrane cuff ), 1. Kalmar AF, et al.: Resuscitation 2012, 83:177-182. Figure 1 (abstract P150). D-value of individual test ventilations. Figure 1 (abstract P150). D-value of individual test ventilations. Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 S57 http://ccforum.com/supplements/17/S2 Figure 1 (abstract P152). Downward leak in PEEP 0 cmH2O without suction. Each point represents the mean and standard deviation of eight repeated measures. with SSD. However, the shape most suitable for preventing dripping onto the subglottis has not been determined. The purpose of this study was to determine whether an ET with tapered-type cuff can reduce the incidence of VAP. Methods After approval from the appropriate ethics committee, we conducted a single-institutional prospective randomized clinical trial on the eff ectiveness of using an ET with a diff erent cuff type. Adult patients (n = 399; ≥18 years old) were screened between April 2010 and March 2012, and 289 patients expected to require mechanical ventilation (MV) for at least 48 hours were randomized. Patients were assigned to intubation with one of the following two HVLP ETs with SSD on the experimental tube: tapered type (Taperguard™ Evac®; Covidien, Dublin, Ireland) or spindle type (Hi-Lo Evac®; Covidien). Cuff pressure measurement was carried out every 6 hours, and cuff pressure was maintained between 20 and 30 cmH2O. The primary outcome was VAP incidence based on semiquantitative bronchoalveolar lavage fl uid culture of 3+ or phagocytosis on Gram staining in patients intubated for at least 48 hours. The other outcome was the rate of achieving an appropriate cuff pressure (20 to 30 cmH2O), time to VAP onset, duration of MV, duration of ICU stay, mortality, and adverse events. Figure 1 (abstract P152). Downward leak in PEEP 0 cmH2O without suction. Each point represents the mean and standard deviation of eight repeated measures. Eff ect of body position changes on endotracheal tube cuff pressure in ventilated patients Fernandez JF, et al.: Chest 2012, 142:231-238. 1. Fernandez JF, et al.: Chest 2012, 142:231-238. P152 y y Results The rate of microbiologically confi rmed VAP was 21.7% (23/106) for the tapered-type ET patients and 21.7% (23/106) for the spindle-type ET patients (P = 1.00). The rate of achieving appropriate cuff pressure was 83.2% (332/1,974) for the tapered type and 82.4% (328/1,867) for the spindle type (P = 0.549). No signifi cant diff erences between groups were observed for time to VAP onset, duration of MV, ICU stay. The incidence of reintubation due to laryngeal edema after extubation was slightly higher in patients with the tapered-type ET (11.5%, 6/52) than in patients with the spindle-type ET (2.0%, 1/49), but the diff erence was not signifi cant (P = 0.113).ff with conventional Portex (PT, globular PVC cuff ) in leakages across cuff s (microaspiration) under simulated clinical situations. It has been shown that globular PVC cuff s protect poorly against leakages due to microchannels formed from infolding of redundant cuff material [1]. We hypothesized that TG and MC better prevent microaspiration, which is a major mechanism of ventilator-associated pneumonia (VAP). Methods Each ETT was inserted into a silicone cylinder of 2  cm wide inclined at 35°. Then 20  ml water was added above the cuff and leakage measured every minute under fi ve diff erent simulated clinical conditions: mechanical ventilation for acute severe asthma (positive end-expiratory pressure (PEEP) 0 cmH2O), normal lungs (PEEP 5 cmH2O) and acute respiratory distress syndrome (PEEP 10 cmH2O), and disconnection from the ventilator with and without spontaneous breathing eff ort. Spontaneous breathing was simulated with a respiratory gas exchange simulator. Suction was applied at 200 cmH2O sustained for 3 minutes at the Murphy eye. Each scenario was repeated with cuff pressures (Pcuff ) 10, 20 and 30 cmH2O maintained by a Pcuff maintenance device. Conclusion Diff erences in cuff type and shape under identical conditions of cuff pressure control have no infl uence on the incidence of VAP. Acknowledgement UMIN-CTR UMIN000003371. Acknowledgement UMIN-CTR UMIN000003371. P154f Eff ect of body position changes on endotracheal tube cuff pressure in ventilated patients Eff ect of body position changes on endotracheal tube cuff pressu in ventilated patients C Lizy1, W Swinnen2, S Labeau3, S Blot1 1Gent University, Ghent, Belgium; 2General Hospital Sint Blasius, Dendermonde, Belgium; 3University College Ghent, Belgium Critical Care 2013, 17(Suppl 2):P154 (doi: 10.1186/cc12092) C Lizy1, W Swinnen2, S Labeau3, S Blot1 1Gent University, Ghent, Belgium; 2General Hospital Sint Blasius, Dendermonde, Belgium; 3University College Ghent, Belgium Critical Care 2013, 17(Suppl 2):P154 (doi: 10.1186/cc12092) Results PT leaked grossly in all scenarios without PEEP and at PEEP 5  cmH2O in the presence of suction irrespective of Pcuff (Pcuff 30 cmH2O: PT 19.7 ± 0.4, TG 0.0 ± 0.1, MC 0.0 ± 0.0, P <0.001; Pcuff 20 cmH2O: PT 19.9 ± 0.4, TG 7.4 ± 6.2, MC 0.0 ± 0.0, P <0.001; Pcuff 10 cmH2O: PT 20.0 ± 0.0, TG 12.7 ± 5.1, MC 0.9 ± 0.8, P <0.001). Leakage under these scenarios can be reduced in TG and prevented in MC by Pcuff ≥20 cmH2O (Figure 1).f Introduction In order to avoid either microaspiration (<20 cmH2O) or tracheal injury (>30 cmH2O), the target for endotracheal tube (ETT) cuff pressure (CP) is between 20 and 30 cmH2O. Our objective was to assess the eff ect of patient positioning changes on CP in adult patients. f 2 Conclusion Microcuff outperformed the others in preventing microaspiration, while Portex leaked grossly even at a recommended Pcuff of 20 to 30 cmH2O whenever PEEP was lost. The eff ect of ETT type on the incidence of VAP warrants further investigation. Reference Methods A sample of 12 orally intubated and sedated patients was selected. Patients received neuromuscular blockade and were positioned in a neutral start position (backrest, head of bed elevation (HoB) 30°, head in neutral position) and CP set at 25 cmH2O. Subsequently 16 diff erent position changes were performed: antefl exion head, hyperextension head, left and right lateral fl exion of head, left and right rotation of the head, semirecumbent position (HoB 45°), recumbent position (HoB 10°), horizontal position, trendelenburg (–10°), left and right lateral positioning over 30°, 45° and 90°. Once the patient was correctly positioned, CP was recorded during an end-expiratory ventilatory hold. CP observed was compared with CP at start position (25 cmH2O). Also the number of values outside the target range (20 to 30 cmH2O) was reported as considered clinically relevant. 1. Fernandez JF, et al.: Chest 2012, 142:231-238. 1. P153 Endotracheal tube with tapered-type cuff for preventing ventilator-associated pneumonia: a randomized clinical trial N Saito, T Yagi, Y Hara, H Matsumoto, K Mashiko Nippon Medical School Chiba Hokusoh Hospital, Inzai, Chiba, Japan Critical Care 2013, 17(Suppl 2):P153 (doi: 10.1186/cc12091) P153 Endotracheal tube with tapered-type cuff for preventing ventilator-associated pneumonia: a randomized clinical trial N Saito, T Yagi, Y Hara, H Matsumoto, K Mashiko Nippon Medical School Chiba Hokusoh Hospital, Inzai, Chiba, Japan Critical Care 2013, 17(Suppl 2):P153 (doi: 10.1186/cc12091) 2 y Results A total of 192 measurements were performed (12 subjects×16 positions). Results are shown in Table 1 (CP reported as median, IQR). In every position a signifi cant deviation in CP was observed. In total, 40.6% of values exceeded the upper limit of 30 cmH2O. No values beneath the lower target limit of 20 cmH2O were observed. In each position the upper target limit was exceeded at least once. Conclusion Simple changes in patient positioning may result in potentially harmful CP (>30 cmH2O). These observations call for strict CP monitoring. 2 Results A total of 192 measurements were performed (12 subjects×16 positions). Results are shown in Table 1 (CP reported as median, IQR). In every position a signifi cant deviation in CP was observed. In total, 40.6% of values exceeded the upper limit of 30 cmH2O. No values beneath the lower target limit of 20 cmH2O were observed. In each position the upper target limit was exceeded at least once. Introduction The most common cause of ventilator-associated pneumonia (VAP) is aspiration of oral secretion through the endotracheal tube (ET). Subglottic suction drainage (SSD) has been recommended as a safety measure against aspiration due to its high eff ectiveness. Currently, two types of cuff shape  – spindle and tapered – are predominant in high-volume, low-pressure (HVLP) ETs Conclusion Simple changes in patient positioning may result in potentially harmful CP (>30 cmH2O). These observations call for strict CP monitoring. Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 S58 Table 1 (abstract P154). P155 Towards safer airway management in the critically ill: lessons from National Audit Project 4 Introduction National Audit Project 4 (NAP4) highlighted the need to improve airway management in ICUs and key recommendations were the continuous use of end-tidal carbon dioxide (ETCO2) monitoring, pre-intubation checklists and diffi cult airway trolleys [1]. This complete cycle audit aimed to quantify the current state of airway management on our ICU and the eff ectiveness of implementing the NAP4 recommendations. Results The studied ETTs remained in place for a median of 7  days (IQR range 4 to 15). The amount of secretions assessed by CT scan was 0.293 ± 0.290 ml (range 0.032 to 0.777 ml). Secretion volumes were not related to patient severity at admission (SAPS 2, P/F ratio) or days of intubation; an inverse correlation with patient’s age was present (P  =  0.032, R2  =  0.46). Bacterial growth was present in 9/11 (82%) ETT fl uids cultures and Candida spp. showed an elevated prevalence (6/11, 55%). In the bench tests, the cleaning device reduced resistance to airfl ow (diff erence before and after cleaning 5.5 (95% CI = 8.9 to 1.6)  cmH2O/l/second, P  =  0.006). After cleaning, resistance resulted higher than intact ETTs, although with a clinically negligible diff erence (diff erence 0.3 (95% CI = 0.2 to 0.6 cmH2O/l/second), P = 0.032). Methods Data collection was carried out prospectively for both phases and included documentation of intubation, use of ETCO2 and the incidence of serious adverse events (SAEs). The contents of the intubation boxes were compared against the Diffi cult Airway Society (DAS) guidelines [2]. The re-audit was carried out 6 months following the introduction of a pre-intubation checklist, a documentation sticker, a diffi cult airway trolley and standardization of the basic bedside airway boxes with a checklist of contents. A training program in airway management for all ICU staff was also introduced. f 2 Conclusion Micro-CT scan is a feasible and promising technique to assess secretions volume in ETTs after extubation. The use of an ETT cleaning device decreases resistance to airfl ow in bench tests; the eff ectiveness of such a device in the clinical setting could be properly assessed by post-extubation CT scan. gf Results The baseline characteristics of both groups were similar. The initial audit included 45 patients and the re-audit 58 patients. In the initial audit 19% of patients had accurate documentation of intubation with the use of ETCO2. The continuous use of ETCO2 was 80%. P153 Endotracheal tube with tapered-type cuff for preventing ventilator-associated pneumonia: a randomized clinical trial N Saito, T Yagi, Y Hara, H Matsumoto, K Mashiko Nippon Medical School Chiba Hokusoh Hospital, Inzai, Chiba, Japan Critical Care 2013, 17(Suppl 2):P153 (doi: 10.1186/cc12091) Endotracheal tube cuff pressure values following patient position changes Cuff P for Δ from Measurements pressure start position >30 cmH2O (cmH2O)a (25 cmH2O) (n (%)) Antefl exion head 33.5 (27.5 to 42.0) 0.005 9 (75) Hyperextension head 30.5 (27.5 to 42.0) 0.004 6 (50) Lateral fl exion head, left 26.5 (25.3 to 28.8) 0.025 1 (8) Lateral fl exion head, right 27.0 (25.3 to 29.8) 0.049 2 (17) Rotation head, left 27.0 (26.3 to 33.8) 0.003 3 (25) Rotation head, right 28.0 (26.3 to 38.8) 0.020 4 (33) Semirecumbent, 45° HoB 28.0 (25.3 to 31.5) 0.009 2 (17) Recumbent, 10° HoB 28.0 (25.3 to 31.5) 0.016 3 (25) Horizontal back rest 28.5 (25.3 to 31.5) 0.011 3 (25) Trendelenburg, 10° 29.0 (25.3 to 32.0) 0.008 5 (42) Left lateral position, 30° 30.5 (25.3 to 32.0) 0.010 6 (50) Left lateral position, 45° 30.5 (27.3 to 32.8) 0.005 6 (50) Left lateral position, 90° 34.0 (27.0 to 36.8) 0.006 7 (58) Right lateral position, 30° 31.0 (27.0 to 34.5) 0.003 6 (50) Right lateral position, 45° 32.5 (28.3 to 35.8) 0.002 8 (50) Right lateral position, 90° 31.0 (29.3 to 35.0) 0.003 7 (58) Total (192 measurements) – – 78 (40.6) HoB, head of bed elevation. aCuff pressure values reported as median (fi rst to third quartile). Table 1 (abstract P154). Endotracheal tube cuff pressure values following patient position changes This audit demonstrated that simple measures in conjunction with a structured training program can help to improve the safety of airway management in critically ill patients. Further work is required to improve compliance with the pre-intubation checklist alongside a continued education program. References 1. The Royal College of Anaesthetists [www.rcoa.ac.uk/nap4] 2. Diffi cult Airway Society Equipment List [http://www.das.uk.com/equipmentlistjuly2005.htm] P156 Quantitative assessment of endotracheal tube (ETT) biofi lm by micro-CT scan: evaluation of the eff ectiveness of ETT cleaning devices A Coppadoro1, G Bellani1, T Mauri1, R Borsa1, V Meroni1, A Barletta1, M Teggia Droghi1, A Lucchini2, R Marcolin2, N Patroniti1, S Bramati2, A Pesenti1 1University of Milan-Bicocca, Monza, Italy; 2San Gerardo Hospital, Monza, Italy Critical Care 2013, 17(Suppl 2):P156 (doi: 10.1186/cc12094) Introduction The goal of this study was to assess the amount of biofi lm within the endotracheal tube (ETT) by CT scan and to evaluate in a bench test a device aimed at ETT biofi lm removal. P157 P157 Early identifi cation of patients at risk of diffi cult intubation in the ICU: development and validation of the MACOCHA score in a multicenter cohort study A De Jong1, N Molinari1, N Terzi2, N Mongardon3, B Jung1, S Jaber1 1Montpellier University Hospital, Montpellier, France; 2Caen University Hospital, Caen, France; 3Cochin University Hospital, Paris, France Critical Care 2013, 17(Suppl 2):P157 (doi: 10.1186/cc12095) P153 Endotracheal tube with tapered-type cuff for preventing ventilator-associated pneumonia: a randomized clinical trial N Saito, T Yagi, Y Hara, H Matsumoto, K Mashiko Nippon Medical School Chiba Hokusoh Hospital, Inzai, Chiba, Japan Critical Care 2013, 17(Suppl 2):P153 (doi: 10.1186/cc12091) Biofi lm grows within the ETT soon after intubation, resulting in increased resistance to airfl ow. Biofi lm detachment from the ETT can result in bacterial colonization of the distal airways. The use of an ETT cleaning device could eff ectively reduce the patient’s airfl ow resistance burden. The quantifi cation and distribution of biofi lm within the ETT might help to evaluate ETT cleaning device eff ectiveness. gf Methods We collected ETTs of 11 critically ill patients after extubation. Micro-CT scan (SkyScan 1176; Bruker, Belgium) was performed using a resolution of 35 μm. Axial sections of the 20 cm above the cuff were reconstructed, and the volume of secretions was assessed by a density criterion. Microbiological cultures of the ETT lavage fl uid were then obtained. Patient’s demographics and clinical data were recorded. In a diff erent set of bench experiments, we injected 1  ml water-based polymer into new ETTs of diff erent sizes. We measured resistance to airfl ow before and after using an ETT cleaning device (Airway Medix Closed Suction System; Biovo Technologies, Tel Aviv, Israel). We also obtained resistance values of intact ETTs as controls. HoB, head of bed elevation. aCuff pressure values reported as median (fi rst to third quartile). P155 Towards safer airway management in the critically ill: lessons from National Audit Project 4 Bedside airway boxes did not contain a checklist of standardized equipment. A diffi cult airway trolley was not located on the ICU. Following introduction of the changes described, a signifi cant improvement in the use of ETCO2 to confi rm intubation (46% vs. 19%, P <0.01) and continuous use of ETCO2 monitoring (100% vs. 80% P ≤0.0004) was observed. The use of the pre- intubation checklist (7%) and stickers (11%) was poor. All bedside boxes had a checklist and the required equipment. A diffi cult airway trolley is located on the ICU with an equipment list and DAS algorithm attached. No SAEs were recorded in either phase. Early identifi cation of patients at risk of diffi cult intubation in the ICU: development and validation of the MACOCHA score in a multicenter cohort study A De Jong1, N Molinari1, N Terzi2, N Mongardon3, B Jung1, S Jaber1 1Montpellier University Hospital, Montpellier, France; 2Caen University Hospital, Caen, France; 3Cochin University Hospital, Paris, France Critical Care 2013, 17(Suppl 2):P157 (doi: 10.1186/cc12095) A De Jong1, N Molinari1, N Terzi2, N Mongardon3, B Jung1, S Jaber1 1Montpellier University Hospital, Montpellier, France; 2Caen University Hospital, Caen, France; 3Cochin University Hospital, Paris, France Critical Care 2013, 17(Suppl 2):P157 (doi: 10.1186/cc12095) Introduction Diffi cult intubation (DI) in the ICU is a challenging issue, associated with severe life-threatening complications [1,2]. The objective was to develop and validate a simplifi ed score for identifying patients with DI in the ICU and to report related complications. Introduction Diffi cult intubation (DI) in the ICU is a challenging issue, associated with severe life-threatening complications [1,2]. The objective was to develop and validate a simplifi ed score for identifying patients with DI in the ICU and to report related complications. Conclusion Serious airway complications are rare. Safer airway manage ment is essential in the ICU to prevent morbidity and mortality. S59 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Methods Data collected in a prospective multicenter-study from 1,000 consecutive intubations from 42 ICUs were used to develop a simplifi ed score of DI, which was then validated externally in 400 consecutive intubation procedures from 18 other ICUs and internally by bootstrap on 1,000 iterations. common indications of intubation were acute respiratory failure (61.0%) and coma (17.1%). The primary outcome occurred in 138 of 246 intubations (56.1%). Within the fi rst hour, the incidence of severe hypotension was 47.2%, of severe hypoxemia was 20.3%, of cardiac arrest was 5.7% and of death was 4.5%. Results In multivariate analysis, the main predictors of DI (incidence = 11.3%) were related to the patient (Mallampati score III or IV, obstructive sleep apnea syndrome, reduced mobility of cervical spine, limited mouth opening), to pathology (severe hypoxia, coma) and to the operator (non-anesthesiologist). From the β-parameter, a seven- item simplifi ed score (MACOCHA score; Table 1) was built, with an area under the curve (AUC) of 0.89 (95% CI = 0.85 to 0.94). Tracheal intubation for a diffi cult airway using Airway scope®, KingVision® and McGRATH®: a comparative manikin study of inexperienced personnel J Itai1, Y Tanabe2, T Nishida1, T Inagawa1, Y Torikoshi1, Y Kida1, T Tamura1, K Ota1, T Otani1, T Sadamori1, K Une1, R Tsumura1, Y Iwasaki1, N Hirohashi1, K Tanigawa1 J Itai1, Y Tanabe2, T Nishida1, T Inagawa1, Y Torikoshi1, Y Kida1, T Tamura1, K Ota1, T Otani1, T Sadamori1, K Une1, R Tsumura1, Y Iwasaki1, N Hirohashi1, K Tanigawa1 g 1Hiroshima University Hospital, Hiroshima-shi, Japan; 2Hiroshima City Hospital, Hiroshima-shi, Japan Critical Care 2013, 17(Suppl 2):P159 (doi: 10.1186/cc12097) g 1Hiroshima University Hospital, Hiroshima-shi, Japan; 2Hiroshima City Hospital, Hiroshima-shi, Japan Critical Care 2013, 17(Suppl 2):P159 (doi: 10.1186/cc12097) g 1Hiroshima University Hospital, Hiroshima-shi, Japan; 2Hiroshima City Hospital, Hiroshima-shi, Japan Critical Care 2013, 17(Suppl 2):P159 (doi: 10.1186/cc12097) Table 1 (abstract P157). MACOCHA score calculation worksheet Points Factors related to patient Mallampati score III or IV 5 Obstructive apnea syndrome 2 Reduced mobility of cervical spine 1 Limited mouth opening <3 cm 1 Factors related to pathology Coma 1 Severe hypoxemia (<80%) 1 Factor related to operator Non-anaesthesiologist 1 Total 12 Conclusion DI in ICU is strongly associated with severe life-threatening complications. A simple score including seven clinical items discriminates diffi cult and non-DI in ICU. References 1. Jaber et al.: Crit Care Med 2006, 34:2355-2361. 2. Jaber et al.: Intensive Care Med 2010, 36:248-255. P158 Incidence of major complications related to endotracheal intubation in ICUs of nonacademic Brazilian public hospitals AB Cavalcanti1, K Normilio-da-Silva1, JC Acarine Mouro1, F Moreira1, AA Kodama1, R Del-Manto2, O Berwanger1 1Hospital do Coração – HCor, São Paulo, Brazil; 2Hospital Militar de Área de São Paulo – HMASP, São Paulo, Brazil Critical Care 2013, 17(Suppl 2):P158 (doi: 10.1186/cc12096) d b d h d f Table 1 (abstract P157). MACOCHA score calculation worksheet Points Factors related to patient Mallampati score III or IV 5 Obstructive apnea syndrome 2 Reduced mobility of cervical spine 1 Limited mouth opening <3 cm 1 Factors related to pathology Coma 1 Severe hypoxemia (<80%) 1 Factor related to operator Non-anaesthesiologist 1 Total 12 Table 1 (abstract P157). MACOCHA score calculation worksheet Table 1 (abstract P157). MACOCHA score calculation worksheet Introduction The Airway scope® (AWS), the KingVision® (KV) and the McGRATH® (MG) are new indirect video laryngoscopes designed to facilitate eff ective and safe tracheal intubation under various conditions. However, there are few comparative studies as for performance in tracheal intubation attempted by inexperienced personnel. Early identifi cation of patients at risk of diffi cult intubation in the ICU: development and validation of the MACOCHA score in a multicenter cohort study In the validation cohort (prevalence of DI = 8%), the AUC was of 0.86 (95% CI = 0.76 to 0.96), with a sensitivity of 73%, a specifi city of 89%, a negative predictive value of 98% and a positive predictive value of 36%. After internal validation by bootstrap, the AUC was 0.89 (95% CI = 0.86 to 0.93). Severe life-threatening events (severe hypoxia, collapse, cardiac arrest or death) occurred in 38% of the 1,000 cases. Patients with DI (n = 113) had signifi cantly higher severe life-threatening complications than those who had a non-DI (51% vs. 36%, P <0.0001). Conclusion Incidence of major complications associated with endotracheal intubation is unacceptably high in Brazilian public ICUs. There is an urgent need for eff ective measures to increase safety of this common procedure in our environment. Tracheal intubation for a diffi cult airway using Airway scope®, KingVision® and McGRATH®: a comparative manikin study of inexperienced personnel The purpose of this study was to evaluate success rates, time to intubation with use of these devices by inexperienced personnel in a simulated manikin diffi cult airway.i Methods Twenty-nine fi fth-year medical students with no previous experience in tracheal intubation participated in this study. We used an advanced patient simulator (SimMan®; Laerdal Medical, Stavanger, Norway) to simulate normal and diffi cult airway scenarios including cervical spine rigidity, swollen tongue, and pharyngeal edema. The sequences in selecting devices and scenarios were randomized. Success rate for tracheal intubation, and the time required for visualization of the glottis (T1), tracheal intubation (T2), and infl ation of the lungs (T3) were analyzed. Also, numbers of audible dental click during the intubation attempt were recorded. The three diff erent intubation devices were tested in four diff erent scenarios by 29 students. Results All three devices had very high success rates of tracheal intubation (AWS 100%; KV 100%; MG 99%). In the normal airway, T1, T2 and T3 were AWS: 4.4 ± 3.3 seconds, 7.5 ± 3.8 seconds, 11.1 ± 3.8 seconds; KV: 6.9 ± 6.9 seconds, 10.3 ± 7.9 seconds, 14.1 ± 8.3 seconds; MG: 4.6  ±  1.3 seconds, 11.4  ±  5.2 seconds, 16.1  ±  5.4 seconds, respectively (NS). In the three diffi cult airway scenarios, T1, T2 and T3 were AWS: 5.3 ± 4.7* seconds, 10.4 ± 6.9* seconds, 14.2 ± 6.9* seconds; KV: 10.9  ±  12.8 seconds, 21.5  ±  18.1 seconds, 25.4  ±  18.3 seconds; MG: 13.0  ±  15.3 seconds, 26.2  ±  24.6 seconds, 31.0  ±  24.3 seconds, respectively (*P  <0.05 AWS vs. KV and MG). The number of audible dental click sounds with the MG was greater than with the AWS and KV (AWS 5%*; KV 8%*; MG 28%*; *P <0.05 AWS and KV vs. MG). 1. Jaber et al.: Crit Care Med 2006, 34:2355-2361. 2. Jaber et al.: Intensive Care Med 2010, 36:248-255. P159 P159 Tracheal intubation for a diffi cult airway using Airway scope®, KingVision® and McGRATH®: a comparative manikin study of inexperienced personnel J Itai1, Y Tanabe2, T Nishida1, T Inagawa1, Y Torikoshi1, Y Kida1, T Tamura1, K Ota1, T Otani1, T Sadamori1, K Une1, R Tsumura1, Y Iwasaki1, N Hirohashi1, K Tanigawa1 1Hiroshima University Hospital, Hiroshima-shi, Japan; 2Hiroshima City Hospital, Hiroshima-shi, Japan Critical Care 2013, 17(Suppl 2):P159 (doi: 10.1186/cc12097) Incidence of major complications related to endotracheal intubation in ICUs of nonacademic Brazilian public hospitals Conclusion The AWS, KV and MG had very high success rates of tracheal intubation and are suitable as intubation devices for inexperienced personnel. In the diffi cult airway, however, the intubation time with AWS was signifi cantly shorter than with KV and MG. These fi ndings suggested that AWS may be most useful device particularly in diffi cult situations such as emergency settings. Further studies in a clinical setting are needed to confi rm these fi ndings. Introduction Our objective is to determine the incidence of major complications related to endotracheal intubations (death, cardiac arrest, severe hemodynamic instability or severe hypoxemia) per- formed in the ICUs of nonacademic Brazilian public hospitals. Methods This is a report of the baseline phase of a cluster randomized trial with two parallel arms. In this baseline phase, we collected data from sequential patients needing endotracheal intubation in 17 ICUs from Brazil. Patients needing endotracheal intubation after cardiac arrest were excluded. In a second ongoing phase, not reported here, we have randomized ICUs to a multifaceted intervention to prevent complications of endotracheal intubation. Primary outcome was defi ned as the occurrence of death, cardiac arrest, severe cardiovascular instability (systolic blood pressure <60 mmHg at least one time, <90 mmHg and persisting >30 minutes or need to initiate vasopressors) or severe hypoxemia (SpO2 <80%) within 1 hour after intubation. P160 NT-proBNP and cardiac cycle effi ciency changes during extubation process in critically ill patients i Results One hundred patients were surveyed. The ET and LT groups included 49 and 51 patients, respectively. Tracheostomy was performed using a percutaneous procedure in 48 patients (ET: 25, LT: 23) and a surgical procedure in 52 patients (ET: 24, LT: 28). Sixty-two patients (ET: 34, LT: 28) survived to discharge and 16 patients died in the ICU (ET: 7, LT: 9). Fifty-six patients (ET: 31, LT: 25) were weaned from ventilator support and tracheostomy cannula was removed in 20 patients (ET: 11, LT: 9). There were no signifi cant diff erences in type of tracheostomy procedure, period from tracheostomy until ICU and hospital discharge, rate of patients who could be weaned from ventilator and removed tracheostomy cannula, and ICU and hospital mortality between the groups. The length of mechanical ventilation and the time to removal of tracheostomy cannula were signifi cantly shorter in the ET group (5 ± 7 vs. 26 ± 41 and 29 ± 24 vs. 94 ± 83 days, respectively). Introduction Several factors can lead to weaning failure from mechanical ventilation (MV). Among these, cardiac dysfunction is one of the main causes. NT-proBNP has been proposed as a biomarker of cardiovascular function during weaning from MV. Unfortunately it does not provide for a continuous monitoring of cardiac function. Pulse wave analysis may serve as a continuous bedside monitoring tool of cardiovascular performance. Cardiac cycle effi ciency (CCE) is an indirect index of left ventricular performance obtained by the pulse contour method MostCare (Vygon, Padova, Italy). The aim of this study was to evaluate the correlation between NT-proBNP and CCE and the potential usefulness of such variables during the weaning process from MV. Conclusion In this retrospective study, early tracheostomy reduced the length of weaning after tracheostomy and the time to removal of tracheostomy cannula, while there were no diff erences in the length of ICU stay and patient outcome. In critically ill adult patients who require mechanical ventilation, a tracheostomy performed at an earlier stage may shorten the duration of artifi cial ventilation. A further randomized clinical trial is essential to determine the eff ectiveness and safety of early tracheostomy. Methods Twenty-two long-term (>48 hours) mechanically ventilated patients capable of performing a weaning trial of spontaneous breathing (SBT) were enrolled in the study. Inclusion criteria were: age >18 years and equipment with a standard arterial catheter line. Resistance of neonatal endotracheal tubes: a comparison of four commercially available types Introduction In mechanically ventilated neonates the fl ow-dependent resistance of the endotracheal tube (ETT) causes a noticeable pressure diff erence between airway and tracheal pressure [1]. This may potentially lead to retardation of the passive driven expiration and dynamic lung infl ation consecutively but more importantly increases Introduction In mechanically ventilated neonates the fl ow-dependent resistance of the endotracheal tube (ETT) causes a noticeable pressure diff erence between airway and tracheal pressure [1]. This may potentially lead to retardation of the passive driven expiration and dynamic lung infl ation consecutively but more importantly increases Results We evaluated 246 intubations. The mean age was 49 ± 21.9 years; the SAPS 3 admission score was 57.4 ± 15.4. The most S60 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 after extubation (T2, T3) compared with T1; conversely, CCE showed an inverse trend. Overall, a negative correlation was found between NT-proBNP and CCE values (R  =  –0.81, P <0.001). Signifi cant inverse correlations were found between NT-proBNP and CCE at T1, T2, and T3 (R  =  –0.91, –0.75 and –0.73 respectively; P  < 0.001). The overall correlation between NT-proBNP and CCE was –0.74 in the SB group and –0.86 in the CPAP group. Standard hemodynamic and ventilatory data did not show signifi cant changes during the study. the work of breathing. The aim of this study was to compare the resistive pressure drop of four commercially available neonatal ETTs with internal diameter (ID) of 2.0 mm. the work of breathing. The aim of this study was to compare the resistive pressure drop of four commercially available neonatal ETTs with internal diameter (ID) of 2.0 mm. Methods The pressure–fl ow relationship of neonatal ETTs (internal diameter 2.0  mm) of four diff erent manufacturers (Mallinckrodt, Hi- Contour, Covidien, Dublin, Ireland; Portex, Cole’s Neonatal Tube, Smith Medical, St Paul, MN, USA; Rüsch, Silko Clear, Telefl ex Medical, Kernen, Germany; and Vygon, Pediatric Endotracheal Tube, Ecouen, France) was determined in a physical model consisting of a tube connector, an anatomically curved ETT and an artifi cial trachea. The model was ventilated with a sinusoidal gas fl ow with an amplitude of ±100 ml/ second and a ventilation rate ranging from 40 to 60 cycles/minute. The coeffi cients of an approximation equation considering ETT resistance were fi tted separately to the measured pressure–fl ow curves for inspiration and expiration. Comparison of outcomes between early and late tracheostomy for critically ill patients Guttmann J, et al.: Crit Care Med 2000, 28:1018-1026. Comparison of outcomes between early and late tracheostomy for critically ill patients Results The pressure drop profi les of all ETTs were nonlinearly fl ow dependent. The expiratory pressure drop (Pexp) slightly exceeded the inspiratory one (Pinsp). The ETT of Portex Cole’s Neonatal Tube had a signifi cant lower pressure drop (Pinsp 8.8  ±  0.3  cmH2O vs. Pexp –11.2 ± 0.2 cmH2O at an air fl ow of 100 ml/second) compared with all other ETTs (Mallinckrodt: Pinsp 19.2 ± 0.4 cmH2O vs. Pexp –24.3 ± 0.7 cmH2O; Rüsch: Pinsp 28.3 ± 0.8 cmH2O vs. Pexp –33.0 ± 0.8 cmH2O; Vygon: Pinsp 19.2 ± 0.4 cmH2O vs. Pexp –22.6 ± 0.6 cmH2O, all P <0.05) for all fl ow and ventilation rates, respectively. K Suzuki1, S Kusunoki1, T Yamanoue1, K Tanigawa2 1Hiroshima Prefectural Hospital, Hiroshima, Japan; 2Hiroshima University Hospital, Hiroshima, Japan Critical Care 2013, 17(Suppl 2):P162 (doi: 10.1186/cc12100) Introduction Tracheostomy is one of the more commonly performed procedures in critically ill patients requiring long-term mechanical ventilation. However, the optimal timing or method of performing tracheostomies in this population remains to be established. In the present study, we compared outcomes of early and late tracheostomy in critically adult patients with diff erent clinical conditions. y Conclusion The ETT resistance highly contributes to the total airway resistance in neonatal ventilation. The fl ow-dependent pressure drop of shouldered Cole’s Neonatal Tube (Portex) was up to 70% less in inspiration (resp. 67% in expiration) compared with straight tubes with an internal diameter corresponding to the narrow part of Cole’s tube. We conclude that neonatal intubation with a Cole’s tube can clearly reduce the resistive load due to the endotracheal tube and thus potentially prevent additionally work of breathing. Reference yf Methods All patients needing tracheostomy in the Critical Care Medical Center of Hiroshima Prefectural Hospital from January 2009 to December 2011 were surveyed. Patients with tracheostomy who were not indicated for mechanical ventilation were excluded from the subjects. Early tracheostomy (ET) was defi ned as <10 days after tracheal intubation and late tracheostomy (LT) was defi ned as ≥10 days after intubation. We compared patient characteristics, type of tracheostomy procedure, length of weaning from ventilator and outcomes between the groups. Data are shown as the mean ± SD, with unpaired t test and Mann–Whitney U test used for statistical analyses. Statistical signifi cance was accepted at P <0.05. 1. Guttmann J, et al.: Crit Care Med 2000, 28:1018-1026. 1. NT-proBNP and cardiac cycle effi ciency changes during extubation process in critically ill patients Exclusion criteria were: neuromuscular disease, tracheotomy, renal failure, and traumatic brain injury. During the weaning process, NT- proBNP plasma levels, CCE, and standard hemodynamic and ventilatory data were collected 30 minutes before extubation (T1), 2 hours (T2) and 12 hours later (T3). After removal of tracheal tube, patients with a history of heart failure received continuous positive airway pressure (CPAP group). Patients with normal cardiac function were maintained with spontaneous breathing (SB group). Reference . Arabi Y, et al.: Crit Care 2004, 8:R347-R352. Arabi Y, et al.: Crit Care 2004, 8:R347-R352. Arabi Y, et al.: Crit Care 2004, 8:R347-R352. Resistance of neonatal endotracheal tubes: a comparison of four commercially available types gi g g y Conclusion NT-proBNP correlated well with CCE. The latter seems to be an additional attractive index of cardiovascular state that, in association with NT-proBNP changes, may provide information about cardiac function on a beat-by-beat basis during weaning process from MV. P162 Ultrasound scanning for percutaneous dilatational tracheostomy: a systematic review y R Pugh, A Slater y R Pugh, A Slater y R Pugh, A Slater R Pugh, A Slater Glan Clwyd Hospital, Bodelwyddan, UK Critical Care 2013, 17(Suppl 2):P163 (doi: 10.1186/cc12101) Results Sixty-six paired NT-proBNP and CCE values were obtained. Patients in the SB group and in the CPAP group were 10 and 12, respectively. In both groups there was a trend towards an increase in NT-proBNP values after extubation, an opposite trend was observed regarding CCE values (P <0.05). NT-proBNP levels showed an increase Glan Clwyd Hospital, Bodelwyddan, UK Critical Care 2013, 17(Suppl 2):P163 (doi: 10.1186/cc12101) Introduction Percutaneous dilatational tracheostomy (PDT) remains a frequently performed procedure in the ICU. However, there is great S61 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Figure 1 (abstract P164). Oxygenation time to oxygen alveolar pressure >100 mmHg. Figure 2 (abstract P164). Mean carbon dioxide alveolar pressure at the start and at the end of the test. variability in the course of blood vessels in the pre-tracheal area. A 5% risk of clinically relevant bleeding was recently reported for patients undergoing PDT [1]. We conducted a systematic review of reports evaluating clinical outcomes following use of ultrasound scanning (US) for PDT. variability in the course of blood vessels in the pre-tracheal area. A 5% risk of clinically relevant bleeding was recently reported for patients undergoing PDT [1]. We conducted a systematic review of reports evaluating clinical outcomes following use of ultrasound scanning (US) for PDT. Figure 1 (abstract P164). Oxygenation time to oxygen alveolar pressure >100 mmHg. Methods Two investigators performed a search of the literature using the following databases: CENTRAL, Embase, MEDLINE and SCOPUS. The following eligibility criteria were used: population including adults >16 years managed in the ICU; use of ultrasound to guide decision- making pre-PDT or guide PDT performance; report of clinically relevant outcome measures. Nonrandomised controlled trials were classifi ed according to Cochrane Non-Randomised Study Methods Group criteria [2] and evaluated for risk of bias. [ ] Results An initial search identifi ed 2,043 reports, of which 10 studies met eligibility criteria: eight case series, one randomised controlled trial (RCT) and one prospective cohort study, incorporating 488 patients. Two studies specifi cally reported data on patients with obesity (n = 29 patients) and one study reported data for a group of patients with spinal cord fi xation (n = 6). Reference 1. Cook TM, Nolan JP, Magee PT, Cranshaw JH: Needle cricothyroidotomy. Anaesthesia 2007, 62:289-291. P Persona, P Diana, A Ballin, F Baratto, M Micaglio, C Ori g Clinica di Anestesia e Medicina Intensiva, Padova, Italy y Critical Care 2013, 17(Suppl 2):P164 (doi: 10.1186/cc12102) Introduction Failed airway situations are potentially catastrophic events and require a correct approach with appropriate tools. Recently, Ventrain has been presented as a manual device for emergency ventilation through a small-bore cannula, which can provide expiratory assistance by applying the Venturi eff ect. y R Pugh, A Slater US was used to guide decision to perform PDT or surgical tracheostomy in fi ve studies, with decision to perform surgical tracheostomy ranging from 0 to 27% of cases. US was used to guide insertion point in seven studies, and used real-time in four studies. Times to perform US-guided PDT were reported in four studies (ranging from 8 to 12 minutes). No studies compared time taken with or without US. Data on complications of procedure were reported in nine studies. Minor bleeding was reported for eight cases (1.6% overall). Prolonged bleeding was reported in two cases (0.4%). There were no episodes of catastrophic bleeding among 488 cases. High risk of bias was identifi ed in fi ve studies in terms of patient selection. An intervention protocol was not defi ned in three reports. No attempt was made at blinding any aspect of the 10 studies. Figure 2 (abstract P164). Mean carbon dioxide alveolar pressure at the start and at the end of the test. g y Conclusion Use of US guidance could theoretically help minimise risk of haemorrhagic complications during PDT and perhaps reduce time taken to perform PDT. However, there is currently inadequate evidence from controlled cohort studies or RCTs to suggest that routine use for PDT in selected or unselected groups improves clinically relevant outcome measure. References 1. Delaney et al.: Crit Care 2006, 10:R55. 2. Higgins JPT, Green S (Eds): Cochrane Handbook for Systematic Reviews of Interventions. Version 5.1.0. The Cochrane Collaboration; 2011. [www.cochrane-handbook.org] 2. Higgins JPT, Green S (Eds): Cochrane Handbook for Systematic Reviews of Interventions. Version 5.1.0. The Cochrane Collaboration; 2011. [www.cochrane-handbook.org] vs. Melker –0.3; vs. Quicktrach –5.9) (Figure 2) and moreover the users judged it more favorably. Conclusion In this manikin study, Ventrain seemed to be able to appropriately oxygenate and ventilate a patient in a CICV situation. When compared with the best available choices, it has shown not to be inferior. R f Evaluation of a new device for emergency transcricoid ventilation in a manikin model Reference g CW Carspecken, D Talmor This may represent a paucity of evidence on the subject, suggesting that further clinical trials on the topic are needed to contribute to the evidence base. This also highlights the need for international consensus on the topic, to reduce duplication of eff orts, standardise practice, and improve outcomes. Reference 1. Appraisal of Guidelines for Research and Evaluation: Instrument. AGREE II [http://www.agreetrust.org/] ble 1 (abstract P166). Summary of results returned following systematic review Total Results with Full texts Guidelines AGREE instrument atabase results limits retrieved found compliant bMed 4,685 1,596 33 EDLINE 3,859 1,337 17 26 2 NAHL 711 172 6 edical societies/guideline websites/clearinghouses N/A N/A N/A 54 3 oogle 17,600,000 497 N/A Introduction Protocol-based care of the tracheostomised patient is important, as adverse events confer a high rate of mortality. Little is known regarding the existence of formal evidence-based guidelines on tracheostomy care. The aim of this study was to perform a systematic review for evidence-based guidelines on adult tracheostomy care. (<$11) electronics were designed such that no power supply would be needed apart from a computer or mobile device. Collection of airfl ow signal and calibration was performed with a standard 3 l syringe with fl ow volume measurement on one adult subject. Results Calibration of the assembled device over range of 0.5 to 3 l/ second enabled conversion of pressure diff erence to air fl ow with a mean measured tube resistance of 0.258 (kPa*second)/l. Robust signal responses to an adult subject’s continuous respiratory maneuvers on the tube itself were demonstrated. Subject performance of the pMDI technique with subsequent tidal volume breathing was recorded and analyzed (Figure 1). Methods A systematic search of PubMed, MEDLINE, guideline clearinghouses, centres of evidence-based practice, and professional societies’ guidelines relating to care of adult patients with a tracheostomy was performed by two reviewers. In addition, a Google search of publicly available tracheostomy care guidelines was performed. Search terms: (tracheostom* OR tracheotom*) AND (protocol* OR guideline* OR standard* OR management OR consensus OR algorithm*). Filters: English language, human, from 1 January 1990 to date, adult patients. Guideline appraisal criteria: the quality of guidelines retrieved was assessed using the Appraisal of Guidelines Research and Evaluation II (AGREE II) instrument [1]. y Conclusion Design and calibration of a novel low-cost monitoring device for bronchodilator delivery monitoring enabled detection and recording of characteristic fl ow volume respiratory patterns for point-of-care diagnostics in resource-limited settings. g CW Carspecken, D Talmor Future work will require clinical testing and automated detection of the correct pMDI patient technique. Results The search results are summarised in Table  1. A total of 80 guidelines were identifi ed. Five were found to satisfy the AGREE II criteria and only three related to the entire spectrum of tracheostomy management. The majority was informal and was not published or evidence based. p References References 1. Ari A, et al.: Respir Care 2012, 57:613-626. 2. Riviello E, et al.: Crit Care Med 2011, 39:860. 2. Riviello E, et al.: Crit Care Med 2011, 39:860. Conclusion Five evidence-based guidelines on adult tracheostomy management were identifi ed. This may represent a paucity of evidence on the subject, suggesting that further clinical trials on the topic are needed to contribute to the evidence base. This also highlights the need for international consensus on the topic, to reduce duplication of eff orts, standardise practice, and improve outcomes. g CW Carspecken, D Talmor Harvard Medical School and Beth Israel Deaconess Medical Center, Boston, MA, USA y yf Methods We used the SimulARTI Human Patient Simulator to evaluate Ventrain. Initially, we studied the eff ectiveness and security in ventilating and oxygenating the patient. In a second phase, the Ventrain performance was compared with what is considered to be the present gold standard (Quicktrach II, Portex Mini-Trach II Seldinger Kit, Melker Emergency Cricothyrotomy Catheter Set). Seven anesthesiologists performed an emergency transcricoid ventilation with each device in the same setting. Critical Care 2013, 17(Suppl 2):P165 (doi: 10.1186/cc12103) Critical Care 2013, 17(Suppl 2):P165 (doi: 10.1186/cc12103) Introduction Eff ective delivery of aerosolized bronchodilators for patients with asthma is crucial for adequate therapy in critical care and emergent settings. Often administered with pressure-metered dose inhalers (pMDIs), bronchodilator delivery depends on the correct patient technique during administration [1] and the ability to measure treatment response, which are diffi cult to monitor at the point of care and particularly so in resource-poor settings where standard in- hospital monitoring is unavailable [2].l Results Ventrain provided an average tidal volume of 334 ml and an average minute volume of 2.4  l in the considered situation, with a modifi cation of PAO2 from 32 to 702 mmHg and of PACO2 from 54.5 to 38.8  mmHg. In the second phase, the time needed to obtain an eff ective oxygenation with Ventrain was found to be shorter than other devices (median diff erence; vs. Minitrach –60 seconds; vs. Melker –35  seconds; vs. Quicktrach –25 seconds) (Figure  1); the ability to remove CO2 resulted bigger (average diff erence: vs. Minitrach –11.9; Methods A point-of-care device for airfl ow measurement during bronchodilator delivery was designed and tested for use in resource- limited settings. The handheld device was constructed from a clinical aerosol delivery tube with a bidirectional sensor for pressure diff erential detection about the aerosol element (Figure 1). The custom low-cost S62 Critical Care 2013, Volume 17 Suppl 2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 http://ccforum.com/supplements/17/S2 Figure 1 (abstract P165). Figure 1 (abstract P165). Figure 1 (abstract P165). Figure 1 (abstract P165). $11) electronics were designed such that no power supply would be eeded apart from a computer or mobile device. Collection of airfl ow gnal and calibration was performed with a standard 3 l syringe with l ow volume measurement on one adult subject. g CW Carspecken, D Talmor esults Calibration of the assembled device over range of 0.5 to 3 l/ econd enabled conversion of pressure diff erence to air fl ow with a ean measured tube resistance of 0.258 (kPa*second)/l. Robust signal sponses to an adult subject’s continuous respiratory maneuvers on e tube itself were demonstrated. Subject performance of the pMDI chnique with subsequent tidal volume breathing was recorded and nalyzed (Figure 1). onclusion Design and calibration of a novel low-cost monitoring evice for bronchodilator delivery monitoring enabled detection nd recording of characteristic fl ow volume respiratory patterns for oint-of-care diagnostics in resource-limited settings. Future work will quire clinical testing and automated detection of the correct pMDI atient technique. eferences Ari A, et al.: Respir Care 2012, 57:613-626. Riviello E, et al.: Crit Care Med 2011, 39:860. 166 vidence-based guidelines and protocols for the management of dult patients with a tracheostomy: a systematic review Siddiqui1, PB Sherren2, MA Birchall3 North West London Hospitals NHS Trust, London, UK; 2Barts and The London HS Trust, London, UK; 3The Royal National Throat Nose and Ear Hospital, ndon, UK itical Care 2013, 17(Suppl 2):P166 (doi: 10.1186/cc12104) Introduction Protocol-based care of the tracheostomised patient is important, as adverse events confer a high rate of mortality. Little is known regarding the existence of formal evidence-based guidelines on tracheostomy care. The aim of this study was to perform a systematic review for evidence-based guidelines on adult tracheostomy care. Methods A systematic search of PubMed, MEDLINE, guideline clearinghouses, centres of evidence-based practice, and professional societies’ guidelines relating to care of adult patients with a tracheostomy was performed by two reviewers. In addition, a Google search of publicly available tracheostomy care guidelines was performed. Search terms: (tracheostom* OR tracheotom*) AND (protocol* OR guideline* OR standard* OR management OR consensus OR algorithm*). Filters: English language, human, from 1 January 1990 to date, adult patients. Guideline appraisal criteria: the quality of guidelines retrieved was assessed using the Appraisal of Guidelines Research and Evaluation II (AGREE II) instrument [1]. Results The search results are summarised in Table  1. A total of 80 guidelines were identifi ed. Five were found to satisfy the AGREE II criteria and only three related to the entire spectrum of tracheostomy management. The majority was informal and was not published or evidence based. Conclusion Five evidence-based guidelines on adult tracheostomy management were identifi ed. Can we keep neck breathers safe? A survey on training adequacy of medical staff caring for tracheostomy/laryngectomy patients Results PDTs with LMA were successful in 99.3% of the patients (n = 149). The ratings were 1 or 2 in 96% of cases with regards to ventilation and to blood gas analysis, in 96.6% for identifi cation of relevant structures and tracheal puncture site, and in 93.3% for the view inside the trachea during PDT. A rating of 5 was assigned to one patient requiring tracheal reintubation for inadequate ventilation. There were no damages to the bronchoscope or reports of gastric aspiration. Can we keep neck breathers safe? A survey on training adequacy of medical staff caring for tracheostomy/laryngectomy patients C Tjen, G Rajendran, S Hutchinson Norfolk and Norwich University Hospital, Norwich, UK Critical Care 2013, 17(Suppl 2):P168 (doi: 10.1186/cc12106) C Tjen, G Rajendran, S Hutchinson j j Norfolk and Norwich University Hospital, Norwich, UK y Critical Care 2013, 17(Suppl 2):P168 (doi: 10.1186/cc12106) Introduction A fatal incident related to a blocked tracheostomy tube prompted a review in our Trust. To provide safe tracheostomy care, changes in staffi ng, education and operational policies were recommended. Training of potential fi rst responders to tracheostomy or laryngectomy emergencies remains outstanding. We aim to quantify the training defi cit. Tracheostomies are common in critical care but these patients require ongoing management of an artifi cial airway on discharge to the ward and even the community. In 2010 our critical care unit cared for 108 tracheostomy patients, of which 30 were transferred to the wards. The 4th National Audit Project highlighted complications including hypoxic brain injury and death [1] and the National Patient Safety Agency recognised a number of avoidable aspects [2]. Existing guidelines for management of these patients including emergencies are not widely known. p p g p Conclusion The Blue Dolphin PDT using a LMA showed defi nite advantages regarding inspection of dilation process. This method improves visualization of the trachea and larynx during a video-assisted procedure and prevents the diffi culties associated with the use of an ET such as cuff puncture, tube transection by the needle, accidental extubation, and bronchoscope lesions. The LMA results as an eff ective and successful ventilatory device during PDT. This may be especially relevant in cases of diffi cult patient anatomy where improved structural visualization optimizes operating conditions. The intensivist performing PDT should be scrupulous when deciding which method to use. References 1. Cook T, et al.: Br J Anaesth 2011, 106:632-642. 1. Cook T, et al.: Br J Anaesth 2011, 106:632-642. 2. McGrath B, et al.: Postgrad Med J 2010, 86:522-525. Results All 11 ICUs questioned perform and manage tracheostomies. Of 22 respondents, 10 knew of guidelines covering all of the emergencies described above and their location. Four respondents thought that these guidelines were accessible in an emergency setting, one-half of which were on computer systems requiring a login and search function. With regards to emergency management, 19 respondents felt competent in a tracheostomy emergency; almost exclusively through experience and in-house teaching. No respondents were aware of any formal emergency tracheostomy management courses. 2. McGrath B, et al.: Postgrad Med J 2010, 86:522-525. Laryngeal mask as a safe and eff ective ventilatory device during Blue Dolphin tracheostomy in the ICU The insertion of the LMA, the quality of ventilation, the blood gas values, the view of the tracheal puncture site, and the view of the balloon dilatation were rated as follows: very good (1), good (2), barely acceptable (3), poor (4), and very poor (5). Results PDTs with LMA were successful in 99.3% of the patients (n = 149). The ratings were 1 or 2 in 96% of cases with regards to ventilation and to blood gas analysis, in 96.6% for identifi cation of relevant structures and tracheal puncture site, and in 93.3% for the view inside the trachea during PDT. A rating of 5 was assigned to one patient requiring tracheal reintubation for inadequate ventilation. There were no damages to the bronchoscope or reports of gastric aspiration. 1. Cook TM, et al.: Results of the 4th National Audit Project of the Royal College of Anaesthetists. Br J Anaesth 2011, 106:632-642. College of Anaesthetists. Br J Anaesth 2011, 106:632-642. 2. McGrath BA, et al.: Multidisciplinary guidelines for the management of tracheostomy and laryngectomy airway emergencies. Anaesthesia 2012, 67:1025-1041. p y g g tracheostomy and laryngectomy airway emergencies. Anaesthesia 2012, 67:1025-1041. tracheostomy and laryngectomy airway emergencies. Anaesthesia 2012, 67:1025-1041. Can we keep neck breathers safe? A survey on training adequacy of medical staff caring for tracheostomy/laryngectomy patients In our ICU the Blue Dolphin PDT with LMA has become the procedure of choice. References Can we keep neck breathers safe? A survey on training adequacy of medical staff caring for tracheostomy/laryngectomy patients C Tjen, G Rajendran, S Hutchinson Norfolk and Norwich University Hospital, Norwich, UK Critical Care 2013, 17(Suppl 2):P168 (doi: 10.1186/cc12106) Can we keep neck breathers safe? A survey on training adequacy of medical staff caring for tracheostomy/laryngectomy patients C Tjen, G Rajendran, S Hutchinson Norfolk and Norwich University Hospital, Norwich, UK Critical Care 2013, 17(Suppl 2):P168 (doi: 10.1186/cc12106) P167 Survey of emergency tracheostomy management in the East of England region N Lawrence, L Oakley, C Swanavelder, M Palmer West Suff olk Hospital, Bury St Edmunds, UK Critical Care 2013, 17(Suppl 2):P167 (doi: 10.1186/cc12105) Results We achieved a response rate of 39% (65/168). Respondents comprised: 33% anaesthesia/critical care, 47% medicine and 14% surgery. Over one-half (36/65) had managed tracheostomy/laryngec- tomy emergencies, with 42% (15/36) of these incidents occurring on the wards and one in an outpatient clinic. Only 20% (13/65) had received any formal training on management of a blocked/misplaced tracheostomy tube and only 18% (12/65) were aware of any guidelines. One-third of responders lacked confi dence in management of these emergencies and 88% felt they would benefi t from formal training including simulation. Introduction The 4th National Audit Project of the Royal College of Anaesthetists [1] concluded that the majority of airway-related signifi cant complications in ICUs resulted from displaced or blocked tracheostomies and recommended together with the Intensive Care Society and the National Tracheostomy Safety Project that each ICU in the UK should have an emergency airway management plan and guidelines [2]. The aim of this survey was to establish whether such guidelines exist and are familiar to those working within the ICUs of the East of England (EoE), their ease of availability in an emergency and the degree of emergency tracheostomy training within the region. Conclusion The population of patients with exteriorised tracheas is increasing and represents a high-risk group. Management of airway emergencies in these patients is not part of standard life-support courses. According to our trainees, these scenarios are relatively common and a signifi cant proportion of fi rst responders are poorly equipped to deal with them. Our Trust will be including specifi c training on the emergency management of neck breathers as part of in-house resuscitation training. We would contend that national resuscitation courses should consider doing the same. f Methods Data collection was via a telephone survey of 11 ICUs in the EoE training region during July 2012 with one senior ICU nurse and one ICU trainee questioned per hospital. Questions related to the existence and accessibility of guidelines for tracheostomy emergencies, and to the respondent’s degree of emergency tracheostomy training and their perceived availability of formal training. P166 P166 Evidence-based guidelines and protocols for the management of adult patients with a tracheostomy: a systematic review 1. Appraisal of Guidelines for Research and Evaluation: Instrument. AGREE II [http://www.agreetrust.org/] 1. Appraisal of Guidelines for Research and Evaluation: Instrument. AGREE II [http://www.agreetrust.org/] Table 1 (abstract P166). Summary of results returned following systematic review Total Results with Full texts Guidelines AGREE instrument Database results limits retrieved found compliant PubMed 4,685 1,596 33 MEDLINE 3,859 1,337 17 26 2 CINAHL 711 172 6 Medical societies/guideline websites/clearinghouses N/A N/A N/A 54 3 Google 17,600,000 497 N/A Table 1 (abstract P166). Summary of results returned following systematic review Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 S63 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 P167 ENT, thoracics and A&E were approached. All completed forms were included. P167 Survey of emergency tracheostomy management in the East of England region N Lawrence, L Oakley, C Swanavelder, M Palmer West Suff olk Hospital, Bury St Edmunds, UK Critical Care 2013, 17(Suppl 2):P167 (doi: 10.1186/cc12105) 2. De Nicola A, Sucre MJ: Abstract 829. Proceedings Book of 40th Congress of the Society of Critical Care Medicine 2011: January 15–19 2011; San Diego, CA. Mount Prospect, IL: Society of Critical Care Medicine. Laryngeal mask as a safe and eff ective ventilatory device during Blue Dolphin tracheostomy in the ICU A De Nicola1, MJ Sucre1, G Donnarumma1, A Corcione2 1San Leonardo Hospital, Castellammare di Stabia, Italy; 2Monaldi Hospital, Naples, Italy A De Nicola1, MJ Sucre1, G Donnarumma1, A Corcione2 1San Leonardo Hospital, Castellammare di Stabia, Italy; 2Monaldi Hospital, Naples, Italy Critical Care 2013, 17(Suppl 2):P169 (doi: 10.1186/cc12107) g y y g Conclusion Despite national guidance within the UK this survey highlights that implementation and awareness of emergency tracheostomy guidelines in ICUs in the EoE region is poor, and when present they are not readily accessible in an emergency. Emergency training has largely been informal and the availability of formal training courses has not been recognised. In order to improve patient safety there is a need to ensure that emergency tracheostomy management including guidelines, equipment and formalised tracheostomy emergency training are adopted and embraced universally. References Introduction Usually percutaneous tracheostomy is accomplished via the tracheal tube. Some severe complications during percutaneous dilatational tracheostomy (PDT) may be related to poor visualization of tracheal structures. The alternative implies extubation and re- insertion of a laryngeal mask (LMA). An accidental extubation as well as an injuring of the vocal cords (because of the infl ated cuff during dislocation) appears impossible in this method. Subjectively, the bronchoscopic view obtained via a LMA seems to be better than that obtained with an endotracheal tube (ET) [1,2]. Cook TM, et al.: Results of the 4th National Audit Project of the Royal 1. Cook TM, et al.: Results of the 4th National Audit Project of the Royal College of Anaesthetists. Br J Anaesth 2011, 106:632-642. Methods In this prospective observational study, the bedside PDT was performed using the Ciaglia Blue Dolphin method in 150 critically ill patients. The patient’s tracheal tube was exchanged for a LMA Fastrach™ before undertaking PDT. The insertion of the LMA, the quality of ventilation, the blood gas values, the view of the tracheal puncture site, and the view of the balloon dilatation were rated as follows: very good (1), good (2), barely acceptable (3), poor (4), and very poor (5). Methods In this prospective observational study, the bedside PDT was performed using the Ciaglia Blue Dolphin method in 150 critically ill patients. The patient’s tracheal tube was exchanged for a LMA Fastrach™ before undertaking PDT. References 1. Linstedt U, et al.: Anesth Analg 2010, 110:1076-1082. Methods An anonymous online survey was sent to all trainees who may respond to a tracheostomy emergency in our organisation. Trainees in anaesthesia/critical care, general medicine, general surgery, 2. De Nicola A, Sucre MJ: Abstract 829. Proceedings Book of 40th Congress of the Society of Critical Care Medicine 2011: January 15–19 2011; San Diego, CA. Mount Prospect, IL: Society of Critical Care Medicine. Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 S64 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 P170 Figure 1 (abstract P171). P170 Real-time ultrasound-guided balloon dilatational percutaneous tracheostomy is a safe procedure in critically ill patients: an evaluation study A Taha, A Shafi e, M Mostafa, P Wallen, H Hon, R Marktanner Sheikh Khalifa Medical City, Abu Dhabi, United Arab Emirates Critical Care 2013, 17(Suppl 2):P170 (doi: 10.1186/cc12108) Introduction Balloon dilatational percutaneous tracheostomy with radial outward dilation minimizes bleeding and injury to tracheal rings [1]. Using the bronchoscope during the puncture of the trachea holds a risk of inaccurate placement, of hypoventilation and of needle puncturing the bronchoscope. This study was conducted in order to evaluate the safety features and feasibility of combining real-time ultrasound-guided puncture of the trachea with visualization of the needle path during tracheal puncture [2]. We provide precise localization of tracheal cartilage and proper site puncture for the stoma by ultrasound, with subsequent performance of balloon dilatational percutaneous tracheostomy using the Blue dolphin technique.i p y g p q Methods Twenty-fi ve patients including 15 males and 10 females, mean age 61 years, with age range from 23 to 102 years, underwent bedside percutaneous tracheostomy combining real-time ultrasound and the Blue Dolphin technique. Results The median time for the procedure was 15 (12 to 20) minutes. Targeted placement for the tracheostoma between the second and third or the third and fourth tracheal ring was achieved in 100%. No signifi cant complications (for example, tracheal bleeding, puncture posterior tracheal wall, misplacement of the tracheal cannula) occurred. One fractured tracheal ring was identifi ed using bronchoscopy after the procedure. No conversion into a bronchoscopically guided or into a surgical open technique was necessary. found, with no signifi cant diff erence between RV systolic function, pulmonary acceleration time and pulmonary velocity time integral between survivors and nonsurvivors. Right ventricular restriction in interventional lung assist for acute respiratory distress syndrome Critical Care 2013, 17(Suppl 2):P172 (doi: 10.1186/cc12110) G Tavazzi1, M Bojan2, S Canestrini2, M White2, S Price2 1University of Pavia Foundation Policlinico San Matteo IRCCS, Pavia, Italy; 2Royal Brompton Hospital, London, UK Introduction Global left ventricular electromechanical dyssynchrony (GLVD) is uncoordinated LV contraction that reduces the extent of intrinsic energy transfer from the myocardium to the circulation leading to a reduction in peak LV pressure rise, prolonged total isovolumic time (t-IVT) and fall in stroke volume [1]. This potentially important parameter is not routinely assessed in critically ill cardiothoracic patients. Introduction Acute cor pulmonale (ACP) is associated with increased mortality in patients ventilated for acute respiratory distress syndrome (ARDS). Interventional lung assist (iLA) allows a lung-protective ventilatory strategy, whilst allowing CO2 removal, but requires adequate right ventricular (RV) function. RV restriction (including presystolic pulmonary A wave) [1] is not routinely assessed in ARDS. p Methods A prospective analysis of retrospectively collected data in cardiothoracic ICU patients who underwent echocardiography was performed. In addition to epidemiological factors, echo data included comprehensive assessment of LV/RV systolic and diastolic function including Doppler analysis of isovolumic contraction/ relaxation, ejection time (ET) and fi lling time (FT). t-IVT was calculated as (60 – (total ET + total FT)) and the Tei Index as (ICT + IRT) / ET. t-IVT >14 second/minute and Tei index >0.48 were used to defi ne GLVD [2]. Data are shown as mean ± SD/median (interquartile range). p y y Methods A prospective analysis of retrospectively collected data in patients with echo during iLA was performed. Data included epidemiologic and ventilatory factors, LV/RV function, evidence of RV restriction and pulmonary hemodynamics. Data are shown as mean ± SD/median (interquartile range). Results Thirty-two patients (45 ± 17 years), 22 male (68%), SOFA score 11.15 ± 2.38 were included. Pulmonary hypertension (PHT) was 53%, and hospital mortality 43%. Mortality was not associated with age, days on iLA, length of ICU stay, inotropic support, nitric oxide or level of ventilatory support, but was associated with pressor requirement (P = 0.005), a worse PaO2:FiO2 ratio (9.4 (7.8 to 12.6) vs. 15.2 (10.7 to 23.9), P = 0.009) and higher pulmonary artery pressures (56.5 mmHg (50 to 60) vs. 44.5 (40.5 to 51.2), P = 0.02). No echo features of ACP were g Results A total of 103 patients (63.5 ± 18.4 years), 65 male (63%), APACHE II score (14.6 ± 7.4) were included. References References 1. Gromann TW, et al.: Anesth Analg 2009, 108:1862-1866. 2. Venkatakrishna Rajajee et al.: Crit Care 2011, 15:R54. 3. Guinot et al.: Crit Care 2012, 16:R40. P172 Left ventricular electromechanical dyssynchrony and mortality in cardiothoracic intensive care G Tavazzi , M Bojan , A Duncan , A Vazir , S Price 1University of Pavia Foundation Policlinico San Matteo IRCCS, Pavia, Italy; 2Necker-Enfants Malades University Hospital, Paris, France; 3Royal Brompton Hospital, London, UK References The incidence of RV restriction was high (43%), and independent of PHT, RV systolic function and level of respiratory support, but correlated with CO2 levels (restrictive 7.1 kPa (7.4 to 8.0) vs. 6.1 (5.8 to 6.8), P = 0.03). See Figure 1. Conclusion This study demonstrates the feasibility and safeness combining real-time ultrasound guidance and balloon dilatational percutaneous tracheostomy (Blue Dolphin technique) in critically ill patients. In our division, this technique has become the standard bedside tracheostomy procedure because it combines excellent patient safety features with avoidance of intraprocedural tube misplacement, hypoventilation and accidental bronchoscopy damage even in technically diffi cult cases [3]. g Conclusion Typical echo features of ACP were not seen in this study, possibly because of the protective ventilatory strategies allowed by use of iLA. The incidence of RV restriction may refl ect more subtle abnormalities of RV function. Further studies are required to elucidate RV pathophysiology in critically ill adult patients with ARDS. Reference 1. Cullen S, et al.: Circulation 1995, 91:1782-1789. P171 P171 Right ventricular restriction in interventional lung assist for acute respiratory distress syndrome G Tavazzi1, M Bojan2, S Canestrini2, M White2, S Price2 1University of Pavia Foundation Policlinico San Matteo IRCCS, Pavia, Italy; 2Royal Brompton Hospital, London, UK Critical Care 2013, 17(Suppl 2):P171 (doi: 10.1186/cc12109) P171 Right ventricular restriction in interventional lung assist for acute respiratory distress syndrome G Tavazzi1, M Bojan2, S Canestrini2, M White2, S Price2 1University of Pavia Foundation Policlinico San Matteo IRCCS, Pavia, Italy; 2Royal Brompton Hospital, London, UK Critical Care 2013, 17(Suppl 2):P171 (doi: 10.1186/cc12109) Right ventricular restriction in interventional lung assist for acute respiratory distress syndrome G Tavazzi1, M Bojan2, S Canestrini2, M White2, S Price2 1University of Pavia Foundation Policlinico San Matteo IRCCS, Pavia, Italy; 2Royal Brompton Hospital, London, UK Critical Care 2013, 17(Suppl 2):P171 (doi: 10.1186/cc12109) Intracardiac ECG for confi rmation of correct positioning of central venous catheters is safe and cost-eff ective M Eriksson, R Dörenberg Surgical Sciences, Uppsala, Sweden Critical Care 2013, 17(Suppl 2):P175 (doi: 10.1186/cc12113) Methods Data for all ultrasound-guided interventions, including complications, are prospectively collected in our department for audit purposes; in this study we involved only CVC insertions in the ICU between February 2011 and November 2012. Electronic medical and laboratory records and paper-based nursing charts were retrospectively studied for all interventions, specifi cally looking for blood results, coagulation abnormalities and intervention-related complications. Introduction About 10 years ago the use of chest radiographs as the golden standard to ensure correct positioning of central venous catheters (CVC) was questioned. The frequent use of CVCs was also challenged. We decided to retrospectively evaluate our routines in a large surgical unit in a Swedish university hospital. Methods All X-rays were centrally registered. Chest X-ray performed in our unit is almost entirely used to confi rm CVC positioning. The Certofi x CVC set for the Seldinger technique in combination with Certodyn – Universaladapter (B Braun, Germany) is now used as the routine equipment and the right jugular vein is our standard approach. Results In the study period, ultrasound guidance was employed for a total of 291 central line insertions in 220 ICU patients. Coagulopathy was detected in 127 cases at the time of CVC placement (43.6%). On the day of CVC insertion, coagulation abnormalities were corrected in 20 cases (15.7%); 33 out of 50 patients with severe coagulopathy (66.0%) and 74 out of 77 patients with coagulopathy of moderate severity (96.1%) had no correction at all. Correction was started only after CVC insertion for reasons unrelated to CVC placement in a further eight and two patients with severe and less severe coagulopathy (16.0% and 2.6%), respectively. No bleeding complications were observed. Results In 2002 the total number of X-rays performed in patients at our unit was 2,306, which corresponds to the approximate number of inserted CVCs at that time, since a confi rmatory X-ray was routine. X-rays were rarely performed on other indications in our unit. X-ray costs were at that time approximately €300,000 (~€130/each). The year after, 1,726 chest X-rays were performed, refl ecting both the use of intracardiac confi rmation of correct CVC position and also a reduced use of CVCs. This trend has continued over time. In 2011 approximately 600 CVCs were inserted at our unit. X-rays were performed in about 20% of these cases. Cyanoacrylate glue prevents early bleeding of the exit site after CVC or PICC placement p G Scoppettuolo, MG Annetta, C Marano, E Tanzarella, M Pittiruti Catholic University, Rome, Italy Critical Care 2013, 17(Suppl 2):P174 (doi: 10.1186/cc12112) p G Scoppettuolo, MG Annetta, C Marano, E Tanzarella, M Pittiruti Catholic University, Rome, Italy C i i l C 2013 17(S l 2) P174 (d i 10 1186/ 12112) y y itical Care 2013, 17(Suppl 2):P174 (doi: 10.1186/cc12112) Intracardiac ECG for confi rmation of correct positioning of central venous catheters is safe and cost-eff ective The cost for a chest X-ray is today ~€200, meaning that X-ray costs were approximately €24,000. We have not experienced any medical problems when intracardiac ECG was used for positioning confi rmation. On the contrary, aspiration of venous blood without apparent p-waves in a patient with sinus rhythm may suggest improper placement of the CVC; for example, the right brachial vein. p y g p Conclusion In patients undergoing CVC insertion in our ICU, coagulopathy is common. We observed uncomplicated CVC placement in all 41 patients with severe uncorrected coagulopathy and in a further 76 patients with coagulopathy of moderate severity. When combined with other studies, our data suggest that ultrasound-guided CVC placement without routine correction of coagulation abnormalities may be safe in the ICU. Safety of ultrasound-guided central venous access in critically ill patients with uncorrected coagulopathy Safety of ultrasound-guided central venous access in critically ill patients with uncorrected coagulopathy G Reusz1, C Langer1, G Egervari1, P Sarkany1, A Csomos2 1Markhot Ferenc Hospital, Eger, Hungary; 2Semmelweis University, Budapest, Hungary Critical Care 2013, 17(Suppl 2):P173 (doi: 10.1186/cc12111) Conclusion Glue is an inexpensive and highly eff ective tool for avoiding the risk of early bleeding of the exit site after catheter placement. We also suggest that in the next future the glue might prove to have benefi cial collateral eff ects on the risk of extraluminal contamination (by reducing the entrance of bacteria in the space between the catheter and the skin), as well as on the risk of dislocation (by increasing the stability of the catheter inside the skin breach). y G Reusz1, C Langer1, G Egervari1, P Sarkany1, A Csomos2 g g y 1Markhot Ferenc Hospital, Eger, Hungary; 2Semmelweis University, Budapest, Hungary g g y 1Markhot Ferenc Hospital, Eger, Hungary; 2Semmelweis University, Budapest, Hungary y Critical Care 2013, 17(Suppl 2):P173 (doi: 10.1186/cc12111) Introduction Correction of coagulopathy before central venous catheter (CVC) insertion is a common practice; however, when ultrasound guidance is used this is controversial as mechanical complications are rare. Studies in oncology patients suggest that CVC placement without prior correction of coagulopathy is safe but no studies are available for critically ill patients and guidelines do not give recommendations [1,2]. We do not routinely correct coagulopathy, even if severe, when ultrasound guidance is used and the purpose of this retrospective study was to evaluate the safety of this practice. P175 Right ventricular restriction in interventional lung assist for acute respiratory distress syndrome Results In 65 consecutive patients (45 PICCs, 11 dialysis catheters and nine CVCs), there was no signifi cant local bleeding at 1 hour or at 24 hours after catheter placement. No local adverse reaction occurred. No damage to the polyurethane of the catheters was detected. Conclusion Glue is an inexpensive and highly eff ective tool for avoiding the risk of early bleeding of the exit site after catheter placement. We also suggest that in the next future the glue might prove to have benefi cial collateral eff ects on the risk of extraluminal contamination (by reducing the entrance of bacteria in the space between the catheter and the skin), as well as on the risk of dislocation (by increasing the stability of the catheter inside the skin breach). Methods The aim of this pilot study was to verify the effi cacy of a cyanoacrylate glue in reducing the risk of early bleeding at the exit site after CVC or PICC placement. We studied a group of adult patients consecutively undergoing placement of polyurethane CVCs or PICCs without reverse tapering in a non-intensive ward of our hospital. All lines were inserted according to the same protocol, which included 2% chlorhexidine antisepsis, maximal sterile barriers, ultrasound guidance, EKG guidance and securement with sutureless device. Two minutes after placement of the glue, the exit site was covered with a temporary gauze dressing, which was replaced by transparent membrane at 24 hours. All patients were assessed at 1 hour and at 24 hours. fi Conclusion GLVD that limits cardiac output is common in the cardiothoracic ICU, and signifi cantly related to mortality. When diagnosed, the underlying cause should be sought and treatment instigated to minimize the t-iVT (pacing optimization/revascularization/ inotrope titration/volaemia optimization). References References 1. Cavanna L, et al.: World J Surg Oncol 2010, 8:91. 2. Lamperti M, et al.: Intensive Care Med 2012; 38:1105-1117. 1. Cavanna L, et al.: World J Surg Oncol 2010, 8:91. 2. Lamperti M, et al.: Intensive Care Med 2012; 38:1105-1117. Conclusion If we had continued to use CVCs at the same frequency as we did 10 years ago, and used X-ray confi rmation in practically all cases, we would have paid approximately €460,000 annually. Reduced use of CVCs, in combination with intracardiac confi rmation of CVC positioning, has not only allowed us to reduce costs associated with CVC insertion by more than €400,000, corresponding to a reduction rate of more than 90%, but also decreased the patient’s exposure to X-ray irradiation. Stas M, et al.: Eur J Surg Oncol 2001, 27:316-320. Joshi A, et al.: Indian Crit Care Med 2008, 12:10-14. p References 1. Duncan A, et al.: J Am Coll Cardiol 2003, 41:121-128. 2. Tei C, et al.: J Am Coll Cardiol 1996, 28:658-664. 1. Duncan A, et al.: J Am Coll Cardiol 2003, 41:121-128. 2. Tei C, et al.: J Am Coll Cardiol 1996, 28:658-664. p Results In 65 consecutive patients (45 PICCs, 11 dialysis catheters and nine CVCs), there was no signifi cant local bleeding at 1 hour or at 24 hours after catheter placement. No local adverse reaction occurred. No damage to the polyurethane of the catheters was detected.f Right ventricular restriction in interventional lung assist for acute respiratory distress syndrome The prevalence of GLVD was high (24/103, 22%) and associated with signifi cantly increased mortality, 7.5% vs. 25% (P = 0.02). There was no diff erence in requirement for cardiorespiratory support between the two populations, but there were signifi cant diff erences (no GLVD vs. GLVD) in requirement for Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 S65 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 patient’s care and logistical problems. In our experience, the rate of signifi cant local bleeding after placement of PICCs without reverse tapering may be as high as 40% at 1 hour and 15% at 24 hours, while the rate of bleeding after placement of a large-bore dialysis catheter is above 50% at 1 hour. pacing (35% vs. 62%, P = 0.02), atrial fi brillation (20% vs. 41%, P = 0.03), QRS duration (92.0 (80.0 to 120.0) vs. 116.5 (95.0 to 154.0), P = 0.01) and QTc (460.0 (416.0 to 498.5) vs. 477.5 (451.2 to 541.0), P = 0.02). There was no signifi cant diff erence in ejection fraction (no GLVD 43.0 (35.0 to 49.5) vs. GLVD 39.6 (29.5 to 49.7), P  =  0.43), mitral regurgitation (40.5% vs. 62.5%, P  =  0.06), or any other measures of LV systolic or diastolic function between the two groups. There was good correlation between the two methods used to assess dyssynchrony (LV t-IVT:LV Tei index correlation coeffi cient = 0.80, P <0.001). Methods The aim of this pilot study was to verify the effi cacy of a cyanoacrylate glue in reducing the risk of early bleeding at the exit site after CVC or PICC placement. We studied a group of adult patients consecutively undergoing placement of polyurethane CVCs or PICCs without reverse tapering in a non-intensive ward of our hospital. All lines were inserted according to the same protocol, which included 2% chlorhexidine antisepsis, maximal sterile barriers, ultrasound guidance, EKG guidance and securement with sutureless device. Two minutes after placement of the glue, the exit site was covered with a temporary gauze dressing, which was replaced by transparent membrane at 24 hours. All patients were assessed at 1 hour and at 24 hours. Is automated brachial cuff measurement of arterial pressure less accurate in cases of arrhythmia? y K Lakhal1, S Faiz2, M Martin1, AS Crouzet1, F Reminiac2, S Ehrmann2, R Cinotti1, X Capdevila3, K Asehnoune4, Y Blanloeil1, B Rozec1, T Boulain2 1Centre Hospitalier Universitaire Laennec, Nantes, France; 2Centre Hospitalier Universitaire Bretonneau, Tours, France; 3Centre Hospitalier Universitaire Lapeyronie, Montpellier, France; 4Centre Hospitalier Universitaire Hotel-Dieu, Nantes, France Introduction In cases of arrhythmia, the beat-to-beat variation of arterial pressure (AP) may impair the accuracy of automated cuff measurements. Indeed, this oscillometric device relies on the detection of arterial wall oscillations. Our aim was to determine, in ICU patients, whether brachial cuff measurements are really less reliable during arrhythmia than during regular rhythm. y g g y Methods Patients with arrhythmia and carrying an intra-arterial catheter were prospectively and consecutively included in this multi- center study. After each arrhythmic inclusion, a regular rhythm patient was included. A second inclusion was possible in case of change in the cardiac rhythm. Three pairs of invasive and brachial cuff (Philips® MP70 monitor) measurements of mean arterial pressure (MAP) were respectively averaged. Some patients underwent a second set of measurements, after a cardiovascular intervention (passive leg raising, volume expansion, initiation of/increase in catecholamine infusion) allowing the assessment of MAP changes. Figure 2 (abstract P177). Use of CO data. Results In the 111 analyzed inclusions (in 103 patients) there was only one failure in displaying a brachial cuff measurement of MAP. Arrhythmic patients (atrial fi brillation 88%, frequent extrasystoles 7%, fl utter 5%) were similar (P >0.3) to patients in regular rhythm for MAP (median 74 (IQR 67 to 80) vs. 75 (69 to 84) mmHg), SAPS II score, BMI, arm circumference, norepinephrine administration (36% vs. 35%), mechanical ventilation (80% vs. 81%), and site of the intra- arterial catheter (radial artery: 80% vs. 85%). Between arrhythmic and regular rhythm patients: the agreement (Bland–Altman analysis) between invasive and brachial cuff measurements of MAP was similar (mean bias –0.4 ± 7.2 (limits of agreement –14/14) mmHg vs. 3.0 ± 8.2 (–13/19) mmHg, respectively); the detection of hypotension (invasive MAP  <65  mmHg) by the brachial cuff was of similar reliability (area under the ROC curve (AUC) = 0.91 (95% CI = 0.80 to 0.97) vs. AUC = 0.98 (0.89 to 1), P = 0.2); and the detection of a response (>10% increase in MAP) to therapy was of similar reliability (AUC = 1 (0.85 to 1) (n = 22) vs. Is automated brachial cuff measurement of arterial pressure less accurate in cases of arrhythmia? AUC = 0.99 (0.78 to 1) (n = 17), P = 0.5). Figure 2 (abstract P177). Use of CO data. Results Fifty-three patients (18.5%) had evidence of CO monitoring. LiDCO was the most popular method (Figure 1). A total of 264 (94%) patients received treatment with vasopressors and/or inotropes. CO data were utilised in a variety of ways (Figure 2). Conclusion The majority of potential donors require vasopressors and/or inotropes post BSD, but it seems only a minority currently have their CO monitored. There is variation in how CO data are utilised to direct haemodynamic management. We welcome the development of standardised bundle-driven donor management. Conclusion These preliminary results suggest that arrhythmia does not impair the reliability of automated cuff measurements of MAP. y References Introduction Early bleeding from the exit site after CVC or PICC placement is a very common event that causes diffi culties in the Stas M, et al.: Eur J Surg Oncol 2001, 27:316-320. Joshi A, et al.: Indian Crit Care Med 2008, 12:10-14. Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 S66 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Figure 1 (abstract P177). CO device. Figure 2 (abstract P177). Use of CO data. P176 Figure 1 (abstract P177). CO device. P176 Is automated brachial cuff measurement of arterial pressure less accurate in cases of arrhythmia? K Lakhal1, S Faiz2, M Martin1, AS Crouzet1, F Reminiac2, S Ehrmann2, R Cinotti1, X Capdevila3, K Asehnoune4, Y Blanloeil1, B Rozec1, T Boulain2 1Centre Hospitalier Universitaire Laennec, Nantes, France; 2Centre Hospitalier Universitaire Bretonneau, Tours, France; 3Centre Hospitalier Universitaire Lapeyronie, Montpellier, France; 4Centre Hospitalier Universitaire Hotel-Dieu, Nantes, France Critical Care 2013, 17(Suppl 2):P176 (doi: 10.1186/cc12114) Reference 1. Murugan R, et al.; HIDonOR Study Investigators: Preload responsiveness is associated with increased interleukin-6 and lower organ yield from brain- dead donors. Crit Care Med 2009, 37:2387-2393. Cardiac output monitoring in brain-stem-dead potential organ donors: an audit of current UK practice CJ Wright1, A Broderick2, G Mandersloot3 1Glasgow Royal Infi rmary, Glasgow, UK; 2National Health Service Blood and Transplant, London, UK; 3The Royal London Hospital, London, UK Critical Care 2013, 17(Suppl 2):P177 (doi: 10.1186/cc12115) P179 Prediction of 28-day mortality by indocyanine green disappearance rate, other markers of hepatic function and transpulmonary thermodilution parameters: a prospective study in 154 patients W Huber, D Ertekin, T Langer, B Saugel, T Lahmer, M Messer, C Spinner, C Schultheiss, RM Schmid Klinikum rechts der Isar, Technical University of Munich, Germany Critical Care 2013, 17(Suppl 2):P179 (doi: 10.1186/cc12117) Methods A prospective observational study was designed and conducted. Inclusion criteria were: patients who had underwent elective or urgent/emergent cardiac surgery, with cardiac index (CI) <2.5 l/minute/m2 estimated by means of a PAC, left ventricle ejection fraction (LVEF) <40%, lactate >2 mmol/l, age >18 years. A central venous catheter (CVC) and a PAC were inserted for each patient before surgery in the same right internal jugular vein in accordance with standard procedure. Proper position of the PAC was confi rmed with pressure tracings and chest X-ray. Mixed and central venous blood samples were collected from the distal ports of the PAC and CVC respectively 30 minutes after ICU admission, and every 6 hours for a total of three samples in a 24-hour period for each patient. All blood samples were analyzed by a co-oximeter (Radiometer ABL800 fl ex; Radiometer, Copenhagen, Denmark). Statistical analysis was performed by Stats Direct (Ver.2.5.8, Cheshire, UK) and GraphPad (Vers. Prism 4.0; San Diego, CA, USA). All data were tested for normal distribution with the Kolmogorov–Smirnov test. Statistical analysis was performed by linear regression analysis. The agreement between absolute values of ScvO2 and SvO2 were assessed by the mean bias and 95% limits of agreement (LOA) ((mean bias ± 1.96)×standard deviation) according to the method described by Bland and Altman [5]. Introduction Hepatic dysfunction has been associated with outcome of ICU patients. However, most scoring systems including APACHE II only marginally refl ect acute liver dysfunction on admission. Indocyanine green (ICG) is eliminated by hepatobiliary excretion. Therefore, the ICG plasma disappearance rate (ICG-PDR) is used as a dynamic liver function test. ICG-PDR has been associated with mortality in several studies. Methods A prospective study to compare prediction of 28-day mortality by ICG-PDR, other markers of liver function and scoring systems (primary endpoint). In the subgroup of patients with trans- pulmonary thermodilution (TPTD) monitoring (PiCCO device; Pulsion, Munich, Germany), predictive capabilities of ICG-PDR were compared with cardiac index (CI), extravascular lung water index (EVLWI), global end-diastolic volume index (GEDVI) and pulmonary vascular permeability index (PVPI). ICG-PDR (i.v. Indocyanine green plasma disappearance rate for assessment of liver function: re-evaluation of normal ranges and impact of biometric data Introduction Signifi cant changes in haemodynamics occur after brain stem death (BSD) and there is evidence that yield of transplantable organs may be decreased in donors who remain preload responsive prior to donation [1], suggesting that optimisation of the cardiac output (CO) may be benefi cial in potential organ donors. We describe current UK practice with regard to CO monitoring in this group. W Huber, M Kranzmayr, C Schultheiss, W Reindl, A Krug, B Saugel, U Mayr, RM Schmid W Huber, M Kranzmayr, C Schultheiss, W Reindl, A Krug, B Saugel, U Mayr, RM Schmid Klinikum rechts der Isar, Technical University of Munich, Germany Critical Care 2013, 17(Suppl 2):P178 (doi: 10.1186/cc12116) Introduction The indocyanine green plasma disappearance rate (ICG-PDR) is a dynamic liver function test that can be non-invasively measured by pulse densitometry. ICG-PDR is associated with mortality and other markers of outcome. Due to predominant use of ICG-PDR in the ICU setting, the normal range is based on scarce data available outside the ICU and given with 18 to 25%/minute. Methods We reviewed a database of 287 brain-stem-dead potential organ donors collected by specialist nurses in organ donation (SN-OD) over a 6-month period (30 April 2011 to 31 October 2011) across multiple UK centres. The database contained data on donor management in the period from initial SN-OD review to immediately prior to transfer to the operating theatre. We analysed data on CO monitoring and vasopressor/inotrope use. Where information was missing/not recorded in the dataset, the treatment referred to was interpreted as not given/not done. Methods To prospectively re-evaluate the normal range and to analyze the potential impact of biometric data on ICG-PDR, we measured ICG-PDR (i.v. injection of 0.25  mg/kg ICG; LiMON, Pulsion, Munich, S67 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 independently associated with age (P = 0.025), but not with any other biometric parameter (gender, weight and height). Germany) in 95 outpatients with ulcerative colitis (UC). Due to a prevalence of primary sclerosing cholangitis (PSC) of 5% in UC, patients were asked regarding previous diagnosis of PSC. Additionally, serum bilirubin, AST, ALT, INR, AP, γGT and cholinesterase were determined. Association of biometric data (age, gender, height, weight) and diagnosis of PSC on ICG-PDR were evaluated using Spearman corre- lation, ROC and multivariate analysis (statistics: IBM SPSS 20). Indocyanine green plasma disappearance rate for assessment of liver function: re-evaluation of normal ranges and impact of biometric data Conclusion ICG-PDR on admission is an independent predictor of 28-day mortality. Predictive capabilities particularly of APACHE II can be improved by combination with ICG-PDR. Among TPTD-derived parameters, only PVPI provides signifi cant prediction of mortality. y Results A total of 95 patients (54 male, 41 female), age 43.2 ± 15.5 (21 to 76) years, weight 74 ± 16 kg, height 175 ± 10 cm, BMI 23.7 ± 4, bilirubin 0.48 ± 0.27 mg/dl, AST 26.8 ± 11.4 U/l, ALT 26.8 ± 14.9 U/l, previous diagnosis of PSC 8/95 (8.4%). ICG-PDR ranged from 13.8 to 44.0 with a mean of 28.2 ± 6.8 and a median of 27.0%/minute. In univariate analysis ICG-PDR was signifi cantly associated with age (r = –0.480; P <0.001), weight (r = –0.262; P = 0.011) and female gender (r = 0.221; P = 0.032), but not to height (P  =  0.681). In multivariate analysis (R  =  0.459) including the variables age, gender, height, weight and diagnosis of PSC, only age was independently (P <0.001) associated with ICG-PDR. With each year in age, ICG-PDR decreased by 0.206%/minute. In ROC analysis, ICG-PDR above the upper normal range (>25%/minute) was signifi cantly associated with young age (AUC 0.746; P <0.001). P180 P180 Mixed and central venous oxygen saturation are not interchangeable in patients with cardiogenic shock after cardiac surgery S Romagnoli, P Balsorano, F Landucci, A De Gaudio AOUC Careggi, Florence, Italy Critical Care 2013, 17(Suppl 2):P180 (doi: 10.1186/cc12118) g y S Romagnoli, P Balsorano, F Landucci, A De Gaudio AOUC Careggi, Florence, Italy Introduction Mixed venous oxygen saturation (SVO2) represents a well-recognized parameter of oxygen delivery (DO2)–consumption (VO2) mismatch and its use has been advocated in critically ill patients in order to guide hemodynamic resuscitation [1] and oxygen delivery optimization. Nevertheless, the pulmonary artery catheter (PAC) is not readily available and its use is not devoid of risks. Furthermore, its use has been decreasing in recent years in surgical and cardiac surgical patients as the benefi t of guiding therapy with this device is unclear [2-4]. Central venous oxygen saturation (ScVO2) has been suggested as an alternative to SVO2 monitoring due to its feasibility in several settings. Unfortunately concerns arise from its capability to correlate with SVO2, the relationship being infl uenced by several factors, such as hemodynamic impairment and pathological process. Hemodynamic instability and shock often complicate cardiac surgery, and the SVO2– ScVO2 relationship has not been specifi cally investigated in this setting. The aim of this study is to compare SVO2 and ScVO2 values in patients with cardiogenic shock after cardiac surgery. i Conclusion ICG-PDR (normal) values should be corrected for age. With a decrease in ICG-PDR of 0.206% per year, the range between the upper and lower normal level (25 to 18%/minute) is passed through within about 35 years of life. These fi ndings are in accordance with functional loss of other organ functions (for example, cardiac output, glomerular fi ltration rate) with increasing age. Normal ranges of innovative markers of organ function mainly derived from ICU populations should be re-evaluated outside the ICU. Utility of transesophageal echocardiography in the ICU: a preliminary US perspective References 1. Rivers E, et al.: Early goal-directed therapy in the treatment of severe sepsis and septic shock. N Engl J Med 2001, 345:1368-1377. 2. Edwards JD: Oxygen transport in cardiogenic and septic shock. Crit Care Med 1991, 19:658-663. 3. Hadian M, et al.: Evidence based of the use of the pulmonary artery catheter: impact data and complications. Crit Care 2006, 10(Suppl 3):S11-S18. 4. Connors AF, et al.: The eff ectiveness of right heart catheterization in the initial care of critically ill patients. JAMA 1996, 276:889-897. 5. Bland JM, Altman DG: Statistical methods for assessing agreement between two methods of clinical measurement. Lancet 1986, 1:307-310. 6. Walley KR: Use of central venous oxygen saturation to guide therapy. Am J Respir Crit Care Med 2011, 184:514-520. Introduction While TEE is providing a direct assessment of the cardiac function and volume status as a diagnostic tool, until recently it has been impractical to be continuously available for monitoring. A new disposable, monoplanar TEE probe (ImaCor) can remain in the patient for up to 72 hours, allowing repeated measures of ventricular function and volume status, parameters needed to monitor response to therapy. Methods We assessed the benefi t these TEE data provided in the assessment of fi ve domains: hypovolemia, right ventricular dysfunction, left ventricular dysfunction, sepsis, and valvular abnormality. Bedside practitioners listed their diagnoses before and after seeing primary TEE images perform by trained physicians. We used a 0 to 5 Likert scale to assess diff erential diagnosis before and after the TEE, comparing changes using a paired t test. 1. Rivers E, et al.: Early goal-directed therapy in the treatment of severe sepsis and septic shock. N Engl J Med 2001, 345:1368-1377. 1. Rivers E, et al.: Early goal-directed therapy in the treatment of severe sepsis and septic shock. N Engl J Med 2001, 345:1368-1377. 2. Edwards JD: Oxygen transport in cardiogenic and septic shock. Crit Care Med 1991, 19:658-663. 3. Hadian M, et al.: Evidence based of the use of the pulmonary artery catheter: impact data and complications. Crit Care 2006, 10(Suppl 3):S11-S18.f 3. Hadian M, et al.: Evidence based of the use of the pulmonary artery catheter: impact data and complications. Crit Care 2006, 10(Suppl 3):S11-S18.f g g p Results All requests for TEE were to access hemodynamic instability. A total of 18 patients were screened and nine were eligible, in which 16 total TEE studies were performed. Utility of transesophageal echocardiography in the ICU: a preliminary US perspective There were no complications with TEE and all patients tolerated the long-term placement of the probe well. Of the fi ve diagnostic domains studied, right ventricular failure was the most commonly underdiagnosed contributor to the hemodynamic instability among patients prior to TEE (P = 0.054) (Figures 1 and 2). 7. Reinhart K, et al.: Comparison of central-venous to mixed-venous oxygen saturation during changes in oxygen supply/demand. Chest 1989, 95:1216-1221. 7. Reinhart K, et al.: Comparison of central-venous to mixed-venous oxygen saturation during changes in oxygen supply/demand. Chest 1989, 95:1216-1221. P179 During circulatory shock, not homogeneous oxygen extraction and regional blood fl ow Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 S68 Figure 1 (abstract P180). Table 1 (abstract P181). Hemodynamic parameters before and after treatment Group 2 compared with Group 1 (P <0.001). The increase in plasma NO level was signifi cantly higher in Group 2 (Table 1). Conclusion L-Arginine i.v. infusion 5 ml/kg during 24 hours increased NO blood level and decreased MPAP in pediatric sepsis, but did not deteriorate hemodynamic system values. Table 1 (abstract P181). Hemodynamic parameters before and after treatment Group 1 Group 1 Group 2 Group 2 Value before after before after MAP (mmHg) 66.9 ± 3.4 68.8 ± 3.0 72.8 ± 2.0 71.8 ± 1.3 MPAP (mmHg) 43.5 ± 2.6 44.3 ± 3.4 50.8 ± 3.0 28.8 ± 2.1 CO (l/minute/m2) 4.2 ± 0.2 5.0 ± 0.4 4.8 ± 0.2 4.8 ± 0.3 SpO2 (%) 93.7 ± 1.2 93.4 ± 1.5 94.6 ± 0.7 98.1 ± 0.3 redistribution make SVO2 a more reliable parameter suggesting the global adequacy of cardiac output rather than ScVO2. In this study we aimed at evaluating SvO2–ScVO2 diff erences in patients with cardiogenic shock, as defi ned by hyperlactatemia, low CI, and LVEF <40%, after cardiac surgery. Our results highlighted a great variability for these two parameters, with a clinically unacceptable mean bias and LOA. As expected, ScVO2 values were consistently higher. References P179 bolus of 0.25 mg/kg ICG; LiMON device, Pulsion) and all other predictors were determined within 48 hours after admission. Statistics: IBM SPSS 20. Results A total of 20 patients were enrolled. In 18 out of 20 cases all three blood samples were collected. In two patients only two blood samples were drawn as they exited the inclusion criteria. Linear regression analysis between the two variables resulted in an r2 of 0.708. Bland–Altman analysis (Figure 1) for the pooled measurements of SvO2 and ScvO2 showed a mean bias and LOA of 6.82% (SD of bias 5.3) and –3.71 to +17.3% respectively. Results A total of 154 patients (46 female, 108 male), age 59 ± 13 years, APACHE II score 16.0 ± 5.7, SOFA score 7.6 ± 4.2. Etiology: sepsis 14.4%, cirrhosis 28.8%, GI bleeding 8.9%, ARDS 17.8%, cardiogenic shock 4.1%, acute renal failure 3.4%, various 22.6%. The 28-day mortality was signifi cantly predicted by APACHE II (ROC-AUC: 0.762; P <0.001) and SOFA (AUC: 0.784; P <0.001). Among markers of hepatic function on admission, ICG-PDR provided the largest AUC (0.742; P <0.001), which was larger than for GOT (AUC: 0.646; P = 0.019), bilirubin (AUC: 0.641; P = 0.023) and INR (AUC: 0.614; P = 0.067). Among TPTD parameters, only PVPI signifi cantly predicted 28-day mortality (AUC: 0.643; P = 0.043), whereas CI, GEDVI and EVLWI were not predictive. Prediction of 28-day mortality by SOFA could not be improved by models including any hepatic parameter. By contrast, ICG-PDR was independently (P = 0.036) associated with mortality when included in a model (R = 0.58) with APACHE II. This model based on APACHE II and ICG-PDR provided the largest of all ROC-AUCs (AUC: 0.804; P <0.001). ICG-PDR itself was y Conclusion ScVO2 has been advocated as an attractive and simple indicator of DO2–VO2 mismatch [6]. Its role as a surrogate of the well- established SVO2 has been investigated in several settings, and it has been purposed in the hemodynamic resuscitation of critically ill septic patients [1]. Nevertheless, the SVO2–ScVO2 relationship can be infl uenced by several factors due to ScVO2 dependency from global blood fl ow redistribution that can occur during hemodynamic impairments. It has been shown previously that in healthy people ScVO2 values tend to underestimate SVO2 values, due to the higher oxygen content from inferior vena cava [7]. P181 Pulmonary hemodynamic disorder in pediatric sepsis and their correction with L-arginine infusion M Georgiyants, V Korsunov Kharkov Medical Academy Post-Graduate Education, Kharkov, Ukraine Critical Care 2013, 17(Suppl 2):P181 (doi: 10.1186/cc12119) g M Georgiyants, V Korsunov Figure 1 (abstract P182). Ventricular function according to the Likert scale. Kharkov Medical Academy Post-Graduate Education, Kharkov, Ukraine Critical Care 2013, 17(Suppl 2):P181 (doi: 10.1186/cc12119) Introduction The acute respiratory distress syndrome and pulmonary hypertension (PH) is one of the factors of septic mortality. Some data demonstrate arginine defi ciency as an important pathogenic factor of septic PH. We suppose that intravenous L-arginine infusion improves NO production and reduce PH. Methods We examined 46 patients with sepsis, severe sepsis and septic shock in accordance with San Antonio Criteria. Organ dysfunction severity was defi ned according to SOFA. The mean pulmonary artery pressure (MPAP) was estimated by Doppler method, cardiac output (CO), and stroke volume (SV)  – by ultrasound M-mode. SpO2, mean arterial pressure (MAP), blood gas, and plasma NO level was evaluated. The patients of Group 1 (n = 21, age 22.1 ± 8.5 months) had respiratory and hemodynamic support, and antibiotics. Group 2 (n  =  25, age 27.0  ±  11.2  months) had the same treatment with continuous i.v. infusion of 5  ml/kg body weight 4.2% L-arginine solution during 24 hours. Results The patients of Groups 1 and 2 did not have statistical diff erence of SOFA (4.1 ± 1.0 vs. 4.4 ± 0.7), need for ventilator support (57.0 ± 11.0% vs. 44.0 ± 10.0%), and dose of inotropes (P >0.05). The reduction of MPAP and increase of SaO2 was signifi cantly higher in Figure 1 (abstract P182). Ventricular function according to the Likert scale. Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 S69 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Figure 2 (abstract P182). Volume status, valve, sepsis according to the Likert scale. ICU in Thailand that was capable of measuring extravascular lung water. None of the Thai ICUs used transcutaneous PCO2, near-infrared spectroscopy, gut mucosal tonometry and sublingual sidestream dark- fi eld for tissue perfusion monitoring. Only four ICUs had transcutaneous PO2. However, measuring the level of lactate as one of the tissue perfusion markers was routinely performed in about 50% of the ICUs. Conclusion There were variations in monitoring performance in Thai ICUs. These vary by type of hospital. Academic ICUs had a tendency for advance monitoring in overall aspects. Establishing a critical care echocardiography laboratory K Yastrebov1, A McLean2 Establishing a critical care echocardiography laboratory K Yastrebov1, A McLean2 1The St George Hospital, Sydney, Australia; 2Nepean Hospital, Sydney, Australia Critical Care 2013, 17(Suppl 2):P184 (doi: 10.1186/cc12122) Introduction Echocardiography is increasingly utilized by inten sive care physicians in everyday practice. Standardization of echocardiographic studies and reporting, quality assurance and medicolegal requirements necessitate establishment of a dedicated system within the critical care setting. We describe the process of setting up a critical care echocardiography (CCE) laboratory based on our experience from three separate ICUs. Figure 2 (abstract P182). Volume status, valve, sepsis according to the Likert scale. Methods A retrospective review and analysis of the process involved in establishment of echocardiography laboratories within ICUs. Methods A retrospective review and analysis of the process involved in establishment of echocardiography laboratories within ICUs. Results Creating a CCE service involves a number of stages and takes several years to achieve. Major components include staffi ng, equipment, quality control, study archiving and networking capability. For staffi ng the objective is to identify and recruit staff with adequate training and expertise in CCE, providing 24/7 specialist cover in addition to supporting and training junior medical and nursing staff . There is further a need to acquire funding for high-quality ultrasound machines and related hardware as well as long-term DICOM-based archiving and reporting systems. This should be based on projections of annual volumes of echo studies and corresponding digital storage. Networking connectivity is highly desirable, including obligatory back-up solutions and site allocations. A business case incorporating all the above should precede any development as identifi able funding sources and administrative approval are essential. The implementation stage requires the presence of a project leader who can organize the trialing of scanners, archiving, reporting and research systems, ensure compatibility with existing hospital and cardiology networks, and who can assist in individualizing archiving and reporting software refl ecting institutional and ICU specifi cs. Coordination with the IT department is very important. Clear contractual vendor obligations for service, maintenance and future upgrades of hardware and software need to be specifi ed. Training and credentialing of staff is best achieved within a systematic framework that includes ongoing competency review, education and QA programs. Partnership with cardiology may benefi t both groups. Major pitfalls are associated with poor initial training, lack of expertise and leadership, and bad vendor contracts. Cardiopulmonary monitoring in Thai ICUs: results of ICU-RESOURCE I surveys K Chittawatanarat1, A Wattanatham2, D Sathaworn2, C Permpikul3, TSCCM Study Group4 1Chiang Mai University, Chiang Mai, Thailand; 2Pramongkudklao Hospital, Bangkok, Thailand; 3Siriraj Hospital, Mahidol University, Bangkok, Thailand; 4Thai Society of Critical Care Medicine, Bangkok, Thailand Critical Care 2013, 17(Suppl 2):P183 (doi: 10.1186/cc12121) Introduction Although rapid progress in ICU monitoring with advanced equipments has been developed, there were limited data on ICU monitoring in Thailand. The objective of this study was to determine the current utilization of monitoring in Thai ICUs. g Methods A self-administered questionnaire was developed by the TSCCM research subcommittee. Data verifi cation was processed by an online medical research tools program (OMERET). p g Results A total of 350 questionnaires were sent to ICUs throughout Thailand. In total, 256 questionnaires were confi rmed after being received at the end of June 2012. Of these, 140 fi lled forms (56.9%) were returned for fi nal analysis. More than 70% of the ICUs had basic hemodynamic monitoring. Less than 10% of general and regional hospitals could perform cardiac output monitoring by thermodilution technique compared with 60% of academic teaching hospitals. New and advanced hemodynamic monitoring techniques such as pulse pressure variation, systolic pressure variation, stroke volume variation, PiCCO, Vigileo-Flo Tract, Pleth variability index device and echocardiography were available only in ICUs of academic teaching hospitals except ultrasound-based techniques including transthoracic and transesophageal echocardiography and USCOM. For respiratory monitoring, all ICUs had a SpO2 monitoring device but only one-half of them had end-tidal CO2 monitoring. Nearly 80% of ventilator support in participating ICUs was capable of displaying graphic waveform monitoring. Only 43.6% of participating ICUs had a ventilator machine that could calculate lung mechanics data. Advanced respiratory monitoring such as EIT and esophageal pressure monitoring are available only in ICUs of academic teaching hospitals. There was no Conclusion Establishment of a CCE laboratory requires careful planning, and allocation of adequate human and fi nancial resources. Many potential problems can be identifi ed and prevented in advance. Strong expert leadership plays an important role. P185 Establishing a critical care echocardiography laboratory K Yastrebov1, A McLean2 Conclusion Our results suggest that having continuously available TEE for monitoring and management of hemodynamically unstable patients increases awareness of right ventricular dysfunction in the ICU. References Conclusion Our results suggest that having continuously available TEE for monitoring and management of hemodynamically unstable patients increases awareness of right ventricular dysfunction in the ICU. References 1. Vieillard-Baron A, et al.: Intensive Care Med 2004, 30:1734-1739. 2. Monnet X, et al.: Intensive Care Med 2005, 31:1195-1201. 1. Vieillard-Baron A, et al.: Intensive Care Med 2004, 30:1734-1739. 2. Monnet X, et al.: Intensive Care Med 2005, 31:1195-1201. P181 Some advance monitoring used in developed countries is also unavailable in Thailand. P187l P187 Infl uence of positive end-expiratory pressure on four-chamber longitudinal strain analysis by speckle tracking echocardiography F Franchi, A Faltoni, M Cameli, S Cecchini, M Lisi, M Contorni, S Mondillo, S Scolletta University of Siena, Italy Critical Care 2013, 17(Suppl 2):P187 (doi: 10.1186/cc12125) Introduction Speckle-tracking echocardiography (STE) has emerged as an ultrasound technique for accurately evaluating myocardial function also in critically ill patients. By tracking the displacement of the speckles during the cardiac cycle, the strain rate can be measured offl ine after adequate image acquisition. The aim of the study was to evaluate the eff ects of the positive end-expiratory pressure (PEEP) on four-chamber longitudinal strain (LS) analysis in critically ill patients. g y y p Methods We enrolled 20 consecutive patients (mean age 64 ± 18) who needed mechanical ventilation and were admitted to the ICU due to heterogeneous causes. Inclusion criteria were: hypoxia requiring PEEP titration, invasive arterial pressure monitoring, age >18. Exclusion criteria were: myocardial dysfunction, cardiac arrhythmias and valvular pathologies. The same operator performed three standard echocardiography measurements (MyLab 70 Xvision; Esaote), each of them after having increased PEEP at 5, 10, and 15 cmH2O (T1, T2, T3, respectively). Blood gas analysis, respiratory, and hemodynamic parameters provided by a pulse contour method were also recorded. STE analysis was performed offl ine (XStrain™MyLab 70 Xvision; Esaote). Results Left peak atrial LS (LA-PALS) was signifi cantly reduced from T1 to T2, and from T2 to T3 (40.2  ±  12%, 35.9  ±  9%, 28.4  ±  8%, T1, T2, T3, respectively; P  <0.05). Right peak atrial LS (RA-PALS) and right ventricular (RV)-LS showed a signifi cant reduction only at T3 (RA-PALS: 44.7 ± 48.5% at T1, 35.9 ± 11% at T3; RV-LS: –20.2 ± 2% at T1, –16.3 ± 1.1% at T3; P <0.05). Left ventricular (LV)-LS did not change signifi cantly during titration of PEEP. Cardiac chambers’ volumes and cardiac output (CO) showed a signifi cant reduction at higher levels of PEEP. Pulse pressure variation was signifi cantly aff ected by higher levels of PEEP (P <0.05). Conclusion Contrast-enhanced ultrasonography clearly improves visualization of the perfusion in various tissues. It is very likely to be superior to standard Doppler ultrasound, and is safe and well tolerated in critically ill patients. P185 Contrast-enhanced ultrasonography in the ICU: promising tool or exciting toy?i Contrast-enhanced ultrasonography in the ICU: promising tool or exciting toy?i Contrast-enhanced ultrasonography in the ICU: promising tool or exciting toy? I Göcze, R Herzog, BM Graf, A Agha, HJ Schlitt, K Pfi ster, E Jung, T Bein University Medical Centre Regensburg, Germany Critical Care 2013, 17(Suppl 2):P185 (doi: 10.1186/cc12123) I Göcze, R Herzog, BM Graf, A Agha, HJ Schlitt, K Pfi ster, E Jung, T Bein University Medical Centre Regensburg, Germany Critical Care 2013, 17(Suppl 2):P185 (doi: 10.1186/cc12123) Introduction Contrast-enhanced ultrasonography (CEUS) is a dynamic digital ultrasound-based imaging technique, which allows quantifi cation of the microvascularisation up to the capillary vessels. As a novel method for assessment of tissue perfusion it is ideally designed for use in the ICU. CEUS is cost-eff ective and safe and can be repeatedly performed at the bedside without radiation and nephrotoxicity. S70 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Methods The frequency of CEUS use in the multidisciplinary surgical ICU was retrospectively evaluated for the period from 1  September 2011 to 1 September 2012. Furthermore, contributions of this novel method to the management of critically ill ICU patients as well as its accuracy were assessed. References Krishnagopalan S, Kumar A, Parrillo JE, Kumar A: Myocardial dysfunction in the patient with sepsis. Curr Opin Crit Care 2002, 8:376-388. 2. Parker MM, McCarthy KE, Ognibene FP, et al.: Right ventricular dysfunction and dilatation, similar to left ventricular changes,characterize the cardiac depression of septic shock in humans. Chest 1999, 97:126-131. y Results In total, 33 CEUS studies were performed in critically ill ICU patients. The most frequent indications included: assessment of the liver perfusion, assessment of the pancreas and kidney perfusion after pancreas and kidney transplantation, assessment of the renal perfusion in acute kidney injury (AKI), assessment of active bleeding and assessment of the bowel perfusion. In all studies, the correct diagnosis was achieved and the transport of critically ill patients to the radiology department for further diagnostic procedures as well as application of iodinated contrast agents was avoided. In 16 cases signifi cant new fi ndings were detected. Twelve of them were missed by conventional standard Doppler ultrasound prior to CEUS. In assessment of seven cases with AKI, impaired or delayed perfusion and microcirculation of the kidney was observed in six patients. In three patients urgent surgical intervention was performed because of CEUS results. In three cases active bleeding was excluded at the bedside due to absence of contrast agent extravasation into hematoma (thigh and perihepatic) or into abdominal cavity, without need for complementary CT imaging or angiography. In one case the regular perfusion of intestinal anastomosis was confi rmed with no need for surgical exploration. None of patients undergoing CEUS manifested any adverse reactions or developed any complications associated with the imaging technique. P186 Hemodynamic disclosure of septic shock patients by intensive care ultrasound P Theerawit, Y Sutherasan, T Hongpanat Ramathibodi Hospital, Mahidol University, Bangkok, Thailand Critical Care 2013, 17(Suppl 2):P186 (doi: 10.1186/cc12124) Hemodynamic disclosure of septic shock patients by intensive care ultrasound P Theerawit, Y Sutherasan, T Hongpanat P Theerawit, Y Sutherasan, T Hongpanat Ramathibodi Hospital, Mahidol University, Bangkok, Thailand Critical Care 2013, 17(Suppl 2):P186 (doi: 10.1186/cc12124) Introduction Even though invasive hemodynamic devices are usually used for assessment of septic shock victims, they cannot evaluate the heart function. LV dysfunction as well as right heart syndrome are not uncommon in sepsis and critical patients. Intensive care ultrasound discloses these data and leads to appropriate treatment. Conclusion In hypoxic patients with normal cardiac function, PEEP titration determined a reduction of LA-LS, RA-LS and RV-LS values. LV-LS values were not infl uenced by PEEP changes. The fall in CO, observed with higher values of PEEP, seemed to be related to the impairment of preload and not of myocardial contractility. Whenever interpreting data on cardiac function obtained with longitudinal strain analysis, attention of the clinician should be drawn to diff erent levels of PEEP. The higher the PEEP, the more the probability of misleading interpretation of STE data. Methods The study was a prospective cross-sectional study. The measurement was performed within 24 hours of ICU admission. We excluded patients with history of COPD and pulmonary hypertension from any diseases. Only good-quality images acquired from subjects were included for analysis. The primary objective was to disclose how the hemodynamic changed in septic patients by ICU-US. y g p p y Results A total of 133 septic patients were measured by ICU-US. Good image quality was acquired in 115 cases (86.47%). The mean ages were 57.48 ± 17.87 years. The three major causes of sepsis were pneumonia, unknown source, abdominal infection and bacteremia. Heart failure at admission was found only in 1.79%. Previous history of hypertension, DM, and coronary artery disease was found in 12.17%, 11.30%, and 1.74% of patients. The mean LV ejection fraction (LVEF) was 54 ± 15.43%. The percentages of patients with LVEF <35%, 35 to 40%, 41 to 45% and >45% were 13.2%, 7.5%, 8.5%, and 70.8% respectively. Diastolic dysfunction defi ned by E/A ratio <1 was observed in 47.5% of patients. A total 44.6% of cases had cardiac output under 4 l/minute whereas CO over 6 l/minute was found in 18.1% of cases. The average mean pulmonary artery pressure was 34.75 ± 15.13 mmHg. The proportion of patients with meanPAP over 25 mmHg was 76.2%. RV to LV ratio >1 was found in 42.4% of septic patients. P187l Promising indications for the use of CEUS in the ICU may be the assessment of kidney microcirculation and assessment of liver perfusion in liver transplant and liver trauma patients. P186 P189 Validation of less-invasive hemodynamic monitoring with Pulsiofl ex in critically ill patients: interim results of a multicentre study K Van de Vijver1, C Pigozzi1, L Vervliet1, V Vanbiervliet1, V Brabers1, I Vos1, H Maes1, N Van Regenmortel1, I De laet1, K Schoonheydt1, H Dits1, J Belda2, Z Molnar3, M Malbrain1 1Ziekenhuis Netwerk Antwerpen, ZNA Stuivenberg, Antwerp, Belgium; 2Hospital Clinico Universitario, Valencia, Spain; 3University of Szeged, Hungary Critical Care 2013, 17(Suppl 2):P189 (doi: 10.1186/cc12127) Results A total of 103 measurements in 77 patients were performed. In seven patients measurement with Nexfi n was not possible. For CO (55 paired measurements), values were 6  ±  2.1  l/minute (range 2.6 to 12). Pearson’s correlation coeffi cient comparing Nexfi n-CO with reference CO showed a good correlation (R2  =  0.52). Bland–Altman analysis comparing both CO techniques revealed a mean bias ± 2SD (LA) of 0.3 ± 3.6 l/minute (58% error). The MAP was 84.2 ± 15.6 mmHg (53 to 131.5) and values obtained with the Nexfi n correlated well with the reference method with an R2 of 0.72. Bland–Altman analysis comparing both MAP techniques revealed a mean bias ± 2SD (LA) of –0.3 ± 18 mmHg (20.9% error). However, Nexfi n-MAP did not correlate well with NIBP (R2 = 0.36). Hemoglobin values obtained with Nexfi n Massimo technique did not correlate well with laboratory values (R2 = 0.26, 33% error). The 26 patients that died in the ICU had higher APACHE II (P = 0.017), SAPS II (P <0.0001), SOFA (P <0.0001) and SOS (P = 0.004) scores and signifi cantly lower MAP (P <0.0001), hemoglobin (P = 0.01) and lower dp/dtmax (P = 0.003), a marker for contractility. There were no outcome diff erences with regard to subgroup analysis in patients with either low or high CO or SVR. Introduction Thermodilution (TD) is considered a gold standard for measurement of cardiac index (CI) in critically ill patients. The aim of this study is to compare intermittent bolus TD CI with intermittent automatic calibration CI (AutoCI) and two continuous CIs obtained by pulse contour analysis with PiCCO2 (PiCCI) and Pulsiofl ex (PuCCI). 2l Methods Interim results of an ongoing prospective multicentre study in 53 patients. Age 58.7 ± 15.4, SAPS II score 51.4 ± 14.7 and SOFA score 10 ± 3.2. P188 Evaluation of a new calibration index suggesting recalibration of the pulse contour cardiac index by transpulmonary thermodilution: a prospective study W Huber, K Waldleitner, S Mair, B Saugel, RM Schmid Klinikum rechts der Isar, Technical University of Munich, Germany Critical Care 2013, 17(Suppl 2):P188 (doi: 10.1186/cc12126) Introduction After calibration by thermodilution (TD), the PiCCO device is able to assess cardiac index (CI) using pulse contour (PC) analysis. The manufacturer suggests recalibration by TD after 8 hours. Recently, we suggested a calibration index indicating a certain probability of a relevant bias and triggering the next calibration. With changes of CIpc compared with the previous CItd being a key predictor of the bias of CIpc compared with the following CItd, the manufacturer implemented a soft alarm indicating changes in CIpc compared with the last CItd (thresholds adjustable to 15%, 25% and 35%). The aim of Conclusion Cardiac dysfunction, namely left ventricle and probably right ventricle, was not uncommon in septic shock patients. Without intensive care ultrasound, all crucial information was delayed until patient deterioration and initial treatment may be harmful. Thus cardiac ultrasound should be used initially to disclose hemodynamic features before routine resuscitation is initiated. S71 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Results In total, 940 CCI and 382 TDCI values were obtained: 940 paired PiCCI and PuCCI; 358 paired AutoCI-TDCI measurements. TDCI values ranged from 1.5 to 6.9 l/minute/m2 (mean 3.6 ± 1.1), AutoCI from 1.8 to 7.2 (3.6 ± 0.9), PiCCI from 1.0 to 7.1 (3.5 ± 1.1) and PuCCI from 1.3 to 7.6 (3.6 ± 1). Pearson’s correlation coeffi cient comparing mean PuCCI and PiCCI values per patient had an R2 of 0.79. Comparison between AutoCI and TDCI had an R2 of 0.51. Changes in AutoCI correlated well with changes in TDCI (R2  =  0.44, concordance coeffi cient  =  95.7), as did changes in PuCCI versus changes in PiCCI (R2 = 0.99, CC = 93.4%). Changes in PiCCI and PuCCI induced by an intervention correlated well with each other (R2  =  0.86, CC  =  100%). The percentage error (PE) obtained by Bland and Altman analysis and R2 for the diff erent comparisons are presented in Table 1. the study was prospective evaluation of predictive capabilities of the 15% alarm regarding a bias of CIpc compared with CItd exceeding critical thresholds of 15%, 20% and 25%. P190 P190 P190 Interim results of an ongoing study on the use of non-invasive hemodynamic monitoring with Nexfi n in critically ill patients K Van de Vijver, V Brabers, C Pigozzi, L Vervliet, V Vanbiervliet, H Maes, I Vos, M Peetermans, N Van Regenmortel, I De laet, K Schoonheydt, H Dits, M Malbrain Ziekenhuis Netwerk Antwerpen, ZNA Stuivenberg, Antwerp, Belgium Critical Care 2013, 17(Suppl 2):P190 (doi: 10.1186/cc12128) Interim results of an ongoing study on the use of non-invasive hemodynamic monitoring with Nexfi n in critically ill patients K Van de Vijver, V Brabers, C Pigozzi, L Vervliet, V Vanbiervliet, H Maes, I Vos, M Peetermans, N Van Regenmortel, I De laet, K Schoonheydt, H Dits, M Malbrain Ziekenhuis Netwerk Antwerpen, ZNA Stuivenberg, Antwerp, Belgium Critical Care 2013, 17(Suppl 2):P190 (doi: 10.1186/cc12128) Conclusion Bias of CIpc compared with the next CItd does not depend on the time since last CItd. Changes in CIpc itself compared with the last CItd are associated with the bias. A 15% change in CIpc trend alarm provided by the new PiCCO algorithm is signifi cantly associated with bias values exceeding 15%, 20% or 25% of CItd, whereas time to last calibration >8 hours is not associated with bias exceeding these critical thresholds. Introduction The Nexfi n monitor (BMEYE, the Netherlands) enables continuous non-invasive analysis of blood pressure (MAP) as well as cardiac output (CO) measurements. The aim of the present study was to validate the Nexfi n in a mixed population of medical ICU patients. Introduction The Nexfi n monitor (BMEYE, the Netherlands) enables continuous non-invasive analysis of blood pressure (MAP) as well as cardiac output (CO) measurements. The aim of the present study was to validate the Nexfi n in a mixed population of medical ICU patients. Introduction The Nexfi n monitor (BMEYE, the Netherlands) enables continuous non-invasive analysis of blood pressure (MAP) as well as cardiac output (CO) measurements. The aim of the present study was to validate the Nexfi n in a mixed population of medical ICU patients. Methods Interim results of a prospective ongoing study in 77 patients admitted to the medical ICU (46 patients mechanically ventilated, M/F ratio 1/1). Age 65.6 ± 15.9, BMI 25.6 ± 4.8, APACHE II score 22.9 ± 10.9, SAPS II 48 ± 20.1, SOFA score 7.5 ± 4.5. A modifi ed outreach score (SOS) was calculated on admission. P190 For all patients, simultaneous recording of arterial pressure by radial line (n = 78), PiCCO (n = 44) or by NIBP with arm cuff (n = 47) was compared with the Nexfi n monitor. Statistical analysis was performed with Student’s t test, Pearson correlation and Bland–Altman analysis. i Methods Interim results of a prospective ongoing study in 77 patients admitted to the medical ICU (46 patients mechanically ventilated, M/F ratio 1/1). Age 65.6 ± 15.9, BMI 25.6 ± 4.8, APACHE II score 22.9 ± 10.9, SAPS II 48 ± 20.1, SOFA score 7.5 ± 4.5. A modifi ed outreach score (SOS) was calculated on admission. For all patients, simultaneous recording of arterial pressure by radial line (n = 78), PiCCO (n = 44) or by NIBP with arm cuff (n = 47) was compared with the Nexfi n monitor. Statistical analysis was performed with Student’s t test, Pearson correlation and Bland–Altman analysis. P188 the study was prospective evaluation of predictive capabilities of the 15% alarm regarding a bias of CIpc compared with CItd exceeding critical thresholds of 15%, 20% and 25%. Methods A prospective analysis of 329 routine TPTD measurements in 70 patients. The CI-trend alarm was set to 15%, and CIpc, trend alarm (yes/no), relative and absolute changes in CIpc were recorded immediately before TD providing exact measurement of CItd. Predictive capabilities of the 15% trend alarm regarding the bias were evaluated using Spearman correlation, chi-square test, ROC analysis and Wilcoxon test (IBM SPSS 20). Results A total of 70 patients (24 female, 46 male), age 62 ± 14 years, APACHE II score 19.5 ± 7.5, SOFA score 8.9 ± 4.4. At 937 ± 904 (50 to 5,795) minutes after the last CItd, CIpc provided a bias of –0.0566 ± 0.761 l/ minute*m2 compared with the next CItd. Percentage error was 34.6%. Absolute and relative bias of CIpc compared with the new CItd did not correlate to time to last TD, but correlated (all P <0.001) to CIpc itself (r = 0.333; r = 0.385) and relative (r = 0.531; r = 0.537) and absolute (r = 0.533; r = 0.529) changes in CIpc compared with last CItd. The 15% CIpc trend alarm was indicated before 83/329 measurements (25.2%). The amount of bias exceeded 15% and 20% in 101 (30.7%) and 79 (24%) of TDs. In TDs with indicated trend alarm (≥15% deviation of CIpc to the last CItd), the amount of bias more frequently exceeded 15% (P = 0.019), 20% (P <0.001) and 25% (P <0.001). Time to last calibration ≥8 hours was not associated with bias exceeding 15% (P = 0.735), 20% (P = 0.888) or 25% (P = 281). Conclusion The preliminary results indicate that in unstable critically ill patients, CI can be reliably monitored with Pulsiofl ex technology via a femoral line. Pulsiofl ex was also able to keep track of changes in CI. P191 Cost-eff ectiveness analysis of stroke volume variation guided perioperative hemodynamic optimization J Benes, J Zatloukal, A Simanova, I Chytra, E Kasal The Medical Faculty and Hospital in Plzen – Charles University Prague, Plzen, Czech Republic Critical Care 2013, 17(Suppl 2):P191 (doi: 10.1186/cc12129) P191 Cost-eff ectiveness analysis of stroke volume variation guided perioperative hemodynamic optimization J Benes, J Zatloukal, A Simanova, I Chytra, E Kasal The Medical Faculty and Hospital in Plzen – Charles University Prague, Plzen, Czech Republic Critical Care 2013, 17(Suppl 2):P191 (doi: 10.1186/cc12129) Introduction Perioperative goal-directed therapy (pGDT) can sub- stantially improve the outcome of high-risk surgical patients [1]. But the approach needs an initial investment and increases the staff workload. Economic factors might participate in the weak adherence to the pGDT concept. Some model studies support pGDT cost-eff ectiveness, but real economic data based on a recent clinical trial are lacking. We performed an economic analysis of hemodynamic optimization using the stroke volume variation trial [2] in order to elucidate this issue. Figure 1 (abstract P192). Correlation between PaO2/FiO2 and EVLWIc. Methods The hospital care invoices of all 120 patients included in the trial were retrospectively extracted. Due to the nature of the data we have adopted the healthcare payer’s perspective. We performed a comparison of induced costs between the Vigileo (n = 60) and Control (n = 60) groups and constructed a cost tree using the study group and complications occurrence as distributive parameters. The incremental cost-eff ectiveness ratio per complication avoided was calculated and, fi nally, diff erent reimbursing categories were assessed as potential cost drivers. (EVLWI) refl ects pulmonary edema [2]. The new EV1000/VolumeView (Edwards Lifesciences) can accurately measure EVLWI corrected for the actual volume of lung parenchyma (EVLWIc). The aim of our study is to prove a stronger correlation between EVLWIc and PaO2/FiO2 compared with EVLWI in patients undergoing pulmonary resection. Methods A prospective observational study. Seven patients with lung cancer undergoing pulmonary resection were monitored using the EV1000 plathform. EVLWI was assessed by thermodilution at the following time points: after intubation (t1); during single-lung ventilation (t2); after lung resection (t3); after ICU admission (t4); 12 hours (t5) and 18 hours after ICU admission (t6). EVLWIc values were also collected at t3 and t4. PaO2/FiO2 was measured at the same time points. Results A decreased rate (18 vs. 35 patients) and number of complications (34 vs. 78) were observed in the original trials Vigileo group. The mean cost of intervened patient was lower (€2,877 ± 2,336 vs. €3,371  ±  3,238; P  =  0.38). According to the cost-tree analysis, patients with complications (n = 53; 44%) consumed signifi cantly more resources (€235,623; 63%). References 1. Pinsky MR: Clin Chest Med 2003, 24:549-560. 2. Sakka SG, et al.: Chest 2002, 122:2080-2086. 1. Pinsky MR: Clin Chest Med 2003, 24:549-560. 2. Sakka SG, et al.: Chest 2002, 122:2080-2086. Acknowledgements The study was supported by the MSM0021620819 and the Charles University Research Fund (project number P36). References P193 1. Hamilton MA, et al.: Systematic review and meta-analysis on the use of preemptive hemodynamic intervention to improve postoperative outcomes in moderate and high-risk surgical patients. Anesth Analg 2011, 112:1392-1402.l Goal-directed resuscitation therapy in high-risk patients undergoing cardiac surgery (GRICS study): a randomized controlled trial – preliminary results E Osawa1, A Rhodes2, J Vincent3, J Almeida1, J Fukushima1, B Pileggi1, C Park1, L Camara1, J Auler Jr1, R Chan1, M Piccioni1, M Lima1, F Galas1, L Hajjar1 1Heart Institute, São Paulo, Brazil; 2St George’s Healthcare NHS Trust and St George’s University of London, UK; 3Universite Libre de Bruxelles, Brussels, Belgium Critical Care 2013, 17(Suppl 2):P193 (doi: 10.1186/cc12131) Goal-directed resuscitation therapy in high-risk patients undergoing cardiac surgery (GRICS study): a randomized controlled trial – preliminary results P191 Cost-eff ectiveness analysis of stroke volume variation guided perioperative hemodynamic optimization J Benes, J Zatloukal, A Simanova, I Chytra, E Kasal The Medical Faculty and Hospital in Plzen – Charles University Prague, Plzen, Czech Republic Critical Care 2013, 17(Suppl 2):P191 (doi: 10.1186/cc12129) A gain of €634 per avoided complications confi rms that the lower complications rate was the most important cost driver. Both the clinical care for patients costs (€505 vs. 912; P = 0.04) and ward stay costs (€244 vs. 402; P  =  0.03) were decreased by the intervention. On the contrary, the intervention increased anaesthesia costs (€880 vs. 688; P = 0.001). Results No signifi cant correlation was found between EVLWI and PaO2/ FiO2 (r  =  –0.3124, P  >0.05), while a signifi cant correlation was seen between EVLWIc and PaO2/FiO2 (r = –0.528, P =0.009; Figure 1). Conclusion Despite the small sample size, this study shows that in patients undergoing pulmonary resection the EVLWIc is more strongly correlated to PaO2/FiO2 than EVLWI. Therefore, the EV1000 may be a valuable tool for more reliable hemodynamic monitoring in this subgroup of patients. R f Results No signifi cant correlation was found between EVLWI and PaO2/ FiO2 (r  =  –0.3124, P  >0.05), while a signifi cant correlation was seen between EVLWIc and PaO2/FiO2 (r = –0.528, P =0.009; Figure 1). 2 2 g Conclusion Despite the small sample size, this study shows that in patients undergoing pulmonary resection the EVLWIc is more strongly correlated to PaO2/FiO2 than EVLWI. Therefore, the EV1000 may be a valuable tool for more reliable hemodynamic monitoring in this subgroup of patients. Conclusion Intraoperative fl uid optimization with the use of stroke volume variation and the Vigileo/FloTrac system showed not only a substantial improvement of morbidity, but was also associated with an economic benefi t. This observed benefi t highly exceed the increased monitoring costs in our trial. P192 P192 EV1000/VolumeView: a new device for a more reliable measurement of extravascular lung water index in patients with lung resections A Donati, C Melia, V Monaldi, R Domizi, E Damiani, A Carsetti, C Scorcella, R Castagnani, P Pelaia Università Politecnica delle Marche, Ancona, Italy Critical Care 2013, 17(Suppl 2):P192 (doi: 10.1186/cc12130) P192 EV1000/VolumeView: a new device for a more reliable measurement of extravascular lung water index in patients with lung resections A Donati, C Melia, V Monaldi, R Domizi, E Damiani, A Carsetti, C Scorcella, R Castagnani, P Pelaia Università Politecnica delle Marche, Ancona, Italy Critical Care 2013, 17(Suppl 2):P192 (doi: 10.1186/cc12130) Goal-directed resuscitation therapy in high-risk patients undergoing cardiac surgery (GRICS study): a randomized controlled trial – preliminary results p y E Osawa1, A Rhodes2, J Vincent3, J Almeida1, J Fukushima1, B Pileggi1, C Park1, L Camara1, J Auler Jr1, R Chan1, M Piccioni1, M Lima1, F Galas1, L Hajjar1 2. Benes J, et al.: Intraoperative fl uid optimization using stroke volume variation in high risk surgical patients: results of prospective randomized study. Crit Care 2010, 14:R118. 2. Benes J, et al.: Intraoperative fl uid optimization using stroke volume variation in high risk surgical patients: results of prospective randomized study. Crit Care 2010, 14:R118. jj 1Heart Institute, São Paulo, Brazil; 2St George’s Healthcare NHS Trust and St George’s University of London, UK; 3Universite Libre de Bruxelles, Brussels, Belgium l l (d ) P189 All patients underwent PiCCO monitoring via a femoral line whilst a radial line was kept in place during four 8-hour time periods (in the fi rst two periods, the Pulsiofl ex was connected to a radial line; in the last two it was connected to a femoral line). In the fi rst and third periods, the Pulsiofl ex was calibrated with TDCI, for the second and fourth periods Pulsiofl ex was calibrated with AutoCI. Simultaneous PiCCI and PuCCI measurements were obtained every 2  hours while simultaneous TDCI and AutoCI were obtained every 8 hours. We also looked at the eff ects of 40 interventions. p g Conclusion In unstable critically ill patients, MAP (and CO) can be monitored with the Nexfi n. The exact patient population for this Table 1 (abstract P189). Results of Bland and Altman analysis Pulsiofl ex AutoCal PE (CCl) (%) n R2 PE (TD-Pi) (%) n R2 PE (TD-Pu) (%) n R2 All All 37.9 940 0.73 22.8 382 0.88 38.5 382 0.66 All Yes 43.4 510 0.50 20.4 210 0.88 42.3 210 0.47 All No 27.8 430 0.83 25.6 172 0.85 32.5 172 0.74 Fem All 30.6 464 0.73 20.2 192 0.88 33.0 192 0.66 Rad All 44.2 476 0.58 25.2 190 0.85 43.7 190 0.56 Table 1 (abstract P189). Results of Bland and Altman analysis S72 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Figure 1 (abstract P192). Correlation between PaO2/FiO2 and EVLWIc. technology has yet to be defi ned and more patients are probably needed for pattern recognition, although the results indicate that low MAP and dp/dtmax are associated with poor outcome. In the future, Nexfi n data could theoretically be incorporated in a new outreach score. P191 Treatment-related cardiac complications associated with goal-directed therapy in high-risk surgical patients: a meta-analysis N Arulkumaran, C Corredor, M Hamilton, M Grounds, J Ball, A Rhodes, M Cecconi Treatment-related cardiac complications associated with goal-directed therapy in high-risk surgical patients: a meta-analysis N Arulkumaran, C Corredor, M Hamilton, M Grounds, J Ball, A Rhodes, M Cecconi St George’s Hospital, London, UK Critical Care 2013, 17(Suppl 2):P195 (doi: 10.1186/cc12133) Treatment-related cardiac complications associated with goal-directed therapy in high-risk surgical patients: a meta-analysis N Arulkumaran, C Corredor, M Hamilton, M Grounds, J Ball, A Rhodes, M Cecconi St George’s Hospital, London, UK Critical Care 2013, 17(Suppl 2):P195 (doi: 10.1186/cc12133) Introduction We hypothesized that goal-directed therapy (GDT) is not associated with an increased risk of cardiac complications in high-risk, noncardiac surgical patients. Patients with limited cardiopulmonary reserve are at risk of mortality and morbidity after major surgery [1]. Augmentation of the oxygen delivery index (DO2I) with a combination of intravenous fl uids and inotropes (GDT) has been shown to reduce the postoperative mortality and morbidity in high-risk patients [2]. However, concerns regarding cardiac complications associated with fl uid challenges and inotropes used to augment cardiac output may deter clinicians from instituting early GDT in the very patients who are more likely to benefi t. Results Twenty patients from the GDT group were compared with 20 control patients. Both groups were comparable concerning baseline characteristics and severity scores, except for a higher prevalence of hypertension and heart failure in the GDT group. Intraoperative data showed no diff erence regarding length of extracorporeal circulation, fl uid balance, transfusion or inotropic requirement. Patients from the GDT group were given more fl uids within the fi rst 8 hours as compared with the conventional group (1,250  ml vs. 500  ml, P  <0.001). GDT patients showed a median ICU stay of 3 days (95% CI: 3 to 4) compared with 5 days in control patients (95% CI: 4 to 7). Moreover, hospital stay was less prolonged in GDT patients (10 days vs. 14 days, P = 0.043). Inotropic dependence was lower in the GDT group (29  hours vs. 55 hours, P = 0.003) as well as the cumulative dobutamine dosage (8 vs. 19 μg/kg/day, P = 0.025). Also, GDT group presented a lower incidence of infections, tachyarrithmias and acute renal failure (RIFLE R) when compared with the control group. yi Methods Systematic search of MEDLINE, Embase and CENTRAL databases for randomized controlled trials of GDT in high-risk surgical patients. Studies including cardiac surgery, trauma, and pediatric surgery were excluded to minimize heterogeneity. References 1. Pearse RM, et al.: Lancet 2012, 380:1059-1065. 2. Hamilton MA, et al.: Anesth Analg 2011, 112:1392-1402. 2. Hamilton MA, et al.: Anesth Analg 2011, 112:1392-1402. Results In the group treated with a restrictive volume approach, patients received fl uids at the rate of 7.0 ± 1.0 ml/kg/hour. PaO2/FiO2 was 288 ± 14 after intubation and 270 ± 22 before extubation. In the group treated with a liberal volume approach, fl uids were replaced at 11.0 ± 2.0 ml/kg/hour. PaO2/FiO2 was 259 ± 24 after intubation and 223 ± 43 before extubation. Surgery combined with OLV was found to signifi cantly aff ect the PaO2/FiO2 value (ANOVA, F1,14 = 15.85a, P = 0.001, partial η2 = 0.531). The average PaO2/FiO2 level was signifi cantly higher in the restrictive-replacement group than in the liberal-replacement group (ANOVA, F1,14  =  9.66, P  =  0.008, partial η2  =  0.408). There was no interaction between the groups (ANOVA, F1,14  =  1.7a, P  =  0.215, partial η2 =0.108). Mean length of stay in the ICU was similar between the restrictive-replacement group (5.2  ±  2.3 days) and the liberal- replacement group (6.3 ± 1.6 days) (ANOVA, F1,14 = 0.814a, P = 0.382, partial η2 = 0.055). Perioperative volume management for esophageal cancer surgery M Karaman Ilic, G Madžarac, J Kogler, D Stančić Rokotov, N Hodoba University Hospital Centre Zagreb, Croatia Critical Care 2013, 17(Suppl 2):P194 (doi: 10.1186/cc12132) Perioperative volume management for esophageal cancer surgery M Karaman Ilic, G Madžarac, J Kogler, D Stančić Rokotov, N Hodoba University Hospital Centre Zagreb, Croatia Critical Care 2013, 17(Suppl 2):P194 (doi: 10.1186/cc12132) Introduction Pulmonary complications are the primary reason for extending a patient’s stay in the ICU. The aim of this study was to investigate whether perioperative volume management can infl uence the PaO2/FiO2 value and total length of stay in the ICU. Conclusion Perioperative, physiologically guided, GDT in high-risk surgical patients is not associated and actually reduces postoperative cardiovascular complications. 2 2 Methods Sixteen patients were divided into two groups: one group was treated with a restrictive approach (≤8 ml/kg/hour), and the other with a liberal approach (> 8  ml/kg/hour). Patients were randomly allocated using sealed envelopes. During the thoracic part of the surgical procedure, all patients received one-lung ventilation (OLV). P195 or extracardiac arteriopathy) were allocated to GDT or conventional hemodynamic therapy. We excluded patients with endocarditis, previous use of dobutamine, need for IABP, high dose of vasopressors and emergency surgery. The GDT protocol involved hemodynamic resuscitation aimed at a target of a cardiac index >3  l/minute/m2 through a three-step approach: fl uid therapy of 250 ml lactated Ringer’s solution, dobutamine infusion up to a dose of 20 μg/kg/minute, and red blood cell transfusion to reach a hematocrit level above 28%. EV1000/VolumeView: a new device for a more reliable measurement of extravascular lung water index in patients with lung resections g Critical Care 2013, 17(Suppl 2):P193 (doi: 10.1186/cc12131) g Critical Care 2013, 17(Suppl 2):P193 (doi: 10.1186/cc12131) Introduction A goal-directed resuscitation therapy (GDT) through optimization of cardiac output reduces complications in noncardiac surgeries. We investigated whether the implementation of a GDT protocol in high-risk cardiac surgery with the use of LiDCO Rapid reduces postoperative complications as compared with the standard of care. Introduction A goal-directed resuscitation therapy (GDT) through optimization of cardiac output reduces complications in noncardiac surgeries. We investigated whether the implementation of a GDT protocol in high-risk cardiac surgery with the use of LiDCO Rapid reduces postoperative complications as compared with the standard of care. A Donati, C Melia, V Monaldi, R Domizi, E Damiani, A Carsetti, C Scorcella, R Castagnani, P Pelaia Università Politecnica delle Marche, Ancona, Italy Critical Care 2013, 17(Suppl 2):P192 (doi: 10.1186/cc12130) Introduction Hemodynamic monitoring is important in high-risk surgical patients in order to detect and correct circulatory instability, thereby improving outcome [1]. The extravascular lung water index Methods We performed a prospective and randomized study whereby consecutive patients fulfi lling one high-risk criteria (EuroSCORE >5, ejection fraction <50%, recent myocardial infarction, unstable angina S73 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 1. Nisanevich V, Felsenstein I, Almogy G, Weissman C, Einav S, Matot I: Eff ect of intraoperative fl uid management on outcome after intraabdominal surgery. Anesthesiology 2005, 103:25-32.l 1. Nisanevich V, Felsenstein I, Almogy G, Weissman C, Einav S, Matot I: Eff ect of intraoperative fl uid management on outcome after intraabdominal surgery. Anesthesiology 2005, 103:25-32. 2. Wei S, Tian J, Song X, Chen Y: Association of perioperative fl uid balance and adverse surgical outcomes in esophageal cancer and esophagogastric junction cancer. Ann Thorac Surg 2008, 86:266-272. Treatment-related cardiac complications associated with goal-directed therapy in high-risk surgical patients: a meta-analysis N Arulkumaran, C Corredor, M Hamilton, M Grounds, J Ball, A Rhodes, M Cecconi We reviewed the rates of all cardiac complications, arrhythmias, acute myocardial ischemia, and acute pulmonary edema. Meta-analyses were performed and forest plots drawn using RevMan software. Data are presented as odd ratios (ORs) (95% CIs), and P values. Conclusion A hemodynamic therapy tailored to an optimized cardiac output reduced the length of ICU stay, vasoactive drug requirement and postoperative complications. Results We identifi ed 23 randomized controlled trials including 2,219 patients, who reported cardiac complications. GDT was associated with a reduction in total cardiovascular complications (OR = 0.55 (0.39 to 0.78), P  =  0.0007), and with a signifi cantly reduced incidence of arrhythmias (OR = 0.59 (0.38 to 0.91), P = 0.02). GDT was not associated with an increase in acute pulmonary edema (OR = 0.68 (0.42 to 1.10), P = 0.11) or acute myocardial ischemia (OR = 0.70 (0.38 to 1.27), P = 0.23). Subgroup analysis of overall cardiovascular complications revealed that the benefi t is most pronounced in patients receiving fl uid and inotrope therapy (OR = 0.55 (0.34 to 0.89), P = 0.01) to achieve a supranormal oxygen delivery target (OR = 0.50 (0.32 to 0.79), P = 0.003), guided by the use of minimally invasive cardiac outmonitoring (OR = 0.50 (0.33 to 0.77), P = 0.002). p p p Acknowledgement Grants received from FAPESP 2011/19987-5. Acknowledgement Grants received from FAPESP 2011/19987-5. Estimation of potential cost-savings related to the implementation of perioperative hemodynamic goal-directed therapy COVIG (T1) 0.31 (CI 0.24 to 1.63) –0.49 –3.41 to 2.43 COECHO vs. COMC (T1) 0.71 (CI 0.56 to 1.08) –0.055 –1.19 to 1.08 COECHO vs. COVIG (T2) 0.27 (CI 0.16 to 1.53) –0.49 –3.24 to 2.34 COECHO vs. COMC (T2) 0.79 (CI 0.66 to 1.107) –0.023 –0.94 to 0.9 CI, confi dence interval; LoA, limits of agreement. between 0.35 and 0.55. Importantly, these odd ratios were not related to the morbidity rates. In the US publication [2], extra costs for treating patients with 1+ complication were $17,949. In the Swiss (CH) publication [3], they were $34,446. See Table 1. y Conclusion Depending on the pre-implementation morbidity rate, the degree of pGDT-induced morbidity reduction and the country, cost-savings ranged between $808 and $13,434 per patient. This large variability suggests that local/hospital estimations are desirable before starting pGDT implementation. These tailored evaluations would also allow more precise cost-saving estimations by taking into account the actual and expected pGDT compliance rates. and compared with those calculated with the echocardiographic standard formulation (stroke volume  =  cross-sectional area×velocity time integral; COECHO  =  SV×heart rate). In every patient CO was measured twice: at baseline (T1) and after volume loading (500  ml lactate Ringer solution) (T2). Agreements between COVIG, COMC, and COECHO were evaluated by means of simple linear regression (r2) and Bland–Altman analysis. and compared with those calculated with the echocardiographic standard formulation (stroke volume  =  cross-sectional area×velocity time integral; COECHO  =  SV×heart rate). In every patient CO was measured twice: at baseline (T1) and after volume loading (500  ml lactate Ringer solution) (T2). Agreements between COVIG, COMC, and COECHO were evaluated by means of simple linear regression (r2) and Bland–Altman analysis. 1. Hamilton MA, et al.: A systematic review and meta-analysis on the use of preemptive hemodynamic intervention to improve postoperative outcomes in moderate and high-risk surgical patients. Anesth Analg 2011, 112:1392-1402. 2. Boltz et al.: Synergistic implications of multiple postoperative outcomes. Am J Med Qual 2012, in press. 3. Vonlanthen R, et al.: The impact of complications on costs of major surgical procedures. Ann Surg 2011, 254:907-913. 3. Vonlanthen R, et al.: The impact of complications on costs of major surgical procedures. Ann Surg 2011, 254:907-913. y Results Twenty patients were enrolled in the study. Values of r2, bias and limit of agreement at T1 and T2 are summarized in Table 1. Estimation of potential cost-savings related to the implementation of perioperative hemodynamic goal-directed therapy p p T Simon, G Marx RWTH University Hospital Aachen, Germany y y Critical Care 2013, 17(Suppl 2):P196 (doi: 10.1186/cc12134) Introduction Many studies have demonstrated the ability of peri- operative hemodynamic goal-directed therapy (pGDT) to decrease post operative morbidity in patients undergoing medium-to-high-risk surgery [1]. As a result, pGDT may be a cost-saving strategy. Our goal was to provide an estimation of potential cost-savings based on recent literature. Methods The largest and most recent meta-analysis [1] on pGDT was used to estimate what could be the reduction of postoperative morbidity if pGDT was to be adopted. Costs related to the treatment of patients developing at least one (1+) postoperative complication were obtained from two recent US [2] and Swiss [3] publications. Potential cost-savings related to the adoption of pGDT were calculated according to the actual morbidity rate, assuming 0% pGDT use so far, and 100% compliance rate after implementation. Conclusion Results from this small sample indicate that esophageal carcinoma surgery by itself had a detrimental eff ect on the PaO2/FiO2 value, which restriction of perioperative volume did not signifi cantly aff ect. Volume restriction also did not aff ect length of stay in the ICU. References 1. Nisanevich V, Felsenstein I, Almogy G, Weissman C, Einav S, Matot I: Eff ect of intraoperative fl uid management on outcome after intraabdominal surgery. Anesthesiology 2005, 103:25-32.l Results The 2011 meta-analysis [1] of 29 RCTs (4,805 patients) showed that pGDT is associated with a reduction in the rate of patients developing 1+ postoperative complications with odd ratios ranging 2. Wei S, Tian J, Song X, Chen Y: Association of perioperative fl uid balance and adverse surgical outcomes in esophageal cancer and esophagogastric junction cancer. Ann Thorac Surg 2008, 86:266-272. Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 S74 Actual morbidity rate no pGDT (%) 10 20 30 40 50 60 Expected morbidity rate pGDT (%) 3.5 to 5.5 7 to 11 11 to 17 14 to 22 18 to 28 21 to 33 Expected cost reduction/US patient ($) 808 to 1,167 1,615 to 2,333 2,423 to 3,500 3,231 to 4,667 4,039 to 5,833 4,846 to 7,000 Expected cost reduction/CH patient ($) 1,550 to 2,239 3,100 to 4,478 4,478 to 6,545 6,200 to 8,956 7,578 to 11,023 9,300 to 13,434 Table 1 (abstract P197) Pearson r2 Bias LoA COECHO vs. Cardiac output estimation: Vigileo and Mostcare versus echocardiography S Romagnoli, D Quattrone, AR De Gaudio S Romagnoli, D Quattrone, AR De Gaudio S Romagnoli, D Quattrone, AR De Gaudio Azienda Ospedaliero Universitaria Careggi, Florence, Italy Critical Care 2013, 17(Suppl 2):P197 (doi: 10.1186/cc12135) g Azienda Ospedaliero Universitaria Careggi, Florence, Italy y Critical Care 2013, 17(Suppl 2):P197 (doi: 10.1186/cc12135) Conclusion Vigileo did not prove to be a substitute to the reference system; pre-loaded data, necessary for vascular impedance estimation, may be one of the main limitations that made Vigileo measurements less accurate than the MostCare ones. On the contrary, MostCare, an uncalibrated totally independent system, was shown to properly estimate the vascular impedance in these hemodynamically stable patients. Further comparisons in unstable conditions are needed to confi rm our observations. Introduction In the present study we analyzed the reliability for cardiac output (CO) measurement of Vigileo (Edwards Lifescience, Irvine, CA, USA) and MostCare (pressure recording analytical method; Vygon-Vytech, Padova, Italy) in comparison with transthoracic Doppler echocardiography (as the reference method) in patients undergoing vascular surgery. Estimation of potential cost-savings related to the implementation of perioperative hemodynamic goal-directed therapy CO values ranged from 3.9 and 8.6 l/minute (echo), from 3.4 to 9.9 (Vigileo) and from 4 to 8.3 (MostCare); the Pearson’s and Bland–Altman methods showed poor agreement between COECHO and COVIG, demonstrating a tendency to overestimation (see Figure 1). The percentage of error (PE) was 51.7% at T1 and 49.3% at T2. On the contrary, MostCare measures showed good agreement with echocardiography (see Table 1) with a PE of 22.4% at T1 and of 17% at T2. Computerized decision support system improves consistency of haemodynamic assessment amongst ICU team members AE Aneman, R Ranganatha Liverpool Hospital, Liverpool, Australia Critical Care 2013, 17(Suppl 2):P199 (doi: 10.1186/cc12137) Computerized decision support system improves consistency of haemodynamic assessment amongst ICU team members AE Aneman, R Ranganatha Liverpool Hospital, Liverpool, Australia Critical Care 2013, 17(Suppl 2):P199 (doi: 10.1186/cc12137) Methods The in vitro set-up consisted of a centrifugal pump, a network of tubes with variable volumes, an electromagnetic fl owmeter to measure and adjust the generated fl ow, heating devices to maintain constant temperature (37°C), two thermistors for thermodilution measurement, an ultrasound transducer and a pressure stabilizer. A small bolus of UCA diluted in cold saline (1 mg SonoVue® in 20 ml saline at 4°C) was injected into the system. The cold UCA passage through a fi rst and a second region of interest (ROI) was measured simultaneously with the ultrasound transducer and the thermistors. The measurements were performed at diff erent fl ows and volumes. BVs were estimated using the two diff erent approaches, namely CEUS and thermodilution. The IDCs were processed and fi tted separately with a dedicated model to estimate the ΔMTT of the cold UCA bolus between the two ROIs and the two thermistors. All the measurements were repeated three times. Results A linear relation between BVs estimated by the two techniques was observed with a correlation coeffi cient of 0.94. The bias of CEUS with respect to the true volumes was –40.1  ml; the bias of thermodilution was 84.3 ml. The most prominent diff erences between the two techniques were observed in case of high volume and low fl ow, possibly due to diff erent transport kinetics between UCAs and heat. AE Aneman, R Ranganatha Liverpool Hospital, Liverpool, Australia p p p Critical Care 2013, 17(Suppl 2):P199 (doi: 10.1186/cc12137) Critical Care 2013, 17(Suppl 2):P199 (doi: 10.1186/cc12137) Introduction This study evaluated the potential of the NAVIGATOR (Applied Physiology) haemodynamic monitor to improve consistency of cardiovascular assessments amongst ICU members with diff erent levels of expertise and experience. Methods Patients (n  =  20) post cardiac surgery were monitored by NAVIGATOR to display heart effi ciency (Eh), mean systemic fi lling pressure (Pms) and vascular resistance (SVR) against targets for mean arterial pressure and cardiac output set by the clinical team. i References y Methods Both Vigileo and MostCare were connected to the FloTrac transducer (Edwards Lifescience) for CO calculation. The data from Vigileo and MostCare were registered (COVIG and COMC respectively) . Romano SM, et al.: Crit Care 2002, 30:1834-1841. . Romano SM, et al.: Crit Care 2002, 30:1834-1841. 2. Geisen M, et al.: Curr Opin Crit Care 2012, 18:377-384. Figure 1 (abstract P197). Bland–Altman analysis (T1). Figure 1 (abstract P197). Bland–Altman analysis (T1). Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 S75 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 P198 Agreement (maximum two steps deviation for each assessment between staff ; number of patients), disagreement (median and interquartile range of steps) and therapeutic agreement (intervention/s to change Eh, Pms and SVR in similar direction/s, number of patients) were recorded and analysed for statistical diff erence BLIND versus OPEN (Fisher’s exact test, Mann–Whitney test, P <0.05). P198 Brachial pulse waveform characteristics predict development of organ failure in septic patients S Kazune, I Jagmane Hospital of Traumatology and Orthopaedics, Riga, Latvia Critical Care 2013, 17(Suppl 2):P198 (doi: 10.1186/cc12136) y Results Eh was commonly overestimated, Pms commonly under- estimated with no clear trend for SVR. Agreement amongst all categories of staff increased and disagreement score decreased for Eh, Pms and SVR (Table 1) comparing BLIND versus OPEN assessments. Agreement for therapeutic interventions also increased signifi cantly from 4/20 (BLIND) to 18/20 patients (OPEN). Introduction Pulse waveform characteristics (pulse wave transit time and augmentation index) are measures of arterial stiff ness. Previous studies have found an association between severity of acute infl ammatory states and increased arterial stiff ness but it is not known whether non-invasive pulse waveform analysis could predict development of multiple organ failure in septic patients. The purpose of this study was to evaluate the photoplethysmographic brachial artery pulse wave transit time and augmentation index and their changes in response to induced forearm ischemia in septic ICU patients and correlate these indices to the development of subsequent end organ damage. Table 1 (abstract P199) BLIND OPEN Eh 6/20, 2 (1 to 3) 17/20*, 1 (0 to 1)* Pms 6/20, 2.5 (2 to 4) 17/20*, 2 (1 to 2)* SVR 8/20, 2 (1 to 2) 16/20*, 1 (1 to 2)* Patients (n/20) and score (median and IQR). *P <0.05. Methods A prospective observational study in patients with sepsis within 24  hours of admission. i References Severity of sepsis was assessed with APACHE II score (median 18.5) and SOFA score (median 7.5). Three- minute signal recording was done concurrently from the brachial artery at the elbow and the radial artery at the wrist with an originally designed photoplethysmograph at rest and after 5 minutes of induced forearm ischemia. Recordings were analyzed to obtain the pulse wave transit time and augmentation index at rest and 60  seconds after induced ischemia. The SOFA score was recalculated at 48 hours post recording. Patients (n/20) and score (median and IQR). *P <0.05. Conclusion The assessment of heart function, intravascular fi lling and resistance state in postoperative cardiac patients was made signifi cantly more consistent amongst diff erent ICU staff members using the NAVIGATOR haemodynamic monitor. Such enhanced consistency could potentially make the haemodynamic management more eff ective with improved clinical outcomes. Results We studied 14 consecutive general ICU patients. There was a negative linear relationship between the pulse wave transit time (median 22.6  ms) at rest and increase in SOFA score in 48  hours (P = 0.02, r = 0.96). The postischemic pulse wave transit time increased in all patients (median 25.7  ms) but no association was found between the proportion of increase and subsequent change in SOFA. Correlation between rest (median 7.6) and postischemic (median 7.2) augmentation index and 48-hour SOFA scores was not statistically signifi cant (r = 0.57, P = 0.46). Reference Reference 1. Vlachopoulos C, Dima I, Aznaouridis K, Vasiliadou C, Ioakeimidis N, Aggeli C, Toutouza M, Stefanadis C: Acute systemic infl ammation increases arterial stiff ness and decreases wave refl ections in healthy individuals. Circulation 2005, 112:2193-2200. Volume quantifi cation by contrast-enhanced ultrasound and thermodilution: an in vitro comparison gi Conclusion This study indicates that in early sepsis pulse waveform characteristics could predict the risk of developing end organ failure. The pulse wave transit time is more robust than the augmentation index and could be easier to use in patients with poor perfusion. Vascular reactivity indices do not seem to have predictive value in this context. Introduction In clinical practice, blood volumes (BV) are typically measured by thermodilution. Recently, contrast-enhanced ultrasound (CEUS) has been proposed as an alternative minimally invasive approach for BV assessment [1]. This method measures BV using a single peripheral injection of a small bolus of ultrasound contrast agent (UCA) detected by an ultrasound scanner. By measuring the acoustic backscatter, two indicator dilution curves (IDCs) can be derived from two diff erent sites in the circulatory system. IDC analysis permits deriving the mean transit time (MTT) the bolus takes to travel between the injection site and two measurement sites. Assessment of the BV between these sites is obtained by multiplying the diff erence in MTT (ΔMTT) by the blood fl ow. In this study, we compare diff erent volumes in an in vitro set-up by CEUS with true set-up volumes and thermodilution acquired volumes. Introduction In clinical practice, blood volumes (BV) are typically measured by thermodilution. Recently, contrast-enhanced ultrasound (CEUS) has been proposed as an alternative minimally invasive approach for BV assessment [1]. This method measures BV using a single peripheral injection of a small bolus of ultrasound contrast agent (UCA) detected by an ultrasound scanner. By measuring the acoustic backscatter, two indicator dilution curves (IDCs) can be derived from two diff erent sites in the circulatory system. IDC analysis permits deriving the mean transit time (MTT) the bolus takes to travel between the injection site and two measurement sites. Assessment of the BV between these sites is obtained by multiplying the diff erence in MTT (ΔMTT) by the blood fl ow. In this study, we compare diff erent volumes in an in vitro set-up by CEUS with true set-up volumes and thermodilution acquired volumes. Fluid responsiveness in pressure support ventilation: role of asynchrony A Messina, D Colombo, G Cammarota, M De Lucia, F Della Corte, P Navalesi Università Maggiore della Carità A. Avogadro, Novara, Italy Critical Care 2013, 17(Suppl 2):P202 (doi: 10.1186/cc12140) Introduction Pulse pressure variation (PPV) is a dynamic indicator of fl uid responsiveness, which is known to have a low sensibility and specifi city in patients ventilated in pressure support (PS) [1]. We aim to investigate patient–ventilator asynchrony as a potential source of hemodynamic interference in PS. Methods Institutional ethical approval was obtained. Intubated major trauma patients were sequentially enrolled. Exclusions included major thoracic burns and children. Bioreactance fl ow monitoring (NICOM; Cheetah) was applied at the same time as ECG leads and the calibration step performed during handover from the prehospital team. Time to availability of oxygen delivery data was recorded and trauma team members surveyed regarding for perceived benefi ts and concerns from this monitoring. The infl uence of fl ow monitoring on fl uid resuscitation, time to CT and defi nitive disposal (to OR/ICU) was measured and compared with a control population matched for injury severity score, age and sex. y Methods We performed a prospective study including PS ventilated patients who met inclusion criteria for fl uid depletion [1]. Patients who showed an asynchrony index (AI) exceeding 10% were included in the asynchrony group (AG). The remaining patients were included in the synchrony group (SG) [2]. Beat-to-beat hemodynamic variables were recorded through PRAM (Mostcare; Vytech Health srl, Padova, Italy). PPV cutoff of 13% was used to identify fl uid responders/nonresponders. A fl uid challenge of 500  ml normal saline was given in 5  minutes. An increase of 15% of cardiac index after 10 minutes indicated fl uid responsiveness. Results Cardiac index was available at mean 10.6  minutes (median 9 minutes; SD 3.9), fl uid responsiveness at mean 35.9 minutes (median 35; SD 11.3) and oxygen delivery calculation at mean 25.3 minutes (median 25; SD 7.7). Passive leg raise was not performed in 63% of patients due to concerns about pelvic or brain injury. Volume of fl uid infused (mean 738 vs. 925 ml; P = 0.124), time to CT (mean 57.4 vs. 68.8 minutes; P = 0.08), and time to defi nitive disposal (mean 124.9 vs. 146.1 minute; P = 0.069) were all reduced in the fl ow monitored group, although not signifi cantly diff erent when compared with a matched Results So far, eights patients showed an AI >10% while 16 did not. Overall sensitivity was 28.6% versus 50% in SG; overall specifi city was 76.5% versus 91.7% in AG. Computerized decision support system improves consistency of haemodynamic assessment amongst ICU team members AE Aneman, R Ranganatha Liverpool Hospital, Liverpool, Australia Critical Care 2013, 17(Suppl 2):P199 (doi: 10.1186/cc12137) Four categories of staff participated: nine consultants (C), eight senior registrars (SR), nine registrars (R) and 11 nurses (N) (median ICU experience 15, 7, 2 and 10  years, respectively) and were asked to score Eh, Pms, and SVR ranging (discrete steps of one) from –5 (grossly subnormal) to 0 (normal) to 5 (grossly supranormal) fi rst without (BLIND) and then given (OPEN) access to the NAVIGATOR display. Recommendations for therapeutic interventions were noted. S76 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 control group (Mann–Whitney U rank sum). Eighty-four percent of trauma team members surveyed felt the fl ow monitoring data to be useful, and only 11% felt it may impair clinical management.l Conclusion Given the good correlation between BVs estimated with CEUS and thermodilution, CEUS is not inferior to thermodilution with the advantage of being minimally invasive. Reference Conclusion Cardiac index, fl uid responsiveness and oxygen delivery data can be obtained inform a primary survey. Rather than introducing delays, the use of fl ow monitoring was associated with a trend towards decreased time to imaging; less fl uid use pre-damage control point and reduced time to defi nitive disposal. Further research is required to confi rm benefi ts and mechanism. 1. Mischi M, et al.: IEEE Trans UFFC 2004, 51:1137-1141. 1. Mischi M, et al.: IEEE Trans UFFC 2004, 51:1137-1141. P202 Fluid responsiveness in pressure support ventilation: role of asynchrony A Messina, D Colombo, G Cammarota, M De Lucia, F Della Corte, P Navalesi and disposal y , , 3. Dunham M, et al.: J Trauma Acute Care Surg 2012, 73:479-485. y 3. Dunham M, et al.: J Trauma Acute Care Surg 2012, 73:479-485. King’s Health Partners AHSC, London, UK Critical Care 2013, 17(Suppl 2):P201 (doi: 10.1186/cc12139) Fluid responsiveness in pressure support ventilation: role of asynchrony A Messina, D Colombo, G Cammarota, M De Lucia, F Della Corte, P Navalesi Fluid responsiveness in pressure support ventilation: role of asynchrony A Messina, D Colombo, G Cammarota, M De Lucia, F Della Corte, P Navalesi Università Maggiore della Carità A. Avogadro, Novara, Italy Critical Care 2013, 17(Suppl 2):P202 (doi: 10.1186/cc12140) Introduction The use of cardiac output monitoring has been shown to be benefi cial in the setting of perioperative medicine and critical illness [1,2]. More recently, its application in the setting of major trauma has been described [3]. Here, we describe our preliminary experience of embedding bioreactance fl ow monitoring within the major trauma primary survey of severely injured patients and the subsequent eff ect on patient management. P203 Comparison of pulse pressure variation with radial arterial systolic, diastolic and pulse transit time interval variation in pediatric patients undergoing liver transplantation YJ Moon, IG Jun, WJ Shin, BH Sang, GS Hwang Asan Medical Center, Seoul, South Korea Critical Care 2013, 17(Suppl 2):P203 (doi: 10.1186/cc12141) P203 Comparison of pulse pressure variation with radial arterial systolic, diastolic and pulse transit time interval variation in pediatric patients undergoing liver transplantation YJ Moon, IG Jun, WJ Shin, BH Sang, GS Hwang Asan Medical Center, Seoul, South Korea Critical Care 2013, 17(Suppl 2):P203 (doi: 10.1186/cc12141) l g p Methods We measured the eff ects after the administration of 50, 100 and 500 ml bolus colloid infusions on CO (Modelfl ow (COm) and LiDCO (COli)), CVP and MAP in 21 patients on mechanical ventilation after elective cardiothoracic surgery. From the data we analysed the smallest volume that was predictive for the eff ects of 500 ml on cardiac output. Results COli and COm increased after 50, 100 and 500 ml fl uid loading. Best results are observed for changes in COm after 100 ml fl uid loading (area under the ROC 0.86, 95% CI between 0.65 and 1.00). A change in Modelfl ow CO of at least 4.3% has a sensitivity of 67% and a specifi city of 100% after 100 ml fl uid loading. Sensitivity is 60% and specifi city 83% for a similar cutoff in CO measured with the LiDCO device after 100 ml fl uid loading. In our patient population, MAP and COli did not predict responsiveness with more accuracy than mathematical chance. See Figure 1.l Introduction In pediatric patients, dynamic preload indices to predict fl uid responsiveness have confl icting results in comparison with adults. A recent study demonstrated that pulse pressure variation (PPV) ≥16% has provided an accurate method for predicting fl uid responsiveness in pediatric congenital heart surgery. We aimed to compare PPV and respiratory systolic, diastolic and pulse transit time interval variation (STV, DTV and PTTV, respectively) as predictors of fl uid responsiveness during pediatric liver transplantation. g p p Methods A total of 61 data from 16 pediatric patients, median age 5.4 years (range 0.1 to 9 years), were retrospectively evaluated from electrically recorded radial arterial and central venous pressure (CVP) waveform. The time from the onset of systolic upstroke to the dicrotic notch was defi ned as the systolic time interval (STI), and the time from dicrotic notch to the beginning of systolic upstroke was defi ned as the diastolic time interval (DTI). The time from peak R wave on electrocardiography to the onset of systolic upstroke was defi ned as the pulse transit time (PTT) interval. P203 Comparison of pulse pressure variation with radial arterial systolic, diastolic and pulse transit time interval variation in pediatric patients undergoing liver transplantation YJ Moon, IG Jun, WJ Shin, BH Sang, GS Hwang Asan Medical Center, Seoul, South Korea Critical Care 2013, 17(Suppl 2):P203 (doi: 10.1186/cc12141) STV was calculated by averaging of three consecutive respiratory cycles with the following: (STImaximum – STIminimum) / STImean. The same method was used for calculating DTV, PTTV and PPV. STV, DTV and PTTV were corrected by cardiac period. Averaged CVP was used as a static preload index. PPV threshold ≥16% was used to discriminate fl uid responsiveness. Receiver operating characteristic (ROC) curves and Pearson’s correlation analysis were used for the comparison. Conclusion Changes in pulse contour CO can be used in a mini-fl uid challenge to assess fl uid responsiveness in our postcardiac surgery patients. Effi cacy of intraoperatory optimisation of fl uids guided with transoesophageal Doppler monitorisation: a multicentre randomised controlled trial S Maeso1, R Villalba2, J Ripollés3, S Asuero2, J Blasco1, J Calvo3 1Agencia Lain Entralgo, Madrid, Spain; 2Hospital Universitario Ramón y Cajal, Madrid, Spain; 3Hospital Infanta Leonor, Madrid, Spain Critical Care 2013, 17(Suppl 2):P205 (doi: 10.1186/cc1243) Fluid responsiveness in pressure support ventilation: role of asynchrony A Messina, D Colombo, G Cammarota, M De Lucia, F Della Corte, P Navalesi Università Maggiore della Carità A. Avogadro, Novara, Italy Critical Care 2013, 17(Suppl 2):P202 (doi: 10.1186/cc12140) Overall Cohen’s k was 33.3% versus 61.2% in AG (see Figure 1). However, because none of the responders in the AG group was detected by PPV, statistical analysis was not feasible within this subgroup. Figure 1 (abstract P202). Figure 1 (abstract P202). S77 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Conclusion The consistency of PPV in predicting fl uid responsiveness during PS seems to be more reliable in the patients with better patient– ventilator synchrony. References References 1. Heenen S, et al.: Crit Care 2006, 10:R102. 2. Vitacca M, et al.: Chest 2004, 126:851-859. Introduction The mini-fl uid challenge is a widely used strategy to manage fl uid loading in the ICU and OR. Although it might be a rational strategy, data on the mini-fl uid challenge and its reliability are very limited. We investigated the value of changes in pulse contour cardiac output as a result of a mini-fl uid challenge of 50 and 100 ml to predict fl uid loading responsiveness. Mini-fl uid challenge: how much fl uid and what parameter to use? B Geerts, RB De Wilde, JJ Maas, LP Aarts, JR Jansen Leiden University Medical Centre, Leiden, the Netherlands Critical Care 2013, 17(Suppl 2):P204 (doi: 10.1186/cc12142) Mini-fl uid challenge: how much fl uid and what parameter to use? B Geerts, RB De Wilde, JJ Maas, LP Aarts, JR Jansen Leiden University Medical Centre, Leiden, the Netherlands Critical Care 2013, 17(Suppl 2):P204 (doi: 10.1186/cc12142) y y References 1. Heenen S, et al.: Crit Care 2006, 10:R102. 2. Vitacca M, et al.: Chest 2004, 126:851-859. Effi cacy of intraoperatory optimisation of fl uids guided with transoesophageal Doppler monitorisation: a multicentre randomised controlled trial S Maeso1, R Villalba2, J Ripollés3, S Asuero2, J Blasco1, J Calvo3 1Agencia Lain Entralgo, Madrid, Spain; 2Hospital Universitario Ramón y Cajal, Madrid, Spain; 3Hospital Infanta Leonor, Madrid, Spain Critical Care 2013, 17(Suppl 2):P205 (doi: 10.1186/cc1243) Results PPV showed correlations with STV, DTV and PTTV (r  =  0.65, 0.57 and 0.60, respectively), but less with CVP (r = –0.30). Area under ROC curves (AUC) of STV, DTV, PTTV and CVP were 0.834, 0.872, 0.832 and 0.613, respectively. Cutoff values of STV, DTV, PTTV and CVP were 7.7% (sensitivity/specifi city, 0.80/0.83), 7.7% (sensitivity/specifi city, 0.70/0.88), 8.7% (sensitivity/specifi city 0.67/1.0) and 3.1% (sensitivity/ specifi city 0.50/0.85), respectively. Introduction The objective is to compare stay in surgery monitored with oesophageal Doppler with unmonitored. Introduction The objective is to compare stay in surgery monitored with oesophageal Doppler with unmonitored. g Methods A randomized trial. We present preliminary results obtained in the fi rst 55 cases. The surgeries were general and urological. ISRCTN93543537. Results There were no diff erences in any of the baseline variables. A total of 69.1% were men. The mean age was 65.80 years. There were 63.6% general surgery and 36.4% urologic surgery. There were 81.5% open surgeries and 18.5% laparoscopic. The results were favorable to the intervention group for most outcomes; these diff erences did not i y y Conclusion This study shows that STV, DTV and PTTV can be used as a surrogate for PPV ≥16%, suggesting that this novel method can be used to predict hemodynamic response during pediatric surgery. Reference 1. Renner J, et al.: Br J Anaesth 2012, 108:108-115. Figure 1 (abstract P204). Cardiac function curve: a fl uid challenge of 50 ml in a (non)responder. gure 1 (abstract P204). Cardiac function curve: a fl uid challenge of 50 ml in a (non)responder. Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 S78 Table 1 (abstract P205). Analysis of the outcome variables ODM Control Outcome Units (n = 32) (n = 23) P value Surgery time Minutes 206.20 225.59 0.532 Total stay Days 13.92 18.19 0.292 Postsurgery Days 10.93 14.36 0.280 Time to enteral Days 2.73 3.13 0.567 Time to deambulation Days 2.52 3.00 0.359 Complications % 34.4 21.7 0.377 Table 1 (abstract P205). Effi cacy of intraoperatory optimisation of fl uids guided with transoesophageal Doppler monitorisation: a multicentre randomised controlled trial Analysis of the outcome variables P207 Prediction of stroke volume response to fl uid bolus in 100 children R Saxena, A Durward, I Murdoch, S Tibby Guy’s & St Thomas’ NHS Trust, London, UK Critical Care 2013, 17(Suppl 2):P207 (doi: 10.1186/cc12145) Figure 1 (abstract P206). Figure 1 (abstract P206). reach statistical signifi cance (Table 1). We emphasize the diff erence in postoperative stay of 3.4 days. Only complications results were against the intervention. Conclusion The preliminary results obtained for the postsurgical length of stay, as for most of the outcomes, were favorable to monitoring by oesophageal Doppler. P206 P206 Looking for the infl exion point of the Frank–Starling curve HD Aya, M Geisen, C Ebm, N Fletcher, M Grounds, A Rhodes, M Cecconi St George’s Healthcare NHS Trust, London, UK Critical Care 2013, 17(Suppl 2):P206 (doi: 10.1186/cc12144) Introduction Fluid overload is associated with poor outcome in the critically ill. Thus, an accurate predictor of a positive haemodynamic response (increase in stroke volume) to fl uid challenge is vital.i l Methods We studied the predictive value (positive response defi ned as change in stroke volume >15% after 10 ml/kg fl uid bolus) of a range of haemodynamic variables: static (CVP, active circulating volume, central blood volume, total end diastolic volume), dynamic (systolic pressure variation, stroke volume variation) and contactility (dp/dt), in a group of 100 ventilated children (median weight 10 kg). Variables were measured using transpulmonary ultrasound dilution and PRAM (an arterial pulse contour method). Introduction Fluid responsiveness is defi ned based on an arbitrary increase of cardiac output (CO) or stroke volume (SV) of 10 to 15%. We hypothesise that the variation of heart effi ciency (Eh) and the slope (S) defi ned by the relative increase of CO over the relative increase of mean fi lling pressure (Pmsa) can be used as alternative defi nitions of fl uid responsiveness. l p Methods Patients admitted to the ICU were monitored with a calibrated LiDCOplus (LiDCO, UK) and Navigator (Applied Physiology, Australia) to estimate Pmsa and Eh (Pmsa – central venous pressure/Pmsa). A 250 ml fl uid challenge was performed over 5 minutes. Categorical data were compared by Pearson chi-square test. Correlation was assessed by Kappa test. The infl exion point of S to defi ne responders was obtained by ROC curve analysis. p Results We performed 168 paired measurements (pre-fl uid and post- fl uid challenge), with a SV response rate of 45%. Overall predictive values were poor, but slightly better for static versus dynamic variables (Table 1). When SV response was analysed as a continuous variable, the two predictive multivariable variables were change in TEDVI and baseline dp/dt (r2 = 0.30, both P <0.001). Table 1 (abstract P207). ROC areas for haemodynamic variables AUC 95% CI CVP 0.50 0.41 0.59 ACVI 0.62 0.54 0.71 CBVI 0.59 0.50 0.68 TEDVI 0.65 0.56 0.73 SPV 0.59 0.50 0.69 SVV 0.48 0.39 0.58 dp/dt 0.59 0.50 0.68 Table 1 (abstract P207). ROC areas for haemodynamic variables Table 1 (abstract P207). P207 d P207 Prediction of stroke volume response to fl uid bolus in 100 children R Saxena, A Durward, I Murdoch, S Tibby Guy’s & St Thomas’ NHS Trust, London, UK Critical Care 2013, 17(Suppl 2):P207 (doi: 10.1186/cc12145) P206 ROC areas for haemodynamic variables AUC CI y y Results A total of 104 fl uid challenges were observed in 40 patients. ROC curve analysis reveals an area under the curve of 0.93 (95% CI = 0.85 to 1, P <0.001). The best cutoff for the slope was 0.76 (sensitivity 0.92, specifi city 0.93). The proportions of responders identifi ed by the ΔEh (Table 1) and by the slope method (Table 2) are smaller compared with the relative increase of SV method. Signifi cant correlation was found between both methods and the ΔSV (ΔEh κ = 0.54, P <0.001; S κ = 0.55, P <0.001). See Figure 1. g Conclusion Moderate agreement is observed between new and current defi nitions of fl uid responsiveness. 1. Michard F, et al.: Chest 2002, 121:2000. Table 1 (abstract P206). Distribution of events according to ΔSV and ΔEh Response by ΔSV ≥10% Response by ΔEh ≥0 P value Nonresponder 62 (59.6%) 76 (73.1%) <0.001 Responder 42 (40.4%) 28 (26.9%) <0.001 Table 2 (abstract P206). Distribution of events according to ΔSV and the slope (S) Response by ΔSV ≥10% Response by S ≥0.76 P value Nonresponder 62 (59.6%) 75 (72.1%) <0.001 Responder 42 (40.4%) 29 (27.9%) <0.001 Table 1 (abstract P206). Distribution of events according to ΔSV and ΔEh Conclusion The predictive ability for typical static and dynamic haemodynamic variables, when taken in isolation, is poor. However, improved prediction is seen when baseline contractility is taken into account. SP De Wolf, J Stens, RJ Van der Zwan, N Koning, C Boer VU University Medical Centre, Amsterdam, the Netherlands Critical Care 2013, 17(Suppl 2):P208 (doi: 10.1186/cc12146) Introduction This study compared the eff ect of pulse pressure variation (PPV) and cardiac index (CI)-guided fl uid therapy versus mean arterial S79 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Conclusion SDF imaging is a feasible, non-invasive bedside method to study the postoperative intestinal microcirculation. The established reference ranges are useful for early detection of postoperative local complications and studies of microcirculatory changes induced by systemic pathologies; for example, in sepsis. pressure (MAP)-guided fl uid therapy on microcirculatory perfusion in patients undergoing abdominal surgery. g g g y Methods Patients undergoing elective abdominal surgery were randomized into a PPV/CI-guided group (n  =  11) or a MAP-guided (n = 12) group. P206 PPV, CI and MAP were measured using the non-invasive fi nger arterial blood pressure measurement device ccNexfi n (Edwards Lifesciences BMEYE, Amsterdam, the Netherlands). Tidal volumes were ≥8  ml/kg with PEEP ≥8  mmHg. In both groups, MAP of 70  mmHg was maintained. In the PPV/CI group, an intraoperative algorithm was used keeping the PPV under 12% and CI above 2.5  l/minute/ m2 using fl uid therapy and dobutamine and noradrenaline infusion, respectively. Sublingual microvascular perfusion was measured after anesthesia induction, and every subsequent hour using sidestream dark-fi eld imaging (Microscan; Microvision Medical, Amsterdam, the Netherlands). The perfused small vessel density (PVD) values were offl ine quantifi ed.i P210 P210 Monitoring microcirculatory blood fl ow with a new sublingual tonometer in a porcine model of haemorrhagic shock P Palagyi, J Kaszaki, Z Molnar University of Szeged, Hungary Critical Care 2013, 17(Suppl 2):P210 (doi: 10.1186/cc12148) Introduction Tissue capnometry has been used to assess organ perfusion but it is not generally available at the bedside [1,2]. Our aim was to test a new sublingual capillary tonometer in haemorrhagic shock. fl qi Results The fi rst hour during surgery, the PPV/CI-guided group tended to receive more fl uids than the MAP-guided group (1,014 ± 501ml vs. 629 ± 463 ml; P = 0.07). At this time point, the PVD was slightly lower in the PPV/CI-guided group (16.7 ± 3.1 mm/mm2) when compared with the MAP-guided group (17.9 ± 3.9 mm/mm2; P = 0.41). In both groups the PVD remained stable during the fi rst 2 hours of surgery. However, 2 hours after the start of surgery, the PVD in the PPV/CI group restored and tended to be higher than in the MAP-guided group (21.1 ± 1.9 vs. 18.1 ± 3.4 mm/mm2; P = 0.09). After 1 hour of surgery, the administered fl uid volume correlated inversely with PVD (r = –0.59, P = 0.011).l Methods Thirty-six mini-pigs were anaesthetised, ventilated and divided into sham operated and shock groups. Instrumentation included: intestinal and sublingual tonometry, haemodynamic monitoring (PiCCO; Pulsion, Germany) and orthogonal polarization spectroscopy (OPS). After baseline measurements (T0) haemorrhagic shock was induced and maintained by reducing mean arterial pressure (MAP) to ~40  mmHg for 60  minutes. Measurements were repeated every 30  minutes (T1 to T6). Fluid resuscitation started after T2 aiming to increase MAP to 75% of the baseline value. OPS imaging was performed at T0, T2 and T6. P211 Methods For this observational prospective cohort study 77 patients were screened. In total, 165 SDF measurements could be obtained. All patients included had bowel surgery for chronic infl ammatory bowel disease or intestinal malignancies and received an ileostomy. Patients were excluded if they had signs of local infection or bleeding, or if they were admitted to the ICU for sepsis or perioperative complications. The SDF device was gently inserted into the stoma at a depth of 3 to 4 cm, and fi ve real-time images were recorded. All videos recorded were analyzed offl ine using AVA® software (Microvision Medical, Amsterdam, the Netherlands). The following parameters were quantifi ed: microvascular fl ow index (MFI), total vessel density (TVD), perfused vessel density (PVD), and proportion of perfused vessels (PPV). Patients were followed for 3 days post surgery with fi ve images captured every day. References 1. Creteur et al.: Curr Opin Crit Care 2006, 12:272-277. 2. Cammarata et al.: Shock 2009, 31:207-211. 1. Creteur et al.: Curr Opin Crit Care 2006, 12:272-277. 2. Cammarata et al.: Shock 2009, 31:207-211. Postoperative imaging of the intestinal microcirculation I Abd 1 R H ll1 D H l 1 V C 2 CH L h 1 Abdo1, R Hall1, D Henzler1, V Cerny2, CH Lehmann1 1Dalhousie University, Halifax, Canada; 2Charles University, Medical Faculty in Hradec Kralove, Czech Republic Conclusion In this model of haemorrhagic shock, sublingual PCO2 gap showed good correlation with RBCV, suggesting that this new sublingual capillary tonometer may be an appropriate tool for monitoring microcirculation at the bedside. f Introduction Sidestream dark-fi eld (SDF) imaging was introduced recently to study the sublingual microcirculation in humans. Patients with ileostomies off er a unique access to the intestinal microcirculation. To date, no reference ranges for standard microcirculatory parameters of the gut are available. Therefore, the aim of our study was to establish a database for postoperative microcirculatory parameters for ileostomies. P209 Postoperative imaging of the intestinal microcirculation I Abdo1, R Hall1, D Henzler1, V Cerny2, CH Lehmann1 1Dalhousie University, Halifax, Canada; 2Charles University, Medical Faculty in Hradec Kralove, Czech Republic Critical Care 2013, 17(Suppl 2):P209 (doi: 10.1186/cc12147) P206 Data are presented as the median (interquartile range), for statistical analysis Friedmann ANOVA, Mann–Whitney and Spearman tests were used as appropriate. l y Conclusion Goal-directed fl uid management resulted in a higher administered fl uid volume in the beginning of surgery, and this was associated with a slightly reduced microcirculatory perfusion when compared with MAP-guided fl uid management. Microcirculatory perfusion tended to improve as surgery progressed in the goal-directed fl uid therapy group. Our fi ndings suggest that goal-directed and MAP- guided fl uid management are associated with distinct patterns in fl uid resuscitation, which may be of consequence for microvascular perfusion. p pp p Results Bleeding resulted in a signifi cant decrease in MAP and cardiac index, and an increase in heart rate. Macrohaemodynamic changes were accompanied by signifi cant changes in red blood cell velocity (RBCV) and a signifi cant increase in the intestinal and sublingual mucosal-to-arterial carbon dioxide partial pressure diff erence (PCO2 gap): from 4 (2 to 11) to 30 (23 to 37) mmHg in the sublingual, and from 25 (17 to 31) to 50 (33 to 64) mmHg in the ileum. RBCV decreased from 1,075 (945 to 1,139) to 520 (449 to 621) μm/second in the sublingual area, and from 646 (596 to 712) to 419 (350 to 451) μm/second in the ileum. There was signifi cant correlation between RBCV and PCO2 gap in sublingual and intestinal regions alike: r = –0.58; r = –0.71, P <0.0001, respectively. Sublingual microcirculatory changes during transient intra-abdominal hypertension: a study in laparoscopic surgery patients Introduction Microvascular alterations play an important role in development of organ failure [1]. It is not known whether increased intra-abdominal pressure (IAP) is associated with microcirculatory perfusion derangements. Our hypothesis was that transiently increased IAP is related to microcirculatory alterations in laparoscopic cholecystectomy patients. Introduction Microvascular alterations play an important role in development of organ failure [1]. It is not known whether increased intra-abdominal pressure (IAP) is associated with microcirculatory perfusion derangements. Our hypothesis was that transiently increased IAP is related to microcirculatory alterations in laparoscopic cholecystectomy patients. Results We were able to capture clear images of the small intestine microvasculature. Distinct villi were visible with a dense network of microvessels (diameter: 6 to 17 μm). Mean TVD (± 2SD) was 19.3 (±  1.0)  mm/mm2, PVD: 18.5 (±  1.1)  mm/mm2, PPV: 94.5 (±  5)% and MFI: 2.8 (± 0.1). Patients’ age, sex and comorbidity had no signifi cant impact on postoperative microvascular parameters. No signifi cant changes of the microvascular parameters were observed during the fi rst 3 postoperative days. Methods Sixteen patients (14 female, two male) who underwent laparoscopic cholecystectomy were studied. Sublingual orthogonal polarization spectral (OPS) imaging was used to detect microcirculatory function. OPS was done before surgery, at least 15  minutes after initiation of pneumoperitoneum and 1 hour after the end of pneumo- peritoneum. The microcirculation cutoff value for vessels was 20 μm. Data are presented as medians with interquartile ranges. S80 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Results Patient median age was 54 (39 to 63) years, ASA score 2 (2 to 3), BMI 29.7 (24.9 to 34.7), haemoglobin concentration 138 (133 to 142) g/l, and hematocrit 42 (39 to 43). IAP was held at 12.5 (12 to 13) mmHg, median duration of pneumoperitoneum was 45 (24 to 55)  minutes. Median MAP was 86 (69 to 93), abdominal perfusion pressure (APP) 73 (57 to 81)  mmHg during the pneumoperitoneum. Median fl uid administration during anesthesia was 1,050 (1,000 to 1,400)  ml. Altogether 448 microcirculation videos were taken. Interobserver variability was 24%. The following microcirculatory parameter values describe before, during and after pneumoperitoneum periods. Total vascular density was 19.4 (17.0 to 21.1); 18.5 (17.0 to 20.9); 19.3 (16.9 to 20.9) n/mm2. Perfused vessels density was 13.3 (10.9 to 15.2); 13.8 (8.9 to 18.0); 13.1 (11.0 to 16.0) n/mm2. Microcirculatory response to experimentally-induced whole body heat stress in healthy humans A reduction in glycocalyx thickness, represented by an increased PBR, correlates with impairment of perfused microvascular vessel density. Our data support the use of intraoperative PBR monitoring as a novel clinical indicator of microcirculatory perfusion. g y y Results The perfused vessel density remained stable during off - pump surgery, while the PBR decreased from 2.6 ± 0.1 μm (baseline) to 2.3  ±  0.1  μm (ICU). Nonpulsatile CPB was associated with loss of microcirculatory perfusion during bypass (15.1  ±  2.7  mm/mm2) and ICU admission (15.3  ±  2.6  mm/mm2) compared with baseline (19.8  ±  2.8  mm/mm2). Pulsatile CPB reduced the perfused vessel density from 20.9 ± 2.4 to 16.7 ± 2.6 mm/mm2, but this was restored towards baseline levels upon ICU admission (20.3  ±  2.3  mm/mm2; P = 0.02 between groups). The PBR increased upon initiation of CPB in both groups, but remained elevated in the nonpulsatile fl ow group only. In the pulsatile group, the PBR started at 2.4 ± 0.1 μm, increased to 2.7 ± 0.1 μm during bypass but restored already during CPB towards a PBR value of 2.3 ± 0.1 μm (ICU). The PBR and perfused vessel density showed a good correlation (r = –0.65; P = 0.002), demonstrating loss of perfused vascular density when the glycocalyx is damaged. y y y y Methods Eight healthy men with no history of cold and/or heat injury were recruited to this study. Passive body heating was performed by continuous immersion up to the waist in the water bath at 44°C and continued until rectal temperature reached 39.5°C. Before, at the end of whole body heating and 1 hour after heating was ended, systemic hemodynamics and direct in vivo observation of the sublingual microcirculation were obtained with sidestream dark-fi eld imaging. Assessment of microcirculatory parameters of convective oxygen transport (microvascular fl ow index (MFI), proportion of perfused vessels (PPV)), and diff usion distance (perfused vessel density (PVD) and total vessel density (TVD)) was done using a semiquantitative method. Vessels were separated into large (mostly venules) and small (mostly capillaries) using a diameter cutoff value of 20 μm.i p y g y y g Conclusion The glycocalyx is damaged after initiation of CPB, and restoration is impaired after exposure to nonpulsatile fl ow during the use of the heart–lung machine. A reduction in glycocalyx thickness, represented by an increased PBR, correlates with impairment of perfused microvascular vessel density. Changes in microcirculatory perfusion during cardiac surgery are paralleled by alterations in glycocalyx integrityf Conclusion Defined cold exposure had no effect on the micro- circulation. Introduction Previous studies demonstrate that loss of glycocalyx integrity is associated with impaired microvascular function. We investigated whether glycocalyx dimensions are reduced in patients undergoing cardiac surgery with or without cardiopulmonary bypass (CPB), and are paralleled by loss of microcirculatory perfusion using in vivo microcirculation measurements. Microvascular response to cold stress in healthy humans A Pranskunas1, R Rasimaviciute1, J Paltanaviciute1, L Daniuseviciute2, V Pilvinis1, M Brazaitis2 Microvascular response to cold stress in healthy humans A Pranskunas1, R Rasimaviciute1, J Paltanaviciute1, L Daniuseviciute2, V Pilvinis1, M Brazaitis2 1Lithuanian University of Health Sciences, Kaunas, Lithuania; 2Lithuanian Sports University, Kaunas, Lithuania Critical Care 2013, 17(Suppl 2):P213 (doi: 10.1186/cc12151) Introduction Cold exposure can be adapted for exercise or therapeutic purposes, but its impact on microcirculation in healthy humans has not been well defi ned. We hypothesize that whole body cold stress may impair microcirculation. Introduction Cold exposure can be adapted for exercise or therapeutic purposes, but its impact on microcirculation in healthy humans has not been well defi ned. We hypothesize that whole body cold stress may impair microcirculation. Methods Seven volunteers were recruited for the water immersion procedure. During the cooling protocol the volunteers every 20 minutes of immersion were asked to step out from the bath and rest for 10  minutes in a room environment and then return to the water bath for the next 20  minutes of immersion. This head-out immersion procedure in bath water at 14°C continued until the rectal temperature was dropped to 35.5°C or the time of 180 minutes was terminated. Maximum cold water immersion time was 120  minutes. Before, at the end of whole body cooling and 1 hour after cooling was ended, systemic hemodynamics and direct in vivo observation of the sublingual microcirculation were obtained with sidestream dark-fi eld imaging. Assessment of microcirculatory parameters of convective oxygen transport (microvascular fl ow index (MFI), proportion of perfused vessels (PPV)), and diff usion distance (perfused vessel density (PVD) and total vessel density (TVD)) was done using a semiquantitative method.i Conclusion Microcirculatory alterations are mild during transient increase of intra-abdominal pressure in laparoscopic surgery patients. References 1. De Backer D, et al.: Crit Care Med 2006, 34:1918-1924. 2. Vellinga NAR, et al.: Crit Care Res Pract 2012. doi:10.1155/2012/121752 1. De Backer D, et al.: Crit Care Med 2006, 34:1918-1924. 2. Vellinga NAR, et al.: Crit Care Res Pract 2012. doi:10.1155/2012/121752 1. De Backer D, et al.: Crit Care Med 2006, 34:1918-1924. 1. De Backer D, et al.: Crit Care Med 2006, 34:1918-1924. 2. Vellinga NAR, et al.: Crit Care Res Pract 2012. doi:10.1155/2012/121752 P212 Changes in microcirculatory perfusion during cardiac surgery are paralleled by alterations in glycocalyx integrity C Boer1, NJ Koning1, J Van Teeff elen2, AB Vonk1, H Vink2 1VU University Medical Center, Amsterdam, the Netherlands; 2Maastricht University, Maastricht, the Netherlands Critical Care 2013, 17(Suppl 2):P212 (doi: 10.1186/cc12150) Sublingual microcirculatory changes during transient intra-abdominal hypertension: a study in laparoscopic surgery patients Proportion of perfused vessels (PPV) was 61 (50 to 69); 64 (45 to 76); 60 (54 to 67)%. Microvascular fl ow index was 2.4 (2.0 to 2.5); 2.5 (2.0 to 3.0); 2.3 (2.0 to 2.9) and heterogeneity index was 0.8 (0 to 0.9); 0.6 (0 to 1.0); 0.6 (0 to 0.8). No signifi cant diff erences in microcirculatory parameters were observed between time points. PPV was somewhat less than that of described in healthy volunteers (61% vs. 90%) [2]. Microcirculatory response to experimentally-induced whole body heat stress in healthy humans A Pranskunas1, E Milieskaite1, I Maraulaite1, L Daniuseviciute2, Z Pranskuniene1, V Pilvinis1, M Brazaitis2 1Lithuanian University of Health Sciences, Kaunas, Lithuania; 2Lithuanian Sports University, Kaunas, Lithuania Critical Care 2013, 17(Suppl 2):P214 (doi: 10.1186/cc12152) Methods Patients undergoing on-pump surgery with nonpulsatile (n = 11) or pulsatile (n = 13) CPB or off -pump surgery (n = 13) underwent sublingual sidestream dark-fi eld imaging at baseline, during coronary grafting and upon ICU admission to assess perfused microvascular vessel density. Glycocalyx integrity was evaluated using the GlycoCheck Measurement Software, and expressed as the perfused boundary region (PBR). An increase in PBR represents deeper penetration of erythrocytes into the glycocalyx, and is indicative for compromised glycocalyx thickness. Introduction Vasodilation and increased skin blood fl ow (also sweating) are infl uential in heat dissipation during heat exposure and exercise. It is unclear how heat stress infl uences microcirculation. Side dark-fi eld imaging visualizes the blood fl ow at the capillary level and helps to assess perfusion heterogeneity. Clinical and experimental data show that the sublingual region is clinically relevant for detecting microcirculatory alterations and more represents central microcirculation than cutaneous perfusion. We hypothesize that whole body heat stress may increase capillary density. g y y Results The perfused vessel density remained stable during off - pump surgery, while the PBR decreased from 2.6 ± 0.1 μm (baseline) to 2.3  ±  0.1  μm (ICU). Nonpulsatile CPB was associated with loss of microcirculatory perfusion during bypass (15.1  ±  2.7  mm/mm2) and ICU admission (15.3  ±  2.6  mm/mm2) compared with baseline (19.8  ±  2.8  mm/mm2). Pulsatile CPB reduced the perfused vessel density from 20.9 ± 2.4 to 16.7 ± 2.6 mm/mm2, but this was restored towards baseline levels upon ICU admission (20.3  ±  2.3  mm/mm2; P = 0.02 between groups). The PBR increased upon initiation of CPB in both groups, but remained elevated in the nonpulsatile fl ow group only. In the pulsatile group, the PBR started at 2.4 ± 0.1 μm, increased to 2.7 ± 0.1 μm during bypass but restored already during CPB towards a PBR value of 2.3 ± 0.1 μm (ICU). The PBR and perfused vessel density showed a good correlation (r = –0.65; P = 0.002), demonstrating loss of perfused vascular density when the glycocalyx is damaged. Conclusion The glycocalyx is damaged after initiation of CPB, and restoration is impaired after exposure to nonpulsatile fl ow during the use of the heart–lung machine. Changes in microcirculatory perfusion during cardiac surgery are paralleled by alterations in glycocalyx integrity C Boer1, NJ Koning1, J Van Teeff elen2, AB Vonk1, H Vink2 1VU University Medical Center, Amsterdam, the Netherlands; 2Maastricht University, Maastricht, the Netherlands Critical Care 2013, 17(Suppl 2):P212 (doi: 10.1186/cc12150) Results During cooling and 1 hour after cooling was ended, a signifi cant increase in cardiac output (P  =  0.028 and P  =  0.043) was observed, but there were no changes in heart rate or mean arterial pressure in comparison with baseline variables. There were no signifi cant changes in PPV, MFI, PVD and TVD of small vessels in comparison with baseline variables during all observational time. Changes in microcirculatory perfusion during cardiac surgery are paralleled by alterations in glycocalyx integrity C Boer1, NJ Koning1, J Van Teeff elen2, AB Vonk1, H Vink2 1VU University Medical Center, Amsterdam, the Netherlands; 2Maastricht University, Maastricht, the Netherlands Critical Care 2013, 17(Suppl 2):P212 (doi: 10.1186/cc12150) P215 Observational study of the eff ects of age, diabetes mellitus, cirrhosis and chronic kidney disease on sublingual microvascular fl ow T Reynolds1, S Jhanji2, A Vivian-Smith1, RM Pearse1 1Royal London Hospital, London, UK; 2Royal Marsden Hospital, London, UK Critical Care 2013, 17(Suppl 2):P215 (doi: 10.1186/cc12153) i Methods In a prospective cohort during 1 year, we followed consecu- tively enrolled patients admitted immediately postoperative to the surgical ICU of Hospital Santa Luzia, Brasília, Brazil. Patients were assigned to two groups: LC >10% and LC ≤10%. The primary outcome measure was mortality at 7 and 28  days. The secondary outcome included hospital and ICU length of stay (LOS). Introduction Sidestream dark-fi eld (SDF) imaging is an important new technology that has been used to demonstrate microcirculatory abnormalities in a variety of critical illnesses [1]. The microcirculation is also aff ected by age and chronic comorbidities. However, the eff ect of these conditions on SDF microcirculatory parameters has not been well described. p g y ( ) Results A total of 417 patients were followed. In total, 50.4% were male and 83% underwent elective surgery. The mean age was 59 ± 16, APACHE II score 8 ± 5, SAPS 2 26 ± 11. The mortality at 7 days was 0.95% (n = 4) and the mortality at 28 days was 2.15% (n = 9), respectively. Hospital mortality was 4.79% (n = 20). Sixty-one percent (n = 255) of the patients had LC >10% versus 39% (n = 162) with LC ≤10%. Those who had LC ≤10% were older (62 ± 16 vs. 57 ± 17, P = 0.00) and had greater APACHE II score (9 ± 6 vs. 7 ± 4, P = 0.00) and SAPS 2 (28 ± 12 vs. 25 ± 10, P = 0.02). There was no diff erence in ICU LOS (5 ± 12 vs. 4 ± 9 days, P = 0.54) and hospital LOS (10 ± 15 vs. 9 ± 11 days, P = 0.48). Initial lactate levels were lower in the group with LC ≤10% (1.1 ± 0.9 vs. 1.9 ± 1.6, P = 0.00); however, mean lactate was higher in 24 hours (2.0 ± 1.8 vs. 1.0 ± 0.7, P = 0.00). P215 All of the patients who died in the fi rst 7 days had LC ≤10% (2.46%, n = 4, P = 0.02); this group also had a higher mortality at 28 days (4.32%, n = 7 vs. 0.78%, n = 2; P = 0.03). The relative risk for mortality LC ≤10% in 7 and 28 days was 1.02 (95% CI: 1.00 to 1.05) and 5.07 (95% CI: 1.17 to 27.09), respectively. Signifi cant diff erence was observed in the Kaplan–Meier survival curves for 7 and 28 days (P = 0.01 and 0.02, respectively). The sensibility of LC ≤10% was 100% (95% CI: 51 to 100%) for 7-day mortality and 78% (95% CI: 45 to 94%) for 28-day mortality. The specifi city was 62% (95% CI: 57 to 66%) for 7-day mortality and 62% (95% CI: 57 to 66%) for 28-day mortality. C l i D i i i i l l l l l l d Methods Sublingual SDF images were obtained from fi ve groups of 20 participants: healthy volunteers under 25 years, healthy volunteers over 55 years, and stable patients over 55 years with one of diabetes mellitus (DM), cirrhosis and stage  5 chronic kidney disease (CKD). Microcirculatory parameters [1] between the groups were then com- pared for signifi cance using ANOVA for parametric data and the Kruskal–Wallis test for nonparametric data. This was approved by the local ethics committee. Results All DM patients were type 2, with mean glycated haemoglobin (HbA1c) of 8.8% (SD 1.7%). Seventeen cirrhotic patients were Child– Pugh–Turcotte score A and one was score B. For CKD, the mean estimated glomerular fi ltration rate was 11.5  ml/minute (SD 2.9). Median microvascular fl ow index (MFI) was 2.85 (IQR 2.75 to 3.0) for participants aged <25, 2.81 (2.66 to 2.97) for those aged >55, 2.88 (2.75 to 3.0) for those with DM, 3.0 (2.83 to 3.0) for those with cirrhosis and 3.0 (2.78 to 3.0) for those with CKD (P for diff erence = 0.14). There were no signifi cant diff erences in the proportion of perfused vessels and perfused vessel density between the groups. See Figure 1. Conclusion Despite initial lactate levels, lactate clearance ≤10% proved to be a good predictor of mortality in 7 and 28 days in surgical patients admitted in the postoperative period to the ICU. References e e e ces 1. Nguyen HB, et al.: Crit Care Med 2004, 32:1637-1642. 2. Jansen TC, et al.: Am J Respir Crit Care Med 2010, 182:752-756. y p y Reference 1. De Backer D, et al.: How to evaluate the microcirculation: report of a round table conference. Crit Care 2007, 11:R101. Figure 1 (abstract P215). Microcirculatory response to experimentally-induced whole body heat stress in healthy humans Our data support the use of intraoperative PBR monitoring as a novel clinical indicator of microcirculatory perfusion. f Results Whole body heating resulted in signifi cantly increased heart rate (P  =  0.012) and cardiac output (P  =  0.046) in comparison with baseline variables. One hour after heating was ended, the heart rate S81 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 remained increased (P = 0.012), but cardiac output returned to baseline values. During all observational time the mean arterial pressure remain unaltered. There was no signifi cant diff erence in MFI and PPV of small vessels at the end of heating and 1 hour after heating in comparison with baseline variables. One hour after heating we observed signifi cant increase in PVD (P  =  0.046) and TVD (P  =  0.028) of small vessels regardless of lack of diff erence at the end of heating (P = 0.753 and P = 0.075, respectively) in comparison with baseline variables. C l i Wh l b d h i i d d i d d h i remained increased (P = 0.012), but cardiac output returned to baseline values. During all observational time the mean arterial pressure remain unaltered. There was no signifi cant diff erence in MFI and PPV of small vessels at the end of heating and 1 hour after heating in comparison with baseline variables. One hour after heating we observed signifi cant increase in PVD (P  =  0.046) and TVD (P  =  0.028) of small vessels regardless of lack of diff erence at the end of heating (P = 0.753 and P = 0.075, respectively) in comparison with baseline variables. P217 Assessment of the mottling score as a mortality predictor in critically ill patients E Bastos de Moura, F Ferreira Amorim, C Darwin Silveira, M Oliveira Maia Hospital Santa Luzia, Brasília, Brazil Critical Care 2013, 17(Suppl 2):P217 (doi: 10.1186/cc12155) P215 y Conclusion Older age, diabetes, and chronic kidney and liver disease need not be considered confounding factors for comparison of SDF microcirculatory parameters in the critically ill. Microcirculatory response to experimentally-induced whole body heat stress in healthy humans Lactate clearance as a determinant of mortality in surgical patients FF Amorim1, EB Moura2, AR Santana1, FB Soares1, LG Godoy1, TA Rodrigues1, LJ Almeida1, GM FIlho1, TA Silva1, AP Amorim3, JA Neto2, MO Maia2 1Escola Superior de Ciências da Saúde, Brasília, Brazil; 2Unidade de Terapia Intensiva Adulto do Hospital Santa Luzia, Brasília, Brazil; 3Liga Academica de Medicina Intensiva do Distrito Federal (LIGAMI), Brasilia, Brazil Critical Care 2013, 17(Suppl 2):P216 (doi: 10.1186/cc12154) Lactate clearance as a determinant of mortality in surgical patients FF Amorim1, EB Moura2, AR Santana1, FB Soares1, LG Godoy1, TA Rodrigues1, LJ Almeida1, GM FIlho1, TA Silva1, AP Amorim3, JA Neto2, MO Maia2 1Escola Superior de Ciências da Saúde, Brasília, Brazil; 2Unidade de Terapia Intensiva Adulto do Hospital Santa Luzia, Brasília, Brazil; 3Liga Academica de Medicina Intensiva do Distrito Federal (LIGAMI), Brasilia, Brazil Critical Care 2013, 17(Suppl 2):P216 (doi: 10.1186/cc12154) Conclusion Whole body heating induced time-dependent changes in capillary density. Introduction Serial measurements of lactate over time may be a better prognosticator than a single lactate concentration [1]. Early lactate- guided therapy also reduces ICU length of stay and ICU and hospital mortality [2]. This study aims to assess the prognostic value of the lactate clearance (LC) in the fi rst 24 hours in surgical patients. Introduction Serial measurements of lactate over time may be a better prognosticator than a single lactate concentration [1]. Early lactate- guided therapy also reduces ICU length of stay and ICU and hospital mortality [2]. This study aims to assess the prognostic value of the lactate clearance (LC) in the fi rst 24 hours in surgical patients. Impact of diff erent haemodynamic resuscitation strategies on brain perfusion and tissue oedema markers in a model of severe haemorrhagic shock pi yf p g y y Results Of the 770 patients included in the analysis, 52 died within 30  days. The standard SI at a cutoff point of 0.7 had the highest predictability and sensitivity of 30-day mortality (area under the curve (AUC) = 0.7, sensitivity = 0.79, specifi city = 0.56). In comparison, one of the modifi ed SIs (pulse/diastolic blood pressure) had 0.65 sensitivity and 0.71 specifi city (AUC = 0.73). A heart rate >100 bpm predicted 30- day mortality with 0.40 sensitivity and 0.82 specifi city (AUC = 0.63). Conclusion To our knowledge, this is the fi rst study to examine the relationship between the SI and mortality in patients with a GI haemorrhage. It appears that the standard SI, when compared with various modifi ed SIs and conventional vital signs, had the highest combined predictability and sensitivity of 30-day mortality in a population of patients suff ering from GI haemorrhage. Further prospective studies are needed to confi rm these fi ndings. Faculdade de Medicina, Universidade de São Paulo, Brazil i yf g y y Results Of the 770 patients included in the analysis, 52 died within 30  days. The standard SI at a cutoff point of 0.7 had the highest predictability and sensitivity of 30-day mortality (area under the curve (AUC) = 0.7, sensitivity = 0.79, specifi city = 0.56). In comparison, one of the modifi ed SIs (pulse/diastolic blood pressure) had 0.65 sensitivity and 0.71 specifi city (AUC = 0.73). A heart rate >100 bpm predicted 30- day mortality with 0.40 sensitivity and 0.82 specifi city (AUC = 0.63).i Faculdade de Medicina, Universidade de São Paulo, Brazil Critical Care 2013, 17(Suppl 2):P218 (doi: 10.1186/cc12156) Critical Care 2013, 17(Suppl 2):P218 (doi: 10.1186/cc12156) Introduction This study aimed to compare the cerebral eff ects of terlipressin (TERLI) with conventional prehospital fl uid resuscitation with lactated Ringer’s solution (LR) in a model of haemorrhagic shock (HS). y y y pi y Conclusion To our knowledge, this is the fi rst study to examine the relationship between the SI and mortality in patients with a GI haemorrhage. It appears that the standard SI, when compared with various modifi ed SIs and conventional vital signs, had the highest combined predictability and sensitivity of 30-day mortality in a population of patients suff ering from GI haemorrhage. Further prospective studies are needed to confi rm these fi ndings. Impact of diff erent haemodynamic resuscitation strategies on brain perfusion and tissue oedema markers in a model of severe haemorrhagic shock Methods Pigs (20 to 30 kg) were randomized into one of the groups: Sham (n = 2), HS (n = 9), LR (3× volume bled; n = 9) or TERLI (2 mg bolus; n  =  9). HS induced to target MAP of 40  mmHg was maintained for 30 minutes. Brain tissue oxygen pressure (PbtO2), intracranial pressure (ICP), cerebral perfusion pressure (CPP), haemodynamics and blood gas analyses were assessed prior to HS (baseline) up to 120 minutes after treatment. Tissue markers of brain oedema (aquaporin-4 (AQP4) and Na–K–Cl cotransporter-1 (NKCC1)), apoptosis (pre-apoptotic protein (Bax)) and oxidative stress (thiobarbituric acid reactive substances (TBARS)) were also measured. Results Sham animals had no signifi cant changes in the variables assessed. HS resulted in a signifi cant decrease in CPP (mean varied from 36 to 39 mmHg), PbtO2 (from 23.6 to 26.6 mmHg), ICP (from 1 to 2 mmHg) and haemodynamics (MAP from 38 to 40 mmHg; CI from 1.8 to 2.1 l/minute/m2), and a signifi cant increase in blood lactate (from 6.7 to 8.9 mmol/l) and cerebral AQP4 (mean ± SE; 167 ± 54% of sham), NKCC1 (237 ± 47% of sham), Bax (167 ± 44% of sham) and TBARS. Fluid resuscitation was followed by an increase in ICP (from 7 to 9 mmHg) and a decrease in CPP (from 41 to 52 mmHg), with an increased expression of cerebral AQP4 (210 ± 56% of sham), NKCC1 (163 ± 32% of sham) and Bax (137 ± 24% of sham). Only TERLI restored baseline values of CPP (from 54 to 61 mmHg) and did not change the cerebral expression of AQP4 (100 ± 6% of sham), NKCC1 (100 ± 1% of sham), Bax (102 ± 6% of sham) and TBARS. Both TERLI and LR recovered baseline levels of PbtO2 (TERLI from 30.0 to 34.2 mmHg; LR from 29.4 to 40.7 mmHg) and MAP (TERLI from 53 to 64 mmHg; LR: 48 to 61 mmHg). Blood lactate levels were not recovered in any group (TERLI from 5.7 to 8.1 mmol/l; LR from 4.5 to 7.7 mmol/l). Inhaled carbon monoxide or nebulized sodium nitrite protect against hemorrhagic shock-induced mitochondrial dysfunction H Gomez, D Escobar, B Ataya, L Gordon, O Ogundele, M Pinsky, S Shiva, B Zuckerbraun University of Pittsburgh, PA, USA Critical Care 2013, 17(Suppl 2):P220 (doi: 10.1186/cc12158) Introduction Shock induces mitochondrial damage, which can lead to tissue injury and infl ammation. Accuracy of the shock index and various modifi ed shock indexes to predict early mortality in patients suff ering from gastrointestinal haemorrhage Introduction The shock index (SI) is an easy-to-use clinical tool that rapidly identifi es patients at risk of haemodynamic decompensation. Previous studies focused primarily on patients suff ering from pneumonia, pulmonary embolism, ruptured ectopic pregnancy and traumatic haemorrhagic shock. Modifi ed SIs have also been studied. Could the SI be accurate in patients suff ering from gastrointestinal (GI) haemorrhage? The aim of this study was to compare the performance of the SI with various modifi ed SIs and conventional vital signs in predicting 30-day mortality in a population of patients with a GI haemorrhage. Conclusion The mottling score was an objective reproducible system to bedside use and a good predictor of septic shock mortality. References 1. Hernandez G, et al.: Evolution of peripheral vs metabolic perfusion 1. Hernandez G, et al.: Evolution of peripheral vs metabolic perfusion parameters during septic shock resuscitation. A clinical-physiologic stu 1. Hernandez G, et al.: Evolution of peripheral vs metabolic perfusion parameters during septic shock resuscitation. A clinical-physiologic study. J Crit Care 2012, 27:283-288. parameters during septic shock resuscitation. A clinical-physiologic study. J Crit Care 2012, 27:283-288. 2. Ait-Oufella H, et al.: Mottling score predicts survival in septic shock. Intensive Care Med 2011, 37:801-807. g Methods A single-center post-hoc analysis was conducted of prospectively collected data from patients diagnosed with a GI haemorrhage episode in an academic emergency department (ED) from March 2008 to December 2011. Data were extracted from two databases used at our ED. The SI (pulse/systolic blood pressure) and nine modifi ed SIs were calculated from the available fi rst documented vital signs. ROC curves were used to determine sensitivity and specifi city of the diff erent SIs in predicting 30-day mortality. References 1. Cavus E, et al.: Resuscitation 2009, 80:567-572. 2. Bayram B, et al.: Am J Emerg Med 2011, 30:1176-1182. y Results One hundred and seventeen patients were analyzed; 20 were excluded (18 terminal illness, two with incomplete data). Ninety-seven patients were included; the mean age was 72.8 years, mean SAPS II score was 46.8 (SD ± 15.7), mean APACHE II score was 19.2 (SD ± 8.1); mean norepinephrine dose was 1.25  μg/kg/minute; mean length of stay in ICU was 19.2 days (1 to 176); mottling score distribution was: score 0: 41 patients; score 1: 33 patients; score 2: 14 patients; score 3: two patients; score 4: three patients; score 5: four patients. The sepsis origin was as follows: 65, pulmonary (67%); 18, abdominal (18.5%); nine, urinary (9.5%); two, osseous (2%); one, mediastinal; one, skin and soft tissue; and one, central nervous system (1% each). Comparing the mortality in Groups 1, 2 and 3, we found a signifi cant diff erence (P = 0.042), even greater when considering 28-day mortality (P = 0.004). The Kaplan–Meier survival method showed P = 0.000. P219 P219 Accuracy of the shock index and various modifi ed shock indexes to predict early mortality in patients suff ering from gastrointestinal haemorrhage J St-Cyr Bourque, J Cliche, J Chauny, R Daoust, J Paquet, É Piette Hôpital du Sacré-Coeur de Montréal, Canada Critical Care 2013, 17(Suppl 2):P219 (doi: 10.1186/cc12157) Assessment of the mottling score as a mortality predictor in critically ill patients y E Bastos de Moura, F Ferreira Amorim, C Darwin Silveira, M Oliveira Maia Hospital Santa Luzia, Brasília, Brazil Critical Care 2013, 17(Suppl 2):P217 (doi: 10.1186/cc12155) Introduction The use of peripheral perfusion objective parameters to anticipate successful resuscitation in septic shock has been recently investigated [1]. The mottling score, a perfusion parameter used for decades, has been proposed to correlate with septic shock survival [2], and was tested in this study as a clinical tool in predicting mortality. Methods A prospective observational study was conducted, with patients consecutively admitted to a tertiary hospital ICU in Brasília, Brazil. From July 2011 to May 2012, all patients diagnosed with septic shock were enrolled. Demographic data, diagnoses, shock origin and severity scores were recorded. After initial resuscitation, the score was registered in the fi rst 3 days by the same observer, considering the score on the lower limb without an arterial catheter, or the worst between the lower limbs, and the worst in the 3 days. Exclusion criteria were terminal illness with no intervention decision and incomplete S82 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 contributed to the cerebral apoptosis. None of the treatments caused cerebral oxidative stress. data. The scores are pooled in Group 1 (scores 0 and 1), Group 2 (scores 2 and 3) and Group 3 (scores 4 and 5) to compare mortality. Statistical analysis was made using the chi-square test. contributed to the cerebral apoptosis. None of the treatments caused cerebral oxidative stress. data. The scores are pooled in Group 1 (scores 0 and 1), Group 2 (scores 2 and 3) and Group 3 (scores 4 and 5) to compare mortality. Statistical analysis was made using the chi-square test. P221 Is norepinephrine more eff ective than other vasopressors for septic shock? A systematic review and meta-analysis F Zhou1, Z Peng2, W Zhang3, J Bishop2, Q Song1 1Chinese People’s Liberation Army General Hospital, Beijing, China; 2University of Pittsburgh, PA, USA; 3University of Pittsburgh School of Medicine, PA, USA Critical Care 2013, 17(Suppl 2):P221 (doi: 10.1186/cc12159) Is norepinephrine more eff ective than other vasopressors for septic shock? A systematic review and meta-analysis y y Results A total of 300 patients underwent randomization, were infused with the study drug (148 patients received vasopressin, and 152 norepinephrine), and were included in the analysis. Patients who received vasopressin had a lower rate of morbidity (23.5% vs. 34%, P = 0.001) as compared with the norepinephrine group. The 30-day mortality rate was 6.1% in the norepinephrine group and 4.6% in the vasopressin group (P = 0.570). There were no signifi cant diff erences in the overall rates of serious adverse events (7.4% and 6.6%, respectively; P = 0.772). Introduction Norepinephrine has been widely used in septic shock. However, its eff ect remains controversial. We conduct a systematic review and meta-analysis to compare the eff ect between norepinephrine and other vasopressors. Methods The PubMed, Embase, and Cochrane Library databases from database inception until October 2012 were searched. We selected randomized controlled trials in adults with septic shock and compared norepinephrine with other vasopressors. The quality of each study included was assessed with Jadad score. After assessing for heterogeneity of treatment eff ect across trials using the I2 statistic, we used a fi xed eff ect model (P ≥0.1) or random-eff ects model (P <0.1) and expressed results as the risk ratio (RR) for dichotomous outcomes or the f Conclusion Vasopressin reduces major morbidity after cardiac surgery as compared with norepinephrine among patients with cardiac surgery with vasoplegic shock. p g Acknowledgement Clinical Trials number NCT01505231. p g Acknowledgement Clinical Trials number NCT01505231. P223 standardized mean diff erence (SMD) for continuous data with 95% CI. Results Eighteen trials (n  =  2,715) met inclusion criteria, which compared norepinephrine with fi ve diff erent vasopressors (dopamine, vasopressin, epinephrine, terlipressin and phenylephrine). The mean Jadad score was 3.11. Overall, there was no diff erence in mortality in the comparisons between norepinephrine and vasopressin, epinephrine, terlipressin and phenylephrine (P  >0.05, respectively). However, norepinephrine had a trend in decreasing mortality compared with dopamine (RR, 0.84; 95% CI, 0.68 to 1.02; P = 0.08). P222 11 mg in PBS×30 minutes, n = 6), and sham (n = 3). CO or NaNO2 were initiated ~30 minutes before resuscitation. Primary endpoints were changes in muscle and platelet mitochondrial respiration between BL and EndObs, quantifi ed by muscle respiratory control ratio (RCR, traditional respirometry), and by the change in proton-leak respiration (PLR) and mitochondrial reserve capacity in platelets. Secondary endpoint was mortality at EndObs. P222 Vasopressin Versus Norepinephrine for the Management of Shock After Cardiac Surgery (VaNCS study): a randomized controlled trial L Hajjar1, JL Vincent2, A Rhodes3, D Annane4, F Galas1, J Almeida1, S Zeferino1, L Camara1, V Santos1, J Pereira1, E Osawa1, E Maciel1, A Rodrigues1, J Jardim1, D Blini1, E Araujo1, F Bergamin5, R Kalil Filho1, J Auler Jr1 1Heart Institute, São Paulo, Brazil; 2Universite Libre de Bruxelles, Brussels, Belgium; 3University of London, UK; 4Faculte Medicine Paris, France; 5Cancer Institute, São Paulo, Brazil Critical Care 2013, 17(Suppl 2):P222 (doi: 10.1186/cc12160) Vasopressin Versus Norepinephrine for the Management of Shock After Cardiac Surgery (VaNCS study): a randomized controlled trial L Hajjar1, JL Vincent2, A Rhodes3, D Annane4, F Galas1, J Almeida1, S Zeferino1, L Camara1, V Santos1, J Pereira1, E Osawa1, E Maciel1, A Rodrigues1, J Jardim1, D Blini1, E Araujo1, F Bergamin5, R Kalil Filho1, J Auler Jr1 1Heart Institute, São Paulo, Brazil; 2Universite Libre de Bruxelles, Brussels, Belgium; 3University of London, UK; 4Faculte Medicine Paris, France; 5Cancer Institute, São Paulo, Brazil Critical Care 2013, 17(Suppl 2):P222 (doi: 10.1186/cc12160) y Results Skeletal muscle RCR decreased in the HSR group (P = 0.04) but not in sham. Decrease in RCR was primarily due to decreased ADP- dependent respiration, without change in state 4 respiration. HSR also resulted in platelet mitochondrial dysfunction as demonstrated by increased PLR and decreased reserve capacity. This correlated with increased platelet activation (%CD62P+ by fl ow cytometry) in HSR. CO or NaNO2 treatment prevented these deleterious changes in both muscle and platelet mitochondrial respiration, as well as limited HSR-induced platelet activation. CO treatment also improved reserve capacity compared with baseline. Mortality was higher in HSR than in CO or NaNO2 (80 vs. 33 and 33%, respectively). Introduction Vasoplegic syndrome is a common complication after cardiac surgery, with negative impact on patient outcomes and hospital costs. Pathogenesis of vasodilatory phenomenon after cardiac surgery remains a matter of controversy. Loss of vascular tone can be partly explained by the depletion of neurohypophyseal arginine vasopressin stores. 1. Jadad AR, et al.: Control Clin Trials 1996, 17:1-12. 2. Higgins JP, et al.: BMJ 2003, 327:557-560. Intraaortic counterpulsation in a second-level institution: indications, management and outcome R Gómez López, P Fernández Ugidos, P Vidal Cortés, M Lorenzo Lage, A Tizón Varela, E Rodríguez Álvarez Complexo Hospitalario Universitario de Ourense, Spain Critical Care 2013, 17(Suppl 2):P223 (doi: 10.1186/cc12161) Introduction Counterpulsation is an important support for patients with cardiac diseases. The use of these devices has been limited typically to hospitals with a cardiac surgery service. The aim of this study is to describe the management of patients with an intraaortic balloon pump (IABP) in our second-level institution. Methods An observational study that includes all patients with IABP in our hospital from January 2010 to September 2012, followed to November 2012. Epidemiological and clinical variables as an indication of IABP, therapeutic management and outcome were collected. Because of the small size sample, statistical analysis was limited to descriptive parameters. Conclusion There is not suffi cient evidence to prove that norepinephrine is superior to vasopressin, epinephrine, terlipressin and phenylephrine in terms of mortality. However, norepinephrine is associated with a decreased HR, cardiac index and an increased SVRI, and appears to have a greater eff ect on decreasing mortality compared with dopamine. Results Seventeen patients (76.5% men, age 66.6 ± 11.6 (43 to 85) years, APACHE score 21.5 ± 14.9 (3 to 46) points) were treated with IABP. The reason for ICU admission was STEMI (n = 6 patients, 35.3%), no-STEMI (n = 5, 29.4%), acute heart failure (n = 4, 23.5%) and out-of-hospital Impact of diff erent haemodynamic resuscitation strategies on brain perfusion and tissue oedema markers in a model of severe haemorrhagic shock Resuscitative adjuncts to limit mitochondrial injury may be eff ective to reduce tissue injury and protect against the sequelae of hemorrhagic shock (HS). Others and we have demonstrated the protective eff ects of inhaled carbon monoxide (CO) or nebulized sodium nitrite (NaNO2) in models of HS. Our aim was to test the hypothesis that CO and NaNO2 protect against hemorrhagic shock-induced tissue injury/infl ammation by limiting mitochondrial damage and preventing bioenergetic failure. Methods Twenty anesthetized female Yorkshire pigs were subjected to severe hemorrhage until unable to compensate or 90 minutes, and were then resuscitated with volume/pressors. Muscle and platelet samples were obtained at baseline (BL) and 2 hours after resuscitation (EndObs). Animals were randomized to: standard of care (HSR, n = 5); HSR+CO (CO; 250 ppm×30 minutes, n  =  6); or HSR+NaNO2 (NaNO2; Conclusion TERLI recovered cerebral perfusion and oxygenation with no signifi cant changes in ICP and cerebral markers of oedema, apoptosis and oxidative stress. LR did not recover CPP probably due to a signifi cant increase in ICP caused by brain oedema, which may have S83 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 P222 Vasopressin is commonly used as an adjunct to catecholamines to support blood pressure in refractory septic shock, but its eff ect on vasoplegic shock is unknown. We hypothesized that the use of vasopressin would be more eff ective on treatment of shock after cardiac surgery than norepinephrine, decreasing the composite endpoint of mortality and severe morbidity. 2 y Conclusion In severe HS, mitochondrial injury in platelets and muscle was limited by CO or NaNO2. Although not powered for a secondary endpoint, mortality was double in HSR versus adjunctive therapies. This suggests that CO and NaNO2 may protect mitochondrial function by maintaining ATP-coupled respiration and reserve capacity, and that this may confer a survival advantage. However, further investigations are required. Methods In this prospective and randomized, double-blind trial, we assigned patients who had vasoplegic shock to receive either vasopressin (0.01 to 0.06 U/minute) or norepinephrine (0.01 to 1 μg/ kg/minute) in addition to open-label vasopressors. All vasopressor infusions were titrated and tapered according to protocols to maintain a target blood pressure. The primary endpoint was major morbidity according to STS (30-day mortality, mechanical ventilation >48 hours, mediastinitis, surgical re-exploration, stroke, acute renal failure). Secondary outcomes were time on mechanical ventilation, ICU and hospital stay, new infection, the time to attainment of hemodynamic stability, occurrence of adverse events and safety. P221 There were a decreased heart rate (HR) (SMD, –2.10; 95% CI, –3.95 to –0.25; P = 0.03), cardiac index (SMD, –0.73; 95% CI, –1.14 to –0.03; P = 0.004) and an increased systemic vascular resistance index (SVRI) (SMD, 1.03; 95% CI, 0.61 to 1.45; P <0.0001) with the treatment of norepinephrine compared with dopamine.fi Intraaortic counterpulsation in a second-level institution: indications, management and outcome R Gómez López, P Fernández Ugidos, P Vidal Cortés, M Lorenzo Lage, A Tizón Varela, E Rodríguez Álvarez Complexo Hospitalario Universitario de Ourense, Spain Critical Care 2013, 17(Suppl 2):P223 (doi: 10.1186/cc12161) 3. Mayo Clin Proc 2012, 87:620-628. 1. J Cardiothorac Vasc Anesth 2011, 25:526-535. P224 Results Our urosepsis population contained 364 patients, of which 129 (35.4%) were either using a BB, a CCB or both before their admission. Mean age was 74.9 years and 48.1% of the patients were women. A total of 36 patients died (9.9%) in a 30-day period. The group taking either BB or CCB had a signifi cantly lower mean SI (0.76, 95% CI: 0.72 to 0.81 vs. 0.93, 95% CI: 0.89 to 0.98, P <0.0001). In our urosepsis population, a SI of 0.7 had a sensitivity of 0.76 and a specifi city of 0.27 (area under the curve (AUC): 0.596) for patients taking neither BB nor CCB. In the group taking either or both medications, a SI of 0.7 had a sensitivity of 0.57 and a specifi city of 0.42 (AUC: 0.578). In both groups, lowering the SI to 0.5 increased the sensitivity to more than 0.95 but lowered specifi city signifi cantly.f Impact of left ventricular ejection fraction and elevated fi lling pressures on mortality in mechanically ventilated patients in severe sepsis or septic shock P225 Eff ect of β-blockers and calcium channel blockers on shock index predictability in patients suff ering from urosepsis J Cliche, J St-Cyr Bourque, R Daoust, J Chauny, J Paquet, E Piette Sacré-Coeur Hospital, Montreal, Canada Critical Care 2013, 17(Suppl 2):P225 (doi: 10.1186/cc12163) Eff ect of β-blockers and calcium channel blockers on shock index predictability in patients suff ering from urosepsis Eff ect of β-blockers and calcium channel blockers on shock index predictability in patients suff ering from urosepsis f J Cliche, J St-Cyr Bourque, R Daoust, J Chauny, J Paquet, E Piette Sacré-Coeur Hospital, Montreal, Canada Critical Care 2013, 17(Suppl 2):P225 (doi: 10.1186/cc12163) Introduction In our busy emergency departments the identifi cation of patients at risk of rapid hemodynamic decompensation is crucial. The shock index (SI) (pulse/systolic blood pressure) is a non-invasive clinical sign associated with more complications if higher than 0.7. The eff ect of β-blockers (BB) and calcium channel blockers (CCB) on the SI has not yet been described. Most studies excluded these patients, owing to the medications’ eff ect on the cardiac pulse. Considering that BB and CCB are commonly prescribed, we studied their eff ect on the SI’s predictability on 30-day mortality in a urosepsis population. Methods This single-center post-hoc analysis of prospectively collected data was conducted in an academic Canadian emergency department (ED) between March 2008 and February 2011. Data were extracted from two institutional databases. We included patients with a fi nal diagnosis of urosepsis, sepsis, pyelonephritis and urinary tract infection. Selected patients also had a documented positive urine culture. The SI predictability on 30-day mortality was calculated for patients taking BB and/or CCB as well as patients taking none of these medications. Sensitivity and specifi city were determined using ROC curves. t tests were used to compare mean SI between both groups. g pp y Conclusion In our hospital the IABP was mainly used in refractory cardiogenic shock because of myocardial ischemia, with an all-cause mortality of 41.2%. Low rates of complications were observed. Transfer of patients with IABP was performed safely. 2. Crit Care Med 2009, 37:441-447. P226 Levosimendan in critically ill adults: a utilisation review J Aron1, S Harrison2, A Milne2, S Patel2, R Maharaj2 1Lewisham NHS Trust, London, UK; 2King’s College Hospital NHS Trust, London, UK ii Results All cause 30-day mortality was 50% (n = 29). Forty-six patients (79%) had normal LV function. Forty-four (76%) patients had normal LV fi lling pressures. Patients who survived had lower E/e’ ratio, (median E/e’ 10.6 ± 4 vs. 13.6 ± 7, P = 0.1) but this was not statistically signifi cant. However, when defi ned as E/e’ >15, the mortality was 71%. On the contrary, patients with low LVEF had a mortality of 41% and LVEF was no diff erent between survivors and nonsurvivors (55 ± 16 vs. 58 ± 14, P = 0.34). Introduction Levosimendan improves haemodynamic performance and may have cardioprotective eff ects. Small trials have demonstrated effi cacy in well-defi ned populations [1]. Given the uncertain benefi ts we describe contemporaneous patient selection, utilisation of levosimedan, haemodynamic eff ects and outcomes. f Methods The study was performed at a single centre’s three ICUs. Data from 38 consecutive admissions, from 2010 to 2012, treated with levosimendan were collected retrospectively. Demographics, illness severity, comorbidity, haemodynamic, metabolic, biochemical, resource utilisation, organ support and hospital outcomes were analysed. Conclusion Myocardial dysfunction is a well-known entity in patients with septic shock. The clinical spectrum of this entity is broad, including LV, RV and diastolic dysfunction. Although the E/e’ ratio has been a known prognostic indicator in other cardiac conditions, its role in these patients is less clear. This study demonstrated that when septal mitral annulus E/e’ was >15, it was a better marker for mortality than LVEF in mechanically ventilated patients with severe sepsis or septic shock. Larger studies incorporating diastolic evaluation and TDI should be performed to further clarify this fi nding. Results Our cohort had a mean age of 58 (95% CI = 53.4 to 64.4). Only 18% underwent cardiothoracic surgery: sepsis, pulmonary emboli and myocardial/pericardial diseases were also treated with levosimendan. Admission characteristics included mean PaO2/FiO2 ratio (PFR) of 186.7 mmHg (95% CI = 160 to 212) and mean cardiac index (CI) of 2.3 (95% CI 1.9 to 2.8). The median APACHE II score was 24 (interquartile range 18.5 to 30). Levosimendan was initiated at a variable point during ICU treatment (days 0 to 14) and was usually the third inotrope References S84 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 cardiac arrest (n = 2, 11.8%). The indications of IABP were refractory cardiogenic shock (n  =  12, 70.6%), high-risk percutaneous coronary intervention (n = 2, 11.8%), refractory angina (n = 1, 5.9%), refractory pulmonary edema (n  =  1, 5.9%) and electrical storm (n  =  1, 5.9%). Five IABPs (27.4%) were inserted in the ICU and the rest in the catheterization laboratory. Six patients (35.3%) suff ered a cardiac arrest prior to hemodynamic stabilization. Three patients (17.6%, the electrical storm and the out-of-hospital cardiac arrest) died before coronary catheterization was performed and the other three were treated with mild therapeutic hypothermia. Thirteen patients (76.5%) needed invasive mechanical ventilation. In six patients (35.3%) invasive hemodynamic monitoring was performed (one pulmonary artery catheter, fi ve PICCO). Transthoracic echocardiography was performed in all patients and transesophageal in six (35.3%). Six of the patients (35.3%) were transferred to the reference centre for immediate coronary artery bypass grafting (CABG). No complications were reported during the transfer. During the ICU stay, femoral artery pseudoaneurysm was reported in one of the patients and inguinal hematoma after IABP withdrawal in two (no transfusion required). Four patients (27.4%) died because of refractory cardiogenic shock despite revascularization. The length of mechanical support was 1.67 ± 1.5 days (0 to 6). Impact of left ventricular ejection fraction and elevated fi lling pressures on mortality in mechanically ventilated patients in severe sepsis or septic shock p p S Gillespie, J Pulido Mayo Clinic, Rochester, MN, USA Critical Care 2013, 17(Suppl 2):P224 (doi: 10.1186/cc12162) S Gillespie, J Pulido Mayo Clinic, Rochester, MN, USA Introduction Myocardial dysfunction in septic shock is common and the presentation is broad. There are confl icting data regarding the prognostic implications of low left ventricular (LV) ejection fraction and elevated E/e’ ratio on mortality in this patient population. We sought to assess the impact of LV dysfunction and elevated E/e’ ratio on 30- day mortality in mechanically ventilated patients with severe sepsis or septic shock. Conclusion To our knowledge this is the only study analysing the eff ect of BB and CCB on the SI predictability of 30-day mortality. Our results indicate that SI cannot be used to accurately predict mortality with patients suff ering from urosepsis. In both our groups, SI performance was poor, as shown by the ROC curves. BB or CCB did not infl uence these results. Methods Fifty-eight mechanically ventilated patients with severe sepsis or septic shock admitted from 1 August 2007 to 31 January 2009 were prospectively evaluated with transthoracic echocardiogram within 24 hours of admission. Left ventricular ejection fraction was assessed using the modifi ed Simpson method as recommended by the American Society of Echocardiography. Normal LV function was defi ned as LVEF 40%. Mitral infl ow pulsed wave Doppler of peak E waves and tissue Doppler imaging (TDI) of the septal mitral annulus peak velocities were measured, the E/e’ ratio was obtained. Elevated LV fi lling pressures was defi ned as E/e’ >15. P227 P227 Levosimendan versus dobutamine in Tako-tsubo cardiomyopathy FR Righetti, MP Parolini, GC Castellano St. Boniface Hospital Verona, Italy Critical Care 2013, 17(Suppl 2):P227 (doi: 10.1186/cc12165) and their interaction. Data are reported as mean ± standard deviation. Results Ivabradine decreased the HR from 223 ± 18 to 196 ± 15 bpm (P <0.05). Contractility and relaxation remained unchanged. Ivabradine reduced the positive chronotropic eff ect at all doses of dobutamine (10 nM: 232 ± 37 vs. 187 ± 19, 100 nM: 265 ± 37 vs. 211 ± 40, 1 μM: 316 ± 35 vs. 250 ± 39, 10 μM: 320 ± 33 vs. 235 ± 40 bpm; P <0.05). It shifted the maximum positive inotropic action of dobutamine to lower dose ranges (100 nM: 2,924 ± 841 vs. 2,978 ± 955, 300 nM: 3,743 ± 925 vs. 4,795 ± 1,298, 1 μM: 4,138 ± 935 vs. 4,896 ± 1,861 mmHg/second; P  <0.05). A comparable shift was seen for relaxation (100  nM: –2,178 ± 686 vs. –2,520 ± 742, 300 nM: –2,615 ± 726 vs. –3,150 ± 888, 1  μM: –2,903  ±  752 vs. –2,972  ±  967  mmHg/second; P  <0.05). Levosimendan increased the HR only at high doses. With ivabradine, no positive chronotropic eff ect of levosimendan was observed (100 nM: 185 ± 30 vs. 162 ± 22, 1 μM: 208 ± 28 vs. 166 ± 29, 10 μM: 242 ± 27 vs. 168 ± 36 bpm; P <0.05). Ivabradine attenuated the positive inotropic eff ect of levosimendan (100 nM: 2,303 ± 303 vs. 1,737 ± 262, 1 μM: 2,977 ± 481 vs. 1,940 ± 449, 10 μM: 3,480 ± 941 vs. 2,189 ± 542 mmHg/ second; P <0.05) but did not signifi cantly alter its lusitropic eff ect. Introduction Tako-tsubo cardiomyopathy is a clinical entity charac- terized by a dysfunction of the left ventricle, usually transient, which manifests itself with symptoms that can mimic an acute coronary syndrome. It is characterized by transient ballooning modifi cation of the left ventricular apex, probably due to neurogenic stimuli resulting in low cardiac output syndrome. The aim of this prospective randomized study was to evaluate, by serial transesophageal echocardiography (TEE), what is the best drug treatment between levosimendan and dobutamine to restore a satisfactory cardiac function in the case of low cardiac output. Methods Twelve adult patients, aged 18  years, were admitted to the ICU with Tako-tsubo cardiomyopathy at entrance. P228 Combined eff ects of ivabradine with dobutamine or levosimendan in isolated perfused hearts D Konrad, R Vicenzi-Moser, M Vicenzi, U Aldenhoff , E Schwarzl, W Toller Medical University Graz, Austria Critical Care 2013, 17(Suppl 2):P228 (doi: 10.1186/cc12166) (range 0 to 5) commenced. Treatment with levosimendan (Table  1) resulted in improved CI and PFR with reductions in EVLW, BE, lactate and creatinine. The ICU length of stay was 15.4 days (95% CI = 13.2 to 17.6) and the hospital mortality was 54%. No signifi cant adverse eff ects were reported. (range 0 to 5) commenced. Treatment with levosimendan (Table  1) resulted in improved CI and PFR with reductions in EVLW, BE, lactate and creatinine. The ICU length of stay was 15.4 days (95% CI = 13.2 to 17.6) and the hospital mortality was 54%. No signifi cant adverse eff ects were reported. Introduction Dobutamine and levosimendan improve cardiac contractility in patients with heart failure, septic cardiomyopathy or cardiac surgery. Tachycardia in these cases is undesired because of aggravating ischemia. Ivabradine, a selective sinus node inhibitor, does not aff ect contractility. A combination of ivabradine with positive inotropic drugs might be favorable. Thus, we compared the cardiac eff ects of dobutamine or levosimendan alone, and combined with ivabradine using the Langendorff method of isolated perfused hearts. Methods Isolated guinea pig hearts (n  =  37) were perfused with incremental doses of dobutamine (10 nM to 10 μM) or levosimendan (30  nM to 10  μM) either alone or combined with 3  μM ivabradine. Heart rate (HR), left ventricular pressures, contractility (+dLVP/dt) and relaxation (–dLVP/dt) were recorded. Data for each drug (dobutamine or levosimendan) were analyzed by two-way ANOVA for repeated measures including the two main eff ects of ivabradine and drug dose and their interaction. Data are reported as mean ± standard deviation. Results Ivabradine decreased the HR from 223 ± 18 to 196 ± 15 bpm (P <0.05). Contractility and relaxation remained unchanged. Ivabradine reduced the positive chronotropic eff ect at all doses of dobutamine (10 nM: 232 ± 37 vs. 187 ± 19, 100 nM: 265 ± 37 vs. 211 ± 40, 1 μM: 316 ± 35 vs. 250 ± 39, 10 μM: 320 ± 33 vs. 235 ± 40 bpm; P <0.05). It shifted the maximum positive inotropic action of dobutamine to lower dose ranges (100 nM: 2,924 ± 841 vs. 2,978 ± 955, 300 nM: 3,743 ± 925 vs. 4,795 ± 1,298, 1 μM: 4,138 ± 935 vs. 4,896 ± 1,861 mmHg/second; P  <0.05). A comparable shift was seen for relaxation (100  nM: –2,178 ± 686 vs. –2,520 ± 742, 300 nM: –2,615 ± 726 vs. P227 The patients were divided randomly into two groups: levosimendan (six patients) treated with levosimendan and standard treatment, and the control group (six patients) with dobutamine and standard treatment. In all patients, serial TEE was performed studying the systolic function, by ejection fraction of the left ventricle with Simpson’s method. The TEEs were performed at the entrance of the patient, after 12 and 24 hours of treatment. The results were expressed as mean with standard deviation. For the comparison between the two groups we used the Fisher test, considered signifi cant with P <0.05. Conclusion Addition of ivabradine to dobutamine attenuates its chronotropic actions without diminishing its inotropic eff ects. A combination of levosimendan with ivabradine does not seem to provide benefi t. Clinical studies are necessary to confi rm these experimental results. P228 P228 Combined eff ects of ivabradine with dobutamine or levosimendan in isolated perfused hearts D Konrad, R Vicenzi-Moser, M Vicenzi, U Aldenhoff , E Schwarzl, W Toller Medical University Graz, Austria Critical Care 2013, 17(Suppl 2):P228 (doi: 10.1186/cc12166) References Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 S85 Table 1 (abstract P226). Treatment outcomes Variable Mean 95% CI CI Δd5 – d0 +1.34 0.4 to 2.2 PFR Δd5 – d0 +284 223 to 345 BE Δd5 – d0 +2.9 0.85 to 5 Lac Δd5 – d0 –1 –2 to 0.2 d0, treatment commenced. Table 1 (abstract P226). Treatment outcomes control group there remains a dysfunction in systolic function. We have shown the drug therapy based on levosimendan contributes to improving the systolic function of the left ventricle compared with treatment with dobutamine despite the initial cardiac stunning. Reference 1. Landoni G, et al.: Eff ects of levosimendan on mortality and hospitalization. A metanalysis of randomized controlled studies. Crit Care Med 2012, 40:634-646. 1. Landoni G, et al.: Eff ects of levosimendan on mortality and hospitalization. A metanalysis of randomized controlled studies. Crit Care Med 2012, 40:634-646. P228 Combined eff ects of ivabradine with dobutamine or levosimendan in isolated perfused hearts D Konrad, R Vicenzi-Moser, M Vicenzi, U Aldenhoff , E Schwarzl, W Toller Medical University Graz, Austria Critical Care 2013, 17(Suppl 2):P228 (doi: 10.1186/cc12166) –3,150 ± 888, 1  μM: –2,903  ±  752 vs. –2,972  ±  967  mmHg/second; P  <0.05). Levosimendan increased the HR only at high doses. With ivabradine, no positive chronotropic eff ect of levosimendan was observed (100 nM: 185 ± 30 vs. 162 ± 22, 1 μM: 208 ± 28 vs. 166 ± 29, 10 μM: 242 ± 27 vs. 168 ± 36 bpm; P <0.05). Ivabradine attenuated the positive inotropic eff ect of levosimendan (100 nM: 2,303 ± 303 vs. 1,737 ± 262, 1 μM: 2,977 ± 481 vs. 1,940 ± 449, 10 μM: 3,480 ± 941 vs. 2,189 ± 542 mmHg/ second; P <0.05) but did not signifi cantly alter its lusitropic eff ect. Conclusion Levosimendan is usually used in patients with cardiogenic shock unresponsive to conventional inotropes or mechanical augmentation. The mortality of this group is high but represents patients with shock refractory to conventional treatment. Its use in sepsis, myocarditis and pulmonary embolism is not well established. In this group levosimendan appears to have a favourable eff ect on gas exchange, renal function and tissue perfusion. Limitations include retrospective analysis and missing data. p y g Reference 1. Landoni G, et al.: Eff ects of levosimendan on mortality and hospitilisation. A meta-analysis of RCT. Crit Care Med 2012, 40:634-646. Intraabdominal hypertension and abdominal compartment syndrome in patients admitted to a medical and surgical ICU at a third referral university hospital Intraabdominal hypertension and abdominal compartment syndrome in patients admitted to a medical and surgical ICU at a third referral university hospital y p N Chindavech1, S Yenarkart2, S Kongsayreepong2 1Siriraj Hospital, Mahidol University, Bangkok, Thailand; 2Division of Critical Care Medicine, Bangkok, Thailand Critical Care 2013, 17(Suppl 2):P231 (doi: 10.1186/cc12169) Introduction Intraabdominal hypertension (IAH), especially abdominal compartment syndrome (ACS), can aff ect organ function leading to multiple organ failure. Appropriate and prompt management could improve survival. Less recognition of this problem in critically ill patients has been reported. The aim of this study was to study the prevalence, predictive factors and clinical outcome of IAH and ACS in a mixed population of critically ill patients by intermittent measuring bladder pressure during the ICU stay. Results The positive inotropic action of dobutamine was least at 37°C, more pronounced at 40°C and best at 34°C (37°C vs. 40°C vs. 34°C: 300 nM 280 ± 71 vs. 301 ± 94 vs. 345 ± 99%, 1 μM 310 ± 74 vs. 327 ± 92 vs. 389 ± 144%, 10 μM 297 ± 69 vs. 339 ± 96 vs. 359 ± 175%; P <0.05). Dobutamine’s positive lusitropic eff ect was not signifi cantly altered by temperature. The positive inotropic action of levosimendan was best at 37°C, in hyperthermia and hypothermia only the three highest doses of levosimendan increased contractility (37°C vs. 40°C vs. 34°C: 100 nM 121 ± 21 vs. 134 ± 15 vs. 87 ± 12%, 300 nM 135 ± 22 vs. 137 ± 17 vs. 94 ± 15%, 1 μM 153 ± 22 vs. 147 ± 21 vs. 108 ± 21%, 3 μM 172 ± 19 vs. 150 ± 24 vs. 115 ± 23%, 10 μM 173 ± 31 vs. 156 ± 28 vs. 117 ± 28%; P <0.05). The positive lusitropic eff ect of levosimendan at 37°C was almost absent in hypothermia and hyperthermia (P <0.05). g y Methods This prospective observational study was done in 130 adult patients (age >18 years) admitted to a medical and general surgical ICU at a third referral university hospital during June to November 2011. Variables about the patient’s profi le laboratory data and clinical outcome as ICU and hospital length of stay, and ICU, in-hospital and 28-day mortality were recorded. y y Results There were 33 (25.4%) medical and 93 (74.6%) surgical ICU patients in this study. Intraabdominal hypertension and abdominal compartment syndrome in patients admitted to a medical and surgical ICU at a third referral university hospital Surgical patients had higher prevalence of IAH than medical patients (57.8% vs. 33.3%, P = 0.015). Medical patients were admitted with severe sepsis/septic shock, AKI, pneumonia and ARDS. Surgical patients were more acutely ill with high ASA (III to IV), severity score, underwent emergency abdominal surgery and received more transfusion but were no diff erent in type of fl uid replacement. Signifi cant risk factors of IAH were coagulopathy (OR  =  2.09, 95% CI = 1.62 to 2.69), intraabdominal infection (OR = 1.87, 95% CI = 1.40 to 2.48), retroperitonium hematoma (OR = 1.82, 95% CI = 1.36 to 2.44), marked ascites (OR = 1.76, 95% CI = 1.32 to 2.36), acidosis (pH <7.2) (OR = 1.82, 95% CI = 1.37 to 2.43), severe sepsis/septic shock (OR = 1.63, 95% CI  =  1.14 to 2.33), and massive transfusion (OR  =  1.51, 95% CI = 1.10 to 2.08). Patients with IAH had more reopened surgeries and had higher ICU, hospital and 28-day mortality. Sixteen (12.3%) patients had ACS, 15 patients underwent emergency surgery, two patients had temporary abdominal closured and one-half of the patients had severe abdominal sepsis and massive transfusion. Fourteen patients died despite temporary abdominal closure. Delayed release abdominal tamponade were most causes of death. Conclusion In isolated perfused hearts, dobutamine has its best positive inotropic eff ect in hypothermia whereas levosimendan increases contractility best at normothermic conditions. Clinical studies are necessary to confi rm these experimental results. Confl icting results between V/Q SPECT and angioscan in pulmonary embolism: what to do? Confl icting results between V/Q SPECT and angioscan in pulmonary embolism: what to do? JM Martel, MN Nadeau, RD Daoust, JP Paquet, JC Chauny Hôpital du Sacré-Coeur de Montréal, Canada Critical Care 2013, 17(Suppl 2):P230 (doi: 10.1186/cc12168) Confl icting results between V/Q SPECT and angioscan in pulmonary embolism: what to do? JM Martel, MN Nadeau, RD Daoust, JP Paquet, JC Chauny Hôpital du Sacré-Coeur de Montréal, Canada Critical Care 2013, 17(Suppl 2):P230 (doi: 10.1186/cc12168) Introduction The objective of this study is to determine the fi nal clinical diagnosis of patients who underwent the double investigation for pulmonary embolism with confl icting results. Pulmonary embolism is a prevalent pathology in the emergency department (ED). A certain proportion of patients undergo a double radiological investigation (V/Q SPECT and angioscan), which incurs higher costs and X-ray doses. However, no study to date has addressed this issue. p Conclusion The prevalence and morbidity/mortality of IAH and ACS were high in this institute. Early appropriate and prompt management, especially fl uid and releasing a tamponade eff ect, were important. P232 P231 Intraabdominal hypertension and abdominal compartment syndrome in patients admitted to a medical and surgical ICU at a third referral university hospital N Chindavech1, S Yenarkart2, S Kongsayreepong2 1Siriraj Hospital, Mahidol University, Bangkok, Thailand; 2Division of Critical Care Medicine, Bangkok, Thailand Critical Care 2013, 17(Suppl 2):P231 (doi: 10.1186/cc12169) Intraabdominal hypertension and abdominal compartment syndrome in patients admitted to a medical and surgical ICU at a third referral university hospital N Chindavech1, S Yenarkart2, S Kongsayreepong2 1Siriraj Hospital, Mahidol University, Bangkok, Thailand; 2Division of Critical Care Medicine, Bangkok, Thailand Critical Care 2013, 17(Suppl 2):P231 (doi: 10.1186/cc12169) P229l i Results Patients in the two groups were statistically comparable with respect to sex (P = 0.31) and age (P = 0.53). The causes of the syndrome of Tako-tsubo were: subarachnoid hemorrhage (six patients) after coronary artery bypass graft (four patients), and polytrauma (two patients). All patients had low cardiac output. In the levosimendan group the ejection fraction at entrance was 25 ± 6%, after 12 hours 36 ± 10%, and 47 ± 5% after 24 hours. In the control group the ejection fraction at entrance was 24 ± 7%, after 12 hours 28 ± 6% and after 24 hours 33 ± 4%. Comparing the two groups we reached statistical signifi cance, P = 0.026. Infl uence of temperature on the cardiac action of dobutamine and levosimendan in isolated perfused hearts U Aldenhoff , R Vicenzi-Moser, M Vicenzi, D Konrad, E Schwarzl, W Toller Medical University Graz, Austria Critical Care 2013, 17(Suppl 2):P229 (doi: 10.1186/cc12167) Introduction Inotropic agents (catecholamines or calcium-sensitizers) are frequently used in hypothermic as well as hyperthermic patient conditions. Divergent results from animal experiments raise doubt as to whether they act to the same extent at diff erent body temperatures. Thus, we studied the infl uence of clinically relevant temperatures on the hemodynamic eff ects of dobutamine and levosimendan using the Langendorff model of isolated perfused hearts. Conclusion Comparing the two groups, we noticed that both started from a low cardiac output. However, in the group who used the drug therapy based on levosimendan we saw a return of systolic function of the left ventricle to near-normal levels within 24 hours, while in the S86 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Methods Isolated guinea pig hearts (n  =  60) were perfused with incremental doses (10 nM to 10 μM) of dobutamine or levosimendan either at normothermic (37°C), hyperthermic (40°C) or hypothermic (34°C) perfusion conditions. Contractility (+dLVP/dt), relaxation (–dLVP/dt), left ventricular pressures and heart rate were recorded. Data with increasing drug dosage were calculated in percent from baseline for each temperature tested. Data for each drug were analysed by two- way ANOVA for repeated measures including the two main eff ects of temperature and drug dose and their interaction. Data are reported as mean ± standard deviation. P232 Methods This retrospective study included patients who underwent a double investigation in the ED of Hôpital du Sacré-Coeur de Montréal from April 2008 to October 2012. Patients were selected from a computerized database of medical prescriptions (MediaMed Technologie). Data were then extracted from the patient’s fi les: patient characteristics, radiology report, diagnosis and treatment. Descriptive statistics were conducted. 3 Red cell distribution width predicts cardiovascular complications after high-risk surgery M Geisen, HD Aya, C Ebm, MA Hamilton, J Ball, M Grounds, A Rhodes, M Cecconi St George’s Healthcare NHS Trust, London, UK Critical Care 2013, 17(Suppl 2):P232 (doi: 10.1186/cc12170) Red cell distribution width predicts cardiovascular complications after high-risk surgery M Geisen, HD Aya, C Ebm, MA Hamilton, J Ball, M Grounds, A Rhodes, M Cecconi St George’s Healthcare NHS Trust, London, UK Critical Care 2013, 17(Suppl 2):P232 (doi: 10.1186/cc12170) Myocardial injury in critically ill patients admitted with noncardiac diagnosesfi Hunziker H, et al.: Crit Care 2012, 16:R89. 1. Bazick HS, et al.: Crit Care Med 2011, 39:1913-1921. 2. Hunziker H, et al.: Crit Care 2012, 16:R89. 1. Bazick HS, et al.: Crit Care Med 2011, 39:1913-1921. 2. Hunziker H, et al.: Crit Care 2012, 16:R89. References 1. Jin R, et al.: Circulation 2005, 111:3359-3365. 2. Stamous SC: Ann Thorac Surg 2011, 91:42-48. 1. Jin R, et al.: Circulation 2005, 111:3359-3365. 2. Stamous SC: Ann Thorac Surg 2011, 91:42-48. Myocardial injury in critically ill patients admitted with noncardiac diagnosesfi g J Lo1, K Lei2, I Webb2, J Coutts2, J Chambers2, A Griffi ths2, J Smith2, E Connell3, P Collinson3, J Peacock1, D Treacher2, M Ostermann2 1King’s College London, London, UK; 2Guys & St Thomas Foundation Hospital, London, UK; 3St George’s Hospital, London, UK Critical Care 2013, 17(Suppl 2):P234 (doi: 10.1186/cc12172) Introduction In the critically ill, the incidence of raised cardiac troponin T (cTnT) levels is high. Although the mechanisms of myocardial injury are not well understood, raised cTnT levels are associated with increased mortality. The aim of our study was to determine the incidence, prevalence and outcome of silent myocardial injury as determined by raised cTnT levels and concomitant ECG changes in critically ill patients admitted for noncardiac reasons. Results A total of 119 patients were included. Seventy-six (63%) patients developed complications postoperatively. Thirty patients (25.2%) developed cardiovascular complications. These patients had a higher median RDW than patients without cardiovascular compli- cations (14.7 vs. 13.8%, P <0.05). RDW on ICU admission was associated with serum lactate concentration on ICU admission: receiver operating characteristic analysis showed an area under the curve of 0.68 (SE = 0.06, P = 0.005; see Figure 1). RDW >14.35% predicted hyperlactataemia with a sensitivity of 76.0% and a specifi city of 71.1%. ad tted o o ca d ac easo s. Methods ECGs were taken and cTnT was measured daily during the fi rst week and on alternate days during the second week until discharge from the ICU or death. After completion of the study, all cTnT levels and ECGs were analysed independently and patients were classifi ed into four groups: defi nite MI (cTnT ≥15 ng/l and defi nite ECG changes of MI), possible MI (cTnT ≥15 ng/l and ischaemic changes on ECG), troponin rise alone (cTnT ≥15 ng/l with no ischaemic ECG changes), or normal. All medical notes were reviewed independently by two ICU clinicians. Results A total of 144 patients were included in the analysis (42% female; mean age 61.9 (SD 16.9); mean APACHE II score 19.4). In total, 121 patients (84%) had at least one cTnT level ≥15 ng/l during their stay in the ICU. Twenty patients (14%) fulfi lled criteria for a defi nite MI, of whom 65% were septic and 50% were on noradrenaline at the time (ICU and hospital mortality: 25% and 30%, respectively). P234 Myocardial injury in critically ill patients admitted with noncardiac diagnoses J Lo1, K Lei2, I Webb2, J Coutts2, J Chambers2, A Griffi ths2, J Smith2, E Connell3, P Collinson3, J Peacock1, D Treacher2, M Ostermann2 1King’s College London, London, UK; 2Guys & St Thomas Foundation Hospital, London, UK; 3St George’s Hospital, London, UK Critical Care 2013, 17(Suppl 2):P234 (doi: 10.1186/cc12172) Consequences of obesity in outcomes after cardiac surgery: analysis of the ARIAM registry EC Curiel-Balsera1, J Muñoz-Bono1, MJ Delgado-Amaya1, , , g y , R Hinojosa-Pérez2, A Reina-Toral3, A Gordillo-Brenes4, R Rivera-Fernández1 1Hospital Regional Carlos Haya, Málaga, Spain; 2Virgen del Rocio Hospital, Seville, Spain; 3Virgen de las Nieves Hospital, Granada, Spain; 4Puerta del mar Hospital, Cádiz, Spain Conclusion Eighty-four per cent of critically ill patients had a raised cTnT level at some stage during their stay in the ICU. More than 40% of patients fulfi lled criteria for a possible or defi nite MI, of whom only 20% were recognised clinically. ICU and hospital outcome were signifi cantly worse in patients with a cTnT rise. The proportion of patients with sepsis was similar between the patients with a defi nite, possible or no MI. Critical Care 2013, 17(Suppl 2):P233 (doi: 10.1186/cc12171) Critical Care 2013, 17(Suppl 2):P233 (doi: 10.1186/cc12171) Introduction Obesity is a disease that aff ects a large part of the population and has been associated with worse outcomes after cardiac surgery. The aim of our study is to evaluate the consequences of obesity related to postoperative complications, hospital length of stay and mortality. Red cell distribution width predicts cardiovascular complications after high-risk surgery Results In all, 125 patients underwent the double investigation. Our sample had a mean age of 63.1 years (SD ±18.6) and was composed of 82 (65.6%) women. One hundred and fi fteen patients (92%) underwent the V/Q SPECT fi rst. The result of 66 (52.8%) SPECT was intermediate or indeterminate. The fi nal diagnosis was pulmonary embolism for 23 patients (18.4%). A signifi cant proportion of patients (19, 38.0%) had confl icting results with the two tests. In this subpopulation, four (21.1%) had a fi nal diagnosis of pulmonary embolism. In the 16 patients with a result of high probability V/Q SPECT and negative angioscan, one (6.3%) had a fi nal diagnosis of pulmonary embolism, but three (100%) with low probability SPECT and positive angioscan were given this fi nal diagnosis. Introduction The red-cell distribution width (RDW) is associated with cardiovascular morbidity and mortality and is a predictor of ICU survival. It was the aim of this study to investigate the potential of RDW to predict postoperative cardiovascular complications (new onset of treated arrhythmias, myocardial ischaemia or initiation of vasopressor support) and its association with markers of tissue perfusion (serum lactate >2.5 mmol/l). Methods Analysis of prospectively recorded data for a register of patients admitted to an 18-bed ICU in a large teaching hospital after undergoing high-risk surgery. Haemodynamic and laboratory parameters during the fi rst 12  hours of ICU stay were recorded as well as demographic characteristics. Assessment for postoperative complications was performed using the postoperative morbidity survey and the Clavien-Dindo classifi cation. In addition, clinical outcome data (hospital mortality, length of ICU stay, length of hospital stay, readmission to the ICU) were recorded. i Conclusion Most patients underwent the double investigation because of intermediate or indeterminate V/Q SPECT results. In case of confl icting results, clinicians based their decision on the angioscan. In future studies, it would be useful to identify contributing factors for this discordance. Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 S87 http://ccforum.com/supplements/17/S2 Figure 1 (abstract P232). Prediction of hyperlactataemia according to RDW. in the development of overall postoperative complications (33% in obese and 35.8% in nonobese, P  =  0.07), although less appreciated were reoperation rate or stroke, as well as further development of postoperative renal failure. Red cell distribution width predicts cardiovascular complications after high-risk surgery After adjusting for severity and length of cardio bypass time, obese patients had lower mortality without being statistically signifi cant, OR = 0.94 (0.79 to 1.04). There were no diff erences in ICU length of stay, but obese patients had greater ward length of stay, 9.04 (10.43) versus 1.18 (9.2) days, P = 0.01. in the development of overall postoperative complications (33% in obese and 35.8% in nonobese, P  =  0.07), although less appreciated were reoperation rate or stroke, as well as further development of postoperative renal failure. After adjusting for severity and length of cardio bypass time, obese patients had lower mortality without being statistically signifi cant, OR = 0.94 (0.79 to 1.04). There were no diff erences in ICU length of stay, but obese patients had greater ward length of stay, 9.04 (10.43) versus 1.18 (9.2) days, P = 0.01. Conclusion Obese patients undergoing cardiac surgery have mortality, rate of complications and length of stay similar to nonobese patients. Obese patients required less reoperation but developed more frequent postoperative renal failure. R f Myocardial injury in critically ill patients admitted with noncardiac diagnosesfi Thirty-nine patients (27%) had a possible MI, of whom 69% were septic and on noradrenaline (ICU and hospital mortality: 31% and 41%, respectively). Sixty-two patients (43%) had a raised troponin without ECG, of whom 69% were septic and 50.7% were on noradrenaline (ICU and hospital mortality: 23% and 31%, respectively). Twenty-three patients had normal cTnT results and serial ECGs, of whom 61% had sepsis. ICU and hospital mortality was 4%. Only 25% of defi nite MIs and 18% of possible MIs were recognised by the clinical teams at the time. Methods ECGs were taken and cTnT was measured daily during the fi rst week and on alternate days during the second week until discharge from the ICU or death. After completion of the study, all cTnT levels and ECGs were analysed independently and patients were classifi ed into four groups: defi nite MI (cTnT ≥15 ng/l and defi nite ECG changes of MI), possible MI (cTnT ≥15 ng/l and ischaemic changes on ECG), troponin rise alone (cTnT ≥15 ng/l with no ischaemic ECG changes), or normal. All medical notes were reviewed independently by two ICU clinicians. y pi y Conclusion RDW is a potential parameter for perioperative risk stratifi cation. Further studies will have to elucidate the ability of acute elevations in RDW to refl ect impaired tissue perfusion. References p y y Results A total of 144 patients were included in the analysis (42% female; mean age 61.9 (SD 16.9); mean APACHE II score 19.4). In total, 121 patients (84%) had at least one cTnT level ≥15 ng/l during their stay in the ICU. Twenty patients (14%) fulfi lled criteria for a defi nite MI, of whom 65% were septic and 50% were on noradrenaline at the time (ICU and hospital mortality: 25% and 30%, respectively). Thirty-nine patients (27%) had a possible MI, of whom 69% were septic and on noradrenaline (ICU and hospital mortality: 31% and 41%, respectively). Sixty-two patients (43%) had a raised troponin without ECG, of whom 69% were septic and 50.7% were on noradrenaline (ICU and hospital mortality: 23% and 31%, respectively). Twenty-three patients had normal cTnT results and serial ECGs, of whom 61% had sepsis. ICU and hospital mortality was 4%. Only 25% of defi nite MIs and 18% of possible MIs were recognised by the clinical teams at the time. 1. Bazick HS, et al.: Crit Care Med 2011, 39:1913-1921. 2. P235 Methods An observational, prospective, multicenter study of patients included in the ARIAM registry of adult cardiac surgery between March 2008 and March 2011. We analyzed clinical variables, the surgical procedure, postoperative complications and mortality, comparing the group of patients with body mass index (BMI) greater or less than 30 kg/m2. P235 Validation of the GRACE score risk in our population L Sayagues1, J Sieira1, E Abbu2, J Chico3, C Pena3, J Gonzalez Juantey2 1HULA, Lugo, Spain; 2CHUS, Santiago de Compostela, Spain; 3CHUVI, Vigo, Spain Critical Care 2013, 17(Suppl 2):P235 (doi: 10.1186/cc12173) P235 Validation of the GRACE score risk in our population L Sayagues1, J Sieira1, E Abbu2, J Chico3, C Pena3, J Gonzalez Juantey2 1HULA, Lugo, Spain; 2CHUS, Santiago de Compostela, Spain; 3CHUVI, Vigo, Spain Critical Care 2013, 17(Suppl 2):P235 (doi: 10.1186/cc12173) 35 Validation of the GRACE score risk in our population Validation of the GRACE score risk in our population L Sayagues1, J Sieira1, E Abbu2, J Chico3, C Pena3, J Gonzalez Juantey2 1HULA, Lugo, Spain; 2CHUS, Santiago de Compostela, Spain; 3CHUVI, Vigo, Spain C iti l C 2013 17(S l 2) P235 (d i 10 1186/ 12173) p p L Sayagues1, J Sieira1, E Abbu2, J Chico3, C Pena3, J Gonzalez Juantey2 1HULA, Lugo, Spain; 2CHUS, Santiago de Compostela, Spain; 3CHUVI, Vigo, Spain Critical Care 2013, 17(Suppl 2):P235 (doi: 10.1186/cc12173 Results The study included 4,712 patients with a mean age of 64.03 (SD ±12.08) years, BMI 28.53 (SD ±4.7) and EuroSCORE 5.58 (SD ±2.91). In 1,940 patients (35.7%) BMI was >30 kg/m2. There were no diff erences Introduction The Global Registry of Acute Coronary Events (GRACE) risk score provides an estimation of the probability of death within S88 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 6  months of hospital discharge in patients with acute coronary syndrome (ACS). Our aim was to assess the validity of this risk score in our contemporary cohort of patients admitted to our third-level hospital. Figure 2 (abstract P236). Incomplete and complete revascularization. p Methods The study involved 1,185 consecutive patients with ACS evaluated between February 2004 and February 2009. Their virtual status was determined 6 months after hospital discharge and the validity of the GRACE risk score was evaluated. y Results In total, 450 (38.8%) patients were admitted for STEMI and 725 (61.2%) for NSTEMI. Percutaneous revascularisation was performed in 846 (71.5%). p Critical Care 2013, 17(Suppl 2):P236 (doi: 10.1186/cc12174) Introduction This is a prospective study in which all ACS cases attending a level 3 hospital were collected consecutively from February 2004 to 2010, and were clinically followed-up until 2012. Introduction In patients with severe and inoperable aortic stenosis, some trials have proven that TAVI excels standard procedures and has also been proven less cost-eff ective. We aim at reviewing short-term results after 2 years since the implementation of this technique in our premises. y Methods Of a total 4,589 cases, only 2,515 were revascularized with ICP (1,742 complete and 907 incomplete; 160 failed). We split the cases into two main groups: STEMI and NSTEMI. p Methods A study of a retrospective cohorts of patients who underwent isolated aortic valve replacement (AVR) by either conventional surgery or TAVI (CoreValve device) from June 2010 to December 2011 at the University Hospital Carlos de Haya (Málaga, Spain). Clinical epidemiologic, complication and short-term outcome variables were registered. Qualitative variables are expressed as percentages, while quantitative variables are expressed as means and SD. Fisher’s exact test and Mann–Whitney’s U-test were used where necessary (5% maximum error ratio). g p Results We observed a 0.6 higher accumulative survival rate in patients with complete ICP over patients who underwent surgery, incomplete ICP or mixed treatment. Those with conservative measures solely have, obviously, worst prognosis. See Figures 1 and 2. Figure 1 (abstract P236). FEVI and prognosis. Results A total number of 27 TAVI and 154 isolated AVR procedures were completed. Intervention typology was chosen according to the recommendations of scientifi c societies, apart from patients’ fulfi llment of the anatomic criteria required for percutaneous implant. Mean age was 67 ± 11 years (54% males) in AVR and 80 ± 6 years (44% males) in TAVI (P <0.05). The additive EuroSCORE in AVR was 7 ± 2 and 9 ± 2 in TAVI (P <0.05). However, 55.6% of the percutaneous-valve patients presented previous coronary-tree alterations with stent implantation, while only 7% of AVR patients showed these alterations (P <0.001). ICU mortality in TAVI and AVR patients was 3.7% and 8.2%, respectively (P  =  NS). Regarding complications, 48.1% of TAVI patients showed altered heart rhythm and 33% required a permanent pacemaker. Electrical disorders were observed in 4% of AVR patients, while 1.9% of these patients required a permanent pacemaker (P <0.001 for both). Reoperation was necessary in 14.8 and 1.9% of TAVI and AVR patients, respectively (P <0.001). P235 The median GRACE risk score was 121 (interquartile range 96/144). Mortality after discharge was 4.4%. The calibration of the GRACE score was Hosmer–Lemeshow P >0.2 and its discriminatory capacity was excellent. Area under the ROC curve was 0.86, 95% CI 0.807 to 0.916, in all patients. See Table 1. Table 1 (abstract P235) GRACE risk Total STEMI NSTEMI Low 270 (22.7) 177 (38.3) 93 (12.8) Medium 334 (28.2) 141 (30.7) 193 (26.7) High 580 (49) 142 (30.9) 438 (60.5) Figure 2 (abstract P236). Incomplete and complete revascularization. Figure 2 (abstract P236). Incomplete and complete revascularization. Conclusion In our study patients undergoing ICP have higher survival rates in comparison with cardiac surgery except those >65 years old and diabetic groups in which cardiac surgery has higher ratios than ICP. In the rest of the groups, no matter how many coronary arteries were aff ected, only those with complete ICP present higher survival rates. It would be important to repeat this study in a multicentric cohort. Conclusion The GRACE risk score for predicting death within 6 months of hospital discharge was validated and can be used in patients with ACS. It would be perfect in the future to include the GRACE risk score in the medical records of this type of patients. Also it would be very interesting to validate this in a multicentric study. Non-invasive valve implantation with TAVI versus conventional aortic valve replacement Non-invasive valve implantation with TAVI versus conventional aortic valve replacement E Trujillo-García, C Joya-Montosa, MJ Delgado-Amaya, E Curiel-Balsera Hospital Regional Carlos Haya, Málaga, Spain Critical Care 2013, 17(Suppl 2):P237 (doi: 10.1186/cc12175) p p L Sayagues1, J Sieira1, E Abbu Assi2, J Chico3, C Pena3, J Gonzalez Juanatey2 1HULA, Lugo, Spain; 2CHUS, Santiago de Compostela, Spain; 3CHUVI, Vigo, Spain aortic valve replacement E Trujillo-García, C Joya-Montosa, MJ Delgado-Amaya, E Curiel-Balsera Hospital Regional Carlos Haya, Málaga, Spain Critical Care 2013, 17(Suppl 2):P237 (doi: 10.1186/cc12175) P238 Time to recover from shock is determinant of a positive fl uid balance in septic shock S Lobo, AL Cunha Faculdade de Medicina de São José do Rio Preto, Brazil Critical Care 2013, 17(Suppl 2):P238 (doi: 10.1186/cc12176) Introduction Excess fl uids may be harmful in critically ill patients. We aimed to evaluate the cumulative fl uid balance during 7 days in patients with septic shock after recovery from shock. Methods A prospective and observational study in septic shock patients. Patients with MAP >65 mmHg and lactate <2.0 mEq/l were included <12  hours after weaning from vasopressors. Daily fl uid balance was registered during 7  days after the enrollment. Patients were divided into two groups according to the full cohort’s median cumulative fl uid balance administered during the period of shock (use of vasopressors) calculated on study day 1: Group 1 ≤4.4 l (n = 20) and Group 2 >4.4 l (n = 20). i Results Seventy-nine patients were enrolled. Fifty-one patients were with moderate UGIB, and 28 patients were with severe UGIB. As indicated in Tables  1 and 2, there was a signifi cant relationship between CLac and sustained bleeding in moderate UGIB (P = 0.02). On the other hand, there was no signifi cant relationship between CLac and sustained bleeding in severe UGIB (P = 0.58). p Results Cumulative fl uid balance was 1.6  ±  1.8 l in Group 1 and 10.2 ± 4.1 l in Group 2 and 8.5 ± 5.3 l in Group 1 and 18.5 ± 7.9 l in Group 2 on study day 8 (P <0.001 for both). Time for recovery from shock was predictive of receiving larger volume of fl uids (OR: 1.38, 95% CI: 1.08 to 1.75, P = 0.009). After zeroing fl uid balance on study day 2, 7 days cumulative fl uid balance continues to increase in both groups (Figure 1). Patients in Group 2 had more prolonged length of stay in the ICU and in hospital than patients in Group 1. Table 1 (abstract P239). Relationship between lactate clearance and sustained bleeding in moderate UGIB (n = 51) Table 1 (abstract P239). Relationship between lactate clearance and sustained bleeding in moderate UGIB (n = 51) Sustained bleeding Nonsustained bleeding (n = 19) (n = 32) P value CLac <0.5 18 (95%) 21 (65%) 0.02 CLac ≥0.5 1 (5%) 11 (34%) Conclusion After recovery from septic shock we notice a huge accu- mulated fl uid balance. A more positive fl uid balance was associated with a more prolonged length of stay in the ICU and in the hospital. P239 Lactate clearance is a predictor of sustained bleeding in emergency room patients with moderate upper gastrointestinal bleeding T Wada1, A Hagiwara2, N Yahagi1, A Kimura2 1University of Tokyo, Japan; 2National Center for Global Health and Medicine, Tokyo, Japan Critical Care 2013, 17(Suppl 2):P239 (doi: 10.1186/cc12177) Figure 1 (abstract P238). Figure 1 (abstract P238). Table 2 (abstract P239). Relationship between lactate clearance and sustained bleeding in severe UGIB (n = 28) Sustained bleeding Nonsustained bleeding (n = 6) bleeding (n = 22) P value CLac <0.5 4 (67%) 18 (82%) 0.58 CLac ≥0.5 2 (33%) 4 (18%) Conclusion If an initial fl uid therapy for moderate UGIB results in high CL h bl di i h l d h d F h i Table 2 (abstract P239). Relationship between lactate clearance and sustained bleeding in severe UGIB (n = 28) Conclusion If an initial fl uid therapy for moderate UGIB results in high CLac, the bleeding might already have stopped. For such a patient, we may save urgent endoscopy. p Critical Care 2013, 17(Suppl 2):P236 (doi: 10.1186/cc12174) y Conclusion Even with our limited experience, TAVI patients are observed to be older, to involve higher surgical risk, and to have undergone previous coronary-tree interventions. Although no Figure 1 (abstract P236). FEVI and prognosis. S89 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 signifi cant diff erences were found regarding mortality, a higher rate of complications was observed in TAVI patients. With no short-term diff erences, a signifi cant rate of postsurgical complications, and a cost- effi ciency handicapped technique, analysis of long-term outcomes seems necessary to assess TAVI’s advantages over conventional AVR. UGIB patient needs an intervention or not. However, the intervention which the GBS mentions includes not only endoscopy but also blood transfusion. Therefore, we cannot determine whether a UGIB patient needs urgent endoscopy or just blood transfusion by GBS alone. We hypothesized that high lactate clearance (CLac) would decrease the likelihood of sustained UGIB. Methods This is a retrospective study. UGIB patients, who visited the emergency department (ED) of the National Center for Global Health and Medicine from April 2011 to March 2012 and received urgent endoscopy in the ED, were enrolled. We collected for each patient the GBS, the blood lactate value on arrival in the ED, the blood lactate value after bolus administration of 20 to 40  ml/kg Ringer’s acetate (initial fl uid therapy) and the report of urgent endoscopy. We classifi ed the severity of UGIB according to GBS. A score ≤12 was classifi ed as moderate, and a score ≥13 was classifi ed as severe. CLac was defi ned as the percentage decrease in blood lactate from the time of arrival in the ED to the time when an initial fl uid therapy was fi nished. CLac <50% was defi ned as low, and CLac ≥50% was defi ned as high. Whether a patient had sustained bleeding or not was determined based on the report of urgent endoscopy. The relationship between CLac and sustained bleeding was examined by Fisher’s exact test, and P <0.05 was considered statistically signifi cant. P238 P238 Time to recover from shock is determinant of a positive fl uid balance in septic shock S Lobo, AL Cunha Faculdade de Medicina de São José do Rio Preto, Brazil Critical Care 2013, 17(Suppl 2):P238 (doi: 10.1186/cc12176) P240 P240 Muscular glucose assessed by microdialysis and blood glucose can predict mortality in septic shock A Massoudi, M Belhadj Amor, C Romdhani, A Ben Gabsia, I Labbene, M Ferjani Military Hospital of Tunis, Tunisia Critical Care 2013, 17(Suppl 2):P240 (doi: 10.1186/cc12178) predict mortality in septic shock A Massoudi, M Belhadj Amor, C Romdhani, A Ben Gabsia, I Labbene, M Ferjani Military Hospital of Tunis, Tunisia Critical Care 2013, 17(Suppl 2):P240 (doi: 10.1186/cc12178) Conclusion After recovery from septic shock we notice a huge accu- mulated fl uid balance. A more positive fl uid balance was associated with a more prolonged length of stay in the ICU and in the hospital. Introduction The aim of our study was to assess the muscular glucose by microdialysis and its association with mortality in septic shock patients. Introduction The aim of our study was to assess the muscular glucose by microdialysis and its association with mortality in septic shock patients. P239 Lactate clearance is a predictor of sustained bleeding in emergency room patients with moderate upper gastrointestinal bleeding T Wada1, A Hagiwara2, N Yahagi1, A Kimura2 1University of Tokyo, Japan; 2National Center for Global Health and Medicine, Tokyo, Japan Critical Care 2013, 17(Suppl 2):P239 (doi: 10.1186/cc12177) Methods We conducted a preliminary prospective study. We included septic shock patients hemodynamically optimized according to international recommendations. A microdialysis catheter was inserted in the femoral quadriceps. Interstitial fl uid samples were collected every 6 hours for 5 days. The determination of muscular glucose was performed by the CMA 600 analyzer (CMA/Microdialysis AB, Sweden). We also performed a dosage of concomitant blood glucose. The study population was divided into two groups according to hospital mortality. Statistic analysis: Mann–Whitney test and chi-squared test: Introduction There is no useful predictor of sustained upper gastro- intestinal bleeding (UGIB). Glasgow–Blatchford scoring (GBS) is based on simple clinical and laboratory variables and can predict whether a S90 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 comparisons between groups. Quantitative variables were expressed as mean  ±  standard deviation or median (interquartile range) as appropriate. Nasogastric tube dislodgement: a problem on our ICU B Morton, R Hall, T Ridgway, O Al-Rawi Liverpool Heart & Chest, Liverpool, UK Critical Care 2013, 17(Suppl 2):P242 (doi: 10.1186/cc12180) Results We included 12 patients with septic shock. The mortality rate was 50%. P240 Demographics were comparable between groups except for age (66 ± 9 vs. 41 ± 12, dead patients vs. survivors, respectively; P = 0.002). Pneumonia was the major cause of septic shock (10 patients). We analysed 167 blood samples and 166 muscular glucose samples. We found a positive association between muscular glucose, blood glucose and mortality. Tissue glucose was signifi cantly higher among dead patients compared with survivors at the 54th hour. Comparing all data, muscular glucose (P = 0.02) and blood glucose (P = 0.007) were signifi cantly higher in dead patients (Table 1). Introduction It was noted on our unit that dislodgement of nasogastric tubes occurred commonly. This can lead to an increased risk of aspiration, interruptions in nutritional support, skin breakdown and radiographic exposure [1]. It is recommended that the position of nasogastric tubes should be confi rmed by aspiration and pH testing, with radiographic confi rmation used only when this is not possible [2]. Methods We performed a retrospective review of chest X-ray (CXR) requests for the 3-month period June to August 2012 using the trust radiology information system. The proportion of CXR requests for confi rmation of position and patient demographics were measured with an estimation of the fi nancial cost performed. Table 1 (abstract P240). Association between muscular glucose, blood glucose and mortality Dead patients Survivors (n = 6) (n = 6) P value Muscular glucose (mmol/l) 9.4 (5.66; 13.71) 7.87 (5.62; 10.41) 0.02 Blood glucose (mmol/l) 10.9 (8.3; 15.5) 8.6 (6.9; 11.4) 0.007 Table 1 (abstract P240). Association between muscular glucose, blood glucose and mortality i Results There were 541 patients admitted to the critical care area in the study period. In total, 207 out of 2,340 (8.8%) CXRs performed were for confi rmation of position. Repeated X-rays were required in some patients (see Table 1); these patients were older and tended to have a longer length of stay. A mobile CXR costs £25 in our trust, if one CXR is accepted per patient with a nasogastric tube; there was an excess of 160 images with a cost of £4,000 in the 3-month period. Conclusion Our data suggest that muscular glucose assessed by microdialysis and blood glucose are associated with mortality in septic shock patients. Therefore, muscular glucose may refl ect the metabolic alterations and microcirculatory dysfunction induced by septic shock. y References References 1. Lorente L, et al.: Accidental catheter removal in critically ill patients: a 1. Lorente L, et al.: Accidental catheter removal in critically ill patients: a prospective and observational study. Crit Care 2004, 8:R229-R233. 2. National Patient Safety Agency: Reducing Harm Caused by the Misplacement of Nasogastric Feeding Tubes. NPSA/2011/PSA002. NHS; 2011. [http://www.nrls.npsa.nhs.uk/alerts/?entryid45=129640] Conclusion There was a high prevalence of constipation among critical care patients with poor adherence to the bowel protocol, which requires improvements in staff awareness and new recommendations (drug chart amendment, and so forth). Duration of mechanical ventilation and failure to feed were greater in constipated than nonconstipated patients. This may indicate a common correlation and further studies are warranted. Due to the possible signifi cant impact of constipation on patients’ recovery, each critical care unit should introduce a bowel protocol or ensure compliance with the existing one before the evidence is clearly established. f Handling of dietary protein in critically ill patients Handling of dietary protein in critically ill patients Handling of dietary protein in critically ill patients F Liebau1, O Rooyackers1, L Van Loon2, J Wernerman1 1Karolinska University Hospital Huddinge, Stockholm, Sweden; 2NUtrIm Handling of dietary protein in critically ill patients F Liebau1, O Rooyackers1, L Van Loon2, J Wernerman1 1Karolinska University Hospital Huddinge, Stockholm, Sweden; 2NUtrIm, Maastricht University, Maastricht, the Netherlands Critical Care 2013, 17(Suppl 2):P243 (doi: 10.1186/cc12181) g y y Liebau1, O Rooyackers1, L Van Loon2, J Wernerman1 , y , , 1Karolinska University Hospital Huddinge, Stockholm, Sweden; 2NUtrIm M i h U i i M i h h N h l d Maastricht University, Maastricht, the Netherlands Critical Care 2013, 17(Suppl 2):P243 (doi: 10.1186/cc12181) y Critical Care 2013, 17(Suppl 2):P243 (doi: 10.1186/cc12181) Introduction Protein turnover measurements by stable isotope techniques can be applied to assess the nutritional/metabolic status in critically ill patients and their response to feeding. Because of uncertain gastrointestinal transport and uptake of nutrients, their contribution needs to be measured separately. We studied whole-body protein kinetics, with special emphasis on the contribution of dietary protein, in ICU patients and healthy controls. Introduction Protein turnover measurements by stable isotope techniques can be applied to assess the nutritional/metabolic status in critically ill patients and their response to feeding. Because of uncertain gastrointestinal transport and uptake of nutrients, their contribution needs to be measured separately. We studied whole-body protein kinetics, with special emphasis on the contribution of dietary protein, in ICU patients and healthy controls. 2. Palácio de Azevedo R: Intestinal constipation in intensive care unit. Rev Bras Ter Intensiva 2009, 21:324-331. P241 Constipation in critically ill patients and its relationship to feeding and weaning from respiratory support g p E Spodniewska, A Guha p Royal Liverpool University Hospital, Liverpool, UK y p y p p Critical Care 2013, 17(Suppl 2):P241 (doi: 10.1186/cc12179) Critical Care 2013, 17(Suppl 2):P241 (doi: 10.1186/cc12179) Introduction Constipation can be defi ned as a failure of the bowels to open for 3 consecutive days. It is a common problem in intensive care settings but not many studies have so far raised the subject. Methods The audit had the Trust Audit Committee’s approval. The existing protocol was used as the benchmark. Patients were studied prospectively to assess compliance with the local bowel protocol, incidence of constipation and relationship to weaning from respiratory support and feeding. All HDU and all mechanically ventilated ICU patients who stayed on the ward for more than 3  days were included, except for patients after bowel surgery and patients with encephalopathy. Conclusion An excess of CXRs were performed for confi rmation of nasogastric tube in our patient population. The recommended methods for position confi rmation were reinforced amongst medical staff . The high number of repeated imaging for some patients indicates that dislodgement of tubes was also a problem. We propose that nasogastric tubes should be bridled after fi rst dislodgement or at tracheostomy insertion to minimise dislodgement in the future. Results Among the 24 HDU and 21 ICU patients audited in the Royal Liverpool University Hospital, 67% and 57% respectively were constipated. Laxatives were prescribed when patients had not opened their bowels for 3 days in 25% HDU and 75% ICU cases. Taking into consideration that the median age, APACHE II score and length of stay for constipated and nonconstipated patients were similar, the relationship to feeding and respiratory support were assessed. Constipated patients required mechanical ventilation for an average of 6.8 days and nonconstipated for 4.3 days. Failure to feed was observed at least once in 58% constipated and 44% nonconstipated ICU patients and 19% constipated and 12.5% nonconstipated HDU patients. 1. Mostafa SM: Constipation and its implications in the critically ill patient. Br J Anaesth 2003, 91:815-819. P240 Table 1 (abstract P242) Table 1 (abstract P242) Mean days CXR for Total NG Total from fi rst to Mean NG Patients CXR CXR Proportion last CXR age 1 47 47 321 0.15 8.45 67.55 2 16 32 181 0.18 15.94 69.63 3 7 21 87 0.24 17.86 70.00 4 10 40 149 0.27 15.90 69.20 5 3 15 52 0.29 23.33 66.00 6 3 18 61 0.30 40.00 76.67 8 2 16 52 0.31 39.50 80.00 9 2 18 46 0.39 29.50 83.00 Confi rmation of nasogastric tube placement in critical care M Moore, R Thomson Confi rmation of nasogastric tube placement in critical care M Moore, R Thomson St Georges Healthcare NHS Trust, London, UK Critical Care 2013, 17(Suppl 2):P244 (doi: 10.1186/cc12182) St Georges Healthcare NHS Trust, London, UK Critical Care 2013, 17(Suppl 2):P244 (doi: 10.1186/cc12182) Conclusion In this population of ICU patients, routine/daily checks of NG pH aspirate appear to be limited. This is almost certainly due to the use of continuous NG feed together with PPIs. The usefulness of pH testing in newly placed NG tubes, however, appears more reliable. Introduction Placement of nasogastric tubes (NGTs) is commonplace in critical care. Misplacement of NGTs is rare and considered a never event [1]. Strategies to avoid never events (by confi rming NGT position) include pH analysis of gastric secretions or chest X-ray confi rmation of tube position. For this reason the authors set out to establish the effi ciencies surrounding safe placement of NGTs in a 17-bed adult cardiothoracic critical care unit in a large teaching hospital. Figure 2 (abstract P245). pH values – new NG tubes. Figure 1 (abstract P245). pH values – routine checks. Figure 1 (abstract P245). pH values – routine checks. Methods This small-scale study of 25 NGT placements during a 5-week period collated data supplied by questionnaire by healthcare workers responsible for NGT placements.i Results Analysis of Adverse Incident Reports identifi ed no never events of misplaced NGTs within the previous 10  years. This audit revealed that the commonest type of NGT was a radio-opaque tube with stylet (corfl o) (92% of placements), with occasional use of the electromagnetic placement system (cortrak) (8% of placements). Sizes 10 (40%) and 12 (56%) were most common. Tube placement was confi rmed by: X-ray (72%); pH of aspirates (35%); electromagnetic tube placement (one patient). The time taken from decision to place NGT to use varied (range 15 to 510 minutes). Little distinction was seen in the time taken to use and NGT confi rmed by aspirate alone (205 minutes) or by X-ray (220  minutes), although the shortest interval was seen in electromagnetic NGT placement (15  minutes). The cost of NGTs confi rmed by aspirate alone was low (approximately £10.00), higher with X-ray confi rmation/electromagnetic placement (approximately £45.00). Figure 1 (abstract P245). pH values – routine checks. Figure 2 (abstract P245). pH values – new NG tubes. pH testing to confi rm nasogastric tube position on the ICU: are we wasting our time? p Results Protein net balance was lower in patients than in the reference group at baseline (–1.8 ± 1.7 vs. 0.6 ± 0.6 mg/kg BW/hour, P = 0.003), and after enteral feeding (–1.1  ±  1.5 vs. 0.6  ±  0.6  mg/kg BW/hour, P  =  0.049). Recovery of labelled Phe from enteral feeding into the systemic circulation was higher in the reference group as compared with patients (20.3 + 11.2% vs. 7.0 + 4.8%, P = 0.005). Enteral feeding did not aff ect protein metabolism in the reference group. In patients, protein breakdown became slightly lower during enteral feeding (10.6 ± 3.3 vs. 11.2 ± 3.3 mg/kg BW/hour, P = 0.086) and protein net balance became slightly higher (–1.1 ± 1.5 vs. –1.8 ± 1.7 mg/kg BW/ hour, P = 0.086). P Temblett, S George Introduction For such a simple procedure, the insertion and position checking of nasogastric (NG) tubes can be very problematic. The UK’s National Patient Safety Agency declared that ‘Placement of NG tubes together with confi rmation of correct placement can carry signifi cant risks’ and recommends that measuring the pH of NG aspirate should be the fi rst-line method of determining correct NG position (safe range ≤5.5) [1]. Methods In order to assess the usefulness of pH testing of NG aspirates in critical care patients, a prospective survey of pH testing of NG tube aspirate was carried out. This was undertaken both in patients who had a newly placed NG tube and in patients who were having regular/ routine checks of their existing NG tube. Conclusion Intrinsically isotope-labelled casein can be used to quantify dietary contribution to protein metabolism in critically ill patients. Hypocaloric enteral feeding marginally improved protein balance in these patients. The low recovery of enterally administered labelled amino acid underlines the need to quantify uptake from the gastrointestinal tract when protein turnover measurements are performed in critically ill patients on enteral nutrition. Results A total of 168 separate pH readings in 41 ICU patients receiving continuous enteral nutrition were recorded. In total, 137/168 patients were receiving proton pump inhibitors (PPIs). Eighteen readings were from newly placed NG tubes and 150 readings from old NG tubes. Fifty- three per cent of routine pH readings were falsely high; that is, pH 6 or above despite the NG tube being in the stomach (Figure 1). References 1. Mostafa SM: Constipation and its implications in the critically ill patient. Br J Anaesth 2003, 91:815-819. 2. Palácio de Azevedo R: Intestinal constipation in intensive care unit. Rev Bras Ter Intensiva 2009, 21:324-331. S91 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Methods Mechanically ventilated, not enterally fed ICU patients (n = 9) were recruited from an interdisciplinary ICU. Healthy, overnight-fasted volunteers (n = 6) served as reference. A primed constant i.v. infusion of 2H-labeled phenylalanine (Phe) and tyrosine was used to quantify whole-body protein metabolism. Patients remained on parenteral nutrition (PN) as clinically indicated; controls received PN starting 2.5 hours before starting enteral feeding. Intrinsically 13C-Phe-labeled casein was infused for 6 hours by nasogastric tube at 0.75 g protein/ hour, together with maltodextrin at 2.73 g/hour. Protein breakdown, synthesis, net balance, and Phe splanchnic extraction were calculated before and at the end of the enteral feeding period, using equations for steady-state whole-body protein kinetics. Comparisons were made by Wilcoxon matched pairs and Mann–Whitney U tests; values are reported as mean ± SD. confi rmations. Even if the frequency and volume of gastric aspiration were greater, there is a belief that pH testing may not be suffi ciently accurate (since many factors alter patients’ gastric pH). It is possible that new technologies such as electromagnetic NGT placement may allow faster/equally safe practices. Further study including cost/benefi t analysis will be needed to confi rm this. Reference 1. National Patient Safety Agency: Patient Safety Alert (5): Reducing the Harm Caused By Misplaced Nasogastric Feeding Tubes. London: NPSA; 2005. [http://www.nrls.npsa.nhs.uk/resources/?EntryId45=59794] pH testing to confi rm nasogastric tube position on the ICU: are we wasting our time? Twenty- eight per cent of newly placed NG tubes had falsely high pH readings (Figure 2). Reference Reference 1. National Patient Safety Agency: Reducing the Harm Caused by Misplaced Nasogastric Feeding Tubes. NPSA/PSA001./PSA002. NPSA; 2005, 2011. [www.nrls.npsa.nhs.uk] P246 Eff ect of probiotic containing lactobacillus, bifi dobacterium and streptococcus thermophilus in critically ill patients M Ebrahimi Mamaghani, S Sanaie, A Mahmoudpour, H Hamishehkar Tabriz University of Medical Sciences, Tabriz, Iran Critical Care 2013, 17(Suppl 2):P246 (doi: 10.1186/cc12184) Introduction Sepsis is the most common cause of death in ICUs [1]. Destruction of intestinal barrier function and increased translocation of bacteria to systemic blood fl ow can lead to sepsis [2]. Probiotics may have benefi cial eff ects in improvement of critically ill patients by modulating intestinal barrier and reduction of infl ammation [3]. The aim of this trial was to determine the eff ect of probiotic on infl ammatory biomarkers and mortality rate of sepsis in critically ill patients in the ICU. p Methods This double-blind, randomized controlled trial was conducted on 40 critically ill patients admitted to the ICU. They were randomly assigned to receive placebo or probiotic for 7  days. The APACHE score, Sequential Organ Failure Assessment (SOFA) and systemic concentrations of IL-6, procalcitonin (PCT) and protein C were measured before initiation of the study and on days 4 and 7. Also, 28- day mortality was evaluated for each patient. y y p Results IL-6 and PCT levels decreased and protein C levels increased signifi cantly in probiotic group over the treatment period (P <0.001). There was a signifi cant diff erence in IL-6, PCT and protein C levels of the 7th day between two groups (P = 0.001, 0.006 and <0.001, respectively). Compared with controls, probiotic was eff ective in improving APACHE and SOFA scores in 7 days (P <0.001). There was signifi cant diff erence between the probiotic and control group in the 28-day mortality rate (20% vs. 55% respectively, P = 0.048).l y Conclusion Probiotics reduce infl ammation and mortality rate in critically ill patients and might be considered as an adjunctive therapy to sepsis. Figure 1 (abstract P247). Selenium levels before and after 10 days of admission. p References Confi rmation of nasogastric tube placement in critical care M Moore, R Thomson Conclusion Despite the small dataset the results demonstrate a concerning delay in the application of enteral feeding and/or drug administration. Whilst reassuring in the steps taken to avoid never events, this study demonstrates that there may be delays in time-critical administration of enteral medicine or optimal nutritional practices. This study reveals a signifi cant problem with aspirating gastric contents for pH testing, necessitating a large number of X-ray position Figure 2 (abstract P245). pH values – new NG tubes. S92 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Table 1 (abstract P247). Mean selenium levels in diff erent sources of sepsis Source Quantity Level (mg/dl) Abdominal 12 0.43 Chest 12 0.71 Skin 3 0.55 CNS 2 0.53 GU 2 0.58 Figure 1 (abstract P247). Selenium levels before and after 10 days of admission. Table 1 (abstract P247). Mean selenium levels in diff erent sources of sepsis Selenium levels in patients with diff erent sources of sepsis L French, P Temblett Morriston Hospital, Swansea, UK Critical Care 2013, 17(Suppl 2):P247 (doi: 10.1186/cc12185) Selenium levels in patients with diff erent sources of sepsis L French, P Temblett Morriston Hospital, Swansea, UK Critical Care 2013, 17(Suppl 2):P247 (doi: 10.1186/cc12185) p References 1. Angus DC, Linde-Zwirble WT, Lidicker J, Clermont G, Carcillo J, Pinsky MR: Epidemiology of severe sepsis in the USA: analysis of incidence, outcome, and associated costs of care. Crit Care Med 2001, 29:1303-1310. 1. Angus DC, Linde-Zwirble WT, Lidicker J, Clermont G, Carcillo J, Pinsky MR: Epidemiology of severe sepsis in the USA: analysis of incidence, outcome, and associated costs of care. Crit Care Med 2001, 29:1303-1310. Conclusion All patients admitted with early septic shock had subnormal selenium levels. Patients with an abdominal source of septic shock had the lowest levels. This survey supports previous studies indicating early supplementation may be benefi cial in septic shock patients. References 2. Hassoun HT, Kone BC, Mercer DW: Post-injury multiple organ failure: the role of gut. Shock 2001, 15:1-10. 2. Hassoun HT, Kone BC, Mercer DW: Post-injury multiple organ failure: the role of gut. Shock 2001, 15:1-10. 3. Morrow LE: Probiotics in the intensive care unit. Curr Opin Crit Care 2009, 15:144-148. 3. Morrow LE: Probiotics in the intensive care unit. Curr Opin Crit Care 2009, 15:144-148. References 1. Berger MM: Nutr Clin Pract 2006, 21:438-449. 2. Angstwurm MW, et al.: Crit Care Med 2007, 35:118-126. 3. Andrews PJ, et al.: BMJ 2011, 342:d1542. References 1. Berger MM: Nutr Clin Pract 2006, 21:438-449. 2. Angstwurm MW, et al.: Crit Care Med 2007, 35:118-126. 3. Andrews PJ, et al.: BMJ 2011, 342:d1542. 1. Berger MM: Nutr Clin Pract 2006, 21:438-449. 1. Berger MM: Nutr Clin Pract 2006, 21:438-449. 2. Angstwurm MW, et al.: Crit Care Med 2007, 35:118-126. 3. Andrews PJ, et al.: BMJ 2011, 342:d1542. P248 Selenium levels in patients with diff erent sources of sepsis L French, P Temblett Morriston Hospital, Swansea, UK Critical Care 2013, 17(Suppl 2):P247 (doi: 10.1186/cc12185) P248 Is a combined i.v. and enteral glutamine regimen superior to a single i.v. glutamine regimen in severe thoracic trauma? D Pavelescu, I Grintescu, I Luca-Vasiliu, L Mirea Emergency Hospital Floreasca, Bucharest, Romania Critical Care 2013, 17(Suppl 2):P248 (doi: 10.1186/cc12186) Morriston Hospital, Swansea, UK Morriston Hospital, Swansea, UK D Pavelescu, I Grintescu, I Luca-Vasiliu, L Mirea D Pavelescu, I Grintescu, I Luca Vasiliu, L Mirea Emergency Hospital Floreasca, Bucharest, Romania g y p Critical Care 2013, 17(Suppl 2):P248 (doi: 10.1186/cc12186) Introduction The aim of this study is to establish whether diff erent types of sepsis have an impact on selenium levels. Selenium is an essential trace element involved in antioxidant and immunological reactions. Selenium levels have been shown to be low in patients with systemic infl ammatory response syndrome and sepsis. Selenium replacement has been recommended in patients with sepsis [1,2]. Greater than 5 days of supplementation may also help to prevent the development of new infections on ICUs [3]. Introduction Glutamine regulates many biological functions in preserving the cell, acts as a key respiratory fuel and nitrogen donor for rapidly dividing cells, and modulates the expression of many genes associated with metabolism, cell defences and repair, and cytokine production. In severe thoracic trauma, glutamine supplementation is essential because the body consumes more than it produces and glutamine eff ects become dependent on its route of delivery. Methods This is a prospective survey where selenium levels were collected from patients admitted with septic shock to a tertiary ICU, for 6 months from October 2010 to March 2011. f Methods Fifty-two patients 19 to 78 years old with surgery for severe thoracic trauma were assessed in two groups: Group A received 0.3 to 0.5  g/kg/day i.v. glutamine  + 20  g enteral glutamine for 7  days, supplementation to enteral nutrition; Group B receive only i.v. glutamine supplementation to enteral nutrition 0.3 to 0.5 g/kg/day for 7 days. Weaning time, the duration of p.o. ileus, incidence and time to resolution of VAP, glycemic level and the percentage decrease of CRP at 96 hours were assessed in both groups. Results Selenium levels were measured in 31 patients with septic shock. Abdominal and chest sepsis were the main sources of infection. Those with an abdominal source of sepsis had the lowest levels, as shown in Table 1. All septic shock patients who had selenium levels taken within the fi rst 10 days of admission had subnormal levels (<0.8 mg/dt), and after 10 days had levels within the normal range, as shown in Figure 1. P248 Is a combined i.v. and enteral glutamine regimen superior to a single i.v. glutamine regimen in severe thoracic trauma? D Pavelescu, I Grintescu, I Luca-Vasiliu, L Mirea Emergency Hospital Floreasca, Bucharest, Romania Critical Care 2013, 17(Suppl 2):P248 (doi: 10.1186/cc12186) Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 S93 Critical Care 2013, Volume 17 Suppl 2 pp http://ccforum.com/supplements/17/S2 Results Weaning time and the duration of p.o. ileus were signifi cantly lower in Group A; although the incidence of VAP is similar in both groups, the time of VAP resolution is lower, the glycemic control is better in Group A. The percentage of CRP decrease is higher in Group A. See Figure 1. Conclusion Glutamine becomes an essential amino acid in severe thoracic trauma and when the patients are fed other than TPN (enteral P249 Clinical eff ect of altered lipid emulsion composition containing fi sh oil in postoperative patients following abdominal surgery: a prospective, randomized, open-label, comparative, multicenter phase 3 clinical study M Keum1, S Hong1, H Han2, D Yoon3, J Seo4, I Yun5 1Asan Medical Center Seoul South Korea; 2Seoul National University Figure 1 (abstract P248). Results. tract P248). Results Results Weaning time and the duration of p.o. ileus were signifi cantly lower in Group A; although the incidence of VAP is similar in both groups, the time of VAP resolution is lower, the glycemic control is better in Group A. The percentage of CRP decrease is higher in Group A. See Figure 1. Conclusion Glutamine becomes an essential amino acid in severe thoracic trauma and when the patients are fed other than TPN (enteral, oral); although hard evidence is lacking, both administration routes may be effi cient as soon as possible. Results Weaning time and the duration of p.o. ileus were signifi cantly lower in Group A; although the incidence of VAP is similar in both groups, the time of VAP resolution is lower, the glycemic control is better in Group A. The percentage of CRP decrease is higher in Group A. See Figure 1. P249 P249 Clinical eff ect of altered lipid emulsion composition containing fi sh oil in postoperative patients following abdominal surgery: a prospective, randomized, open-label, comparative, multicenter phase 3 clinical study M Keum1, S Hong1, H Han2, D Yoon3, J Seo4, I Yun5 1Asan Medical Center, Seoul, South Korea; 2Seoul National University Bundang Hospital, Seongnam, South Korea; 3Gangnam Severance Hospital, Seoul, South Korea; 4Samsung Medical Center, Seoul, South Korea; 5Konkuk University Medical Center, Seoul, South Korea Critical Care 2013, 17(Suppl 2):P249 (doi: 10.1186/cc12187) P249 Clinical eff ect of altered lipid emulsion composition containing fi sh oil in postoperative patients following abdominal surgery: a prospective, randomized, open-label, comparative, multicenter phase 3 clinical study g Conclusion Glutamine becomes an essential amino acid in severe thoracic trauma and when the patients are fed other than TPN (enteral, oral); although hard evidence is lacking, both administration routes may be effi cient as soon as possible. Introduction Omega-3 fatty acids have been shown to decrease infl ammatory responses after trauma and surgery resulting in S94 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 vs. 16.2 ± 19.4 IE, P = 0.155; EN: 52.9 ± 25.6 vs. 61.7 ± 25%, P = 0.304; day 3: gluc: 129 ± 17 vs. 133 ± 21 mg/dl, P = 0.578; ins: 35.1 ± 30.9 vs. 32.3 ± 30.5 IE, P = 0.802; EN: 78.3 ± 28.9 vs. 83.4 ± 29%, P = 0.617; day 5: gluc: 124 ± 16 vs. 132 ± 17 mg/dl, P = 0.276; ins: 25.7 ± 37 vs. 33 ± 26.4 IE, P = 0.562; EN: 73.9 ± 41.4 vs. 87.1 ± 23%, P = 0.354). Interestingly, the calorie achievement was not associated with insulin demand (day 1: R = 0.241, P = 0.156) or average blood glucose (day 1: R = 0.248, P = 0.14) throughout the study. reduction of morbidity and mortality in postoperative patients. The n-6:n-3 polyunsaturated fatty acid (PUFA) ratio in parenteral nutrition (PN) also infl uences the immune modulation. The aim of this study was to evaluate the optimal ratio of PUFA in postoperative patients following abdominal surgery. Methods In a prospective, randomized, open-label, comparative, multicenter, phase 3 clinical study, we compared the safety and effi cacy of a 2.1:1 ratio of n-6:n-3 fatty acid compared with 2.5:1 in postoperative patients requiring PN. Eff ect of hypocaloric versus normocaloric parenteral nutrition on whole body protein kinetics in critically ill neurosurgical patients O Rooyackers, A Berg, J Wernerman y O Rooyackers, A Berg, J Wernerman y , g, Karolinska Institutet and University Hospital, Huddinge, Sweden Critical Care 2013, 17(Suppl 2):P251 (doi: 10.1186/cc12189) y , g, arolinska Institutet and University Hospital, Huddinge, Sweden y g Karolinska Institutet and University Hospital, Huddinge, Sweden Critical Care 2013, 17(Suppl 2):P251 (doi: 10.1186/cc12189) y ritical Care 2013, 17(Suppl 2):P251 (doi: 10.1186/cc12189) Table 1 (abstract P249). Lipid emulsion composition of CFO and KAB Table 1 (abstract P249). Lipid emulsion composition of CFO and KAB CFO KAB Table 1 (abstract P249). Lipid emulsion composition of CFO and KAB CFO KAB Table 1 (abstract P249). Lipid emulsion composition of CFO and KAB CFO KAB LCT (g/l) 60 60 MCT (g/l) 50 60 Olive oil (g/l) 50 50 Fish oil (g/l) 40 30 n-6:n-3 2.1:1 2.5:1 Introduction The optimal feeding of critically ill patients treated in the ICU is controversial. Present guidelines for protein feeding are based on weak evidence obtained with suboptimal methods. Whole body protein kinetics is an attractive technique to assess optimal protein intake by measuring the eff ect of protein feeding strategies on protein synthesis rates, protein degradation rates and protein balance. Here protein kinetics were measured in critically ill neurosurgical patients during hypocaloric and normocaloric parenteral nutrition. g y Methods Neurosurgical patients on mechanical ventilation (n  =  16) were studied. Energy expenditure was measured with indirect calorimetry. After that, the patients were randomized to receiving 24 hours of 50% of measured energy expenditure followed by 24 hours of 100% or 100% before 50%. Whole body protein kinetics were measured during the last half hour of the feeding periods using stable isotope-labeled phenylalanine as a tracer. During a continuous infusion of labeled phenylalanine and tyrosine, plasma samples were obtained and later analyzed for the content of the labeled amino acids using mass spectrometry. Protein kinetics were calculated using standard steady-state kinetics. In addition, amino acid concentrations were analyzed by HPLC. Student’s t test was used for statistical analyses. Conclusion CFO containing low n-6:n-3 PUFA ratio was comparable with KAB in safety and effi cacy in postoperative patients requiring PN and also had advantages with regard to lipid metabolism aspect. References References 1. Simopoulos AP: Biomed Pharmacother 2002, 56:365-379. 2. Heller AR, et al.: Crit Care Med 2006, 34:972-979. References 1. Simopoulos AP: Biomed Pharmacother 2002, 56:365-379. 2. Heller AR, et al.: Crit Care Med 2006, 34:972-979. P249 Fifty-four patients were assigned to receive PN with Combifl ex Omega peripheral® (CFO, low ratio group, n = 28) or SMOFKabiven peripheral® (KAB, high ratio group, n = 26) for 3 days after abdominal surgery. Safety and effi cacy were monitored daily with laboratory parameters, vital signs, and adverse events from the operation day (day 0) until the end of the study (day 4). Conclusion Medical critically ill patients with fat-based or glucose- based EN achieve similar glucose control. EN was not associated with glucose concentrations or insulin demand. P250 Infl uence of fat-based versus glucose-based enteral nutrition formulas on glucose homeostasis y y y Results The patients received 0.5 ± 0.1 and 1.1 ± 0.2 g amino acids/ kg/day (P <0.001) on the days with 50 and 100% of measured energy expenditure respectively. Energy expenditures were 23.4  ±  2.7 and 24.5 ± 2.3 kcal/kg/day (P = 0.05) on the 50 and 100% days respectively. Plasma amino acids concentrations were 2.8 ± 0.5 and 2.9 ± 0.4 mM (P = 0.085) on the 2 days respectively. Whole body protein synthesis was 12% lower when 50% of energy expenditure was given, 11.7 ± 3.0 versus 13.3 ± 2.2 mg/kg/hour (P = 0.025), whilst protein degradation was unaltered 13.6 ± 3.5 versus 14.0 ± 2.6 mg/kg/hour (P = 0.56). Also protein oxidation was unaltered 3.0 ± 2.1 versus 2.9 ± 1.4 mg/kg/hour (P = 0.85). This resulted in a 60% higher whole body protein balance with the normocaloric nutrition, –1.9 ± 2.1 versus –0.7 ± 1.3 mg/kg/ hour (P = 0.014). M Wewalka, A Drolz, C Zauner Introduction Trauma patients who receive fat-based parenteral nutrition (PEN) achieve better glucose control compared with those fed with glucose-based PEN [1,2]. Therefore, we determined whether fat- based enteral nutrition (EN) has the same benefi t on glucose control and exogenous insulin demand in medical intensive care patients compared with glucose-based EN. Here we present preliminary data for this ongoing randomized controlled cohort study. Methods Medical critically ill patients with need for mechanical ventilation and without contraindications for EN are included in the study. Patients are randomly assigned to receive either fat-based (n = 30) or glucose-based (n = 30) EN. To evaluate the individual calorie demand, indirect calorimetry is performed after an overnight fast. The determined amount of EN is administered continuously for 5  days. Glucose concentrations are measured at least three times per day and averaged. Furthermore, exogenous insulin demand per 24 hours and calorie achievement per 24 hours are evaluated daily. Conclusion The protein kinetics measurements and the protocol used were useful to assess the effi cacy of nutritional support in critically ill patients. In the critically ill neurosurgical patients treated in the ICU, hypocaloric feeding was associated with a more negative protein balance, while the amino acid oxidation was not diff erent. References 1. Tappy L, et al.: Crit Care Med 1998, 26:860-867. 1. Tappy L, et al.: Crit Care Med 1998, 26:860-867. 2. Huschak G, et al.: Intensive Care Med 2005, 31:1202-1208. 2. Huschak G, et al.: Intensive Care Med 2005, 31:1202-1208. Results Total cholesterol (TC) and low-density lipoprotein-cholesterol (LDL-C) levels were less changed signifi cantly in the low ratio group (3 ± 18 vs. 16 ± 23 mg/dl, P = 0.027 for TC, 4 ± 12 vs. 12 ± 18 mg/dl, P = 0.026 for LDL-C) compared with the high ratio group in postoperative patients. Other laboratory parameters and adverse events did not show statistically signifi cant diff erences between the groups. See Table 1. P252 Withholding parenteral nutrition for 1 week reduces ICU-acquired weakness Results So far, 37 patients, 16 with fat-based, 21 with glucose-based EN have been included in the study. Both groups had similar age (62 ± 10 vs. 58 ± 16 years, P = 0.44), body mass index (26.7 ± 5.8 vs. 28.4 ± 4.4 kg/ m2, P  =  0.302), SAPS II score (62.4  ±  12.7 vs. 64  ±  12.3, P  =  0.697), and fasting plasma glucose (132 ± 34 vs. 121 ± 26 mg/dl, P = 0.269). Furthermore, resting energy expenditure was similar in both groups (1,522 ± 365 vs. 1,573 ± 313 kcal/day, P = 0.647). Throughout the entire study period, average blood glucose, exogenous insulin demand, and calorie achievements per day were similar between the groups (day 1: gluc: 139 ± 30 vs. 127 ± 20 mg/dl, P = 0.143; ins: 27.8 ± 28.4 G Hermans, B Clerckx, T Vanhullebusch, F Bruyninckx, M Casaer, P Meersseman, D Mesotten, S Vancromphaut, P Wouters, R Gosselink, A Wilmer, G Van den Berghe UZ Leuven, Belgium Critical Care 2013, 17(Suppl 2):P252 (doi: 10.1186/cc12190) G Hermans, B Clerckx, T Vanhullebusch, F Bruyninckx, M Casaer, P Meersseman, D Mesotten, S Vancromphaut, P Wouters, R Gosselin A Wilmer, G Van den Berghe Critical Care 2013, 17(Suppl 2):P252 (doi: 10.1186/cc12190) Critical Care 2013, 17(Suppl 2):P252 (doi: 10.1186/cc12190 Introduction ICU-acquired weakness (ICUAW) is a frequent and important complication of critical illness [1]. A large randomized S95 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 intake. In the abdominal muscle compartments, changes were similar, albeit smaller. Femoral and abdominal subcutaneous adipose tissue compartments were unaff ected by disease and nutritional strategy. Conclusion Early PN did not prevent the pronounced wasting of skeletal muscle observed over the fi rst week of critical illness. Moreover, early PN increased the amount of adipose tissue within the muscle compartments. R f controlled trial (EPaNIC: clinicaltrials.gov: NCT00512122) [2] showed that withholding parenteral nutrition during the fi rst week of ICU stay whereby tolerating substantial caloric defi cit (late PN) accelerated recovery and shortened weaning time as compared with early parenteral substitution for defi cient enteral feeding (early PN). We examined the impact of late PN, as compared with early PN, on incidence and recovery of ICUAW. y Methods A preplanned subanalysis of adult patients included in the EPaNIC trial. P254 P254 Withholding parenteral nutrition during the fi rst week of critical illness increases plasma bilirubin but lowers the incidence of cholestasis and gallbladder sludge YM Vanwijngaerden1, L Langouche1, M Gielen1, Y Debaveye1, M Casaer1, C Liddle2, S Coulter2, R Brunner1, P Wouters1, A Wilmer1, G Van den Berghe1, D Mesotten1 1University Hospitals KU Leuven, Belgium; 2University of Sydney, Australia Critical Care 2013, 17(Suppl 2):P254 (doi: 10.1186/cc12192) 5 Withholding parenteral nutrition during the fi rst week of critical illness increases plasma bilirubin but lowers the incidence of cholestasis and gallbladder sludge YM Vanwijngaerden1, L Langouche1, M Gielen1, Y Debaveye1, M Casaer1, C Liddle2, S Coulter2, R Brunner1, P Wouters1, A Wilmer1, G Van den Berghe1, D Mesotten1 1University Hospitals KU Leuven, Belgium; 2University of Sydney, Australia Critical Care 2013, 17(Suppl 2):P254 (doi: 10.1186/cc12192) Introduction Cholestatic liver dysfunction (CLD) during critical illness, defi ned by hyperbilirubinemia, often occurs and is associated with poor outcome. Parenteral nutrition (PN) is assumed to aggravate CLD. However, hyperbilirubinemia more frequently occurred when the start of PN was delayed until day 8 in the ICU (late PN) [1]. Late PN accelerated recovery as compared with early initiation of PN. Results Clinical ICUAW evaluation was performed in 600 patients (matched n  =  558), electrophysiological testing in 730 (matched n = 684). Late PN reduced the incidence of ICUAW at fi rst evaluation from 43.1% to 34.4%, P = 0.03 (matched: early PN 41.6%, late PN 33.3%, P = 0.04). Signifi cantly fewer patients in the late PN group developed weakness at any time during ICU stay (late PN 37.0%, early PN 46.4%, P = 0.02; matched: late PN 36.2%, early PN 45.2%, P = 0.03). ICUAW may have recovered faster with late PN than with early PN (P = 0.05, matched P = 0.06). Other outcomes were not diff erent. y y Methods This was a preplanned subanalysis of a large randomized controlled trial on early versus late initiation of PN (n = 4,640) [1]. Plasma total bilirubin was quantifi ed in all patients daily while in the ICU. Liver enzymes ALT, AST, GGT and ALP were quantifi ed twice weekly in all patients while in the ICU. In a random predefi ned subset of patients, circulating bile salts were also quantifi ed with MS-HPLC at baseline and on day 3, day 5 and the last day in the ICU (n = 280). Impact of early parenteral nutrition on muscle and adipose tissue compartments during critical illness L Langouche1, MP Casaer2, W Coudyzer2, D Vanbeckevoort2, B De Dobbelaer2, FG Güiza1, PJ Wouters2, D Mesotten2, G Van den Berghe2 1KU Leuven, Belgium; 2University Hospitals Leuven – KU Leuven, Belgium Critical Care 2013, 17(Suppl 2):P253 (doi: 10.1186/cc12191) g y Conclusion Tolerating substantial caloric defi cit by withholding PN until day 8 of critical illness increased circulating levels of bilirubin but reduced the occurrence of gallbladder sludge and lowered GGT, ALP and ALT levels. These results suggest that hyperbilirubinemia during critical illness dies not necessarily refl ect cholestasis and instead may be an adaptive response. Additional analyses on a propensity score- matched patient population are ongoing. Introduction The goal of enhanced nutrition in critically ill patients is to improve outcome by reducing lean tissue wasting. However, such eff ect has not been proven. This study aimed to assess the eff ect of early administration of parenteral nutrition (PN) on muscle volume and composition by repeated quantitative computer tomography (qCT). Introduction The goal of enhanced nutrition in critically ill patients is to improve outcome by reducing lean tissue wasting. However, such eff ect has not been proven. This study aimed to assess the eff ect of early administration of parenteral nutrition (PN) on muscle volume and composition by repeated quantitative computer tomography (qCT). p y p q p g p y (q ) Methods We performed a preplanned substudy of a randomized controlled trial (EPaNIC) that compared early initiation of PN when enteral nutrition was insuffi cient (early PN) with tolerating a pronounced nutritional defi cit for 1 week in the ICU (late PN) [1]. Late PN prevented infections and accelerated recovery. We studied 15 EPaNIC study neurosurgical patients requiring prescheduled repeated follow-up CT scans and six healthy volunteers matched for age, gender and BMI. Repeated abdominal and femoral qCT images were obtained in a standardized manner on median ICU day 2 (IQR 2 to 3) and day 9 (8 to 10). Intramuscular, subcutaneous and visceral fat compartments were delineated manually. Muscle and adipose tissue volume and composition were quantifi ed using standard Hounsfi eld Unit ranges. Results Critical illness evoked substantial loss of femoral muscle volume in 1 week, irrespective of the nutritional regimen. Early PN reduced the quality of the muscle tissue, as refl ected by the attenuation, revealing increased intramuscular water/lipid content. Early PN also increased the volume of adipose tissue islets within the femoral muscle compartment. P252 Withholding parenteral nutrition for 1 week reduces ICU-acquired weakness The study was performed between October 2008 and November 2010 and included those patients who required intensive care for ≥8 days as well as a computer-generated, admission category- matched, random sample of short-stay ICU patients, the latter to correct for possible bias evoked by earlier ICU discharge in one of the two study groups. Assessors blinded for treatment allocation evaluated muscle strength clinically three times weekly from awakening onward and performed nerve conduction studies and electromyography (NCS and EMG) weekly. The primary outcome was the incidence of ICUAW, diagnosed clinically by the Medical Research Council (MRC) sum score (<48/60) [3] at fi rst evaluation. Secondary outcomes included ICUAW at worst and last MRC evaluation, recovery from ICUAW and incidence of abnormal fi ndings on NCS and EMG. All analyses were performed on the total dataset and on a for-baseline characteristics propensity score-matched sample to correct for possible imbalances between the groups. Reference 1. Casaer MP, et al.: N Engl J Med 2011, 365:506-517. 1. Casaer MP, et al.: N Engl J Med 2011, 365:506-517. 1. Casaer MP, et al.: N Engl J Med 2011, 365:506-517. Impact of early parenteral nutrition on muscle and adipose tissue compartments during critical illness These changes in skeletal muscle integrity correlated with caloric p y p q p g p y q Methods We performed a preplanned substudy of a randomized controlled trial (EPaNIC) that compared early initiation of PN when enteral nutrition was insuffi cient (early PN) with tolerating a pronounced nutritional defi cit for 1 week in the ICU (late PN) [1]. Late PN prevented infections and accelerated recovery. We studied 15 EPaNIC study neurosurgical patients requiring prescheduled repeated follow-up CT scans and six healthy volunteers matched for age, gender and BMI. Repeated abdominal and femoral qCT images were obtained in a standardized manner on median ICU day 2 (IQR 2 to 3) and day 9 (8 to 10). Intramuscular, subcutaneous and visceral fat compartments were delineated manually. Muscle and adipose tissue volume and composition were quantifi ed using standard Hounsfi eld Unit ranges. Reference 1. Casaer et al.: N Engl J Med 2011, 365:506-516. 1. Casaer et al.: N Engl J Med 2011, 365:506-516. P254 Gallbladder sludge was evaluated by ultrasound on ICU day 5 by blinded assessors (n = 776). f Conclusion As compared with early PN, late PN reduced the incidence of ICUAW and may have accelerated recovery thereof. References Results From day 1 after randomization until the end of the 7-day intervention window, plasma bilirubin was higher in the late PN than in the early PN group (all P <0.001). In the late PN group, as soon as PN was started on day 8, plasma bilirubin also fell and the two groups became comparable. Maximum levels of GGT, ALP and ALT during the ICU stay were higher in the early PN group (all P <0.01). Compared with baseline, the circulating glycine and taurine conjugated primary bile salts were elevated on day 3, day 5 and last day of the ICU stay (P <0.01 for all). However, there was no diff erence between the two groups. More patients in the early PN than in the late PN group had gallbladder sludge on day 5 (45% vs. 37%; P = 0.04).i 1. Stevens et al.: Int Care Med 2007, 33:1876-1891. 2. Casaer et al.: N Engl J Med 2011, 365:506-516. 3. De Jonghe et al.:, JAMA 2002, 288:2859-2867. 1. Stevens et al.: Int Care Med 2007, 33:1876-1891. 2. Casaer et al.: N Engl J Med 2011, 365:506-516. 3. De Jonghe et al.:, JAMA 2002, 288:2859-2867. References the duration of renal replacement therapy (RRT) [1]. The impact of the intervention on early markers of catabolism has not been investigated. Methods We studied the impact of early versus late PN on daily markers of catabolism in the ICU in the total study population and in propensity score-matched subgroups of long-stay patients. In addition, we calculated the net incorporation rate of the extra amino acids supplied by early PN. 1. Debaveye, Van den Berghe: Annu Rev Nutr 2006, 26:513-538. 2. Masiero et al.: Cell Metab 2009, 10:507-515. 3. Vanhorebeek et al.: J Clin Endocrinol Metab 2011, 96:E633-E645. 4. Derde et al.: Endocrinology 2012, 153:2267-2276. 5. Casaer et al.: N Engl J Med 2011, 365:506-517. Results Plasma urea, the urea/creatinine ratio and nitrogen excretion increased over time in the ICU. Early PN further increased these markers of catabolism, from the fi rst day of amino acid infusion onward, and only marginally improved the nitrogen balance. Also in the group that received PN only after the fi rst week in the ICU, ureagenesis was increased by infusing amino acids. Over the fi rst 2 weeks, approximately two-thirds of the extra amino acids supplied by early PN were net wasted in urea. The above fi ndings were confi rmed in propensity score- matched subgroups of long-stay patients. The higher urea levels with early PN, rather than the kidney function as such, may have driven the observed longer duration of RRT, as supported by multiple regression analysis. P255 Impact of early parenteral nutrition on catabolism J Gunst1, I Vanhorebeek1, MP Casaer1, G Hermans1, PJ Wouters1, J Dubois2, K Claes1, M Schetz1, G Van den Berghe1 1KU Leuven, Belgium; 2Jessa Hospital, Hasselt, Belgium Critical Care 2013, 17(Suppl 2):P255 (doi: 10.1186/cc12193) Introduction Prolonged critically ill patients enter a state of hypercatabolism and muscle weakness, which has been associated with increased morbidity and mortality. Early, full feeding of ICU patients has been advocated to counteract catabolism. However, a large, multicenter study found that early parenteral nutrition (PN) had no signifi cant impact on mortality and even increased dependency on intensive care with, among others, a signifi cant prolongation of Introduction Prolonged critically ill patients enter a state of hypercatabolism and muscle weakness, which has been associated with increased morbidity and mortality. Early, full feeding of ICU patients has been advocated to counteract catabolism. However, a large, multicenter study found that early parenteral nutrition (PN) had no signifi cant impact on mortality and even increased dependency on intensive care with, among others, a signifi cant prolongation of S96 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Overcrowding estimation in the emergency department: is the simplest score the best? Overcrowding estimation in the emergency department: is the simplest score the best? V Winkin, V Pinckaers, V D’Orio, A Ghuysen CHU Liège, Belgium Critical Care 2013, 17(Suppl 2):P258 (doi: 10.1186/cc12196) p V Winkin, V Pinckaers, V D’Orio, A Ghuysen CHU Liège, Belgium Critical Care 2013, 17(Suppl 2):P258 (doi: 10.1186/cc12196) V Winkin, V Pinckaers, V D’Orio, A Ghuysen CHU Liège, Belgium Critical Care 2013, 17(Suppl 2):P258 (doi: 10.1186/cc12196) Results As compared with controls, muscle from critically ill patients showed reduced myofi ber density, a shift to smaller (especially type I) myofi bers, lower myosin and actin mRNA, upregulated mRNA of the ubiquitin ligases muscle-ring-fi nger-1 and atrogin-1, a small increase in the autophagosome formation marker LC3-II/LC3-I, and increases in the autophagic substrates ubiquitin and p62 (all P ≤0.006). Late-PN, resulting in a larger caloric defi cit than early-PN, had no substantial impact on atrophy markers. In contrast, late-PN increased LC3-II/LC3-I (P  =  0.02), which coincided with less accumulation of ubiquitinated proteins/aggregates (P = 0.05). Fewer patients on late-PN developed muscle weakness as compared with early-PN (42% vs. 68%, P = 0.05). In multivariable analysis, a lower LC3-II/LC3-I ratio (P = 0.05) and higher myofi ber density (P = 0.04) were independently associated with muscle weakness. Introduction Emergency department (ED) overcrowding is a major international problem with a negative impact on both patient care and providers. Among validated methods of measurement, emergency physicians have to choose between simple and complex scores [1,2]. The aim of the present study was to compare the complex National Emergency Department Overcrowding Scale (NEDOCS) with the simple occupancy rate (OR) determination. We further evaluated the correlation between these scores and a qualitative assessment of crowding. Methods The study was conducted in two academic hospitals and one county hospital in Liège, Chênée and Verviers; each with an ED census of over 40,000 patient visits per year. Samplings occurred over a 2-week period in January 2011, with fi ve sampling times each day.f Methods The study was conducted in two academic hospitals and one county hospital in Liège, Chênée and Verviers; each with an ED census of over 40,000 patient visits per year. Samplings occurred over a 2-week period in January 2011, with fi ve sampling times each day. Results ED staff considered overcrowding as a major concern in the three EDs. Median OR ranged from 68 to 100, while the NEDOCS ranged from 64.5 to 76.3. P256 f Results In the period studied, the gross attendance fi gure increased by 20,480 (33.72%), whilst the admission rate rose from 22 to 27%. Following closure of the neighbouring ED, the proportion of high- acuity patients attending our institution increased dramatically, with the proportion of category one and two patients (Manchester Triage Scale) increasing by 8.33% (P = 0.076) and 18.80% (P <0.001), respectively. Likewise, ambulance arrivals increased out of proportion to the total increase in attendances (P = 0.016). Admissions from the ED to the ICU increased by 63.04%. Consultants workloads now include 50% more category 1 and 2 patients (P = 0.001).i g p Reference g p Reference 1. Casaer et al.: N Engl J Med 2011, 365:506-517. P257 Impact of closing an emergency department on a neighbouring teaching hospital: the concentrate eff ect J Millar, R Wilson, P O’Connor, R McLaughlin Royal Victoria Hospital, Belfast, UK Critical Care 2013, 17(Suppl 2):P257 (doi: 10.1186/cc12195) P257 Impact of closing an emergency department on a neighbouring teaching hospital: the concentrate eff ect J Millar, R Wilson, P O’Connor, R McLaughlin Royal Victoria Hospital, Belfast, UK Critical Care 2013, 17(Suppl 2):P257 (doi: 10.1186/cc12195) Introduction Closure of an acute hospitals emergency department (ED) has important ramifi cations for those centres expected to take up the resultant workload. The continued reconfi guration of emergency care is likely to produce an increasing number of these scenarios. Little evidence is available to support planning of such initiatives and thus the implications are diffi cult to anticipate. This study aims to demonstrate one hospital’s experience of the rationalisation of emergency care and its eff ect on workload. y Conclusion The extra amino acids supplied by early PN appeared ineffi cient to reverse the negative nitrogen balance, not because of insuffi cient amino acid delivery, but rather because of insuffi cient incorporation with, instead, increased degradation into urea. The substantial catabolism of the extra amino acids, leading to pronounced urea generation, may have prolonged the duration of RRT in the early PN group. Methods This retrospective study was conducted in a large teaching hospital. Activity data were analysed for a 12-month period following the closure of a neighbouring ED. The results were subsequently compared against the year prior to closure. Attendance, triage data, admission rates and waiting times were compared across the two periods, as were workload data for all grades of physician. The chi- squared test was used to examine diff erences between groups.i Impact of early versus late parenteral nutrition on morphological and molecular markers of atrophy and autophagy in skeletal muscle of critically ill patients Impact of early versus late parenteral nutrition on morphological and molecular markers of atrophy and autophagy in skeletal muscle of critically ill patients I Vanhorebeek, MP Casaer, F Güiza, S Derde, I Derese, PJ Wouters, Y Debaveye, J Gunst, G Hermans, G Van den Berghe KU Leuven, Belgium Critical Care 2013, 17(Suppl 2):P256 (doi: 10.1186/cc12194) Introduction Muscle weakness of critical illness is associated with prolonged dependency on ventilatory support and delayed rehabilitation. Muscle wasting related to poor nutrition has long been considered a major determinant, whereas the importance of myofi ber integrity only recently emerged [1-4]. We hypothesized that nutrient restriction early during illness aggravates atrophy while preserving myofi ber integrity by activating the crucial cellular quality control pathway autophagy. The latter could be important to preserve muscle function. Conclusion Reconfi guration of emergency care can have dramatic implications for existing services; these may not always be anticipated. Rationalisation of ED’s may result in a concentration of high-acuity patients accompanied by a downturn in the numbers of patients whose presentations are amenable to care delivered in other settings. This abrupt change in case mix requires a re-examination of existing workforces and their seniority. Methods Critically ill patients (n = 122) were randomized to early (early- PN) or late (late-PN) initiation of parenteral nutrition to complete failing enteral nutrition, while maintaining normoglycemia (80 to 110  mg/ dl) with insulin, in the EPaNIC study [5]. Vastus lateralis biopsies were harvested after 1 week and compared with matched controls (n = 20). P259 GP-led walk-in centre in the UK: another way for urgent healthcare provision M Arain, J Nicholl, M Campbell The University of Sheffi eld, UK Critical Care 2013, 17(Suppl 2):P259 (doi: 10.1186/cc12197) M Arain, J Nicholl, M Campbell The University of Sheffi eld, UK Critical Care 2013, 17(Suppl 2):P259 (doi: 10.1186/cc12197) Introduction It is evident that accident and emergency departments are overloaded with patients, which results in delays in healthcare provision [1]. A large proportion of patients consist of patients with minor illness that can be seen by a healthcare provider in a primary care setting. The aim of the study was to determine the characteristics of patients using GP walk-in services, patients’ satisfaction and the eff ect on emergency department (ED) services. Introduction It is evident that accident and emergency departments are overloaded with patients, which results in delays in healthcare provision [1]. A large proportion of patients consist of patients with minor illness that can be seen by a healthcare provider in a primary care setting. The aim of the study was to determine the characteristics of patients using GP walk-in services, patients’ satisfaction and the eff ect on emergency department (ED) services. Figure 1 (abstract P260). GP patient visits before and after the reform of emergency services. has a negative impact on patient safety [1]. The aim of this study was to analyse whether a reform of emergency care can reduce patient fl ow into the ED. y Methods The survey was conducted in Sheffi eld and Rotherham walk-in centres over 3  weeks during September and October 2011. A self-reported, validated questionnaire was used to conduct survey on the patients presenting at these centres. We estimated that a sample size of around 400 patients from each centre was required to achieve statistically robust results. A post-visit, short questionnaire was also sent to those who agreed for the second questionnaire and provided contact details. ED data were also obtained from April 2008 to March 2010, 1 year before and 1 year after the opening of the GP walk-in centre. Data were entered and analysed in PASW Statistics 18. Ethical approval of the study was obtained from the NHS ethical review committee. Methods A substantial reform of emergency care took place in the province of Kanta-Häme in Southern Finland. Three separate out-of- hours services in primary healthcare (PHC) and one ED in the hospital were combined into one large ED in April 2007. P259 GP-led walk-in centre in the UK: another way for urgent healthcare provision Basic principles of the new ED were: the ED is only for those patients who are seriously ill or injured, and need immediate care; PHC (healthcare centres) take care of acute ordinary illnesses and nonserious injuries during offi ce hours. To achieve these principles a regional fi ve-scale triage system was planned and implemented. The information plan was established. Citizens were systematically informed about the principles of the new ED by mail, articles in the newspapers and interviews in the radio and television. The ED’s Internet pages were planned and established. The number of patient visits (Hämeenlinna region) was analyzed 2 years before and after establishing the new ED. Results A total of 1,030 patients participated in the survey (Rotherham 501; Sheffi eld 529). The mean age of the participants was 32.1 years at Sheffi eld and 30.88 years at Rotherham. A higher proportion of users were female, around 59% at both centres. Most of the patients rated high for convenience of the centre opening hours and location (above 85%, apart from the location of Sheffi eld centre, which was rated high by around 72% of the research participants). Overall 93% patients were satisfi ed with the service at Rotherham centre and around 86% at the Sheffi eld centre. Based on the estimation of the monthly counts of patients attending ED and the GP walk-in centre, around 14% monthly reduction in minor attendances at ED was expected. However, ED routine data did not show any signifi cant reduction in minor attendances as a result of the opening of the GP walk-in centre. g Results During the 2-year period before the establishment of the new ED the mean number of GP patient visits was 1,845 ± 43/month. During the 2-year period after the reform the number was diminished to 1,364 ± 21/month. This change was not associated with the increase of the patient visits taken care of by specialists and hospital residents. See Figure 1. Conclusion An extensive reform of the emergency services can notably reduce patient fl ow into the ED. Reference 1. Boyle A, et al.: Emerg Med Int 2012:838610. [Epub ahead of print] 1. Boyle A, et al.: Emerg Med Int 2012:838610. [Epub ahead of print] p g Conclusion These walk-in centres have been shown to increase accessibility to healthcare service through longer opening hours and walk-in facility. P259 P259 GP-led walk-in centre in the UK: another way for urgent healthcare provision M Arain, J Nicholl, M Campbell The University of Sheffi eld, UK Critical Care 2013, 17(Suppl 2):P259 (doi: 10.1186/cc12197) Reference 1. Gerard K, Lattimer V: Preferences of patients for emergency services available during usual GP surgery hours: a discrete choice experiment. Fam Pract 2005, 22:28-36. Introduction Rio de Janeiro’s Fire Squad is responsible for EMS in the city. During 2010 we implemented 10 ambulances with EKG transmission capability in our city. Our intention was to access the prevalence of acute myocardial infarction in the prehospital setting. Methods We used the Aerotel HeartView EKG system to acquire patient examination and a blackberry phone to transmit and receive the PDF EKG trace. The PDF comes with the cardiologist’s interpretation from a remote hospital, the HCOR São Paulo. Introduction Rio de Janeiro’s Fire Squad is responsible for EMS in the city. During 2010 we implemented 10 ambulances with EKG transmission capability in our city. Our intention was to access the prevalence of acute myocardial infarction in the prehospital setting. P259 GP-led walk-in centre in the UK: another way for urgent healthcare provision Although the eff ect on the reduction of patients’ load at the ED is not visible as these centres cover a fraction of the population, the centre has a potential to divert patients from the ED. P261 Prehospital EKG evaluation in Rio de Janeiro ambulances RV Vasconcellos, FE Erthal, RV Vargas Instituto Nacional de Cardiologia, Rio de Janeiro, Brazil Critical Care 2013, 17(Suppl 2):P261 (doi: 10.1186/cc12199) P260 P260 Establishing a new emergency department: eff ects on patient fl ow V Rautava1, T Valpas1, M Nurmikari2, A Palomäki1 1Kanta-Häme Central Hospital, Hämeenlinna, Finland; 2City of Hämeenlinna, Finland Critical Care 2013, 17(Suppl 2):P260 (doi: 10.1186/cc12198) P260 Establishing a new emergency department: eff ects on patient fl ow V Rautava1, T Valpas1, M Nurmikari2, A Palomäki1 1Kanta-Häme Central Hospital, Hämeenlinna, Finland; 2City of Hämeenlinna, Finland Critical Care 2013, 17(Suppl 2):P260 (doi: 10.1186/cc12198) Overcrowding estimation in the emergency department: is the simplest score the best? We found a signifi cant correlation between Conclusion Early-PN did not counteract muscle atrophy whereas it suppressed autophagy and aggravated weakness. Statistically, muscle weakness was not explained by atrophy or wasting but rather by impaired autophagy and preservation of muscle density. Thus, tolerating nutrient restriction early during critical illness may preserve myofi ber integrity by activating autophagy. i Results ED staff considered overcrowding as a major concern in the three EDs. Median OR ranged from 68 to 100, while the NEDOCS ranged from 64.5 to 76.3. We found a signifi cant correlation between Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 S97 the OR and the NEDOCS (Pearson R = 0.973, 0.974 and 0.972), as well as between the OR, the NEDOCS and the subjective evaluation (P = 0.001). Conclusion Crowding evaluation in the ED requires validated, easy-to- use, scores. Our study indicates that the simple OR is as accurate as the complex NEDOCS, allowing continuous crowding assessment. References Figure 1 (abstract P260). GP patient visits before and after the reform of emergency services. the OR and the NEDOCS (Pearson R = 0.973, 0.974 and 0.972), as well as between the OR, the NEDOCS and the subjective evaluation (P = 0.001). Conclusion Crowding evaluation in the ED requires validated, easy-to- use, scores. Our study indicates that the simple OR is as accurate as the complex NEDOCS, allowing continuous crowding assessment. References 1. Hwang U, McCarthy ML, Bernstein SL, et al.: Measures of crowding in the emergency department: a systematic review. Acad Emerg Med 2011, 18:527-538. 1. Hwang U, McCarthy ML, Bernstein SL, et al.: Measures of crowding in the emergency department: a systematic review. Acad Emerg Med 2011, 18:527-538. 2. Hoot NR, Zhou C, Jones I, Aronsky D: Measuring and forecasting emergency department crowding in real time. Ann Emerg Med 2007, 49:747-755. 2. Hoot NR, Zhou C, Jones I, Aronsky D: Measuring and forecasting emergency department crowding in real time. Ann Emerg Med 2007, 49:747-755. Establishing a new emergency department: eff ects on patient fl ow V Rautava1, T Valpas1, M Nurmikari2, A Palomäki1 y Methods We used the Aerotel HeartView EKG system to acquire patient examination and a blackberry phone to transmit and receive the PDF EKG trace. The PDF comes with the cardiologist’s interpretation from a remote hospital, the HCOR São Paulo. Establishing a new emergency department: eff ects on patient fl ow V Rautava1, T Valpas1, M Nurmikari2, A Palomäki1 1Kanta-Häme Central Hospital, Hämeenlinna, Finland; 2City of Hämeenlinna, Finland Critical Care 2013, 17(Suppl 2):P260 (doi: 10.1186/cc12198) p Results We realized 503 EKG examinations in total. Of these, 248 (49%) had as the chief complaint chest pain, 101 (20%) shortness of breath, 47 (9%) syncope, 36 (7%) palpitation; other complaints were 15%. We detected 32 examinations (6.36%) with ST elevation MI and Critical Care 2013, 17(Suppl 2):P260 (doi: 10.1186/cc12198) Introduction Overcrowding in emergency departments (EDs) is a widely known problem. It causes problems and delays in the ED and S98 Critical Care 2013, Volume 17 Suppl 2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum com/supplements/17/S2 http://ccforum.com/supplements/17/S2 Figure 1 (abstract P261). Acute inferior MI with complete heart block. Figure 2 (abstract P261). Paroxysmal supraventricular tachycardia. Figure 1 (abstract P261). Acute inferior MI with complete heart block. Figure 1 (abstract P261). Acute inferior MI with complete heart block. Figure 1 (abstract P261). Acute inferior MI with complete heart block. Figure 2 (abstract P261). Paroxysmal supraventricular tachycardia. Figure 2 (abstract P261). Paroxysmal supraventricular tachycardia. Figure 2 (abstract P261). Paroxysmal supraventricular tachycardia. S99 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 leg and patient satisfaction were used to assess the outcome. The assessments were made before the FIB, at 15 minutes, 30 minutes and 45 minutes. 44 examinations (8.75%) with ST depression. Atrial fi brillation was detected in 43 examinations (8.5%). See Figures 1 and 2. 44 examinations (8.75%) with ST depression. Atrial fi brillation was detected in 43 examinations (8.5%). See Figures 1 and 2. Conclusion This experience gave us an idea of the prevalence for acute ST elevation MI in the prehospital setting, so that we can better develop our prehospital thrombolysis protocol and focus our training for cardiology care. Results A convenience sample of 19 patients was enrolled in this study. The compartment block was placed by three mergence ultrasound trainers. The mean age was 58.5 years. There were 13 females and six males. P264 Abdominal pain in adolescent females: a single-centre audit and review of management Abdominal pain in adolescent females: a single-centre audit and review of management Introduction Abdominal pain in adolescent females has undergone recent changes with regards to its management under various specialities. The authors report a single-centre audit looking at the correct investigation and management of 12-year-old to 16-year-old girls with abdominal pain in the emergency department setting. Introduction Abdominal pain in adolescent females has undergone recent changes with regards to its management under various specialities. The authors report a single-centre audit looking at the correct investigation and management of 12-year-old to 16-year-old girls with abdominal pain in the emergency department setting. Methods A single-centre audit and retrospective analysis of patients took place using case notes and computerised records. Documentation was analysed using statistical analysis and minimum standards were set and reviewed. Results In 1  year, 76 long peripheral catheters were inserted in emergency conditions, using ultrasound guidance. The success rate was 100%; most lines lasted >1 week and were used for diff erent purposes, including contrast medium injection. Results After exclusion criteria 62 females between the ages of 12 and 16 presented to the paediatric emergency department in Leicester with abdominal pain as the predominant admission symptom during a 12-month period. Documentation of the gynaecological history was poor (menstrual history 47%, sexual history 14%, contraception 8%), as was the performance of basic investigations (urine dipsticks 65%, pregnancy test 42%). Documentation was analysed with regard to discharge diagnosis. Ultrasound investigation was performed on seven of the patients but only once admitted to various specialities. No ultrasound was undertaken upon admission. p p g j Conclusion The direct Seldinger technique allows a rapid and safe placement of the catheter in a vein of the arm or of the forearm, even when the vessel cannot be palpated or seen, as long as it can be visualized by ultrasound and it is not deeper than 2 cm. The long life of this type of peripheral line (up to 2 weeks) is guaranteed by the material (polyurethane being more biocompatible than Tefl on) and by the length of the catheter (which reduces the risk of dislodgment). Also, these catheters are particularly cost-eff ective if compared with a central line or with a midline catheter, since a complete kit including catheter, 20 G needle and 20 cm guidewire costs between €15 and €20. Conclusion Improvement in documentation of minimum standards for these patients is needed. P265 Introduction The lumbar plexus block provides excellent analgesia after hip and knee surgery. One approach to the three major nerves (femoral, lateral cutaneous and obturator) of the lubar plexus is the fascia iliaca compartment block, fi rst described by Dalens and colleagues. Using this blind approach, complete nerve block in the distribution of the lumbar plexus may be achieved only in 38% of cases. Ultrasound-guided regional techniques off er a number of advantages including real-time needle guidance and direct observation of local anesthetic spread within tissue planes. Establishing a new emergency department: eff ects on patient fl ow V Rautava1, T Valpas1, M Nurmikari2, A Palomäki1 The mean pain score was 9.5 at time 0. The mean pain score had improved to 5.5 at 15 minutes, 4.5 at 30 minutes and 3 at 45 minutes, respectively. The patient satisfaction was scored 4.5 on a scale of 1 to 5 where 5 was the most satisfi ed. No patient required any further analgesia up to 90 minutes and no issues were raised about the pain or discomfort upon patient transfer. We have no documented complications or side eff ects. Technique of ultrasound-guided peripheral venous access in the emergency room f Conclusion FIB is now widely used by non-anaesthetic trainees to combat pain in preoperative care due to its safe landmarks. Our results have shown that it can be used safely and eff ectively for pain management in hip fracture by emergency physicians, who are trained in the technique, on the shop fl oor. MG Annetta, G Scoppettuolo, M Biancone, F Toni, M Pittiruti Catholic University, Rome, Italy MG Annetta, G Scoppettuolo, M Biancone, F Toni, M Pittiruti Catholic University, Rome, Italy Critical Care 2013, 17(Suppl 2):P262 (doi: 10.1186/cc12200) Catholic University, Rome, Italy Critical Care 2013, 17(Suppl 2):P262 (doi: 10.1186/cc12200) Critical Care 2013, 17(Suppl 2):P262 (doi: 10.1186/cc12200) Introduction In emergency situations, patients may need a fast and reliable peripheral venous access, which sometimes may be diffi cult to obtain, because of poor visualization of the superfi cial veins due to edema, obesity, hypovolemia or local abnormalities. In such cases, insertion of a central line is potentially time consuming and possibly associated with complications. Furthermore, central lines inserted in emergency are known to be at high risk of infection, so guidelines recommend that they should removed within 24 to 48 hours. In this setting, ultrasound-guided placement of a peripheral venous access might be more rapid, safer and more cost-eff ective than a central line. Methods We have reviewed retrospectively our experience with the emergency use of 18 G or 20 G polyurethane catheters, 8 to 10 cm long, inserted by direct Seldinger technique. P264 Reference 1. Ioannidis JP, Salem D, Chew PW, Lau J: Accuracy and clinical eff ect of out-of- hospital electrocardiography in the diagnosis of acute cardiac ischemia: a meta-analysis. Ann Emerg Med 2001, 37:461-470. P264 Abdominal pain in adolescent females: a single-centre audit and review of management A multidisciplinary care pathway could improve outcome. Consideration should be given to whether early ultrasound investigation is appropriate and there is a further need for investigation as to whether this would improve longer term outcomes. P263 Multidisciplinary approach to improving documentation of visua acuity in patients presenting with ocular trauma L Low, M Johnston NHS Tayside, Dundee, UK Critical Care 2013, 17(Suppl 2):P265 (doi: 10.1186/cc12203) Critical Care 2013, 17(Suppl 2):P265 (doi: 10.1186/cc12203) Introduction This study aimed to implement a multidisciplinary quality improvement project in Perth Royal Infi rmary A&E department to improve documentation of visual acuity (VA) in patients presenting with ocular injury. Methods We hypothesized that real-time UFIB could be successfully performed in the ED and would provide an excellent adjunct or alternative to repeat doses of IVMS for pain control in patients with HFx. The study was conducted at University Hospital, Birmingham, where we see about 90,000 patients every year. All patients with confi rmed femoral (neck/shaft) fractures and pain score >7 were included in the study. Patients with local wounds, or suspected signifi cant pelvic injury were excluded. A combination of 15 ml lignocaine 1% and 15 ml bupvivcaine 0.25% was used. VAS (0 to 10), pain on movement of the Methods The improvement project involves a three-pronged multi- disciplinary approach: ensure that equipment required for VA testing (Snellen chart and pinhole mask) was readily available; encourage VA testing at fi rst point of contact with A&E staff , both nursing/medical staff ; and refresher online course on how to test for VA quickly and accurately, in the 6/X format. We compared the pre-intervention (2 September to 2 October 2012) and post-intervention (11  October to 19 November 2012) rates of VA documentation using the chi-square test. S100 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Figure 1 (abstract P265). Table 1 (abstract P266). Comparison of children receiving BiPAP for status asthmaticus by severity of illness Table 1 (abstract P266). Multidisciplinary approach to improving documentation of visua acuity in patients presenting with ocular trauma L Low, M Johnston NHS Tayside, Dundee, UK Critical Care 2013, 17(Suppl 2):P265 (doi: 10.1186/cc12203) Comparison of children receiving BiPAP for status asthmaticus by severity of illness Moderate Severe (n = 206) (n = 197) P value Age (years) 6.8 ± SD 2.8 7.1 ± SD 4.2 X Gender 62% male 61% male X History of asthma 92% 87% X Prior PICU admission 21% 32% X Prior history of BiPAP use 19% 24% X Prior history of intubation 6% 7% X Mean triage for BiPAP start (hours) 2.8 ± SD 2.1 1.2 ± SD 1.25 <0.0001 Mean time on BiPAP (hours) 3.9 ± SD 3.6 5.6 ± SD 5.2 <0.0001 Median time on BiPAP (hours) 2.7 3.8 X Admission to PICU 58% 75% X Total length of hospital stay 42 ± SD 26 50 ± SD 33 <0.06 Serious complications None None X 72-hour returns n = 1 (0.5%) – n = 4 (2%) – X no admits no admits Results During the pre-intervention period, of a total of 44 patients who presented to A&E with eye injury, only 36 patients (95%) had their VA tested. Following intervention, there was signifi cant improvement in VA testing, where all 43 patients presenting to A&E with eye injuries had their VA tested (100%, P = 0.02). Documentation of VA in the correct (6/X) format increased from 82 to 84% following intervention. There was a 15% improvement in documentation of best-corrected VA, from 48 to 63% post intervention. See Figure 1. Results During the pre-intervention period, of a total of 44 patients who presented to A&E with eye injury, only 36 patients (95%) had their VA tested. Following intervention, there was signifi cant improvement in VA testing, where all 43 patients presenting to A&E with eye injuries had their VA tested (100%, P = 0.02). Documentation of VA in the correct (6/X) format increased from 82 to 84% following intervention. There was a 15% improvement in documentation of best-corrected VA, from 48 to 63% post intervention. See Figure 1. g Conclusion Through a multidisciplinary approach, we were successful in achieving our target of 100% VA documentation rate in all patients presenting with eye injury to PRI A&E. P267 Report of an outbreak of toxicity from a novel drug of abuse in the UK: Eric-3 C Morden, S Haig, C Kelly Conclusion All patients recovered without any sequelae. Three patients’ hair started to grow 3 months from ingestion of thallium, and after half a year their hair was restored to their former state. We had diffi culty ordering iron(III)hexacyanoferrate(II) because this is also known as an antidote for cesium. On 11 March 2011 a megathrust earthquake and tsunami hit Japan and the giant tsunami gave rise to an accident at a nuclear power generation plant. Because the rumor of radioactive substances including cesium might be spread was the talk in the city near the nuclear power plant, the authorities put the antidote under heavy supervision. We could also collect the data for the course of thallium concentration. Thallium concentration of the patients who had an antidote was reduced more rapidly but these patients had a loose stool, thought to be a side eff ect of this antidote. Great Western Hospital, Swindon, UK Critical Care 2013, 17(Suppl 2):P267 (doi: 10.1186/cc12205) Introduction We present a case series of toxicity due to a novel sub- stance in the UK: Eric-3. Novel drugs of abuse are becoming more common throughout the world, and they represent particular diffi culties in their acute management. A recent report from the European Monitoring Centre for Drugs and Drug Addiction and Europol has reported 49 new psychoactive substances reported via its early warning system. Methods This was a retrospective case-note review over a 6-month period. Patients were included if their presentation was due to recent ingestion of Eric-3. Physiological data, symptoms, outcome and destination of the patient from the emergency department were collected. Postmortem toxicological analysis was obtained for one of the two patients who died.i Reference 1. Centers for Disease Control and Prevention: Thallium poisoning from eating contaminated cake – Iraq, 2008. MMWR Morb Mortal Wkly Rep 2008, 57:1015-1018. 1. Centers for Disease Control and Prevention: Thallium poisoning from eating contaminated cake – Iraq, 2008. MMWR Morb Mortal Wkly Rep 2008, 57:1015-1018. Results Forty-one attendances were identifi ed from 18 patients. Two patients died and fi ve were admitted to the ICU. Heart rate and temperature on arrival tended to be above normal (mean heart rate was 112 bpm, with an SD of 18; mean temperature was 37.45°C with an SD of 0.95). In total, 63.4% of attendances included agitation and 34.1% choreiform movements. α-Methyltryptamine and 3-/4-fl uoroephedrine were found in the blood of one of the patients who died. P270 Heart rate variability in children with tricyclic antidepressant intoxication BiPAP for treating moderate and severe asthma exacerbations in a PED A Williams, T Abramo A Williams, T Abramo Introduction BiPAP utilization for the treatment of severe refractory status asthmaticus patients has become an accepted therapy but is not well described for moderate exacerbations. We sought to analyze outcomes from our BiPAP quality database for children presenting in status asthmaticus at varying levels of severity. Methods PED status asthmaticus patients requiring BiPAP from 1  January 2010 to 31  August 2012 had a bedside interview and documentation of information at the time therapies were given. Incomplete data were collected retrospectively. All data were stored and analyzed using a RedCap database. Subjects were stratifi ed into severity groups based on asthma score at the time of BiPAP placement. Results There were 206 subjects in the moderate severity group and 197 in the severe group. Table  1 shows the groups were well matched and compares other pertinent data. Children with severe presentations were placed on BiPAP sooner (P <0.001) and remained on BiPAP longer (P <0.001). The moderate group had a longer wait until BiPAP placement. Tables 2 and 3 demonstrate higher initial BiPAP (IPAP/EPAP) settings with increasing age and severity. Figure 1 trends initiation and termination asthma scores stratifi ed by severity at BiPAP matched and compares other pertinent data. Children with severe presentations were placed on BiPAP sooner (P <0.001) and remained on BiPAP longer (P <0.001). The moderate group had a longer wait until BiPAP placement. Tables 2 and 3 demonstrate higher initial BiPAP (IPAP/EPAP) settings with increasing age and severity. Figure 1 trends initiation and termination asthma scores stratifi ed by severity at BiPAP Figure 1 (abstract P266). Initiation and termination asthma scores grouped by severity. Figure 1 (abstract P266). Initiation and termination asthma scores grouped by severity. S101 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 placement. More of the severe group was admitted to the PICU and had overall longer hospitalizations (P <0.06). None experienced severe complications. Conclusion BiPAP is a benefi cial therapy for children presenting to the PED with severe asthma exacerbations. It may have utility for less severe asthma exacerbations. Table 2 (abstract P266). BiPAP for treating moderate and severe asthma exacerbations in a PED Three patients consented to oral administration of an antidote. Two patients rejected administration because their symptoms were mild and getting better. All symptoms of all patients gradually disappeared by August. We also followed up the course of blood concentration of thallium. The concentration in three patients who took the antidote was reduced more rapidly than the two patients who did not take it. p Conclusion BiPAP is a benefi cial therapy for children presenting to the PED with severe asthma exacerbations. It may have utility for less severe asthma exacerbations. P267 BiPAP for treating moderate and severe asthma exacerbations in a PED Mean settings by age: moderate group Age (years) IPAP EPAP <3 (n = 22) 12.1 ± SD 2.1 6.4 ± SD 1.3 3 to 4 (n = 48) 12.1 ± SD 1.6 6.4 ± SD 1.2 5 to 6 (n = 51) 13.0 ± SD 2.3 6.6 ± SD 1.3 7 to 8 (n = 24) 13.9 ± SD 2.1 7.2 ± SD 1.2 9 to 10 (n = 28) 13.9 ± SD3.2 7.4 ± SD 2.0 11 to 12 (n = 25) 13.8 ± SD 1.8 7.0 ± SD 1.3 13 to 14 (n = 10) 13.8 ± SD 3.2 7.4 ± SD 1.4 15 to 17 (n = 8) 17.0 ± SD 2.4 9.5 ± SD 0.9 Table 3 (abstract P266). Mean settings by age: severe group Age (years) IPAP EPAP <3 (n = 21) 12.7 ± SD 2.0 6.8 ± SD 1.3 3 to 4 (n = 50) 13.3 ± SD 2.8 6.8 ± SD 1.4 5 to 6 (n = 26) 13.9 ± SD 2.2 7.3 ± SD 1.3 7 to 8 (n = 29) 15.0 ± SD 2.4 7.7 ± SD 1.5 9 to 10 (n = 26) 15.7 ± SD 3.5 8.0 ± SD 1.8 11 to 12 (n = 22) 14.5 ± SD 3.2 7.6 ± SD 1.6 13 to 14 (n = 9) 15.3 ± SD 3.2 8.0 ± SD 1.7 15 to 17 (n = 14) 17.5 ± SD 3.0 9.5 ± SD 1.8 References 1. EMCDDA: Europol Annual Report on the Implementation of Council Decision 2005/387/JHA. Lisbon: EMCDDA/Europol; 2011. 2. Brandt SD, et al.: Drug Test Anal 2010, 2:377-382. 3. Sikk K, et al.: Acta Neurol Scand 2007, 115:385-389. 4. Stepens A, et al.: N Engl J Med 2008, 358:1009-1017. 5. Murphree HB, et al.: Clin Pharmacol Ther 1961, 2:722-726. P268 Abstract withdrawn P269 Thallium group poisoning incident in Japan 2011 Y Namba, R Suzuki, J Sasaki, M Takayasu, K Watanabe, D Kenji, M Hayashi, Y Kitamura, M Kawamo, H Masaki, E Kyuuno, M Hayashi, M Yamaguchi, A Maeda Showa University Fujigaoka Hospital, Yokohama, Japan Critical Care 2013, 17(Suppl 2):P269 (doi: 10.1186/cc12207) Introduction Thallium is an odorless, tasteless, heavy metal that has been often used for intentional poisonings. In severe patients, thallium poisoning produces neuromuscular symptoms such as extreme pain and muscle weakness. Introduction Thallium is an odorless, tasteless, heavy metal that has been often used for intentional poisonings. BiPAP for treating moderate and severe asthma exacerbations in a PED In severe patients, thallium poisoning produces neuromuscular symptoms such as extreme pain and muscle weakness. Methods Five case reports. p Results All patients worked at a pharmaceutical factory. They joined a tea party held at their workplace at the end of April 2011. The fi ve patients drank tea from a teapot someone had put thallium in. A few days later, they complained of femoral numbness and pain caused by pressure. About a week later, three of fi ve patients had profound hair loss. Three weeks after the party, they came to our ER. We thought that their symptoms might be caused by some chemicals. We searched the keywords: ‘lower extremity pain’, ‘hair loss’ and ‘poison’ in the Internet. As a result, thallium, mercury, lead, and so forth, were suspicious metals. In those metals, thallium was most likely because it was used in their factory. We immediately examined the blood concentration of several metals and ordered iron(III)hexacyanoferrate(II) that is known as the antidote for thallium poisoning. Only thallium was positive in the blood metal concentration test. Three patients consented to oral administration of an antidote. Two patients rejected administration because their symptoms were mild and getting better. All symptoms of all patients gradually disappeared by August. We also followed up the course of blood concentration of thallium. The concentration in three patients who took the antidote was reduced more rapidly than the two patients who did not take it. Results All patients worked at a pharmaceutical factory. They joined a tea party held at their workplace at the end of April 2011. The fi ve patients drank tea from a teapot someone had put thallium in. A few days later, they complained of femoral numbness and pain caused by pressure. About a week later, three of fi ve patients had profound hair loss. Three weeks after the party, they came to our ER. We thought that their symptoms might be caused by some chemicals. We searched the keywords: ‘lower extremity pain’, ‘hair loss’ and ‘poison’ in the Internet. y y p p As a result, thallium, mercury, lead, and so forth, were suspicious metals. In those metals, thallium was most likely because it was used in their factory. We immediately examined the blood concentration of several metals and ordered iron(III)hexacyanoferrate(II) that is known as the antidote for thallium poisoning. Only thallium was positive in the blood metal concentration test. Heart rate variability in children with tricyclic antidepressant intoxication Heart rate variability in children with tricyclic antidepressant intoxication EC Dinleyici, Z Kilic, S Sahin, R Tutuncu-Toker, M Eren, ZA Yargic, P Kosger B Ucar Eskisehir Osmangazi University Faculty of Medicine, Eskisehir, Turkey Critical Care 2013, 17(Suppl 2):P270 (doi: 10.1186/cc12208) EC Dinleyici, Z Kilic, S Sahin, R Tutuncu-Toker, M Eren, ZA Yargic, P Kosger, B Ucar Conclusion In this outbreak in the UK, Eric-3 gave symptoms similar to other stimulants known as legal highs, including death. It may have been a novel substance, 3-/4-fl uoroephedrine. This underlines the need for prospective data collection and early national and international information sharing. Eskisehir Osmangazi University Faculty of Medicine, Eskisehir, Turkey Critical Care 2013, 17(Suppl 2):P270 (doi: 10.1186/cc12208) Eskisehir Osmangazi University Faculty of Medicine, Eskisehir, Turkey Critical Care 2013, 17(Suppl 2):P270 (doi: 10.1186/cc12208) Introduction Tricyclic antidepressant (TCA) intoxication is among the most common causes of poisonings in our country. The TCA group of Introduction Tricyclic antidepressant (TCA) intoxication is among the most common causes of poisonings in our country. The TCA group of S102 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 demonstrated edematous wall thickening of gastric mucosa, and ascites was observed in 44.4% (12/27) of these patients. The patients with small intestinal anisakiasis demonstrated limited edematous wall thickening of the intestinal tract, and ascites was observed in 90.9% (40/44) of these patients. Furthermore, phlegmon of mesentery fat was observed in 72.7% (32/44) of the small intestinal anisakiasis patients. Conclusion When the cause of abdominal pain cannot be determined by initial assessment, anisakiasis should be considered, especially if the patient has a history of raw or undercooked seafood ingestion. In the ED, certain methods of diagnosis are evaluation of the time to develop symptoms and CT imaging, and a history of raw or undercooked seafood ingestion should be considered in the diagnosis. References drugs aff ects the central nervous and cardiovascular systems, resulting in severe arrhythmia and death. Heart rate variability (HRV) analysis is a non-invasive assessment method that allows evaluation of the cardiac autonomic (sympathetic and parasympathetic) activity. The aim of this study was to evaluate HRV in children requiring ICU stay due to TCA poisoning. Methods Twenty children with isolated TCA poisoning aged between 3 and 16 years who were hospitalized in the pediatric ICU, between March 2009 and July 2010, and 20 healthy children as a control group were enrolled. Diagnosing anisakiasis in the emergency department St. Luke’s International Hospital, Tokyo, Japan p y p Critical Care 2013, 17(Suppl 2):P271 (doi: 10.1186/cc12209) Introduction Humans can be incidentally parasitized by third-stage Anisakis larvae after ingestion of raw or undercooked seafood. Although the clinical symptom of anisakiasis is abdominal pain, the clinical fi nding is nonspecifi c and may be misdiagnosed as appendicitis, ileus, and so forth. Every year, >40,000 patients visit our ED and our hospital near the Tsukiji market (famous seafood market in Tokyo). We often observed cases of anisakiasis in patients who visited the hospital with abdominal pain as the chief complaint. Thus, we researched to determine factors useful to diagnose anisakiasis. Methods We retrospectively reviewed data of 83 patients (58 men, 25 women) diagnosed with anisakiasis in our ED (22 July 2003 to 22 July 2012) and examined the usefulness of clinical history, blood test, diagnostic imaging, and so forth, for anisakiasis diagnosis. Diagnosis was made after (A) endoscopically proven Anisakis polypide and (B) a hematologically positive Anisakis antibody (IgG, IgA) and CT diagnostic imaging. y Conclusion The implementation of the TM Project at a community hospital has a major impact in the eICU and eED Sepsis Management Program, and improved compliance with recommended care bundles. f Heart rate variability in children with tricyclic antidepressant intoxication Clinical and electrocardiographic (ECG) fi ndings were noted in the TCA poisoning group. In both groups, 24-hour time domain HRV analysis (SDNN, SDANN, SDNNi, RMSDD, NN50, and pNN50) was performed. We also recorded frequency domain analysis results at the fi rst 5 minutes and the last 5 minutes of the 24-hour record (VLF, nLF, nHF, LF/HF ratio). 1. Valle et al.: J Med Case Rep 2012, 6:114. 2. David Hwang et al.: Chonnam Med J 2012, 48:73-75. 1. Valle et al.: J Med Case Rep 2012, 6:114. Results The average level of TCA in the study group was 1,116 ± 635 and TCA levels were positively correlated with the duration of QRS interval (P  <0.01). In time-domain nonspectral evaluation, SDNN (P  <0.001), SDNN (P  <0.05), RMSDD (P  <0.01), and pNN50 (P  <0.01) were found signifi cantly lower in the TCA intoxication group compared with the control group, while NN50 (P <0.01) was signifi cantly higher in value. The spectral analysis (frequency domain) of data recorded at fi rst 5 minutes after intensive care admission showed that the values of the nLF (P <0.05) and LF/HF ratio (P = 0.001) were signifi cantly higher in the TCA intoxication group than the controls, while nHF (P = 0.001) values were signifi cantly lower. The frequency domain spectral analysis of data recorded at the last 5 minutes showed a lower nHF (P = 0.001) in the TCA intoxication group than the controls, and the LF/HF ratio was signifi cantly higher (P  <0.05) in the intoxication group. SDNN (P <0.001), RMSDD (P <0.01), SDNNi (P <0.01), and pNN50 (P <0.01) levels were higher in patients with positive ECG fi ndings than those without positive ECG fi ndings. The LF/HF ratio was higher in seven children with seizures (P <0.001).i P272 Implementation of a sepsis protocol in a community hospital using a Telemedicine Program M Steinman1, CA Abreu Filho1, A Andrade2, R Cal1, N Akamine1, J Teixeira2, E Silva1, A Kanamura1, M Cenderoglo1, C Lottenberg1 1Hospital Israelita Albert Einstein, São Paulo, Brazil; 2Hospital Municipal Dr. Moysés Deutsch, São Paulo, Brazil Critical Care 2013, 17(Suppl 2):P272 (doi: 10.1186/cc12210) Implementation of a sepsis protocol in a community hospital using a Telemedicine Program Introduction Sepsis has high incidence around the world, approximately 400,000 new cases occur annually in Brazil. In developing countries it is still an important cause of death due to poor adherence to best practice medicine protocols, mainly in community hospitals. The Brazilian mortality rate of septic shock is around 60%. The aim of this study is to describe the fi rst Brazilian initiative of implementation of a Telemedicine (TM) Project for therapeutic support of septic patients in a community hospital in São Paulo, Brazil. Conclusion Existing fi ndings give us an idea about HRV’s value to determine arrhythmia and predict convulsion risk in TCA poisonings. HRV can be used as a non-invasive method in determining the treatment and prognosis of TCA poisoning. y p , Methods Since May 2012, a TM Program has been implemented at two hospitals in São Paulo – Hospital Municipal Dr. Moysés Deutsch (HMMD), a public, secondary hospital, and Hospital Israelita Albert Einstein (HIAE), a tertiary private philanthropic entity  – due to a partnership with Brazilian Health Ministry. A TM Central Command was located at HIAE with Endpoint 97 MXP Cisco® Solution and a mobile Intern MXP ISDN/IP Cisco® for the remote hospital (HMMD) via dedicated GB/sec connection. Imaging examinations were evaluated using PACS technology. At HMMD, the Sepsis Protocol, based on the Surviving Sepsis Campaign, was started for every recruited patient admitted to the emergency department (ED) or the ICU, and assessed by the Central Command through TM with an experienced consultant. Results Over a 6-month period, 33 patients with diagnosis of sepsis (including severe sepsis and septic shock) admitted to HMMD were evaluated by skilled doctors of HIAE via TM, and the Surviving Sepsis Campaign protocol was instituted. In total, 87.8% of the consultations originated from the ICU and 12.2% from the ED; 16 patients were male (48.4%), mean age was 44.2 years old. TM improved diagnosis in 12.1% and infl uenced clinical management in 87.9% of the cases; in 11 patients (33.3%) TM consultation led to antibiotic change. Hospital mortality rate was 33.3%. P271 Diagnosing anisakiasis in the emergency department T Takabayashi, S Ishimatsu, N Otani, T Mochizuki, R Miyamichi St. Luke’s International Hospital, Tokyo, Japan Critical Care 2013, 17(Suppl 2):P271 (doi: 10.1186/cc12209) Reference 1. Beale R, et al.: Promoting Global Research Excellence in Severe Sepsis (PROGRESS): lessons from an international sepsis registry. Infection 2009, 37:222-232. Results Of the 83 patients, 39 had gastric anisakiasis and 44 had small intestinal anisakiasis. All gastric and small intestinal anisakiasis patients were diagnosed by methods A and B, respectively. A blood test was unable to show the specifi c infl ammatory reaction. A history of raw or undercooked seafood ingestion was noted in 95.2% (79/83) of the patients. This was observed in 100% (39/39) of the gastric anisakiasis patients and 91% (40/44) of the small intestinal anisakiasis patients. With regard to the development of symptoms, symptoms for gastric anisakiasis developed within 48 hours and reached a peak in less than 6 hours, whereas the symptoms for small intestinal anisakiasis reached a peak in 48 hours and persisted for a maximum of 5 days. Diagnostic CT imaging revealed that all the patients with gastric anisakiasis P273 Reform of emergency services: immediate eff ects on cardiac care unit and ICU patient intake AO Alaspää, T Valpas, V Rautava, A Palomäki Kanta-Häme Central Hospital, Hämeenlinna, Finland Critical Care 2013, 17(Suppl 2):P275 (doi: 10.1186/cc12213) Introduction Early onset eff ective care in the emergency department (ED) has been reported to have a great infl uence on the intensive care patients’ morbidity and mortality [1]. Little is known about the infl uence of the reorganisation of the ED on patient intake to the ICUs. The aim of this study was to analyse monthly intake of patients from the ED to the cardiac care unit (CCU) and ICU before and after the reform of emergency services. Results HMMD receives an average of 30 cases of stroke monthly, and thrombolysis did not occur before the implementation of the TM Project, because of the lack of neurologists available to conduce the cases. After implementation of the TM Program, six cases of ischemic stroke were thrombolyzed with alteplase; only one case (16%) progressed to death from septic shock, and one case (16%) presented symptomatic intracranial hemorrhage. g y Methods In Kanta-Häme Central Hospital, a new ED started on 1 April 2007. Four older emergency rooms were combined into one bigger emergency department and an observation ward was introduced with continuous follow-up of vital signs. This study is a retrospective analysis of the patient intake to the CCU and ICU 1 year before and after the reorganisation. Using as data the Finnish Intensive Care Quality Consortium (Intensium, Finland) database and the cardiac database of the hospital, patient transfer from ED to the ICU and CCU was collected and analysed. Monthly pre/post comparisons were carried out statistically by a nonparametric Wilcoxon signed-rank test. Conclusion Thrombolysis in ischemic stroke reduces 30% the risk of disability and 18% the mortality rate. This procedure has been only feasible to be done in the community setting because of the implementation of the TM Project, which permits the presence of a real time consultation with a specialized neurological team from a tertiary center. P274 P274 Analysis of emergency calls achieved in a French emergency dispatching centre: what resources for which patients O Tilak, J Cuny, N Assez, P Goldstein, E Wiel Lille University Hospital Center, Lille Cedex, France Critical Care 2013, 17(Suppl 2):P274 (doi: 10.1186/cc12212) Figure 1 (abstract P275). Combined patient fl ow to the ICU and CCU before and after the reformation of the ED. Initial Brazilian experience of Telestroke for thrombolysis in a community hospital CA Abreu Filho1, A Caluza1, M Steinman1, G Silva1, R Cal1, N Akamine1, J Teixeira2, A Andrade2, E Silva1, A Kanamura1, M Cenderoglo1, C Lottenberg1 CA Abreu Filho1, A Caluza1, M Steinman1, G Silva1, R Cal1, N Akamine1, J Teixeira2, A Andrade2, E Silva1, A Kanamura1, M Cenderoglo1, C Lottenberg1 1Hospital Israelita Albert Einstein, São Paulo, Brazil; 2Hospital Municipal Dr. Moysés Deutsch, São Paulo, Brazil C l C (S l ) (d / ) S103 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 analyses, and then returned home. In total, 25.50% of patients were hospitalized in a medical or surgical department, and 12.42% in the short-term hospitalised unit of the emergency department (stay duration <24  hours). Some 5.10% of patients worsened and were oriented in the ICU. A total 3.77% of patients in a cardiac ICU. In total, 73.84% of patients had stay duration less than 6 hours in the ED, 24.45% <24 hours. Forty percent of patients supported by fi remen and 54% supported by private ambulance left the hospital after a single medical consultation. Introduction Stroke is the leading cause of death in Brazil, the number of stroke patients receiving thrombolysis therapy in the developing world is extremely low. Prehospital delay, fi nancial constraints, and lack of infrastructure are the main barriers for thrombolysis therapy in developing countries. In this way, telemedicine (TM) allows knowledge transfer of medical expertise to remote community hospitals, where there is a shortage of specialized doctors. The aim of this study is to describe the fi rst Brazilian initiative of a Telestroke Program for therapeutic support and monitoring cases of acute stroke, and venous thrombolysis of eligible cases in a community hospital in São Paulo, Brazil. analyses, and then returned home. In total, 25.50% of patients were hospitalized in a medical or surgical department, and 12.42% in the short-term hospitalised unit of the emergency department (stay duration <24  hours). Some 5.10% of patients worsened and were oriented in the ICU. A total 3.77% of patients in a cardiac ICU. In total, 73.84% of patients had stay duration less than 6 hours in the ED, 24.45% <24 hours. Forty percent of patients supported by fi remen and 54% supported by private ambulance left the hospital after a single medical consultation. Conclusion Nearly 70% of patients calling the French emergency medical dispatching centre are sent to hospital. Initial Brazilian experience of Telestroke for thrombolysis in a community hospital Those transportations are supported for two-thirds of cases by a private ambulance or fi remen ambulance. One out of two patients only receive a simple medical consultation in the ED, and go back home. This may concur to the defi ciency of using general medicine in town. They prefer using emergency services for free. Only one patient out of four was hospitalized more than 24 hours. Methods Since May 2012 a TM Program has been implemented at two hospitals in São Paulo – Hospital Municipal Dr. Moysés Deutsch (HMMD), a public, secondary hospital, distant about 60  minutes from the nearest tertiary center, and Hospital Israelita Albert Einstein (HIAE), a tertiary private philanthropic entity – due to a partnership with Brazilian Health Ministry. Patients admitted to the community hospital’s emergency department with acute ischemic stroke as possible diagnosis are submitted to brain imaging examinations, and considered for remote consultation. A TM Central Command was located at HIAE with Endpoint 97 MXP Cisco® Solution and a mobile Intern MXP ISDN/IP Cisco® for the remote hospital (HMMD). Imaging examinations were evaluated using PACS technology. At HIAE the TM Center has skilled doctors, including neurologists, available for consultation 24 hours, 7 days a week. They discussed the neurological cases via TM, and selected stroke patients for intravenous thrombolysis when timely (3 hours symptom onset). P275 P275 Reform of emergency services: immediate eff ects on cardiac car unit and ICU patient intake AO Alaspää, T Valpas, V Rautava, A Palomäki Kanta-Häme Central Hospital, Hämeenlinna, Finland Critical Care 2013, 17(Suppl 2):P275 (doi: 10.1186/cc12213) P277 1. Rivers E, et al.: N Engl J Med 2001, 345:1368-1377. Reform of emergency services: immediate eff ects on cardiac care unit and ICU patient intake Introduction Following patients referred by the French emergency dispatching centre (SAMU 15) in diff erent hospital services (emergency or ICU) is not always easy. The purpose of this study was to analyze each call arriving in the emergency dispatching centre, defi ne resources for transportation, the host hospital department, and analyze the short- term outcome of these patients. p Methods We conducted a prospective, observational study, by collecting all data recorded at the emergency dispatching centre in a continuous 24-hour period. Each call creating a medical fi le was included. We identifi ed the type of call, various administrative information, the purpose of call, resources triggered, medical advice, hospital department admission, and clinical evolution. p p Results A total of 877 patients were included; 67.80% calling from home, 15.63% calling from a public place. Firemen ambulances were sent in 38.89%, private ambulance in 24.65% and an emergency medical ambulance (SMUR) in 11.23%. Simple medical advice without transportation was given in 13.19% of cases. In total, 22.18% of patients were entrusted to the family; 2.89% of patients refused transportation; 69.94% of triggered fi remen ambulances were done by a centre 18 call, without any medical regulation by an emergency physician. A total of 68.59% patients were referred to the emergency department, 1.2% in ICU, and 1.8% in cardiac ICU. In the emergency room, 50.78% of patients received a simple medical consultation with biological Figure 1 (abstract P275). Combined patient fl ow to the ICU and CCU before and after the reformation of the ED. S104 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Results The total decrease in monthly patient infl ow from ED to the ICU and CCU was 30.1% (P = 0.003); that is, from the mean of 47.7 ± 8.2 to 33.3 ± 8.3 patients (Figure 1). The 7.1% decrease in patients taken into the ICU (12.9 ± 4.1 to 12.0 ± 4.6 patients) was not statistically signifi cant. However, the intake from the ED to the ICU decreased by 38.6% (from 34.8 ± 6.5 to 21.3 ± 5.1 patients) (P = 0.002). Conclusion Overall there were no statistical diff erences between the observation years either for overtriage or undertriage. L Escobar Alvaro1, J Jimenez Gonzalez1, A Pelaez Ballesta1, A Corbatón Anchuelo2 Overtriage and undertriage in a prehospital system over 7 years L Carenzo, F Barra, A Messina, D Colombo, T Fontana, F Della Corte Universitè del Piemonte Orientale A. Avogadro, Novara, Italy Critical Care 2013, 17(Suppl 2):P276 (doi: 10.1186/cc12214) Introduction On 11 May 2011 two moderate magnitude earthquakes hit the city of Lorca, southeast Spain. They caused 11 deaths, including two pregnant women and their babies, more than 350 injured, and moderate or severe damage to 80% of the building in the city, including Lorca’s public hospital that had to be evacuated.i Introduction The Novara 118 emergency medical system (EMS) dispatch center manages medical emergency calls coming from a region that spreads out over 1,400 km2 and includes 88 towns and a population of 385,000 people; inhabitant density is 275 inhabitants/km2. p p y Methods Data collection from 1 January 2005 to 31 December 2011 was obtained (EMS software SaveOnLine® Suite 4; REGOLA s.r.l., Turin, Italy). Triage used was the Medical Priority Dispatch System and severity was defi ned by color codes. Methods A descriptive study of the fi les of Lorca’s hospital and the clinical records of patients that attended our service in the 20 hours following the second earthquake on 11 May. i y Results During the study period the Novara 118 dispatch center managed a total of 122,384 EMS interventions. Median (interquartile) overestimation (regardless of severity) was 10.0% (2.4 to 14.1%) while median underestimation was 1.5% (0.8 to 2.5%). See Tables 1 and 2. Results A total of 225 patients were relocated. Simultaneously 224 patients were treated by the emergency service, until the evacuation was completed and after then, from 10:00 pm to 3:00 pm the next day, 47 more in a fi eld hospital placed just outside the hospital building. See Figures 1 and 2. Table 1 (abstract P276). Overtriage (%) Overtriage 2005 2006 2007 2008 2009 2010 2011 Other 3.03 2.47 5.30 4.95 4.06 4.58 4.17 Cardiac 13.23 15.30 27.06 23.72 19.28 19.17 20.14 Drunk 0.00 0.00 0.00 0.00 0.00 0.00 0.00 Poisoning 10.19 18.92 17.91 8.47 8.85 15.20 11.81 Oncological 0.00 2.78 1.05 1.03 3.03 2.47 0.00 Neurologic 9.97 10.05 15.59 15.18 14.57 13.32 13.32 Not identifi ed 13.03 13.15 11.84 11.07 10.33 11.79 7.23 Mental 1.30 0.41 2.09 2.34 2.30 1.27 2.35 Respiratory 10.67 10.89 22.09 18.63 16.29 18.23 14.12 Trauma 12.97 12.68 10.42 8.59 6.78 7.74 7.56 Table 2 (abstract P276). Reform of emergency services: immediate eff ects on cardiac care unit and ICU patient intake When observed individually, trauma showed the only signifi cant overtriage reduction over time; there were no individual modifi cations in undertriage over time. Reference Reference Reference 1. Neely KW, Eldurkar JA, Drake ME: Acad Emerg Med 2000, 7:174-180. 1. Neely KW, Eldurkar JA, Drake ME: Acad Emerg Med 2000, 7:174-180. Conclusion According to our results the reform of the ED may have a signifi cant role in the total infl ow of patients to the CCU and ICU. Reference P278 P278 War injuries and civilian accidents in Afghan children R Kedzierewicz1, P Ramiara2, M Puidupin3, S Mérat4 1CMA des Alpes, Barby, France; 2HIA Sainte Anne, Toulon, France; 3HIA Desgenettes, Lyon, France; 4HIA Begin, Saint Mandé, France Critical Care 2013, 17(Suppl 2):P278 (doi: 10.1186/cc12216) P278 War injuries and civilian accidents in Afghan children R Kedzierewicz1, P Ramiara2, M Puidupin3, S Mérat4 1CMA des Alpes, Barby, France; 2HIA Sainte Anne, Toulon, France; 3HIA Desgenettes, Lyon, France; 4HIA Begin, Saint Mandé, France Critical Care 2013, 17(Suppl 2):P278 (doi: 10.1186/cc12216) p Conclusion The large number of casualties during a major disaster is a global problem, even in the developed countries. When the role of the intensivist is reviewed, many roles were verifi ed to be important; that is, as a leader of a medical team or triage offi cer as well as a professional in the fi eld of specifi c intensive care. However, there are many problems to be solved in the fi elds of disaster medicine. In order to solve the diversifi cation or the various medical problems, it is necessary to compile or systematize a disaster medicine of the world version. The concept of the compendium and our process of trial are shown in relation to intensive care. Introduction We intended to compare paediatric traumatic injuries due to war and others related to civilian accidents (CA). Methods We conducted an observational epidemiologic retrospective study on paediatric cases (<18 years old) seen at the emergency room of Kabul International Airport NATO role 3 medical treatment facility between 2009 and 2012. Introduction We intended to compare paediatric traumatic injuries due to war and others related to civilian accidents (CA). Methods We conducted an observational epidemiologic retrospective study on paediatric cases (<18 years old) seen at the emergency room of Kabul International Airport NATO role 3 medical treatment facility between 2009 and 2012. Results During 3 years, 341 children were admitted to the emergency room (ER) with a mean age of 10 years (9.49 to 10.51). Eighty-eight per cent of children (301) were traumatized, 39% of them (118) due to war injuries (WI) and 61% (183) due to CA. Forty-three per cent of WI (gunshot wound 34%, explosion 66%) but only 19% of injuries due to CA (falls 24%, transport accidents 56%, burns 9%, penetrating injuries 9%, unknown cause 2%) involved polytraumas defi ned as a New Injury Severity Score (NISS) >15 (P <0.001). References Intensive care and disaster medicine: the role of a compendium Y Haraguchi1, Y Tomoyasu1, H Nishi1, M Hoshino1, T Tsubata2, M Sakai1, E Hoshino1 1Disaster Medicine Compendium Team, Japan, Tokyo, Japan; 2Keiyo Hospital, Tokyo, Japan Critical Care 2013, 17(Suppl 2):P279 (doi: 10.1186/cc12217) 1. Hill AD, Vingilis E, Martin CM, Hartford K, Speechley KN: Interhospital transfer of critically ill patients: demographic and outcomes comparison with nontransferred intensive care unit patients. J Crit Care 2007, 22:290-295. 2. Flabouris A, Hart GK, George C: Outcomes of patients admitted to tertiary intensive care units after interhospital transfer: comparison with patients admitted from emergency departments. Crit Care Resusc 2008, 10:97-105. 3. Duke GJ, Green JV: Outcome of critically ill patients undergoing interhospital transfer. Med J Aust 2001, 174:122-125. 1. Hill AD, Vingilis E, Martin CM, Hartford K, Speechley KN: Interhospital transfer of critically ill patients: demographic and outcomes comparison with nontransferred intensive care unit patients. J Crit Care 2007, 22:290-295. 1. Hill AD, Vingilis E, Martin CM, Hartford K, Speechley KN: Interhospital transfer of critically ill patients: demographic and outcomes comparison with nontransferred intensive care unit patients. J Crit Care 2007, 22:290-295. p 2. Flabouris A, Hart GK, George C: Outcomes of patients admitted to tertiary intensive care units after interhospital transfer: comparison with patients admitted from emergency departments. Crit Care Resusc 2008, 10:97-105. 3. Duke GJ, Green JV: Outcome of critically ill patients undergoing interhospital transfer. Med J Aust 2001, 174:122-125. 3. Duke GJ, Green JV: Outcome of critically ill patients undergoing interhospital transfer. Med J Aust 2001, 174:122-125. Introduction There were several catastrophes in the last decades. The make-up of systematic measuring and life-saving medical systems, including intensive care, is thought to be an urgent and essential issue. Our eff orts for establishing a disaster medicine and education system are presented. L Escobar Alvaro1, J Jimenez Gonzalez1, A Pelaez Ballesta1, A Corbatón Anchuelo2 Undertriage (%) Undertriage 2005 2006 2007 2008 2009 2010 2011 Other 1.01 0.77 2.87 1.66 1.44 1.48 1.31 Cardiac 2.16 1.44 1.64 1.60 2.15 1.60 1.14 Drunk 0.00 1.89 0.00 0.00 0.00 0.00 0.00 Poisoning 8.33 0.00 0.00 1.69 0.00 1.60 0.00 Oncological 95.74 0.56 0.00 1.03 3.03 2.47 0.00 Neurologic 2.42 2.32 2.96 3.67 3.05 1.40 2.46 Not identifi ed 1.72 2.13 1.87 2.20 2.04 2.74 3.50 Mental 1.74 0.83 1.05 1.17 0.00 0.42 0.39 Respiratory 2.30 4.16 3.76 3.75 2.57 2.72 2.46 Trauma 1.07 0.92 1.50 1.41 1.36 1.31 1.46 Table 1 (abstract P276). Overtriage (%) Table 1 (abstract P276). Overtriage (%) g Figure 1 (abstract P277). Hospital evacuation. Figure 2 (abstract P277). Emergency service taking care of patients during evacuation. Table 1 (abstract P276). Overtriage (%) Overtriage 2005 2006 2007 2008 2009 2010 2011 Other 3.03 2.47 5.30 4.95 4.06 4.58 4.17 Cardiac 13.23 15.30 27.06 23.72 19.28 19.17 20.14 Drunk 0.00 0.00 0.00 0.00 0.00 0.00 0.00 Poisoning 10.19 18.92 17.91 8.47 8.85 15.20 11.81 Oncological 0.00 2.78 1.05 1.03 3.03 2.47 0.00 Neurologic 9.97 10.05 15.59 15.18 14.57 13.32 13.32 Not identifi ed 13.03 13.15 11.84 11.07 10.33 11.79 7.23 Mental 1.30 0.41 2.09 2.34 2.30 1.27 2.35 Respiratory 10.67 10.89 22.09 18.63 16.29 18.23 14.12 Trauma 12.97 12.68 10.42 8.59 6.78 7.74 7.56 Table 2 (abstract P276). Undertriage (%) Figure 1 (abstract P277). Hospital evacuation. Figure 1 (abstract P277). Hospital evacuation. Figure 1 (abstract P277). Hospital evacuation. Figure 2 (abstract P277). Emergency service taking care of patients during evacuation. Figure 2 (abstract P277). Emergency service taking care of patients during evacuation. Undertriage 2005 2006 2007 2008 2009 2010 2011 Other 1.01 0.77 2.87 1.66 1.44 1.48 1.31 Cardiac 2.16 1.44 1.64 1.60 2.15 1.60 1.14 Drunk 0.00 1.89 0.00 0.00 0.00 0.00 0.00 Poisoning 8.33 0.00 0.00 1.69 0.00 1.60 0.00 Oncological 95.74 0.56 0.00 1.03 3.03 2.47 0.00 Neurologic 2.42 2.32 2.96 3.67 3.05 1.40 2.46 Not identifi ed 1.72 2.13 1.87 2.20 2.04 2.74 3.50 Mental 1.74 0.83 1.05 1.17 0.00 0.42 0.39 Respiratory 2.30 4.16 3.76 3.75 2.57 2.72 2.46 Trauma 1.07 0.92 1.50 1.41 1.36 1.31 1.46 Figure 2 (abstract P277). Emergency service taking care of patients during evacuation. L Escobar Alvaro1, J Jimenez Gonzalez1, A Pelaez Ballesta1, A Corbatón Anchuelo2 S105 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Results Our research indicated that disaster medicine should be established systematically or it is necessary to compile a compendium of disaster medicine from a broad perspective or from a bird’s-eye and long-term view. The Japanese version was tentatively completed with 22 volumes as of the fi nancial year 2005, of which nearly three-quarters are written in Japanese. Although this worked partly during the above- shown catastrophe in Japan 2011, several problems are left to be solved; that is, the insuffi cient operation system of the Japan DMAT or Disaster Medical Assistant Team that seemed to have caused a large number of preventable deaths. Results Our research indicated that disaster medicine should be established systematically or it is necessary to compile a compendium of disaster medicine from a broad perspective or from a bird’s-eye and long-term view. The Japanese version was tentatively completed with 22 volumes as of the fi nancial year 2005, of which nearly three-quarters are written in Japanese. Although this worked partly during the above- shown catastrophe in Japan 2011, several problems are left to be solved; that is, the insuffi cient operation system of the Japan DMAT or Disaster Medical Assistant Team that seemed to have caused a large number of preventable deaths. Conclusion In less than 3 hours 225 patients were evacuated and 224 were given attention by the emergency service of Lorca’s hospital, with the support of personnel from other services of the hospital. The emergency service of the hospital continues to be operative in the building until evacuation is completed and in a fi eld hospital later. P278 Patients experiencing a traumatic injury related to war had lower Paediatric Trauma Scores (PTS) than those injured in a CA (PTS = 6.4 (5.7 to 7.0) vs. PTS = 9.0 (8.7 to 9.4) respectively, P <0.00001), higher ISS (ISS = 16.7 (13.8 to 19.6) vs. ISS = 8.9 (7.5 to 10.2) respectively, P <0.00001), NISS (P <0.0001), mortality rate predicted by the Trauma Injury Severity Score (TRISS, P <0.02) or A Severity Characterization of Traumas (ASCOT, P <0.001). The result is longer overall hospitalization of patients having WI (P < 0.001) and a higher number of surgeries (P <0.02). After the ER, 54% of patients with WI were hospitalized in the ICU (86% of them after surgery) but only 26% of patients involved in a CA (71% after surgery). As many patients with WI as involved in a CA (40%) were admitted to the ward (89% of patients with WI after surgery but only 63% of patients with injuries due to a CA). Thirty-three per cent of patients involved in a CA returned home and one was transferred, whereas only three patients with WI returned home after being in the ER, three patients were transferred and one died in the operating room. Observed paediatric mortality in our medical treatment facility was 2.9% (10 children out of 341): three children died of WI, three due to a CA and one of septic shock due to a medical cause. Factors involved in ICU mortality following medical retrieval: identifying diff erences between ICU survivors and nonsurvivors M Kennedy1, P Visser1, L Harriss2 y 1Ambulance Victoria, Melbourne, Australia; 2Monash University, Melbourne, Australia y 1Ambulance Victoria, Melbourne, Australia; 2Monash University, Melbourne, Australia l l d Critical Care 2013, 17(Suppl 2):P280 (doi: 10.1186/cc12218) Introduction The study aimed to determine factors related to ICU mortality in critically ill patients transferred by Adult Retrieval Victoria (ARV) medical staff . Patients who died in the ICU after interhospital transfer were compared with those who survived. Methods This is a retrospective cohort study of ARV cases between 1 January 2009 and 30 June 2010. Retrieval data were linked with data from the ANZICS CORE APD (Australia and New Zealand Intensive Care Society Centre for Outcome and Resource Evaluation Adult Patient Database). Victoria Data Linkage (VDL) performed linkage of the data. Data included demographic and clinical data obtained during transfer and data related to outcome measures in the ICU. Results Of the 601 cases transferred by ARV during the study period, 549 were eligible for linkage to 25,543 ANZICS APD case records for the same period. VDL matched 460 of these cases (83.8%). Logistic regression analysis revealed the variables associated with mortality were advanced age (OR = 1.02, 95% CI = 1.00 to 1.04, P = 0.02), cardiac principal referral problems (OR = 1.84, 95% CI = 1.02 to 3.32, P = 0.04), lower mean arterial blood pressure (OR = 0.97, 95% CI = 0.95 to 0.99, P = 0.005) and tachycardia (OR = 1.02, 95% CI = 1.00 to 1.03, P = 0.008) on arrival at the destination hospital. Conclusion War injuries are more prone to cause polytrauma than CA. According to the PTS, ISS, NISS, TRISS and ASCOT, children experiencing WI have higher severity scores and predicted mortality rate than others, stay longer in the hospital and have more surgeries. Conclusion Advanced age, lower mean arterial blood pressure and tachycardia towards the completion of transfer were associated with increased ICU mortality in this population. Clinicians should be aware of the additional risk for cardiac patients. P282 P282 Are physicians required during HEMS winch rescue missions? PB Sherren, C Hayes-Bradley, C Reid, B Burns, K Habig Greater Sydney Area HEMS, Sydney, Australia Critical Care 2013, 17(Suppl 2):P282 (doi: 10.1186/cc12220) Introduction A winch-capable helicopter emergency medical service (HEMS) off ers several advantages over standard rescue operations. Little is known about the benefi t of physician winching in addition to a highly trained paramedic. We analysed the mission profi les and interventions performed during rescues involving the winching of a physician in the Greater Sydney Area HEMS (GSA-HEMS). y g y p y p Methods A suggested algorithm for TCA was developed based on the Greater Sydney Area Helicopter Emergency Medical Service’s standard operating procedures and current available evidence. p g p Results An algorithm for the general management of TCA can be seen in Figure 1. In TCA, priority should be given to catastrophic haemorrhage control (tourniquets, direct pressure, haemostatic agents, pelvic and long bone splintage) and volume resuscitation. Simultaneous oxygena tion optimisation should occur with proactive exclusion of tension pneumothoraces with bilateral open thoracostomies. Cardiac ultrasound (US) should be used to help exclude cardiac tamponade and assist in prognostication. The US presence of true cardiac standstill versus low pressure state/pseudo-PEA, and an ETCO2 <1.3 kPa carries a grave prognosis in TCA. Given the high incidence of hypovolaemia, hypoxia and obstructive shock prior to TCA, the role of adrenaline and chest compressions are limited. Figure 2 shows a suggested algorithm for the management of penetrating TCA requiring prehospital thoracotomy. y y y Methods All winch missions involving a physician from August 2009 to January 2012 were identifi ed from the prospectively completed GSA-HEMS electronic database. A structured case-sheet review for a predetermined list of demographic data and physician-only interventions (POI) was conducted. Results We identified 130 missions involving the winching of a physician, of which 120 case sheets were available for analysis. The majority of patients were traumatically injured (90%) and male (85%) with a median age of 37 years. Seven patients were pronounced life extinct on the scene. A total of 63 POI were performed on 48 patients. Administration of advanced analgesia was the most common POI making up 68.3% of interventions. Patients with abnormal RTSc2 scores were more likely to receive a POI when compared with those with normal RTSc2 (P = 0.03). The performance of POI had no effect on median scene times (45 vs. 1. Lockey D, et al.: Ann Emerg Med 2006, 48:240-244. Algorithm for the resuscitation of traumatic cardiac arrest patients in a physician-staff ed helicopter emergency medical service PB Sh C R d b B B Methods Mega-disasters or catastrophes are researched basically on actual medical experience; that is, the 9/11 attack in 2001, Hurricane Katrina in 2005, Indian Ocean earthquake with tsunami in 2004, the Chernobyl incident in 1986 as well as the Higashinihon earthquakes with tsunami, which was followed by the severest degree of nuclear disaster in 2011. f PB Sherren, C Reid, K Habig, B Burns g Greater Sydney Area HEMS, Sydney, Australia y y , y y, Critical Care 2013, 17(Suppl 2):P281 (doi: 10.1186/cc12219) Introduction Survival rates following traumatic cardiac arrest (TCA) are known to be poor but resuscitation is not universally futile [1]. S106 Critical Care 2013, Volume 17 Suppl 2 htt // f / l t /17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 http://ccforum.com/supplements/17/S2 Figure 1 (abstract P281). Traumatic cardiac arrest algorithm. Figure 1 (abstract P281). Traumatic cardiac arrest algorithm. There are distinct diff erences in the pathophysiology between medical cardiac arrests and TCA. Traumatic pathologies associated with an improved chance of successful resuscitation include hypoxia, tension pneumothorax and cardiac tamponade [1]. The authors believe a separate algorithm is required for the management of out-of-hospital TCA attended to by a highly trained physician and paramedic team. P282 43 minutes; P = 0.51). See Tables 1 and 2. Conclusion The suggested algorithm is designed for a highly trained physician-led prehospital team and aims to maximise the number of neurologically intact survivors in out-of-hospital TCA. Reference 1. Lockey D, et al.: Ann Emerg Med 2006, 48:240-244. Conclusion Our high POI rate of 40% coupled with long rescue times and the occasional severe injuries supports the argument for winching doctors. Not doing so would deny a signifi cant proportion of patients time-critical interventions, advanced analgesia and procedural sedation. S107 Critical Care 2013, Volume 17 Suppl 2 http://ccforum com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 http://ccforum.com/supplements/17/S2 Figure 2 (abstract P281). Thoracotomy algorithm. Figure 2 (abstract P281). Thoracotomy algorithm. Figure 2 (abstract P281). Thoracotomy algorithm. P283 Improving maintenance of critical care land and aeromedical transfer equipment D Ashton-Cleary, N Boyd Royal Cornwall Hospital, Truro, UK Critical Care 2013, 17(Suppl 2):P283 (doi: 10.1186/cc12221) Introduction The aim was to assess the content and state of repair of equipment carried for transfer of critical care patients to other hospitals. By chance, several items of date-expired stock were identifi ed in the transfer kit whilst moving a patient to a tertiary centre. This raised the possibility of a more extensive problem with the equipment bags. Due to the geographical location of our district general hospital we undertake around 70 transfers of critical care patients to other hospitals per year (16% by air) and it is clearly important that our equipment is well maintained for these journeys. Table 1 (abstract P282). Demographic data, timings and Coded Revised Trauma Score (RTSc2) Total (n = 120) Male 102 (85) Median (IQR) age (years) 37 (26 to 53) Received a physician-only intervention 48 (40) Pronounced life extinct on arrival 7 (5.8) RTSc2 7.8408 104 (86.7) 7.0001 to 7.8407 2 (1.7) 6.0001 to 7.0000 4 (3.3) 5.0001 to 6.0000 4 (3.3) 4.0001 to 5.0000 0 (0) <4.0001 6 (5) Table 2 (abstract P282). Interventions performed Physician-only intervention Number of interventions (n = 63) Analgesia/procedural sedation Intravenous ketamine 42 (66.7) Intravenous fentanyl 1 (1.6) Fascia iliaca compartment block 1 (1.6) Airway management Rapid sequence induction and intubation 4 (6.3) Surgical airway 1 (1.6) Circulatory support Adult intraosseous access 1 (1.6) Blood transfusion 2 (3.2) Orthopaedic manipulation of joint/limb 6 (9.5) Thoracostomy 1 (1.6) Diagnostic ultrasound 1 (1.6) Hypertonic saline administration 3 (4.8) Data presented as n (%). Safety standards for intrahospital transfer of critical care patients D Ashton-Cleary Royal Cornwall Hospital, Truro, UK Critical Care 2013, 17(Suppl 2):P284 (doi: 10.1186/cc12222) Introduction The aim was to assess care of patients during intrahospital transfer. The UK Royal College of Anaesthetists has defi ned auditable standards for the care of patients and the training of escorting medical and nursing staff in this context [1]. p y y Conclusion Prehospital hyperoxemia did not infl uence the functional neurological outcome. One of the reasons for this fi nding could be the short arrival time to the trauma center where repeated analyses of arterial blood gases were performed. Therefore, correction of fraction of inspired oxygen according to the arterial blood gas analysis shortens the time of hyperoxemia, thus reducing neuronal brain damage. References f Methods Patients in a 27-bed combined general and neurosurgical critical care unit were studied in January 2011 and May 2012. Patients undergoing radiology department imaging or intervention were identifi ed from the electronic imaging library. Records of these transfers were sought in the critical care electronic notes and the standards of documentation graded on a fi ve-point scale (very good, good, average, minimal, absent). Documentation of the grade and training of escorting staff was also sought. Between the two study periods, a transfer safety checklist was introduced. 1. Beynon et al.: Brain tissue oxygen monitoring and hyperoxic treatment in patients with traumatic brain injury. J Neurotrauma 2012, 29:2109-2123. 1. Beynon et al.: Brain tissue oxygen monitoring and hyperoxic treatment in patients with traumatic brain injury. J Neurotrauma 2012, 29:2109-2123. 1. Beynon et al.: Brain tissue oxygen monitoring and hyperoxic treatment in patients with traumatic brain injury. J Neurotrauma 2012, 29:2109-2123. 2. Brenner et al.: Association between early hyperoxia and worse outcomes after traumatic brain injury. Arch Surg 2012, 16:1-5. p j y , 2. Brenner et al.: Association between early hyperoxia and worse outcomes after traumatic brain injury. Arch Surg 2012, 16:1-5. Results A total of 20.9% of 143 patients underwent one or more transfers in January 2011 (40 transfers). In May 2012, 26.4% of 151 patients underwent 57 transfers. In the fi rst period, documentation was graded as minimal (limited to a statement that the patient had left the critical care unit) or absent in 77.5% of transfers. In the 62.5% of patients transferred whilst on invasive ventilation, 88.0% had no documentation by the doctor and in 84.0% it was not known which doctor had escorted the patient. P285 Prehospital hyperoxemia does not infl uence the functional neurological outcome in polytraumatized patients with traumatic head injury V Vujanovic Popovic1, T Pelcl1, M Spindler1, Z Klemenc Ketis2, M Strnad1 1ZD dr. Adolfa Drolca Maribor, Slovenia; 2University of Maribor, Slovenia Critical Care 2013, 17(Suppl 2):P285 (doi: 10.1186/cc12223) g g g Results A total of 13.9% of drug items and 29.2% of equipment items had expired or would do so within 30 days of the initial assessment. The combined weight of one equipment and one drug bag was reduced from 14 to 9 kg (36% reduction) by introducing the new inventory. At reassessment in November 2012, only 10 items of equipment (3.2%) were expired or near to expiry and there were no expired drug items (4.1% near to expiry). In total, 0.3  kg (26 small items) of extraneous equipment had been added through over-restocking and was removed. Conclusion These bags are designed for a clinician to manage a patient when an emergency arises during transfer of a critical care patient. By the introduction of simple measures, the risks posed by expired items or cluttered equipment bags have almost been eradicated. Signifi cant weight savings have been made; this off ers improved ergonomics for staff and is also an important consideration for aeromedical operations. Our department was surprised to discover the extent of decline of our equipment and it may be that other departments would fi nd themselves in a similar position. The anaesthetic registrars who routinely escort the transfer patients have a vested interest to maintain this equipment and this has secured their buy-in to the new checking procedure with clear results. P284 Safety standards for intrahospital transfer of critical care patients D Ashton-Cleary Royal Cornwall Hospital, Truro, UK Critical Care 2013, 17(Suppl 2):P284 (doi: 10.1186/cc12222) P286 Innate immune response-mediated late increase in SuPAR in multi-trauma patients K Timmermans, M Kox, M Vaneker, P Pickkers Radboud University Nijmegen Medical Center, Nijmegen, the Netherlands Critical Care 2013, 17(Suppl 2):P286 (doi: 10.1186/cc12224) P282 Table 2 (abstract P282). Interventions performed Physician-only intervention Number of interventions (n = 63) Analgesia/procedural sedation Intravenous ketamine 42 (66.7) Intravenous fentanyl 1 (1.6) Fascia iliaca compartment block 1 (1.6) Airway management Rapid sequence induction and intubation 4 (6.3) Surgical airway 1 (1.6) Circulatory support Adult intraosseous access 1 (1.6) Blood transfusion 2 (3.2) Orthopaedic manipulation of joint/limb 6 (9.5) Thoracostomy 1 (1.6) Diagnostic ultrasound 1 (1.6) Hypertonic saline administration 3 (4.8) Data presented as n (%). Table 2 (abstract P282). Interventions performed Table 1 (abstract P282). Demographic data, timings and Coded Revised Trauma Score (RTSc2) transfer kit whilst moving a patient to a tertiary centre. This raised the possibility of a more extensive problem with the equipment bags. Due to the geographical location of our district general hospital we undertake around 70 transfers of critical care patients to other hospitals per year (16% by air) and it is clearly important that our equipment is well maintained for these journeys. Introduction The aim was to assess the content and state of repair of equipment carried for transfer of critical care patients to other hospitals. By chance, several items of date-expired stock were identifi ed in the S108 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Methods We maintain two identical sets of equipment (syringes, fl uid, airway management items, and so forth) and drug bags to take on transfers; one equipment and one drug bag taken on each trip. The contents of all four bags were checked and itemised. By careful consideration of the aims of the bags (to provide emergency equipment and drugs for managing one patient during an en-route emergency) a new inventory was devised. Excess items were removed to lighten the bags and improve accessibility to the essential items. Expired stock was removed. A daily checking procedure and tamper-proof seals on the bags were instigated and the bags were reassessed 12 months later. hospital protocols and specifi c training courses to improve standards of transfer medicine locally. The study also identifi ed our portable head CT scanner to have the potential to reduce transfers by 52% and so this has been strongly promoted. Reference Safety standards for intrahospital transfer of critical care patients D Ashton-Cleary Royal Cornwall Hospital, Truro, UK Critical Care 2013, 17(Suppl 2):P284 (doi: 10.1186/cc12222) There was only slight improvement in the second period (71.9% minimal or absent documentation, 80.0% no documentation by the doctor, 72.0% not known which doctor escorted). In the documentation available, six severe complications were noted during the second period (including episodes of severe bradycardia, hypotension and pupil dilatation). 3. Davis et al.: Both hypoxemia and extreme hyperoxemia may be detrimental in patients with severe traumatic brain injury. J Neurotrauma 2009, 26:2217-2223. Prehospital hyperoxemia does not infl uence the functional neurological outcome in polytraumatized patients with traumatic head injury y V Vujanovic Popovic1, T Pelcl1, M Spindler1, Z Klemenc Ketis2, M Strnad1 1ZD dr. Adolfa Drolca Maribor, Slovenia; 2University of Maribor, Slovenia Critical Care 2013, 17(Suppl 2):P285 (doi: 10.1186/cc12223) Introduction The association between hyperoxemia and neurological outcome in trauma patients is not clear. We examined the association between prehospital hyperoxemia and neurological outcome in polytraumatized patients. Introduction The association between hyperoxemia and neurological outcome in trauma patients is not clear. We examined the association between prehospital hyperoxemia and neurological outcome in polytraumatized patients. p y p Methods This was a retrospective study of polytraumatized patients with traumatic head injury who were endotracheal intubated and ventilated with supplemental oxygen (100%) in the prehospital emergency setting. Arterial partial oxygen pressure (PaO2) was measured after arrival at the hospital trauma center. We included patients with initial PaO2 above 160  mmHg (hyperoxemia group). The severity of the trauma was determined upon the admission to the hospital by the Injury Severity Scale (ISS) and the outcome was assessed at the discharge from the hospital using the Glasgow Coma Scale (GCS), Glasgow Outcome Scale (GOS) and Cerebral Performance Categories scale (CPC). Mann–Whitney’s test was used for data analysis. Results Sixty patients were involved in the study. Forty-eight (80%) of them were men and 86.7% sustained blunt trauma. Hyperoxemia was present in 41.6% of patients. Initial average ISS was 38, in patients with normoxemia 32.5 and in patients with hyperoxemia 35.4. Discharge GCS, GOS and CPC in the hyperoxemia group compared with the normoxemia group were 9.86 versus 9.33 (P  =  0.503), 2.52 versus 2.24 (P = 0.613) and 3.10 versus 3.19 (P = 0.936) with the duration of hospitalization of 26.64 days versus 27.72 days (P = 0.984).l Reference 1. Morgan G: Critical care services. In Raising the Standard: A Compendium of Audit Recipes. 2nd edition. London: Royal College of Anaesthetists; 2006. P287 Single-dose estrogen infusion can amplify brain levels of Sonic hedgehog, a signal protein for neuro stem cells and repair following the indirect brain injury resulting after severe torso burns PE Pepe1, JG Wigginton1, JW Gatson1, J Simpkins2, D Maass1, K AbdelFattah1, AH Idris1, V Warren1, JP Minei1 1University of Texas Southwestern Medical Center, Dallas, TX, USA; 2University of North Texas, Fort Worth, TX, USA Critical Care 2013, 17(Suppl 2):P287 (doi: 10.1186/cc12225) Introduction Many proposed resuscitative therapies for cardiac arrest and trauma will require the earliest possible intervention and would occur under volatile circumstances, making true informed consent for clinical trials unfeasible. The purpose here was to report our experience using exception to informed consent during the inaugural pilot study of infusing estrogen for acute injury, the so-called RESCUE Shock study. Methods Fifty patients were enrolled in RESCUE Shock in which estrogen or placebo was infused as soon as possible in the emergency department for trauma patients with a low systolic blood pressure (<90 mmHg) at two level I trauma centers. They were all treated with a single-dose estrogen or placebo infusion within 2  hours using exception from informed consent following US federal guidelines. Introduction Severe burn patients are often noted to have subsequent neurocognitive problems. Experimentally, we have found striking, prolonged elevations of infl ammatory markers in the brain (for example, IL-6) even when the injury occurs in a remote anatomic location. This neuroinfl ammatory response can also be signifi cantly blunted by a single post-burn dose of estrogen. Sonic hedgehog (SHH), an important signaling protein found in the brain, controls and directs diff erentiation of neural stem cells, infl uencing brain regeneration and repair by generating new neurons throughout life. As estrogens not only blunt infl ammation but also exert an infl uence on a variety of stem cells, we hypothesized that 17β-estradiol (E2) might aff ect levels of SHH in the post-burn rat brain. Results Investigator-initiated exception from informed consent studies is feasible, with our FDA IND approval obtained in 31  days, IRB 1 approval in 25 days, and IRB 2 approval in 24 days. Community consultation/notifi cation was successfully accomplished with no one opting out and 47/50 enrolled patients or their legal representatives were notifi ed of participation (one died unidentifi ed, two died with no known contact). Innate immune response-mediated late increase in SuPAR in multi-trauma patients p K Timmermans, M Kox, M Vaneker, P Pickkers Radboud University Nijmegen Medical Center, Nijmegen, the Netherlands Critical Care 2013, 17(Suppl 2):P286 (doi: 10.1186/cc12224) K Timmermans, M Kox, M Vaneker, P Pickkers Radboud University Nijmegen Medical Center, Nijmegen, the Netherlands Critical Care 2013, 17(Suppl 2):P286 (doi: 10.1186/cc12224) Introduction The soluble form of urokinase-type plasminogen activator (suPAR) has been identifi ed as a marker for immune activation and is demonstrated to accurately predict outcome in patients with sepsis or infectious diseases. In multi-trauma patients a considerable immunological response also occurs that is related to multiple organ failure and patient outcome. We investigated the kinetics of suPAR, correlation with the immune response and outcome in multi-trauma patients. Conclusion On average our unit conducts nearly two critical care transfers each day. Severe complications seem to complicate at least 10% of these, stressing the risk, need for good care and ongoing training. The intervention made in this audit had little impact on the standard of documentation. However, it has raised the issue within the consciousness of the staff . It is important to identify interventions that have failed to reach a gold standard to provide the impetus to seek other solutions. As a result of this study, the author has devised new Methods Blood was obtained from adult multi-trauma patients (n = 63) on arrival at the emergency room (ER) of the Radboud University S109 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Nijmegen Medical Centre and days 1, 3, 5, 7, 10 and 14 following trauma. Plasma concentrations of TNFα, IL-6, IL-10, IFNγ, IL-8 and MCP-1 were determined by Luminex, and SuPAR concentrations using ELISA. Clinical data were collected from electronic patient fi les. Concentrations, areas under the curve (AUC) and regression coeffi cients were statistically analyzed. Spearman correlation coeffi cients were calculated and diff erences between survival/nonsurvival groups were analyzed using unpaired Student t tests. Methods Male rats (n = 44) were assigned randomly into three groups: controls/no burn (n = 4); burn/placebo (n = 20); and burn/E2 (n = 20). Burned rats received a 40% 3° TBSA dorsal burn, fl uid resuscitation and one dose of E2 or placebo (0.5 mg/kg intraperitoneally) 15 minutes post burn. Innate immune response-mediated late increase in SuPAR in multi-trauma patients Eight animals from each of the two burn groups (burn/placebo and burn/E2) were sacrifi ced at 24 hours and at 7 days, respectively (sham group at 7 days only), with four each of the two burn groups sacrifi ced at 45 days. Brain tissue samples were analyzed by ELISA for SHH. p Results SuPAR values at admission to the ER were higher in nonsurvivors compared with survivors (n = 16, mean ± SEM 4.1 ± 0.6 ng/ml vs. n = 40, 3.0 ± 0.2 ng/ml, P = 0.03). SuPAR levels increased in time. An increase of suPAR did not predict or precede death, however. SuPAR AUC from ER to day 5 tended to correlate with injury severity score (r = 0.5, P = 0.07). Plasma cytokines in the ER did not correlate with suPAR measured at the same time (for example, TNFα: r = 0.2, P = 0.37, IL-10: r = –0.02, P = 0.91), while cytokine concentrations at the ER did correlate with suPAR levels at days 3 (TNFα: r = 0.6, P <0.01, IL-10: r = 0.5, P = 0.02) and 5 (TNFα: r = 0.7, P <0.01). Results Mean levels of SHH levels were signifi cantly elevated within 24 hours as much as 45 days post injury in burned animals receiving the 17β-estradiol (>1,200  pcg/mg) as compared with the placebo- treated burned animals (<700 pg/mg) and controls (<300 pcg/mg). See Figure 1. g Conclusion Early, single-dose estrogen administration following severe burn injury signifi cantly elevated levels of SHH in brain tissue. This fi nding may represent an extremely novel and important pathway for both neuroprotection and neuroregeneration in burn patients. Conclusion Plasma concentrations of SuPAR measured at admission to the ER are associated with overall survival of multi-trauma patients. Furthermore, suPAR concentrations increased during hospital admission, with most pronounced increases found in patients that suff ered more serious injury and related to the innate immune response determined in the ER. These results indicate that suPAR is an innate immune response-induced late mediator in multi-trauma patients. Feasibility and experience of using exception from informed consent in a pilot study of immediate estrogen infusion for hypotensive trauma patients hypotensive trauma patients JG Wigginton1, PE Pepe1, V Warren1, K AbdelFattah1, JW Gaston1, J Simpkins2, JP Minei1, D Maass1, AH Idris1 1University of Texas Southwestern Medical Center, Dallas, TX, USA; 2University of North Texas, Fort Worth, TX, USA Critical Care 2013, 17(Suppl 2):P288 (doi: 10.1186/cc12226) P288 Feasibility and experience of using exception from informed consent in a pilot study of immediate estrogen infusion for hypotensive trauma patients JG Wigginton1, PE Pepe1, V Warren1, K AbdelFattah1, JW Gaston1, J Simpkins2, JP Minei1, D Maass1, AH Idris1 1University of Texas Southwestern Medical Center, Dallas, TX, USA; 2University of North Texas, Fort Worth, TX, USA Critical Care 2013, 17(Suppl 2):P288 (doi: 10.1186/cc12226) P287 The average number of days to verbal notifi cation of patients or advocates was 6.55 days (range 0 to 51 days) as the study team began notifi cation only after the patient or family was able to reasonably understand information about the study. No one decided against continued follow-up. Overall, patients and their families were very enthusiastic about participation and the data safety monitoring board had no safety concerns after reviewing all study data. Figure 1 (abstract P287). y y Conclusion Although delayed notice of participation occurs for many justifi able reasons, the use of exception from informed consent for novel, time-sensitive resuscitation studies is not only crucial, but can be feasible, and well accepted by patients, their advocates and communities at large. Level of lipopolysaccharide-binding protein and microbial landscape with account of severity of sepsis syndromes in polytrauma Level of lipopolysaccharide-binding protein and microbial landscape with account of severity of sepsis syndromes in polytrauma β Methods A total of 168 male rats were assigned randomly to one of three groups: (1) Sham burn (no actual injury) group (n = 8); (2) Placebo (no treatment) burn group (n = 80); and (3) E2 (estrogen treatment) burn group (n = 80). Groups 2 and 3 had 40% TBSA third-degree dorsal burns, early fl uid resuscitation and, 15 minutes post burn, 0.5 mg/kg intraperitoneal estrogen (Group 3) or placebo (Group 2). From each group of 80, eight animals were sequentially sacrifi ced (and cardiac tissue was sampled for IL-6, TNFα, IL-1β) at one of 10 respective time points: 0, 0.5, 1, 2, 4, 6, 8, 18, 24 hours and day 7 post burn (at day 7 only for the eight shams). The markers were measured by ELISA method.i Introduction The aim was estimation of the clinical and predictive signifi cance of the level of lipopolysaccharide-binding protein (LBP) in blood serum and examination of microbial landscape with account of severity of sepsis syndromes in polytrauma. Methods Clinical examination was performed, which included 99 patients with polytrauma according to sepsis syndrome: SIRS (n = 18), local infection (n = 36), sepsis (n = 27), severe sepsis (n = 12), septic shock (n = 6) to the criteria of АССР/SССМ. The microorganism identifi cation was performed using iEMS Reader MF (Labsystems) with the La Chema multimicrotests. The content of LBP in blood serum was assessed with IMMULITE ONE (USA) with the reagents DPC (USA). Statistical analysis of the data was performed with Statistica 6.0. The numerical characteristics of variables are presented as Me (LQ to UQ). The analysis of diff erences was carried with the Kruskal–Wallis test for multiple comparison of independent groups, with Friedman one-way analysis of variance. The diff erences were statistically signifi cant with P <0.05. g y Results In the burned rats, 17β-estradiol signifi cantly decreased the cardiac levels of TNFα at all time points through 45 days post burn, with the sham animal levels (30 pg/mg) more comparable with the estradiol group (70  pg/mg), and signifi cantly less than the placebo animals (332 pg/mg). Similarly, IL-6 levels in the sham animals (70 pg/mg) were comparable with the estradiol group (86.5 pg/mg), and signifi cantly less than the placebo group (730 pg/mg), even at 45 days post burn. Risk factors for the development of complications following blunt chest trauma: a new risk stratifi cation tool C Battle1, H Hutchings2, S Lovett2, P Evans2 1Morriston Hospital, ABMU Health Board, Swansea, UK; 2Swansea University, Swansea, UK Critical Care 2013 17(Suppl 2):P292 (doi: 10 1186/cc12230) C Battle , H Hutchings , S Lovett , P Evans 1Morriston Hospital, ABMU Health Board, Swansea, UK; 2Swansea University, Swansea, UK , Critical Care 2013, 17(Suppl 2):P292 (doi: 10.1186/cc12230) p p Results One hundred patients with BAT presented; 71 had complete data. The accuracy of FAST in BAT was 59.2%; in these, 31 (43.7%) were confi rmed by CT and 11 (15%) by laparotomy. There were 29 (40.8%) inaccurate FAST scans, all confi rmed by CT. FAST had a specifi city of 94.7% (95% CI: 0.75 to 0.99) and sensitivity of 46.2% (95% CI: 0.33 to 0.60). A positive predictive value of 0.96 (0.81 to 0.99) and negative predictive value of 0.39 (0.26 to 0.54). Fisher’s exact test shows positive FAST is signifi cantly associated with intra-abdominal pathology (P  =  0.001). Cohen’s chance corrected agreement was 0.3. Twenty- one out of 28 who underwent laparotomies had positive FAST results, indicating accuracy of 75% (95% CI: 57 to 87%). Critical Care 2013, 17(Suppl 2):P292 (doi: 10.1186/cc12230 Introduction The aim of the study was to investigate the risk factors for the development of complications following blunt chest trauma and to develop a risk stratifi cation tool to assist in the management of this patient group. The diffi culties in the management of this patient group in the emergency department (ED) due to the development of late complications are well recognised in the literature [1]. g Methods Between 2009 and 2011 a total of 276 patients were admitted to hospital from the ED of a regional trauma centre in Wales, with the primary diagnosis of blunt chest trauma. Patients with immediate life- threatening injuries were excluded. Data were collected retrospectively and included risk factors (age, number of rib fractures, comorbidity, pre-injury anticoagulant use, smoking status, oxygen saturations and respiratory rate on initial assessment in the ED), and outcomes (mortality, any pulmonary morbidity, length of stay of 7 days or more and need for ICU admission). Development of complications was defi ned as the occurrence of one or more of the outcomes investigated. Multivariable logistic regression using fractional polynomials was used to identify Conclusion Patients with false negative scans requiring therapeutic laparotomy is concerning. In unstable patients, FAST may help in triaging and identifying those requiring laparotomy. Negative FAST scans do not exclude abdominal injury. Further randomised control trials are recommended if the role of FAST is to be better understood. Reference 1. P289 Early administration of a single dose of parenteral estrogen decreases infl ammation in the heart for 45 days after severe burns PE Pepe, JG Wigginton, JW Gatson, AH Idris University of Texas Southwestern Medical Center, Dallas, TX, USA Critical Care 2013, 17(Suppl 2):P289 (doi: 10.1186/cc12227) Introduction Patients with severe burn injury experience a rapid elevation in multiple circulating proinfl ammatory cytokines, with the levels correlating with both injury severity and outcome. In animal S110 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Level of lipopolysaccharide-binding protein and microbial landscape with account of severity of sepsis syndromes in polytrauma f y gi Results In 81% of the critically ill patients with polytrauma the post- traumatic period was accompanied with development of infectious complications, Gram-negative (K. pneumoniae, Acinetobacter spp., E. coli) and Gram-positive (S. Epidermidis, S. aureus). Sepsis was diagnosed on 8 to 10 days in 45% of the patients. The signifi cant increase of LPS-BP was found in the fi rst 3 days of the follow-up, compared with the control values (6.7 times higher in SIRS group (χ2(n = 18, df = 3) = 52.8666, P <0.001); 9.9 times higher in the group with local infection (χ2(n = 36, df = 3) = 91.6629, P <0.001); 15.2 times higher in the sepsis group; 20.5 times higher in the severe sepsis group; 47.3 times higher in the septic shock group (χ2(n = 6, df = 3) = 11.0339, P = 0.0115)), whereas the fi rst positive results of the microbiological examination were obtained only on 5 to 7 days. The diagnostic sensitivity of threshold concentration of LBP in blood serum (335 mkg/ml) was 84%, diagnostic specifi city was 88% (ROC curve: 0.88). Conclusion Following severe burn injury in an animal model, an early single dose of estrogen can decrease the prolonged let alone the early onset of cardiac infl ammation. Based on these data, clinical studies of estrogen infusions should be seriously entertained as estrogen may not only be an inexpensive, simple adjunctive therapy in burn management, it may also obviate the need for many subsequent interventions altogether and even diminish mortality. P290 P290 Accuracy of FAST scan in blunt abdominal trauma in a major London trauma centre S Fleming1, R Bird2, K Ratnasingham3, S Sarker3, M Walsh2, B Patel3 1Royal National Orthopaedic Hospital Stanmore, London, UK; 2Royal London Hospital, London, UK; 3Barts Cancer Institute, London, UK Critical Care 2013, 17(Suppl 2):P290 (doi: 10.1186/cc12228) Conclusion The high incidence of the diagnostic levels of LBP in blood serum in patients with sepsis in the early term, before microbiological confi rmation of infection, allows one to use this parameter as an early marker of development of purulent septic complications conditioned by Gram-negative microfl ora. Introduction Blunt abdominal trauma (BAT) is a leading cause of morbidity and mortality. Rapid diagnosis and treatment with the Advanced Trauma Life Support guidelines are vital, leading to the development of focused assessment with sonography in trauma (FAST). Methods A retrospective study carried out from January 2007 to 2008 on all patients who presented with BAT and underwent FAST scan. All patients subsequently had a CT scan within 2 hours of admission or a laparotomy within 2 days. The presence of intraperitoneal free fl uid was interpreted as positive. P291 models, accumulations of these cytokines have been observed in remote organs, including the heart, brain and lungs. However, data are lacking regarding the long-term levels of cytokines in the heart following severe burn injury and also how infusion of parenteral estrogen, a powerful anti-infl ammatory agent, would aff ect these levels. Using a rat model, we studied the eff ects of a full-thickness third- degree burn on cardiac levels of IL-6 and TNFα over 45 days with and without 17β-estradiol infusion. Level of lipopolysaccharide-binding protein and microbial landscape with account of severity of sepsis syndromes in polytrauma IM Ustyantseva, OI Khokhlova, OV Petukhova, YA Zhevlakova Federal Scientifi c Clinical Center of Miners’ Health Protection, Leninsk- Kuznetsky, Russia Critical Care 2013, 17(Suppl 2):P291 (doi: 10.1186/cc12229) Prediction score of indications for whole body computed tomography in blunt trauma patients Prediction score of indications for whole body computed tomography in blunt trauma patients H Oda, R Sasaki, A Hagiwara, A Kimura National Center For Global Health and Medicine, Tokyo, Japan Critical Care 2013, 17(Suppl 2):P294 (doi: 10.1186/cc12232) Prediction score of indications for whole body computed tomography in blunt trauma patients g p y p H Oda, R Sasaki, A Hagiwara, A Kimura National Center For Global Health and Medicine, Tokyo, Japan Critical Care 2013, 17(Suppl 2):P294 (doi: 10.1186/cc12232) g p y p H Oda, R Sasaki, A Hagiwara, A Kimura National Center For Global Health and Medicine, Tokyo, Japan Critical Care 2013, 17(Suppl 2):P294 (doi: 10.1186/cc12232) Introduction Whole body computed tomography (WBCT) appears to be useful for the early detection of clinically occult injury, although its indications have been controversial. The purpose of this study was to develop a clinical prediction score to clarify the indications for blunt trauma patients with multiple injuries (MI) who require WBCT. g g p p Conclusion The results of this study highlight the risk factors for the development of complications following blunt chest trauma. A risk stratifi cation tool has also been developed that could assist in the prediction of poor outcomes in this patient group. The next stage is to complete a prospective validation study. p p j q Methods We conducted a retrospective study of 173 patients with blunt trauma who underwent WBCT at our emergency center between June 2011 and July 2012. We chose the presence or absence of MI (Injury Severity Score ≥15) in need of surgical intervention as the outcome variable. We used bivariate analyses to identify variables potentially predicting the presentation of MI. The predictor variables were confi rmed by multivariate logistic regression analyses. We assigned a score based on the corresponding coeffi cients. Reference g gfi Results Among the 173 patients enrolled, 53 were in the MI group. Four predictors were found to be independently signifi cant by the logistic analysis: (1)  body surface wound ≥3 regions, (2)  positive focused assessment with sonography for trauma, (3)  white blood cell count ≥11,000/μl, and (4) D-dimer ≥8 μg/ml. Score 1 was assigned to predictor (1), score 2 was assigned to predictors (2), (3) and (4). A prediction score was calculated for each patient by adding these scores. The area under the receiver operating characteristic curve was 0.89. Prediction score of indications for whole body computed tomography in blunt trauma patients No patients with a score of 1 or less had MI (Figures 1 and 2). P293 P293 Guidelines for chest drain insertion do not protect relevant anatomical structures J Bowness1, P Kilgour1, S Whiten1, I Parkin1, J Mooney2, P Driscoll1 1St Andrews University, St Andrews, Fife, UK; 2University of Salford, UK Critical Care 2013, 17(Suppl 2):P293 (doi: 10.1186/cc12231) Guidelines for chest drain insertion do not protect relevant anatomical structures Conclusion In patients with a score of 1 or 0, the presence of MI is less likely. These patients may not require WBCT, and selective CT scans of body parts based on clinical presentation should be considered. Figure 1 (abstract P294). Prediction versus MI probability (validation group). Figure 2 (abstract P294). Clinical scorings. Multivariate, logistic regression analyses. Introduction We have reported the risk of chest drain insertion inferior to the diaphragm when using current international guidelines [1]. Another complication is damage to signifi cant peripheral nerves, such as the long thoracic nerve causing winging of the scapula [2]. We assessed these risks using: the European Trauma Course method, a patient’s handbreadth below their axilla just anterior to the midaxillary line; the British Thoracic Society safe triangle [3]; and the Advanced Trauma Life Support (ATLS) course guidance [4]. Figure 1 (abstract P294). Prediction versus MI probability (validation group). Methods We used the above guidelines to place markers (representing chest drains) in the thoracic wall of 16 cadavers bilaterally (32 sides), 1 cm anterior to the midaxillary line. Subsequent dissection identifi ed the course and termination of the long thoracic nerve, the site of lateral cutaneous branches of intercostal nerves, and their relation to the markers. Results The long thoracic nerve was found in the fi fth intercostal space in 16 of 32 cases, always in or posterior to the midaxillary line. Contrary to the description in Grays’ Anatomy (40th edition) it terminated before the inferior border of serratus anterior. Most commonly it was found to end by branching in the fourth (right) or fi fth (left) intercostal space (range third to sixth). Lateral cutaneous branches of intercostal nerves were found in the fi fth intercostal space in 25 of 32 cases. Contrary to the description in Last’s Anatomy (12th edition) they always passed anterior to the midaxillary line (and marker). Figure 1 (abstract P294). Prediction versus MI probability (validation group). Figure 2 (abstract P294). Clinical scorings. Multivariate, logistic regression analyses. P294 risk factors and develop a risk stratifi cation tool. The signifi cant risk factors in the model were selected using backward elimination with the Akaike Information Criterion (AIC) at a signifi cance level of 0.05. The c index and the Hosmer–Lemeshow (H–L) test were calculated to assess discrimination and calibration of the risk stratifi cation tool respectively. Results A total of 161 patients out of the 276 admitted developed complications following blunt chest trauma. Implementation of backward elimination using AIC values resulted in a fi nal model based on the signifi cant risk factors; age, oxygen saturations, number of rib fractures, presence of chronic lung disease and pre-injury anticoagulant use (all P <0.05). The c index for the tool was 0.80 and the H–L score was 9.22 (P = 0.32), indicating good predictive capabilities of the tool. Risk factors for the development of complications following blunt chest trauma: a new risk stratifi cation tool C Battle1, H Hutchings2, S Lovett2, P Evans2 1Morriston Hospital, ABMU Health Board, Swansea, UK; 2Swansea University, Swansea, UK Critical Care 2013 17(Suppl 2):P292 (doi: 10 1186/cc12230) Smith J: Focused assessment with sonography in trauma (FAST): should its role be reconsidered? Postgrad Med J 2010, 86:285-291. S111 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 1. Ahmad MA, et al.: Assessment of severity of chest trauma. Injury 2010, 41:981-983. P296 P296 Severe hyperkalemia is prevalent and can be detected by quantitative electrocardiographic fi ndings and medication history of calcium channel blocker among the patients with symptomatic or extreme bradycardia SB Chon Kangwon National University Hospital, Chuncheon, South Korea Critical Care 2013, 17(Suppl 2):P296 (doi: 10.1186/cc12234) P296 Severe hyperkalemia is prevalent and can be detected by quantitative electrocardiographic fi ndings and medication history of calcium channel blocker among the patients with symptomatic or extreme bradycardia SB Ch Severe hyperkalemia is prevalent and can be detected by quantitative electrocardiographic fi ndings and medication history of calcium channel blocker among the patients with symptomatic or extreme bradycardia h Conclusion Despite the controversies over the role of fl uids in the prehospital environment, the carriage and use of blood is both feasible and safe in a physician-led helicopter emergency medical service. Reference Kangwon National University Hospital, Chuncheon, South Korea Critical Care 2013, 17(Suppl 2):P296 (doi: 10.1186/cc12234) Introduction Severe hyperkalemia (serum potassium ≥6.0 mmol/l with electrocardiography (ECG) change) should be detected and treated immediately [1,2]. Among symptomatic or extreme bradycardia patients, we sought its prevalence and prediction rule by history, vital sign, and ECG [3,4]. 1. Davenport R, et al.: J Trauma 2011, 70:90-95. Table 1 (abstract P295). Demographics of patients receiving a prehospital blood transfusion Table 1 (abstract P295). P296 Demographics of patients receiving a prehospital blood transfusion Total (n = 147) Male (%) 102 (69.3) Age (years), median (IQR) 34.5 (22 to 52) Mechanisms of injury (%) Motor vehicle collision 87 (59.1) Motor bike collision 20 (13.6) Pedestrian versus car 9 (6.1) Gunshot wound/stabbing 9 (6.1) Fall from a height 5 (3.4) Recreational 6 (4.1) Other 11 (7.5) Number of patients trapped on arrival (%) 45 (30.6) Scene time (minutes), mean (SD) 49.9 (27.8) Time from tasking to arrival at hospital (minutes), mean (SD) 126.5 (51.3) Heart rate, median (IQR) 115 (90 to 130) Systolic blood pressure (mmHg), median (IQR) 80 (65 to 105) RTSo 2, median (IQR) 5.967 (4.083 to 6.904) Total number of PRBC units transfused 382 Total number of PRBC units wasted 66 Volume of crystalloid (ml), median (IQR) 500 (0 to 1,500) Pronounced life extinct on scene 22 (15.0) Methods A retrospective cross-sectional study was performed on patients with symptomatic (heart rate (HR) ≤50/minute with dyspnea, chest pain, altered mentality, dizziness/syncope/presyncope, general weakness, oliguria, or shock) or extreme (HR ≤40/minute) bradycardia at an ED of an academic hospital from June 2008 to March 2012. Risk factors of severe hyperkalemia were chosen by multiple logistic regression analysis among history (gender, age, comorbidity, and current medication), vital sign, and ECG (maximal precordial T wave amplitude, PR and QRS intervals). Scoring index was derived by summing up of simplifi ed regression coeffi cients of independent risk factors. Results A total of 169 cases were enrolled. Mean age was 71.2 years (SD, 12.5 years). Females numbered 87 (51.5%). Thirty-six cases (21.3%) had severe hyperkalemia. Four variables were independent risk factors of severe hyperkalemia (simplifi ed scores in parentheses): medication of calcium channel blocker (CCB: 2); maximal precordial T ≥8.5 mV (2); PR (atrial fi brillation or junctional bradycardia: 1); and HR ≤40/minute (1). (Nagelkerke R2 = 0.503, AUC = 0.849 (95% CI 0.786 to 0.899).) Sensitivity and specifi city reached 0.75 and 0.83 when total score was ≥3. For score ≥4, positive likelihood ratio reached 5.54 (sensitivity 0.50, specifi city 0.91). Conclusion Severe hyperkalemia is prevalent among symptomatic or extreme bradycardia patients and could be detected immediately by a scoring index composed of quantitative ECG parameters and history of medication of CCB. Prediction score of indications for whole body computed tomography in blunt trauma patients Conclusion Placement 1 cm anterior to the midaxillary line minimises risk to the long thoracic nerve and lateral cutaneous branches of intercostal nerves. We therefore conclude that not all areas of the British Thoracic Society safe triangle are indeed safe, and anteroposterior placement should follow the European Trauma Course and ATLS guidelines: just anterior to the midaxillary line (for example, 1 cm). References 1. Bowness J, et al.: It’s higher than you think: chest drains and the 5th ICS. Clin Anatomy, in press. 2. Kesienne EB, et al.: Tube thoracostomy: complications and its management. Pulm Med 2012:256878. 3. Laws D, et al.: BTS guidelines for the insertion of a chest drain. Thorax 2003, 58:ii53-ii59. 4. American College of Surgeons Committee on Trauma: Advanced Trauma Life Support for Doctors, Course Manual. Chicago: American College of Surgeons; 2008:108. Figure 2 (abstract P294). Clinical scorings. Multivariate, logistic regression analyses. Figure 2 (abstract P294). Clinical scorings. Multivariate, logistic regression analyses. S112 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Table 2 (abstract P295). Interventions performed P295 Prehospital blood transfusion: 5-year experience of an Australian helicopter emergency medical service PB Sherren, B Burns Greater Sydney Area HEMS, Sydney, Australia Critical Care 2013, 17(Suppl 2):P295 (doi: 10.1186/cc12233) Introduction There is an emerging body of evidence suggesting that early packed red blood cell transfusion accompanied by fresh frozen plasma, while limiting crystalloids, confers a survival benefi t in major trauma [1]. Prehospital blood transfusion has been infrequently described, and concerns over expense, transfusion reactions, risk of disease transmission, short shelf half-life and diffi cult storage have limited the interest of prehospital providers. p p p Methods All Greater Sydney Area HEMS (GSA-HEMS) prehospital missions involving a blood transfusion over a 66-month period were identifi ed and reviewed. The prospectively completed GSA-HEMS electronic database was utilised to identify patients and extract data.i Results We identifi ed 158 missions involving a prehospital blood transfusion, of which 147 patient datasets were complete. The majority of patients had a blunt mechanism of injury (93.9%) and were male (69.3%) with a median (IQR) age of 34.5 (22 to 52) years (Table 1). The majority of patients were haemodynamically unstable, with a median (IQR) heart rate and systolic blood pressure of 115 (90 to 130) and 80 (65 to 105)  mmHg, respectively. Twenty-two patients (15.0%) were pronounced life extinct on the scene. A total of 382 units of packed red blood cells were transfused, with a median of 3 units (range 1 to 6). No early transfusion reactions were noted. A variety of prehospital interventions accompanied the transfusions, ranging from rapid sequence intubation through to thoracotomies (Table 2).l References References 1. Huber-Wgner S, et al.: Lancet 2009, 373:1455-1461. 2. Kimura A, et al.: Acad Emerg Med 2012, 19:734-735. References 1. Huber-Wgner S, et al.: Lancet 2009, 373:1455-1461. 2. Kimura A, et al.: Acad Emerg Med 2012, 19:734-735. Table 2 (abstract P295). Interventions performed Intervention Total (n = 147) Rapid sequence intubation 96 (65.3) Cold endotracheal intubation 15 (10.2) Surgical airway 1 (0.7) Thoracostomy (open or tube) 59 (40.1) Thoracotomy 3 (2.0) Pelvic binder or fracture splintage 89 (60.5) Intraosseous insertion sites 22 (15.0) Humerus 19 Tibia 10 Femur 1 Tourniquet application 15 (10.2) Hypertonic saline administration 16 (10.9) E-FAST performed 27 (18.4) Negative results 9 Positive results (fi ndings) 18 Abdominal free fl uid 14 Pneumothorax 2 Haemothorax 1 Thoracic ultrasound only; all negative 2 (1.3) Cardiac ultrasound only; no cardiac motion 1 (0.7) Focused cardiac ultrasound in resuscitation E Fish, L Fuchs, G Gulati, D Talmor, A Oren-Grinberg E Fish, L Fuchs, G Gulati, D Talmor, A Oren Grinberg Beth Israel Deaconess Medical Center, Boston, MA, USA g Beth Israel Deaconess Medical Center, Boston, MA, USA Results Multivariate logistic regression analysis indicated the following three prehospital variables as signifi cant high-ranking factors for predicting favorable 1-month outcomes: shockable initial rhythm (odds ratio (OR), 5.87; 95% CI, 5.23 to 6.60), witnessed arrest (OR, 3.05; 95% CI, 2.83 to 3.28), and age (≥18 to <71 years; OR, 3.24; 95% CI, 2.97 to 3.53). Using recursive partitioning analysis for development cohort data (2005 to 2008, n = 307,896), we stratifi ed prehospital risk: if OHCA with shockable initial rhythm was witnessed, the probability of CPC 1 to 2 was 20.0% (age, ≥18 to <71 years; grade 1) or 10.3% (age, ≥71 years; grade 2); if OHCA with shockable initial rhythm was unwitnessed, the probability was 6.8% (age, <81 years; grade 3) or 1.8% (age, ≥81 years; grade 4a); if OHCA with unshakable initial rhythm was witnessed, the probability was 1.4% (grade 4b); and if OHCA with unshakable initial rhythm was unwitnessed, the probability was 0.3% (grade  5). The c-statistics for risk stratifi cation of the development and validation cohorts (2009 external data, n = 82,330) were 0.853 (95% CI, 0.846 to 0.859) and 0.875 (95% CI, 0.865 to 0.885), respectively. The odds ratios for CPC 1 to 2 at 1 month between patients at very high and very low were 79.5 (95% CI, 72.5 to 87.4) and 122.1 (95% CI, 102.0 to 147.3), respectively, in these cohorts. , , , Critical Care 2013, 17(Suppl 2):P299 (doi: 10.1186/cc12237) Introduction Focused cardiac ultrasound is recognized as a vital tool in critical care medicine. Few studies, however, have examined the utility of this modality in resuscitation. While integration of ultrasound can supplement resuscitation by potentially establishing the etiology of cardiac arrest, it must be done safely to avoid interruption of compressions. The aim of this study was to examine the integration of focused cardiac ultrasound into resuscitations in our hospital. Methods We performed a retrospective observational study of patients undergoing resuscitation at Beth Israel Deaconess Medical Center, Boston, USA, from 2009 to 2012. Inclusion criteria were age >17 years Figure 1 (abstract P299). Ultrasound fi ndings. References . Nyirenda MJ, et al.: Hyperkalaemia. BMJ 2009, 339:b4114. . Alfonzo AV, et al.: Potassium disorders – clinical spectrum and emergency Alfonzo AV, et al.: Potassium disorders – clinical spectrum and emergency S113 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 management. Resuscitation 2006, 70:10-25. management. Resuscitation 2006, 70:10-25. Methods We performed a prospective study between January 2011 and December 2012 at a university cancer reference hospital in Brazil. Consecutive patients undergoing cardiac arrest and receiving CPR were identifi ed, and a database was used to register baseline characteristics of patients, data for CPR and 30-day survival. During the fi rst 12 months of the study (phase 1), patients received CPR according to the institution’s protocol guided by diff erent physicians. During the last 11 months of the study (phase 2), a multidisciplinary Medical Emergency Team System consisting of a specialized team of ICU nurses, physiotherapists and physicians using an electronic calling system was responsible for the CPR. management. Resuscitation 2006, 70:10 25. 3. Neumar RW, et al.: Part 8: adult advanced cardiovascular life support: 2010 American Heart Association Guidelines for Cardiopulmonary Resuscitation and Emergency Cardiovascular Care. Circulation 2010, 122:S729-S767. 4. Severi S, et al.: Heart rate response to hemodialysis-induced changes in potassium and calcium levels. J Nephrol 2001, 14:488-496. Focused cardiac ultrasound in resuscitation p y Conclusion Prehospital risk stratifi cation (grade 1 to 5) of patients after OHCA using three prehospital factors (shockable initial rhythm, witnessed arrest, and age) accurately classifi es the severity of OHCA and may help clinicians guide in-hospital strategies. Reference 1. Rerisfeld GM, et al.: Survival in cancer patients undergoing in-hospital cardiopulmonary resuscitation: a meta-analysis. Resuscitation 2006, 71:152-160. i p Methods We analyzed data for 390,226 adult patients (age ≥18 years) after nontraumatic OHCA, from a prospectively recorded nationwide Utstein-style Japanese database for 2005 to 2009. The endpoint was 1-month survival with favorable neurological outcome (Cerebral Performance Category (CPC) 1 to 2). Prehospital risk stratifi cation following out-of-hospital cardiac arrest Prehospital risk stratifi cation following out-of-hospital cardiac arrest Y Goto1, T Maeda1, Y Goto2 1Kanazawa University Hospital, Kanazawa, Japan; 2Yawata Medical Center, Komatsu, Japan Critical Care 2013, 17(Suppl 2):P297 (doi: 10.1186/cc12235) p Results A total of 182 patients experienced in-hospital cardiac arrest and received CPR. Most patients were in hospital due to medical admission (82%), had solid tumors (85%), and had localized disease (60%). Pulseless electric activity was the predominant arrest rhythm (54%) and 45% of patients were resuscitated on the ICU. Overall 30-day survival was 9.3%. In the phase 1 of the study, 30-day survival was 3.7%, and in phase 2, survival was 14% (P = 0.017). There were no diff erences in the two phases of the study regarding baseline characteristics of patients, rhythm and place of CPR. Introduction Multivariate analyses have identifi ed factors that have enabled the development of sophisticated equations and scoring models with the ability to predict outcomes following out-of-hospital cardiac arrest (OHCA). However, implementation of such outcome prediction models in research and clinical practice has been slow. The more crucial aspect of these predictions is the lack of prehospital risk stratifi cation for OHCA patients. Prehospital risk stratifi cation for patients after OHCA may help clinicians guide in-hospital strategies, particularly in the emergency department. The purpose of this study was to develop a simple and generally applicable prehospital risk- stratifi cation scheme for patients after OHCA. Conclusion Overall survival from CPR in cancer patients compares favorably with survival rates in noncancer patients. A multidisciplinary Medical Emergency Team System increases survival in cancer patients undergoing cardiopulmonary resuscitation. P298 P298 Multidisciplinary Medical Emergency Team System increases survival in cancer patients undergoing cardiopulmonary resuscitation L Hajjar1, F Galas2, S Vieira2, J Almeida2, E Osawa2, C Park2, J Fukushima2, E Angelo2, A Pinheiro2, J Auler Jr2 1Heart Institute, São Paulo, Brazil; 2Cancer Institute of University of São Paulo, Brazil Critical Care 2013, 17(Suppl 2):P298 (doi: 10.1186/cc12236) Prehospital epinephrine administration and survival among patients with unshockable initial rhythm after out-of-hospital cardiac arrest g Methods Our patient, with a medical history of a laparoscopic repair of a symptomatic diaphragmatic hernia 6  months prior, presented herself at the emergency department with pain in the upper abdomen and nausea. The physical examination, laboratory tests and X-ray of the thorax were normal and she was sent home. Twenty-four hours later paramedics were summoned to our patient because of increased complaints. On arrival of the paramedics she had a normal electrocardiogram (ECG) and during the transfer from her bed to the stretcher she collapsed due to pulseless electric activity (PEA), for which cardiopulmonary resuscitation was started. Sinus rhythm and output was regained after several minutes and the patient was transported to the hospital. At arrival in the hospital, the X-ray of the thorax showed an intrathoracic stomach and a signifi cant mediastinal shift to the right. Results After emergency laparotomy, which concerned correcting the gastric herniation and resection of an ischemic part of stomach, the patient remained hemodynamically stable. Cardiac ischemia was ruled out based on ECG, laboratory fi ndings, cardiac ultrasound and cardiac computed tomography. The ultrasound in the emergency department did show a distended right ventricle and normal left function, which disappeared later (after repositioning the stomach), which is evidence for the mediastinal shift as a cause for the PEA.i Y Goto1, T Maeda1, Y Goto2 Y Goto1, T Maeda1, Y Goto2 Y Goto1, T Maeda1, Y Goto2 1Kanazawa University Hospital, Kanazawa, Japan; 2Yawata Medical Center, Komatsu, Japan 1Kanazawa University Hospital, Kanazawa, Japan; 2Yawata Medical Center, Komatsu, Japan Critical Care 2013, 17(Suppl 2):P302 (doi: 10.1186/cc12240) Critical Care 2013, 17(Suppl 2):P302 (doi: 10.1186/cc12240) Introduction Epinephrine has been a cornerstone of cardiac resuscitation and advanced cardiac life support since the 1960s. However, there is little evidence from clinical trials that epinephrine administration after out-of-hospital cardiac arrest (OHCA) improves long-term survival. There would be subsets of patients for whom epinephrine administration is in fact benefi cial. Our objective was to determine whether prehospital epinephrine administration would improve survival at 1 month in OHCA patients with unshockable initial rhythm. y Methods We analyzed data for 383,045 adult OHCA patients with unshockable initial rhythm, from a prospectively recorded nationwide Utstein-style Japanese database for 2007 to 2010. We divided these patients into two cohorts: prehospital epinephrine administration cohort (n  =  30,237) and non-epinephrine administration cohort (n  =  352,808). Multidisciplinary Medical Emergency Team System increases survival in cancer patients undergoing cardiopulmonary resuscitation Methods We designed a before-and-after study based on a 3-month retrospective assessment of the adult victims of OOH-CA in 2009, before the implementation of the ALERT protocol in the Liege dispatching centre, and the prospective evaluation of the same 3-month period in 2011, immediately after the implementation of this protocol. Data were extracted from ambulance, paramedical and medical intervention teams fi les, as well as the audio recordings of the dispatching centre. Results There were 233 OOH-CAs detected in the fi rst period and 235 in the second. Victims were predominantly male (59%, both periods), aged 66 and 64 years, respectively. Callers were family members in 52% in 2009 and 64% in 2011. In 2009, only 9.9% victims benefi ted from bystander CPR, while there were 22.5% in 2011 (P < 0.0002). Reasons for protocol underuse were: assistance not off ered (42.3%), caller remote from the victim (20.6%) or emotionally distressed (15.5%). Mean no- fl ow time decreased from 253 seconds in 2009 to 168 seconds in 2011 (NS). Ten victims were admitted in ROSC to hospital in 2009 and 13 in 2011 (P = 0.09). p p g p g Methods We designed a before-and-after study based on a 3-month retrospective assessment of the adult victims of OOH-CA in 2009, before the implementation of the ALERT protocol in the Liege dispatching centre, and the prospective evaluation of the same 3-month period in 2011, immediately after the implementation of this protocol. Data were extracted from ambulance, paramedical and medical intervention teams fi les, as well as the audio recordings of the dispatching centre.i i g p g Results There were 233 OOH-CAs detected in the fi rst period and 235 in the second. Victims were predominantly male (59%, both periods), aged 66 and 64 years, respectively. Callers were family members in 52% in 2009 and 64% in 2011. In 2009, only 9.9% victims benefi ted from bystander CPR, while there were 22.5% in 2011 (P < 0.0002). Reasons for protocol underuse were: assistance not off ered (42.3%), caller remote from the victim (20.6%) or emotionally distressed (15.5%). Mean no- fl ow time decreased from 253 seconds in 2009 to 168 seconds in 2011 (NS). Ten victims were admitted in ROSC to hospital in 2009 and 13 in 2011 (P = 0.09). Multidisciplinary Medical Emergency Team System increases survival in cancer patients undergoing cardiopulmonary resuscitation Conclusion Focused cardiac ultrasound is a feasible adjunct to ALS resuscitation and may assist in the early identifi cation of reversible causes of cardiac arrest. Care must be taken to ensure no interruptions to cardiac compressions are made by performance during pulse checks. Further studies are needed to examine the outcomes associated with its integration into resuscitations. Further studies are needed to examine the outcomes associated with its integration into resuscitations. Conclusion Using the ALERT protocol in the Liege dispatching centre signifi cantly improved the numbers of patients in whom bystander CPR was attempted. Dispatchers must embrace this new opportunity to help callers and be encouraged to accept the responsibility of initiating such assistance. Reference Acute intrathoracic gastric herniation as a rare cause of cardiac arrest DW Hoelen, AL Van Duijn, CL Meuwese, JP Ruurda, MA Sikma UMC Utrecht, the Netherlands Critical Care 2013, 17(Suppl 2):P300 (doi: 10.1186/cc12238) Multidisciplinary Medical Emergency Team System increases survival in cancer patients undergoing cardiopulmonary resuscitation L Hajjar1, F Galas2, S Vieira2, J Almeida2, E Osawa2, C Park2, J Fukushima2, E Angelo2, A Pinheiro2, J Auler Jr2 1Heart Institute, São Paulo, Brazil; 2Cancer Institute of University of São Paulo, Brazil Critical Care 2013, 17(Suppl 2):P298 (doi: 10.1186/cc12236) Introduction In the last years, ICU admission has been high in cancer patients due to increased survival related to advances in treatment. Patients with cancer reportedly have poor outcomes from cardiopulmonary resuscitation (CPR). The goal of this study was to evaluate the eff ectiveness of a multidisciplinary Medical Emergency Team System in the CPR outcomes in cancer patients. Figure 1 (abstract P299). Ultrasound fi ndings. S114 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Introduction Early bystander cardiopulmonary resuscitation (CPR) is a key factor in improving survival from out-of-hospital cardiac arrest (OOH-CA). The ALERT algorithm, a simple and eff ective compression-only telephone CPR protocol, has the potential to help bystanders initiate CPR. This study evaluates the eff ectiveness of the implementation of this protocol in the Liege dispatching centre. and performance of focused cardiac ultrasound during resuscitation. Recorded variables included admission data, code demographics and diagnosis, therapy outside standard ALS protocol, time to return of spontaneous circulation (ROSC), and outcomes data. p Results Of 33 eligible patients, 12 were excluded due to incomplete cardiac ultrasound reports; 21 patients were enrolled. Cohort demographics included: 57.1% male, average age 64.3  years, average BMI 28.5  kg/ m2, average Charlson score 5.5. Resuscitations took place on the wards (52.4%), ICU (42.9%), or operating room (4.8%). Most patients had an initial unshockable rhythm (71.5%). The most common ultrasound fi nding was cardiac standstill (47.6%) (Figure 1). The most common intervention as a result of the ultrasound was initiation of a pressor infusion (33.3%), of which 71.4% were ionotropes. Additional therapies included blood transfusion (4.8%), heparin (9.5%), tPA (4.8%), cardiac catheterization (4.8%), and surgery (9.5%). ROSC was achieved in 37.5% of patients; average time to ROSC was 13 minutes. A total 33.3% of patients who underwent ALS were alive at hospital discharge and 28.6% at 1 year. p e e tat o o t s p otoco t e ege d spatc g ce t e. References 1. Deakin CD, et al.: Resuscitation 2010, 81:1305-1352. 1. Deakin CD, et al.: Resuscitation 2010, 81:1305-1352 2. Robson R. Resuscitation 2010, 81:1453-1454. 3. Breitkreutz R. et al.: Crit Care Med 2007, 35:S150-S161. Acute intrathoracic gastric herniation as a rare cause of cardiac arrest DW Hoelen, AL Van Duijn, CL Meuwese, JP Ruurda, MA Sikma UMC Utrecht, the Netherlands Critical Care 2013, 17(Suppl 2):P300 (doi: 10.1186/cc12238) 1. Ghuysen A, Collas D, Stipulante S, Donneau AF, Hartstein G, Hosmans T, Vantroyen B, D’Orio V: Dispatcher-assisted telephone cardiopulmonary resuscitation using a French-language compression-only protocol in volunteers with or without prior life support training: a randomized trial. Resuscitation 2011, 82:57-63. Introduction In this case report, we describe a patient who presented with a cardiac arrest as a result of an obstructive shock, which progressed into cardiac arrest, caused by an acute para-esophageal gastric herniation. P302 Prehospital epinephrine administration and survival among patients with unshockable initial rhythm after out-of-hospital cardiac arrest The endpoints were 1-month survival after OHCA, prehospital return of spontaneous circulations (ROSCs), and 1-month survival with favorable neurological outcome (Cerebral Performance Category (CPC) scale, categories 1 to 2) at 1 month. Conclusion We are the fi rst to describe a patient requiring cardiopulmonary resuscitation for progressive obstructive shock, due to an intrathoracic stomach. Especially after a laparoscopic repair of a diaphragmatic hernia, this is a rare cause for shock and cardiac arrest, which requires a diff erent medical approach. y Results The rate of 1-month survival was 3.72% for the epinephrine administration cohort and 2.49% for the non-epinephrine adminis- tration cohort, 17.9% versus 3.0% for prehospital ROSC, and 0.57% versus 0.77% for CPC 1 to 2 (all P <0.0001). Positive associations were observed between epinephrine administration and 1-month survival (adjusted odds ratio (aOR), 1.18; 95% CI, 1.11 to 1.27), and prehospital ROSC (aOR, 5.50; 95% CI, 5.29 to 5.72; all P <0.0001). Negative association was observed between epinephrine administration and CPC 1 to 2 (aOR, 0.56; 95% CI, 0.48 to 0.66; P <0.0001). Multivariate logistic analysis revealed that age (<66 years; aOR, 4.31; 95% CI, 2.47 to 8.01), total dose Impact of termination of resuscitation for out-of-hospital cardiopulmonary arrest in Japan Results A total of 430 cardiopulmonary arrest events occurred in- hospital between January 2010 and December 2011. A total of 326 patients were excluded because 252 occurred in the ICU, 70 in the coronary care unit and four in the stork unit. In total, 104 patients were enrolled; 50.9% where female and 49.1% male. Median age was 61  years. A total 41.3% of the arrests were due to respiratory arrest and 58.7% due to cardiac arrest. Out of 104 patients who developed cardiopulmonary arrest, 47 (45.19%) occurred during regular working hours, and 57 (54.81%) occurred after regular working hours (P = 0.0081). The event survival was 87.23% during regular working hours compared with 47.37% for the patients who developed cardiopulmonary arrest after regular working hours (P <0.0001). In total, 53.19% of those who developed cardiopulmonary arrest during regular working hours were discharged alive from the ICU compared with 26.32% of those who developed cardiopulmonary arrest after regular working hours.i Introduction What can one anticipate from the introduction of termination of resuscitation (TOR) for patients suff ering out-of-hospital cardiopulmonary arrest (OHCA) in Japan? Irrespective of whether patients have made a living will requesting that medics do not attempt resuscitation, eff orts are made to resuscitate over 90% of OHCA patients in Japan [1,2]; the number of people resuscitated exceed 120,000 every year. The 2010 American Heart Association (AHA) Guidelines for cardiopulmonary resuscitation (CPR) and emergency cardiovascular care (ECC) defi ned the criteria for TOR; this initiative may help reduce the number of unnecessary hospital transports by 40 to 60% and hold down medical costs [3]. p p y g g Conclusion Event survival and survival to discharge were signifi cantly higher in patients who developed cardiopulmonary arrest during regular working hours. Methods This was a single-center retrospective cohort study of patients who suff ered OHCA and were transported to our hospital between April 2009 and March 2011. We investigated the patients’ characteristics, whether they met the TOR criteria, and their outcome at the time of hospital discharge. g Reference 1. Peberdy MA,Ornato JP, Larkin L, et al.: Survival from in-hospital cardiac arrest during nights and weekends. JAMA 2008, 299:785-792. P305 Results A total of 195 patients (mean age, 69 years), 67% of whom were male, were transported to our hospital after suff ering OHCA. Cardiopulmonary arrest was witnessed in 52 cases (27%). P301 Implementation of dispatcher-assisted cardiopulmonary instructions using the ALERT protocol: preliminary results in Belgium A Ghuysen1, S Stipulante1, M El FAssi1, A Donneau2, V D’Orio1, R Tubes1 1CHU – Ulg Liège, Belgium; 2Liège University, Liège, Belgium Critical Care 2013, 17(Suppl 2):P301 (doi: 10.1186/cc12239) A Ghuysen1, S Stipulante1, M El FAssi1, A Donneau2, V D’Orio1, R Tubes1 1CHU – Ulg Liège, Belgium; 2Liège University, Liège, Belgium Critical Care 2013, 17(Suppl 2):P301 (doi: 10.1186/cc12239) S115 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 of epinephrine (1 mg; aOR, 3.65; 95% CI, 2.61 to 5.18), call-response time (<5 minutes; aOR, 3.58; 95% CI, 1.98 to 6.69), witnessed arrest (aOR, 2.17; 95% CI, 1.51 to 3.16), and pulseless electrical activity as an initial rhythm (aOR, 2.02; 95% CI, 1.46 to 2.80) were signifi cantly associated with CPC 1 to 2 at 1 month in the epinephrine administration cohort. of epinephrine (1 mg; aOR, 3.65; 95% CI, 2.61 to 5.18), call-response time (<5 minutes; aOR, 3.58; 95% CI, 1.98 to 6.69), witnessed arrest (aOR, 2.17; 95% CI, 1.51 to 3.16), and pulseless electrical activity as an initial rhythm (aOR, 2.02; 95% CI, 1.46 to 2.80) were signifi cantly associated with CPC 1 to 2 at 1 month in the epinephrine administration cohort. detection and resuscitation of cardiopulmonary arrest are crucial for better clinical outcome. We conducted our study to evaluate event survival of in-hospital cardiopulmonary arrest after regular working hours in nonmonitored areas of a tertiary-care center. of epinephrine (1 mg; aOR, 3.65; 95% CI, 2.61 to 5.18), call-response time (<5 minutes; aOR, 3.58; 95% CI, 1.98 to 6.69), witnessed arrest (aOR, 2.17; 95% CI, 1.51 to 3.16), and pulseless electrical activity as an initial rhythm (aOR, 2.02; 95% CI, 1.46 to 2.80) were signifi cantly associated with CPC 1 to 2 at 1 month in the epinephrine administration cohort. Conclusion In OHCA patients with unshockable initial rhythm, prehospital epinephrine administration signifi cantly increased the rate of survival at 1 month after cardiac arrest. The best single predictor for favorable neurological outcomes at 1  month following prehospital epinephrine administration after cardiac arrest was age (<66 years) followed by total dose of epinephrine (1 mg) and then by call-response time (<5 minutes). p p References 1. Berdowski J, et al.: Resuscitation 2010, 81:1479-1487. 2. Fukuda T: J Jpn Assoc Acute Med 2010, 23:101-108. 3. Morrison LJ, et al.: N Engl J Med 2006, 355:478-487. 4. Fukuda T: J Jpn Soc Intensive Care Med 2012, 19:275. Results TH was initiated in 93% (64/69) of OOHCA patients and 87% (40/46) with ischaemic cardiac aetiology underwent PCI. ICU survival was 58% (40/69); 65% (26/40) scoring CPC 1 to 2 and 35% (14/40) CPC 3 to 4 at ICU discharge. Two patients with CPC 2 improved to CPC 1 during their hospital stay. All patients with CPC 1 to 2 survived to hospital discharge; two required general rehabilitation before returning home. Only 43% (6/14) of CPC 3 to 4 patients survived to hospital discharge; none returned home. Two went into hospice care, one was repatriated to another hospital and three went to neuro-rehab. No CPC 3 to 4 patients improved CPC scores after ICU discharge. Overall hospital survival was 46%. See Table 1. Cognitive performance and cap out-of-hospital cardiac arrestl Introduction Before the introduction of primary percutaneous coronary intervention (PCI) and therapeutic hypothermia (TH) to out-of-hospital cardiac arrest (OOHCA) management, survival to hospital discharge with intact neurological function was poor [1,2]. We aimed to quantify the survival and degree of neurological impairment in OOHCA patients admitted to our ICU since the adoption of post-OOHCA bundles. Conclusion In our study, 65% of the patients who were transported to the hospital after OHCA met the criteria for TOR. Outcomes for patients who met the TOR criteria were signifi cantly worse than those who did not meet the criteria (2.4% vs. 14.5%, P <0.005). In Japan, eff orts are made to resuscitate almost all individuals who suff er OHCA, but 75% of those patients die within a day. In light of the fact that even the medical cost for each of these patients who die within a day amounts to US$1,500 [4], the introduction of TOR will have a particularly strong impact in Japan. Methods Sixty-nine consecutive OOHCA patients admitted to the ICU at Hammersmith Hospital from 1 January 2011 to 30 June 2012 were identifi ed and reviewed from hospital databases. Cognitive status was scored using Cerebral Performance Category (CPC); 1 to 2 normal–mild and 3 to 4 moderate–severe neurological impairment. Scores were determined from ICU summaries, occupational and physiotherapy reports. Hospital discharge outcomes were determined from hospital databases. Impact of termination of resuscitation for out-of-hospital cardiopulmonary arrest in Japan The 2010 AHA Guidelines for CPR and ECC regarding the criteria for TOR were applied in 126 cases (65%), of whom 113 (90%) were dead on arrival, and 13 were successfully resuscitated and admitted. The outcomes for these 13 patients were as follows: 10 died in the hospital, two patients were discharged with a Glasgow Pittsburgh Cerebral Performance Category (CPC) score of 1, and one patient was transferred to another hospital with a CPC score of 3. P301 Methods A retrospective chart review of all adult patients who developed in-hospital cardiopulmonary arrest between January 2010 and December 2011. Working hours are defi ned as 07:00 to 17:00 Saturday to Wednesday. Event survival is defi ned as return of spontaneous circulation (ROSC) for more than 20  minutes. Adult patients 18 years and above who suff ered from in-hospital cardiopulmonary arrest were included. Patients were excluded if they had cardiopulmonary arrest in the emergency department, had implantable cardiovascular device, arrested in monitored areas or had pre-existing DNR orders. Data analysis was accomplished using SAS, version 9.3 (SAS institute, Inc., Cary, NC, USA). Conclusion In OHCA patients with unshockable initial rhythm, prehospital epinephrine administration signifi cantly increased the rate of survival at 1 month after cardiac arrest. The best single predictor for favorable neurological outcomes at 1  month following prehospital epinephrine administration after cardiac arrest was age (<66 years) followed by total dose of epinephrine (1 mg) and then by call-response time (<5 minutes). P303 Impact of termination of resuscitation for out-of-hospital cardiopulmonary arrest in Japan T Fukuda, N Ohashi, M Gunshin, T Matsubara, S Nakajima, Y Kitsuta, N Yahagi Graduate School of Medicine, The University of Tokyo, Japan Critical Care 2013, 17(Suppl 2):P303 (doi: 10.1186/cc12241) References 1. Stub et al.: Am J Cardiol 2011, 107:522-527. 2. Girotra et al.: N Engl J Med 2012, 367:1912-1920. 3. Herlitz et al.: Heart 2003, 89:25-30. 4. Cronier et al.: Crit Care 2011, 15:122. 1. Stub et al.: Am J Cardiol 2011, 107:522-527. 2. Girotra et al.: N Engl J Med 2012, 367:1912-1920. 3. Herlitz et al.: Heart 2003, 89:25-30. 4. Cronier et al.: Crit Care 2011, 15:122. 2. MacLeod et al.: J Cardiothorac Vasc Anesth 2012, 26:1007-1014. patients: does it mean something? p g C Genbrugge, I Meex, S Scheyltjens, J Dens, C De Deyne ZOL, Genk, Belgium Critical Care 2013, 17(Suppl 2):P306 (doi: 10.1186/cc12244) p g C Genbrugge, I Meex, S Scheyltjens, J Dens, C De Deyne ZOL, Genk, Belgium Critical Care 2013, 17(Suppl 2):P306 (doi: 10.1186/cc12244) p g C Genbrugge, I Meex, S Scheyltjens, J Dens, C De Deyne Introduction During cardiopulmonary resuscitation (CPR) monitoring possibilities are limited. Parnia and colleagues investigated the feasibility and role of near-infrared spectroscopy (NIRS) during CPR in cardiac arrest patients (CA) [1]. NIRS could have a role in predicting return of spontaneous circulation (ROSC). Recently, the Equanox® with four wavelengths sensor was validated to provide absolute data on regional cerebral saturation [2]. We measured cerebral oxygenation (rSO2) during CPR with NIRS technology and analyzed the diff erences between initial cerebral saturations in patients achieving ROSC compared with patients without ROSC. p g y Conclusion As previously reported [2], IHCA was associated with a worse prognosis than OHCA. The OHCA survival rate was better than reported elsewhere [3]. The percentage of IHCA attributed to MI was low. Only one OHCA patient was referred for emergency PCI. Routine coronary angiography with ad hoc PCI in VF OHCA has been associated with increased survival [4]. Greater availability of PCI post OHCA could further improve mortality in patients with a primary cardiac pathology. Further investigation should include management of noncardiogenic cardiac arrest. p p Methods With IRB approval, rSO2 was measured with NIRS during resuscitation in 18 out-of hospital CA patients. The Equanox® Advance (NONIN), a NIRS monitoring device that measures absolute rSO2 values, was applied on the right side of the patient’s forehead when the medical emergency team arrived in a resuscitation setting. Placement of the probe did not interfere with the advanced life support algorithm. The sensor remained on the patient’s forehead during resuscitation, and if ROSC was reached the probe was removed on arrival at the emergency department. If ROSC was not achieved, the probe was removed prehospital. ROSC was defi ned as ROSC during more than 20  minutes. The Mann–Whitney test was utilized for comparison of survivor and nonsurvivor data. Student’s t  test was performed to compare the initial rSO2. 1. Parnia et al.: Resuscitation 2012, 83:982-985. P307 P307 Intensive care admission after cardiac arrest: cardiac versus noncardiac causes and consequences for treatment T Hargreaves1, H Kingston1, M Crews1, M Mogk2, I Welters3 1Royal Liverpool University Hospital, Liverpool, UK; 2Moredata GmbH, Giessen, Germany; 3Liverpool University, Liverpool, UK Critical Care 2013, 17(Suppl 2):P307 (doi: 10.1186/cc12245) Intensive care admission after cardiac arrest: cardiac versus noncardiac causes and consequences for treatment T Hargreaves1, H Kingston1, M Crews1, M Mogk2, I Welters3 1Royal Liverpool University Hospital, Liverpool, UK; 2Moredata GmbH, Giessen, Germany; 3Liverpool University, Liverpool, UK Critical Care 2013, 17(Suppl 2):P307 (doi: 10.1186/cc12245) Introduction In-hospital (IHCA) and out-of-hospital cardiac arrest (OHCA) are associated with high mortality [1]. Studies suggest that up to 68% of OHCA is due to acute coronary syndrome, with 38% requiring percutaneous coronary intervention (PCI) [1]. However, revascularisation may not always be available or address the underlying pathology. This study aimed to establish the prevalence of diff erent aetiologies for IHCA and OHCA, and the use of emergency treatment for these patients. disability (CPC 3 to 4) was seen in 19%, greater than previously reported [1]. A higher proportion (35%) of patients discharged from the ICU had moderate–severe neurological disability; most subsequently died in hospital (62%). These fi gures may represent better ICU outcomes subsequent to adoption of OOHCA bundles but suggest further work is required in neuro-disabled survivors. Methods A retrospective case-note review of all patients admitted between 2008 and 2011 to the ICU of an inner-city university hospital after OHCA or IHCA. Biometric data, presenting cardiac rhythm, presumed cause of arrest, management and outcomes were recorded. The Kruskal–Wallis test was used for numerical data analysis and chi- square test for categorical data. Survival from cardiopulmonary arrest after regular working hours in a tertiary-care hospital: retrospective study Introduction Detection and treatment of cardiopulmonary arrest and their antecedents may be less eff ective at night and weekend than weekdays because of hospital staffi ng and response factors [1]. Early Conclusion OOHCA patients admitted to our ICU had a 46% chance of surviving to hospital discharge. Most patients left hospital with good neurological status (CPC 1 to 2); moderate–severe neurological S116 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 disability (CPC 3 to 4) was seen in 19%, greater than previously reported Table 1 (abstract P305). Discharge and neurological status of OOHCA patients Discharge Hospital discharge destination Neuro- Score ICU Hospital Home rehab Repatriation Hospice CPC 1 20 22 19 0 3 0 CPC 2 6 4 4 0 0 0 CPC 3 4 3 0 3 0 0 CPC 4 10 3 0 0 1 2 Total 40 32 23 3 4 2 disability (CPC 3 to 4) was seen in 19%, greater than previously reported [1]. A higher proportion (35%) of patients discharged from the ICU had moderate–severe neurological disability; most subsequently died in hospital (62%). These fi gures may represent better ICU outcomes subsequent to adoption of OOHCA bundles but suggest further work is required in neuro-disabled survivors. References 1. Cobbe SM, et al.: BMJ 1999, 312:1633-1637. 2. Holzer M, et al.: N Engl J Med 2002, 346:549-556. P306 Start value of cerebral saturation in prehospital cardiac arrest patients: does it mean something? C Genbrugge, I Meex, S Scheyltjens, J Dens, C De Deyne ZOL, Genk, Belgium Critical Care 2013, 17(Suppl 2):P306 (doi: 10.1186/cc12244) Table 1 (abstract P305). Discharge and neurological status of OOHCA patients Discharge Hospital discharge destination Neuro- Score ICU Hospital Home rehab Repatriation Hospice CPC 1 20 22 19 0 3 0 CPC 2 6 4 4 0 0 0 CPC 3 4 3 0 3 0 0 CPC 4 10 3 0 0 1 2 Total 40 32 23 3 4 2 Table 1 (abstract P305). Discharge and neurological status of OOHCA patients Discharge Hospital discharge destination 1. Parnia et al.: Resuscitation 2012, 83:982-985. References 1. Cobbe SM, et al.: BMJ 1999, 312:1633-1637. 1. Cobbe SM, et al.: BMJ 1999, 312:1633-1637. 2. Holzer M, et al.: N Engl J Med 2002, 346:549-556. q g Results Data were analysed for 64 patients – 44 (69%) following OHCA and 20 (31%) after IHCA. The median APACHE score for OHCA was 17 and for IHCA was 23.5 (P = 0.001). Hospital survival rate was 10% (n = 2) for IHCA and 38.6% (n = 17) for OHCA (P <0.02). A total of 34.1% (n = 15) OHCA were due to myocardial infarction (MI) compared with 10% (n = 2) of IHCA (P <0.05). The most prevalent aetiologies were MI (n = 17), hypoxia (n = 10), cardiac other (n = 5), sepsis (n = 4), arrhythmia (n = 3) and PE (n = 3). In two IHCA patients more than one likely cause of arrest was reported and in 19 cases no cause was identifi ed. The presenting rhythm was ventricular fi brillation (VF) in 45.3% (n = 29), pulseless electrical activity in 32.8% (n  =  21) and asystole in 20.3% (n = 13). A total of 9.4% (n = 6) were thrombolysed and one (1.6%) patient was referred for emergency PCI. 2. Holzer M, et al.: N Engl J Med 2002, 346:549-556. 1. Parnia et al.: Resuscitation 2012, 83:982-985. 2. MacLeod et al.: J Cardiothorac Vasc Anesth 2012, 26:1007-1014. P308 P308 Comparison of 1-year neurological outcome between intra-hospital and extra-hospital cardiac arrest survivors submitted to mild therapeutic hypothermia in a community-based setting in Brazil CA Abreu Filho1, A Andrade2, A Neto2, S Santos2, M Bracco2, E Silva1, A Baruzzi1, J Teixeira2 1Hospital Israelita Albert Einstein, São Paulo, Brazil; 2Hospital Municipal Dr. Moysés Deutsch, São Paulo, Brazil Critical Care 2013, 17(Suppl 2):P308 (doi: 10.1186/cc12246) 2 Results Of the 18 patients, nine patients had ROSC (survivors). The initial rhythm was the same in both groups, six patients in each group had asystole as initial rhythm. In the group of survivors were six female patients, in the nonsurvivors were two female patients. The mean age in ROSC and no-ROSC groups is respectively 75.8 years (SD ±12.8) and 69.4 years (SD ±22.9, P = 0.48). The mean rSO2 at arrival of the emergency medical team was 31.56% (SD ±29.4) and 12.78% (SD ±12.7) respectively in the ROSC group and no-ROSC group (P = 0.1). The mean time between collapse and start of CPR (basic life support of bystanders) was 6.9 minutes (SD ±8.2) in the no-ROSC group and 8.2 minutes (SD ±7.08, P = 0.69) in the ROSC group. Introduction Mild therapeutic hypothermia (MTH) is the most powerful therapy to improve survival and neurologic outcome after out-of- hospital cardiac arrest. Such benefi t may also occur for unconscious patients after in-hospital cardiac arrest. The aim is to compare 1-year evolution of neurological outcomes of patients treated with MTH after in-hospital versus out-of-hospital cardiac arrest. Conclusion Initial rSO2 values in out-of hospital CA patients with ROSC showed a tendency towards higher values compared with nonsurvivors, but no signifi cant diff erence could be demonstrated, probably related to the small number of patients included in this preliminary report. References Methods A prospective study of patients treated with MTH after cardiac arrest in a community hospital in São Paulo, Brazil. After return of spontaneous circulation, unconscious survivors received MTH using topical ice and cold saline infusions in order to achieve a 32 to 34°C goal temperature, within 6  hours of cardiac arrest, and maintained P310 P310 Infrared pupillometry for outcome prediction after cardiac arrest and therapeutic hypothermia T Suys, N Sala, AO Rossetti, M Oddo Lausanne University Hospital, Lausanne, Switzerland Critical Care 2013, 17(Suppl 2):P310 (doi: 10.1186/cc12248) Conclusion Midterm neurological outcome of MTH after in hospital cardiac arrest seems to be not as good as after out-of-hospital cardiac arrest. Delay in hypothermia initiation, older age and associated comorbidities could explain the worse evolution of this group of patients. Introduction Sedation and therapeutic hypothermia (TH) modify neurological examination and alter prognostic prediction of coma after cardiac arrest (CA). Additional tools, such as EEG and evoked potentials, improve prediction of outcome in this setting, but are not widely available and require signifi cant implementation. i Methods Using a new device for infrared pupillometry, we examined the value of quantitative pupillary light reactivity (PLR) to predict outcome in comatose post-CA patients treated with TH. Twenty-four comatose CA patients treated with TH (33°C, 24  hours) were prospectively studied. The percentage variation in PLR was measured during TH (12 hours from CA), using the NeuroLight Algiscan® (IDMED, Marseille, France). For each patient, three consecutive measures were performed and the best value was retained for analysis. The relationship of PLR P309 Primary percutaneous coronary angioplasty and therapeutic hypothermia in out-of-hospital cardiac arrest R Hunt, M Holl, A Bailey, P Macnaughton Derriford Hospital, Plymouth, UK Critical Care 2013, 17(Suppl 2):P309 (doi: 10.1186/cc12247) Introduction The benefi t of primary percutaneous intervention (PCI) in the management of out-of-hospital cardiac arrest (OHCA) is not clear cut [1]. It has historically been used in patients with ST elevation on post-resuscitation electrocardiogram (ECG) although this is a poor predictor of acute coronary occlusion after cardiac arrest [2]. This study investigates the benefi t of PCI regardless of post-resuscitation ECG. Benefi t is widely claimed for therapeutic hypothermia, so cooling parameters were included. Table 1 (abstract P310). False-positive and false-negative rates for outcome (% of patients) p Methods We analysed all 41 consecutive adults admitted post OHCA to a university hospital ICU between January 2010 and December 2011. Patients received PCI regardless of ECG changes. A Cox proportional hazards model was used to determine the relationship between PCI, cooling and survival to discharge. Routinely collected data such as demographics and details of resuscitation (OHCA Utstein data) were also included. Results Survival to hospital discharge was 41% with 29% of survivors discharged to a neurological rehabilitation centre. References 1. Kern KB: Optimal treatment of patients surviving out-of-hospital cardiac arrest. JACC Cardiovasc Interv 2012, 5:597-605. 2. Spaulding CM, et al.: Immediate coronary angiography in survivors of out- of-hospital cardiac arrest. N Engl J Med 1997, 336:1629-1633. 2. Spaulding CM, et al.: Immediate coronary angiography in survivors of out- of-hospital cardiac arrest. N Engl J Med 1997, 336:1629-1633. 3. Dumas F, et al.: Immediate percutaneous coronary intervention is associated with better survival after out-of-hospital cardiac arrest. Circ Cardiovasc Interv 2010, 3:200-207. 3. Dumas F, et al.: Immediate percutaneous coronary intervention is associated with better survival after out-of-hospital cardiac arrest. Circ Cardiovasc Interv 2010, 3:200-207. References S117 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 with other studies suggesting benefi t for primary PCI regardless of the post-resuscitation ECG [3]. Cooling was not found to improve survival to discharge but further analysis is required to determine impact on neurological function. for 24  hours. Esophageal temperature was monitored; continuous intravenous sedation-analgesia was maintained for 48  hours after initiation of MTH. The Glasgow Outcome Scale (GOS) was used to analyze the neurological outcomes after hospital discharge. for 24  hours. Esophageal temperature was monitored; continuous intravenous sedation-analgesia was maintained for 48  hours after initiation of MTH. The Glasgow Outcome Scale (GOS) was used to analyze the neurological outcomes after hospital discharge. Results From January 2009 to April 2012, 84 patients submitted to MTH were divided into two groups: Group 1, 54 patients presented out- of-hospital cardiac arrest; and Group 2, 30 patients had intra-hospital cardiac arrest. Both groups were similar regarding to gender; Group 2 tended to be older (mean age 44.3 years vs. 33.5 years, P = 0.07), and had more frequently asystole as the cardiac arrest rhythm (45% vs. 15 %, P  =  0.10). Group 2 had shorter duration of resuscitation (12.3 minutes vs. 33.7 minutes, P = 0.03), longer time to hypothermia initiation (309.3 minutes vs. 212.8 minutes, P = 0.04), longer hospital stay after cardiac arrest (50.7 days vs. 32.4 days, P = 0.01) and worse neurological outcome, characterized by the presence of GOS ≤3 at 30 days (68.4% vs. 35.1%, P = 0.03). Hospital mortality was 5.5% in Group 1 and 13.3% in Group 2 (P = 0.21). The 1-year survival rate was 85.1% in Group 1 and 83.3% in Group 2 (P = 0.43); after 1 year follow-up, GOS ≤3 was present in 30.4% of Group 2 patients and in 11.1% of Group 1 patients (P = 0.04). Eff ect of moderate hyperventilation and induced hypertension on cerebral tissue oxygen saturation in comatose post-cardiac arrest patients treated with hypothermia T Suys, N Sala, M Oddo Lausanne University Hospital, Lausanne, Switzerland Critical Care 2013, 17(Suppl 2):P311 (doi: 10.1186/cc12249) l Methods We performed an observational study in 10 comatose patients after cardiac arrest. Patients were treated with hypothermia for 24 hours. Blood viscosity was measured ex vivo using a Contraves LS300 viscometer. Mean fl ow velocity in the middle cerebral artery (MFVMCA) was measured by transcranial Doppler (TCD) at the same time points. T Suys, N Sala, M Oddo T Suys, N Sala, M Oddo Lausanne University Hospital, Lausanne, Switzerland Critical Care 2013, 17(Suppl 2):P311 (doi: 10.1186/cc12249) Lausanne University Hospital, Lausanne, Switzerland y p Critical Care 2013, 17(Suppl 2):P311 (doi: 10.1186/cc12249) Introduction Maintenance of adequate brain perfusion is an essential component of post-resuscitation care. Mean arterial pressure (MAP) and PaCO2 are important determinants of brain perfusion; however, no precise guidelines exist for optimal MAP and PaCO2 targets in comatose post-cardiac arrest (CA) patients. an essential ssure (MAP) however, no n comatose ive cerebral erventilation ents treated dy including hospital CA face cooling NIRS system erventilation PaCO2 from zation, MAP infusion of SctO2 were 19 minutes) Results are nt reduction 5), P <0.001). and IH tests (n = 10 patients). Figure 1 (abstract P312). Figure 1 (abstract P312). p p Methods Using NIRS, we examined changes in non-invasive cerebral tissue oxygen saturation (SctO2) following moderate hyperventilation (HV) and induced hypertension (IH) in comatose CA patients treated with therapeutic hypothermia (TH). A prospective pilot study including comatose patients successfully resuscitated from out-of-hospital CA treated with TH (33°C for 24 hours, using cold saline and surface cooling device), monitored for continuous SctO2 with the Foresight NIRS system (CAS Medical Systems, Branford, CT, USA). Moderate hyperventilation was induced for approximately 30 minutes by decreasing PaCO2 from ~40 to ~30  mmHg, at stable MAP. After PaCO2 normalization, MAP was increased from ~70 to ~90  mmHg by intravenous infusion of norepinephrine, at stable PaCO2. Eff ects of MV and IH on SctO2 were analyzed with a paired t test. Figure 1 (abstract P312). Figure 1 (abstract P312). y p Results Ten patients (mean age, 69.5; mean time to ROSC, 19 minutes) were studied during the stable TH maintenance phase. Results are summarized in Figure 1. MV was associated with a signifi cant reduction of SctO2 from baseline (75% (73 to 76) to 69% (67.5 to 71.5), P <0.001). 0 patients). Figure 1 (abstract P311). Changes in SctO2 after moderate HV and IH tests (n = 10 patients). Figure 1 (abstract P311). P312f Eff ects of viscosity on cerebral blood fl ow after cardiac arrest L Bisschops, G Pop, S Teerenstra, J Van der Hoeven, C Hoedemaekers Radboud University Nijmegen Medical Centre, Nijmegen, the Netherlands Critical Care 2013, 17(Suppl 2):P312 (doi: 10.1186/cc12250) p g Conclusion Quantitative PLR appears highly accurate and superior to standard neurological examination to predict outcome in patients with post-CA coma. Further study is warranted to confi rm these promising fi ndings. Acknowledgements Supported by Grants from the Swiss National Science Foundation (FN 320030_138191) and the European Critical Care Research Network (ECCRN). Introduction After cardiac arrest, microcirculatory reperfusion dis orders develop despite adequate cerebral perfusion pressure. Increased blood viscosity strongly hampers the microcirculation, resulting in plugging of the capillary bed, arteriovenous shunting and diminished tissue perfusion. The aim of the present study was to assess blood viscosity in relation to cerebral blood fl ow in patients after cardiac arrest. P310 Multivariate analysis using a Cox proportional hazards model showed PCI to be an independent predictive factor of survival, unrelated to ECG (hazards ratio, 0.0583; 95% CI, 0.0076 to 0.4485). Cooling had no signifi cant impact on patient survival. See Figure 1. p p g Conclusion In this small retrospective study primary PCI appears to be an independent predictor of survival after OHCA. This is consistent Figure 1 (abstract P309). Comparison of hazards ratios post Cox analysis. Figure 1 (abstract P309). Comparison of hazards ratios post Cox analysis. S118 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 No signifi cant changes in SctO2 were found after IH (74 (72 to 76) vs. 75 (73 to 75.5), P = 0.24). with survival and neurological outcome (CPC scores) at 3  months was analyzed, and the predictive value of PLR was compared with that of standard clinical examination (motor response and brainstem refl exes) performed at 48 hours from CA. Conclusion Moderate HV was associated with signifi cant reduction in cerebral saturation, whilst IH may be detrimental after CA and TH, whilst increasing MAP to supranormal levels with vasopressors does not improve cerebral oxygenation. These data stress the importance of strict control of PaCO2 following CA and TH to avoid secondary cerebral ischemic insults. Results Quantitative PLR was strongly associated with survival (median left-eye PLR 14% (11 to 16%) variation in survivors vs. 5.5% (4 to 8.5%) in nonsurvivors, P < 0.0001) and 3-month neurological outcome (14% (11 to 21%) in patients with CPC 1 to 2 vs. 5.5% (4 to 8.5%) in those with CPC 3 to 5, P <0.0001). Comparable fi ndings were obtained using right-eye PLR. A PLR >10% was 100% predictive of patient prognosis, with false-positive and false-negative rates of 0% for outcome. Clinical examination was signifi cantly associated with outcome; however, motor response (MR) and brainstem refl exes (BRS) yielded higher false- positive and false-negative rates than PLR (Table 1). Acknowledgements MO is supported by Grants from the Swiss National Science Foundation (FN 320030_138191) and The European Critical Care Research Network (ECCRN). P312f Eff ect of moderate hyperventilation and induced hypertension on cerebral tissue oxygen saturation in comatose post-cardiac arrest patients treated with hypothermia Eff ect of moderate hyperventilation and induced hypertension on cerebral tissue oxygen saturation in comatose post-cardiac arrest patients treated with hypothermia T Suys, N Sala, M Oddo Lausanne University Hospital, Lausanne, Switzerland Critical Care 2013, 17(Suppl 2):P311 (doi: 10.1186/cc12249) Association between oxygenation and 6-month mortality during post-cardiac arrest care p M Skrifvars1, J Vaahersalo1, M Reinikainen2, S Bendel3, J Kurola3, M Tiainen1, R Raj1, V Pettilä1, T Varpula1, FinnResusci Study Group1 1Helsinki University Hospital, HUS, Finland; 2North Karelia Central Hosital, Joensuu, Finland; 3Kuopio University Hospital, Kuopio, Finland Critical Care 2013, 17(Suppl 2):P315 (doi: 10.1186/cc12253) M Skrifvars1, J Vaahersalo1, M Reinikainen2, S Bendel3, J Kurola3, M Tiainen1 R Raj1, V Pettilä1, T Varpula1, FinnResusci Study Group1 1Helsinki University Hospital, HUS, Finland; 2North Karelia Central Hosital, Joensuu, Finland; 3Kuopio University Hospital, Kuopio, Finland Critical Care 2013, 17(Suppl 2):P315 (doi: 10.1186/cc12253) g g y g g y Results We had 297 CA patients (168 SR, 129 NR) in whom CPR was done. Return of spontaneous circulation was achieved in 90 patients. TMH was induced in 57 patients (33 SR, 24 NR). Survival to hospital discharge was observed in 27 patients (18/33 (54.5%) SR, 9/24 (37.5%) NR), out of which 18 patients (10/33 (30%) SR, 8/24 (33%) NR) had good neurological outcome. On analysis, TMH was found to be associated with increased odds of survival to hospital discharge (although statistically not signifi cant) in SR patients compared with NR patients (odds ratio (OR) = 2.00; 95% CI = 0.68 to 5.85; P = 0.2837), but it was not associated with any better neurological outcome in terms of CPC score in patients presenting with SR rather than NR (OR = 0.87; 95% CI = 0.28 to 2.68; P = 1.0000). Rather, the odds for good neurological outcome were more in favour of NR (pulseless electrical activity/asystole). Introduction Optimal oxygenation level during post-cardiac arrest (PCA) care is currently undefi ned, and studies have suggested harm from hyperoxia exposure [1]. We aimed to assess the optimal oxygenation level and possible associations of time-weighted exposure to hyperoxia on outcome in patients during PCA care. Introduction Optimal oxygenation level during post-cardiac arrest (PCA) care is currently undefi ned, and studies have suggested harm from hyperoxia exposure [1]. We aimed to assess the optimal oxygenation level and possible associations of time-weighted exposure to hyperoxia on outcome in patients during PCA care. Methods We conducted a prospective observational cohort study in 21 ICUs in Finland between 2009 and 2010. The Utstein Guidelines were used for collecting resuscitation and PCA care data, such as initial rhythm and delay to return of spontaneous circulation (ROSC). P313 p Results Agreement between the diff erent sources of temperature measurement was generally high, with less than 10% outliers beyond ±1.96 SD. The best agreement was seen between intravascular tempera ture measurement and bladder temperature measurement (SD  =  0.14°C), with vaginal measurements showing less agreement (SD = 0.23°C), and rectal measurements showing the least (SD = 0.31°C) with intravascular. Eff ect of moderate hyperventilation and induced hypertension on cerebral tissue oxygen saturation in comatose post-cardiac arrest patients treated with hypothermia T Suys, N Sala, M Oddo Lausanne University Hospital, Lausanne, Switzerland Critical Care 2013, 17(Suppl 2):P311 (doi: 10.1186/cc12249) Changes in SctO2 after moderate HV and IH tests (n = 10 patients). S119 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Results The median viscosity on admission was 9.12 (8.19 to 11.19) mPa. second, and remained stable at 3 and 6 hours after admission. From 6 hours after admission, viscosity decreased signifi cantly to 3.66 (3.12 to 4.04) mPa.second (P <0.001). Median MFVMCA was low (27.0 (23.8 to 30.5) cm/second) on admission, and signifi cantly increased to 63.0 (51.0 to 80.0) cm/second at 72 hours (P <0.001). There was a signifi cant association between viscosity and the MFVMCA (P  =  0.0019). See Figure 1. where temperature is measured. Measurements of temperature can be made rectally, intravascularly, vaginally, or from the bladder, but agreement between these sites is often uncertain. We measured temperature at multiple sites during experimental induction and reversal of hypothermia in swine with a novel esophageal cooling device, hypothesizing that agreement between sites would fall within standard acceptance criteria (average diff erence  ±  1.96 standard deviation of the diff erence) in Bland–Altman analyses. g Conclusion Viscosity decreases in the fi rst 3 days after cardiac arrest and is strongly associated with an increase in cerebral blood fl ow. Since viscosity is a major determinant of cerebral blood fl ow, repeated measurements may guide therapy to help restore cerebral oxygenation after cardiac arrest. f y Methods Five female Yorkshire swine (mean weight 65  kg) were anesthetized and cooled for 24 hours, then gradually rewarmed, with a novel esophageal heat transfer device powered by an external chiller (Gaymar MediTherm III). Swine temperature was measured intra- vascularly, rectally, and either vaginally or in the bladder. Comparisons between temperature readings were then made via Bland–Altman plots. Reference 1. Arrich J, Holzer M, Herkner H, Mullner M: Hypothermia for neuroprotection in adults after cardiopulmonary resuscitation. Cochrane Database Syst Rev 2009, 4:CD004128. Methods We reviewed medical records of all CA patients (in-hospital or out-of-hospital arrest) in whom cardiopulmonary resuscitation (CPR) was performed at our hospital from 1 February 2011 to 31 January 2012 (12 months). The following information was collected: fi rst documented rhythm, whether TMH done or not, and two outcome measures including survival to hospital discharge and neurological outcome at the time of hospital discharge. A measure of good neurological outcome was Cerebral Performance Category score 1 or 2 (CPC, fi ve- point scale; 1 = good cerebral performance to 5 = brain death). Then we quantifi ed the association of TMH with SR as well as NR for both the parameters of outcome – that is, survival to hospital discharge and good neurological outcome – by logistic regression analysis. Association between oxygenation and 6-month mortality during post-cardiac arrest care Measured arterial blood oxygen values during the fi rst 24 hours from admission to the ICU were divided into the following predefi ned oxygenation categories: low (<10  kPa), normal (10 to 19  kPa), intermediate (20 to 29 kPa), and high (>30 kPa). Exposure to hyperoxia was defi ned as paO2 levels higher than 40 kPa [1]. Time spent in diff erent oxygenation categories and the highest, lowest and median oxygen values during the fi rst 24 hours were calculated and included in separate multivariate regression models along with age, delay to ROSC, initial rhythm and the use of therapeutic hypothermia for the prediction of 6-month mortality. Conclusion Although TMH might be associated with better survival chances in patients presenting with SR, neurological outcome was no better (rather worse) in this group of patients when compared with patients with NR as the fi rst documented rhythm. Therapeutic mild hypothermia after cardiac arrest in shockable and nonshockable rhythms: does it improve both survival and neurological outcome? A Gupta Fortis Escorts Heart Institute, New Delhi, India Critical Care 2013, 17(Suppl 2):P313 (doi: 10.1186/cc12251) Introduction Although therapeutic mild hypothermia (TMH) after resuscitation from cardiac arrest (CA) has been postulated and studied to be associated with good outcome of the patients, there is no dearth of data that does not favour TMH. Our aim was to fi nd out whether TMH is associated with good outcome after CA in shockable rhythm (SR) compared with nonshockable rhythm (NR), in terms of survival as well as neurological outcome. Conclusion Bladder temperature measurements demonstrated the best agreement with intravascular temperature measurements during cooling with an esophageal heat transfer device; however, reasonable agreement was demonstrated with rectal and vaginal temperature measurements, suggesting that these sites are also acceptable for use. Reference Diff erences of calcium binding protein immunoreactivities in the young hippocampal CA1 region from the adult following transient ischemic damage Results NSE >45 occurred in 24/68 patients (35.3%) and invariably correlates with bad outcome. The positive predictive value (PPV) NSE >45 for bad outcome is 100%. No patient in this group ever had a GCS ≥12. NSE >33 and <45 occurred in 16/68 patients (23.5%). Thirteen out of 16 patients (81.2%) had bad outcome. However, 7/16 patients (43.8%) woke up at some time (GCS ≥12). NSE <33 occurred in 28/68 patients (41.2%), 17/28 patients (60.7%) had good outcome and 23/28 patients (88.4%) had GCS ≥12 at some time. The PPV NSE <33 for good outcome is 60.7%. The BIS and SR were measured in only 28 patients. Initial BIS ≤10 occurred in 13/28 patients (46.4%) and correlates with bad outcome in 12/13 patients (92.3%). BIS >30 occurred in nine patients, 6/9 (66.7%) had good outcome. Initial SR ≥75 occurred in 11/28 patients (39.3%) and invariably correlates with bad outcome. NSE >25 and SR >60 occurred in 15/28 patients (53.6%) and invariably correlates with bad outcome. g CW Park, HY Lee, JH Cho, MH Won Introduction It has been reported that the young are much more resistant to transient cerebral ischemia than the adult. Introduction It has been reported that the young are much more resistant to transient cerebral ischemia than the adult. Methods In the present study, we compared the chronological changes of calcium binding proteins (CBPs) (calbindin 28k (CB-D 28k), calretinin (CR) and parvalbumin (PV)) immunoreactivities and levels in the hippocampal CA1 region of the young gerbil with those in the adult following 5 minutes of transient cerebral ischemia induced by the occlusion of both the common carotid arteries. Results In the present study, we examined that about 90% of CA1 pyramidal cells in the adult gerbil hippocampus died at 4 days post ischemia; however, in the young hippocampus, about 56% of them died at 7 days post ischemia. We compared immunoreactivities and levels of CBPs, such as CB-D 28k, CR and PV. The immunoreactivities and protein levels of all the CBPs in the young sham were higher than those in the adult sham. In the adult, the immunoreactivities and protein levels of all the CBPs were markedly decreased at 4 days post ischemia; however, in the young, they were apparently maintained. At 7 days post ischemia, they were decreased in the young; however, they were much higher than those in the adult. p References Introduction Neuron-specifi c enolase (NSE) values >33 μg/ml [1] and low bispectral index (BIS) [2] values correlate with bad outcome after cardiac arrest (CA). 1. Wijdicks et al.: Neurology 2006, 67:203-210. 2. Al Thenayan et al.: Neurology 2008, 71:1535-1537. 3. Rossetti et al.: Ann Neurol 2010, 67:301-307. 4. Bisschops et al.: Resuscitation 2011, 82:696-701. 5. Cronberg et al.: Neurology 2011, 77:623-630. 6. Samaniego et al.: Neurocrit Care 2011, 15:113-119. 7. Fugate et al.: Ann Neurol 2010, 68:907-914. 1. Wijdicks et al.: Neurology 2006, 67:203-210. 2. Al Thenayan et al.: Neurology 2008, 71:1535-1537. 3. Rossetti et al.: Ann Neurol 2010, 67:301-307. 4. Bisschops et al.: Resuscitation 2011, 82:696-701. 5. Cronberg et al.: Neurology 2011, 77:623-630. 6. Samaniego et al.: Neurocrit Care 2011, 15:113-119. 7. Fugate et al.: Ann Neurol 2010, 68:907-914. Methods In this nonblinded prospective study, we observed all CA patients from February 2011 until September 2012 surviving at least 24 hours. NSE was measured between 24 and 72 hours after CA. From October 2011 onward, we recorded BIS and suppression ratio (SR) values as soon as possible after arrival in the ICU. Patients treated with therapeutic hypothermia (TH) (33°C for 24  hours) received cisatracurium. Cerebral Performance Category (CPC) [3] 1 and 2 were considered good outcome, CPC 3 to 5 bad outcome and were recorded after 3 months. Statistical analysis was performed using SPSS statistics 19. Diff erences of calcium binding protein immunoreactivities in the young hippocampal CA1 region from the adult following transient ischemic damage Conclusion NSE >45 uniformly correlates with bad outcome after CA. However, we urge caution for the use of intermediate values (33 to 45). In preliminary data, we report that SR >75 might correlate with bad outcome and that combining NSE and SR might improve the predictive value. Also, low NSE and good initial BIS values correlate with preserved cerebral potential and should encourage the clinician. 1. Zandbergen et al.: Neurology 2006, 66:62-68. 1. Zandbergen et al.: Neurology 2006, 66:62-68. 2. Leary et al.: Resuscitation 2010, 81:1133-1137. Conclusion In brief, the immunoreactivities and levels of CBPs were not decreased in the ischemic CA1 region of the young 4 days after transient cerebral ischemia. This fi nding indicates that the longer maintenance of CBPs may contribute to a less and more delayed neuronal death/ damage in the young. 3. Ajam et al.: Scand J Trauma Resusc Emerg Med 2011, 19:38. Neuron-specifi c enolase and bispectral index/suppression ratio for prognostication after cardiac arrest i Conclusion Motor scores at day  3 post cardiac arrest of extension or worse do not reliably predict poor neurological outcome when therapeutic hypothermia has been used. Clinical neurological fi ndings may not be valid predictors of poor neurological outcome after therapeutic hypothermia. g Van Laer1, K Deschilder2, P Lormans2, J Gillet1, W Stockman2 , , , , 1UZ Leuven, Belgium; 2HHRM, Roeselare, Belgium 1UZ Leuven, Belgium; 2HHRM, Roeselare, Belgium g g Critical Care 2013, 17(Suppl 2):P316 (doi: 10.1186/cc12254) g g Critical Care 2013, 17(Suppl 2):P316 (doi: 10.1186/cc12254) Introduction Neuron-specifi c enolase (NSE) values >33 μg/ml [1] and low bispectral index (BIS) [2] values correlate with bad outcome after cardiac arrest (CA). Agreement between temperature measurements during cooling and warming through the esophagus Conclusion Motor scores at day  3 post cardiac arrest of extension or worse do not reliably predict poor neurological outcome when therapeutic hypothermia has been used. Clinical neurological fi ndings may not be valid predictors of poor neurological outcome after therapeutic hypothermia. 2 Conclusion In this multicentre observational study we were unable to defi ne an optimal oxygenation level during PCA care, but hypoxia seemed to be more harmful than hyperoxia. Exposure to hyperoxia was less common than in previous trials, and we were unable to confi rm previous fi ndings indicating an association with mortality. Reference Methods A review of existing literature was undertaken to examine whether the utility of motor scores to predict poor neurological outcome is infl uenced by the use of therapeutic hypothermia.i l Results Six studies were identifi ed [2-7] that investigated the use of motor scores on day 3 post cardiac arrest in patients who had received therapeutic hypothermia. False positive rates (defi ned as 1 – specifi city) for predicting poor neurological outcome were calculable in fi ve of the six studies [2-6] and were 14%, 24%, 11%, 25% and 12% respectively. In all studies the FPR for motor scores of extension or worse were signifi cantly higher than the 0% (0 to 3% 95% CIs) in the AAN guidelines. 1. Kilgannon JH, et al.: JAMA 2010, 303:2165-2171. Motor scores, therapeutic hypothermia and neurological outcome after cardiac arrest M Davidson Agreement between temperature measurements during cooling and warming through the esophagus False positive rates (defi ned as 1 – specifi city) for predicting poor neurological outcome were calculable in fi ve of the six studies [2-6] and were 14%, 24%, 11%, 25% and 12% respectively. In all studies the FPR for motor scores of extension or worse were signifi cantly higher than the 0% (0 to 3% 95% CIs) in the AAN guidelines. Conclusion Motor scores at day  3 post cardiac arrest of extension or worse do not reliably predict poor neurological outcome when therapeutic hypothermia has been used. Clinical neurological fi ndings may not be valid predictors of poor neurological outcome after therapeutic hypothermia. (22 kPa vs. 20 kPa, P = 0.054). In separate multivariate models neither time spent in the low or the intermediate categories, or the median, highest or lowest paO2 was found to correlate with mortality. widespread use of therapeutic hypothermia. The American Association of Neurology guidelines [1] currently recommend that absent or extensor motor scores on day 3 post arrest are reliable indicators or poor neurological outcome with a false positive rate of 0 to 3%. widespread use of therapeutic hypothermia. The American Association of Neurology guidelines [1] currently recommend that absent or extensor motor scores on day 3 post arrest are reliable indicators or poor neurological outcome with a false positive rate of 0 to 3%. desp ead use o t e apeut c ypot e a. e e ca ssoc at o of Neurology guidelines [1] currently recommend that absent or extensor motor scores on day 3 post arrest are reliable indicators or poor neurological outcome with a false positive rate of 0 to 3%. Methods A review of existing literature was undertaken to examine whether the utility of motor scores to predict poor neurological outcome is infl uenced by the use of therapeutic hypothermia. Results Six studies were identifi ed [2-7] that investigated the use of motor scores on day 3 post cardiac arrest in patients who had received therapeutic hypothermia. False positive rates (defi ned as 1 – specifi city) for predicting poor neurological outcome were calculable in fi ve of the six studies [2-6] and were 14%, 24%, 11%, 25% and 12% respectively. In all studies the FPR for motor scores of extension or worse were signifi cantly higher than the 0% (0 to 3% 95% CIs) in the AAN guidelines. Agreement between temperature measurements during cooling and warming through the esophagus p y Results A total of 489 patients were included. The average number of paO2 measurements during the fi rst 24 hours was eight per patient. A total of 6% of patients experienced paO2 values higher than 40 kPa at any time during the fi rst 24  hours. Average times spent in each time oxygenation category during the fi rst 24 hours were as follows: low 14%, normal 69%, intermediate 14%, and high 2% of the time. Survivors spent less time in the low band (P = 0.029) and more time in the intermediate band (P = 0.029) compared with nonsurvivors. The median paO2 during the fi rst 24 hours was higher in survivors than in nonsurvivors (15 kPa vs. 14 kPa, P = 0.016) but there was no diff erence in lowest (11  kPa vs. 10  kPa, P  =  0.162) or the highest paO2 values and warming through the esophagus EB Kulstad1, DM Courtney2, P Shanley3, A Metzger3, T Matsuura3, J Rees4, K Lurie3, S McKnite5 1Advocate Christ Medical Center, Oak Lawn, IL, USA; 2Northwestern University, Chicago, IL, USA; 3University of Minnesota Medical Center, Minneapolis, MN, USA; 4Advanced Circulatory Systems, Minneapolis, MN, USA; 5Minneapolis Medical Research Foundation, Minneapolis, MN, USA Critical Care 2013, 17(Suppl 2):P314 (doi: 10.1186/cc12252) Introduction Measurement of temperature during treatment with therapeutic hypothermia can yield variable values depending on Introduction Measurement of temperature during treatment with therapeutic hypothermia can yield variable values depending on S120 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 (22 kPa vs. 20 kPa, P = 0.054). In separate multivariate models neither time spent in the low or the intermediate categories, or the median, highest or lowest paO2 was found to correlate with mortality. widespread use of therapeutic hypothermia. The American Association of Neurology guidelines [1] currently recommend that absent or extensor motor scores on day 3 post arrest are reliable indicators or poor neurological outcome with a false positive rate of 0 to 3%. Methods A review of existing literature was undertaken to examine whether the utility of motor scores to predict poor neurological outcome is infl uenced by the use of therapeutic hypothermia. Results Six studies were identifi ed [2-7] that investigated the use of motor scores on day 3 post cardiac arrest in patients who had received therapeutic hypothermia. P319 Plymouth Hospitals NHS Trust, Plymouth, UK Plymouth Hospitals NHS Trust, Plymouth, UK P319 Early hypothermia improves survival and reduces the rise of serum biomarkers after traumatic brain injury in swine M Kumar, AD Goldberg, M Kashiouris, L Keenan, A Rabinstein Mayo Clinic, Rochester, MN, USA Critical Care 2013, 17(Suppl 2):P319 (doi: 10.1186/cc12257) y p y Critical Care 2013, 17(Suppl 2):P317 (doi: 10.1186/cc12255) Critical Care 2013, 17(Suppl 2):P317 (doi: 10.1186/cc12255) Introduction Accurate prediction of neurological outcome after cardiac arrest is desirable to prevent inappropriate withdrawal of life- sustaining therapy in patients who could have a good neurological outcome, and to limit active treatment in patients whose ultimate neurological outcomes are poor. Established guidelines to predict neurological outcome after cardiac arrest were developed before the y , , , Critical Care 2013, 17(Suppl 2):P319 (doi: 10.1186/cc12257) Introduction Poor outcomes in clinical trials on the use of therapeutic hypothermia following traumatic brain injury (TBI) may be due to the S121 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 delay in reaching target temperature [1]. We hypothesize that early and rapid induction of hypothermia will mitigate neuronal injury and improve survival in a swine model of TBI. Results Table 1 shows the magnitude of the problem, highlighting the leading causes of TBI in Egypt in 2010. Male sex was predominantly aff ected, 79% of cases. Moderate and severe injuries account for 17.2% of all TBI presented cases. delay in reaching target temperature [1]. We hypothesize that early and rapid induction of hypothermia will mitigate neuronal injury and improve survival in a swine model of TBI. Methods Twenty domestic cross-bred pigs (34 to 35 kg) were subjected to a 5 ATM (100 ms) lateral fl uid percussion TBI. The brain temperature and ICP were measured using Camino®. Serum biomarkers for neuronal injury  – S-100β, neuron-specifi c enolase, glial fi brillary acid protein (GFAP), and neurofi laments heavy chain – were measured daily using enzyme-linked immunosorbent assay. Twelve of the injured animals were rapidly cooled to 32°C within 90  minutes of the injury using a transpulmonary hypothermia technique [2]. Hypothermia was maintained for 48 hours. Eight injured control animals were maintained at 37°C. In both groups, anesthesia (isofl urane 1%) was discontinued and the animals were weaned off the ventilator after 48 hours. Five days post injury, the surviving animals were euthanized and necropsied. Alcohol intoxication impedes the recognition of traumatic brain injury in the prehospital setting and may worsen 6-month outcome R Raj, J Siironen, R Kivisaari, M Kuisma, M Skrifvars Helsinki University Central Hospital, Helsinki, Finland Critical Care 2013, 17(Suppl 2):P321 (doi: 10.1186/cc12259) Alcohol intoxication impedes the recognition of traumatic brain injury in the prehospital setting and may worsen 6-month outcome R Raj, J Siironen, R Kivisaari, M Kuisma, M Skrifvars Helsinki University Central Hospital, Helsinki, Finland Critical Care 2013, 17(Suppl 2):P321 (doi: 10.1186/cc12259) y g g Results Ten of the 12 hypothermia and four of the eight normothermia animals survived to the end of the 5-day study (χ2  =  2.597, df  =  1, P = 0.1071). Although the probability of type I error between survival curves was 11%, the study was clinically signifi cant and showed a clear trend toward improved survival with hypothermia. The intracranial pressures were signifi cantly (P <0.05) lower in the hypothermia group. Both interventions  – that is, general anesthesia and hypothermia  – mitigated the rise of serum biomarkers following TBI. However, the suppression of biomarkers was sustained during the recovery period only in the hypothermia group. With the exception of the GFAP levels, the curves of all biomarkers were signifi cantly diff erent between the groups.i Introduction Transport directly to a neurosurgical trauma center has shown to reduce mortality in patients with traumatic brain injury (TBI). We hypothesized that alcohol intoxication may impede prehospital recognition of TBI, resulting in transportation to a non-neurosurgical hospital. Methods A retrospective analysis of TBI patients admitted to a designated neurosurgical trauma center’s ICU in 2009/10 and primarily treated by the Emergency Medical Service (EMS). Patients were divided into two categories based either direct or indirect trauma transfer by the EMS. Directly transferred patients are directly transported to the neurosurgical trauma center from the injury scene while indirectly transported patients are initially transported to another non- neurosurgical hospital before re-transfer to the trauma center. Data from patient journals and EMS forms were extracted. The blood alcohol level (BAL) was measured by the EMS using an alcohol breath-test. Logistic regression modeling was used to identify variables present at scene associated with transport destination. Conclusion Our preliminary fi ndings show early initiation, rapid induction, and prolonged maintenance (48  hours) of cerebral hypothermia to lower intracranial pressure, blunt the rise in serum biomarkers, and improve survival following TBI. 1. Clifton GL, et al.: Lancet Neurol 2011, 10:131-139. 2. Kumar M., et al.: Anesth Analg 2012, 114:S-160. 1. Clifton GL, et al.: Lancet Neurol 2011, 10:131-139. 2. Kumar M., et al.: Anesth Analg 2012, 114:S-160. P319 The data were analyzed using a log-rank (Mantel–Cox) test, and ANOVA. Conclusion TBI is a serious public health problem in Egypt. Further data interpretation over wider periods of time should be conducted for better understanding of TBI prevalence is highly recommended to develop eff ective injury prevention programs. 1. Clifton GL, et al.: Lancet Neurol 2011, 10:131-139. 2. Kumar M., et al.: Anesth Analg 2012, 114:S-160. Alcohol intoxication impedes the recognition of traumatic brain injury in the prehospital setting and may worsen 6-month outcome R Raj, J Siironen, R Kivisaari, M Kuisma, M Skrifvars Helsinki University Central Hospital, Helsinki, Finland Critical Care 2013, 17(Suppl 2):P321 (doi: 10.1186/cc12259) Results Totally 470 patients met the inclusion criteria; 60% were transported directly and 40% indirectly. In the direct group 15% of patients had a positive BAL, compared with 26% for those indirectly transported. In the logistic regression model, factors associated with direct transport were: BAL ≥2.3‰ (OR: 0.06, CI: 0.01 to 0.36), male gender (OR: 0.35; CI: 0.16 to 0.76), GCS 13 to 15 (OR: 0.28; CI 0.10 to 0.74), high-energy trauma (OR: 9.42; CI 2.15 to 41.20), major extracranial injury (OR: 7.92; CI 2.57 to 24.41), EMS physician telephone consultation (OR: 6.02; CI 2.51 to 14.11) or presence on scene (OR: 8.63; CI 3.50 to 21.26) and incident at a public place outside (OR: 3.05; CI: 1.34 to 6.4) and inside (OR: 2.92; CI: 1.07 to 8.01). Median time delay to trauma center admission was 1:07  hours (IQR: 0.52 to 1:28) for directly transported patients and 4:06 (IQR: 2.54 to 5:43) for those indirectly transported (P  <0.001). There was a clear trend towards poorer neurological outcome for patients with delayed trauma center admission in univariate analysis (P = 0.001). P320 P320 Epidemiology of moderate and severe traumatic brain injury in Cairo University Hospital in 2010 T Montaser1, A Hassan2 1Shobra General Hospital, Cairo, Egypt; 2King Khaled University Hospital, Riyadh, Saudi Arabia Critical Care 2013, 17(Suppl 2):P320 (doi: 10.1186/cc12258) P320 Epidemiology of moderate and severe traumatic brain injury in Cairo University Hospital in 2010 T Montaser1, A Hassan2 1Shobra General Hospital, Cairo, Egypt; 2King Khaled University Hospital, Riyadh, Saudi Arabia Critical Care 2013, 17(Suppl 2):P320 (doi: 10.1186/cc12258) P320 Epidemiology of moderate and severe traumatic brain injury in Cairo University Hospital in 2010 T Montaser1, A Hassan2 1Shobra General Hospital, Cairo, Egypt; 2King Khaled University Hospital, Riyadh, Saudi Arabia Critical Care 2013, 17(Suppl 2):P320 (doi: 10.1186/cc12258) P322 Predictive ability of the IMPACT-TBI prognostic model is improved by adding markers of coagulation R Raj, J Siironen, R Kivisaari, L Handolin, J Hernesniemi, M Skrifvars Helsinki University Central Hospital, Helsinki, Finland Critical Care 2013, 17(Suppl 2):P322 (doi: 10.1186/cc12260) Predictive ability of the IMPACT-TBI prognostic model is improved by adding markers of coagulation Methods A prospective cohort of STBI patients monitored with cerebral microdialysis (CMD) and brain tissue oxygen (PbtO2) was studied. Brain lactate metabolism was assessed by quantifi cation of elevated CMD lactate samples (>4  mmol/l). These were matched to pyruvate and PbtO2, and dichotomized as hyperglycolytic (CMD pyruvate >119 μmol/l) versus nonhyperglycolytic or as hypoxic (PbtO2 <20 mmHg) versus nonhypoxic. Data were expressed as percentages per patient. Global brain perfusion (categorized as oligemic, normal or hyperemic) was assessed with CT perfusion (CTP). Introduction One of the most used prognostic models for traumatic brain injury is the IMPACT-TBI model, which predicts 6-month mortality and unfavorable outcome. Our aim was to study whether adding markers of coagulation improves the model’s predictive power when accounting for extracranial injury. yp p Results Twenty-four patients (total 1,782 CMD samples) were studied. Samples with elevated CMD lactate were frequently observed (41 ± 8% SEM of individual samples). Brain lactate elevations were predominantly hyperglycolytic (73 ± 8.2%), whilst only 14 ± 6.3% of them were hypoxic. Trends over time of both lactate patterns are shown in Figure 1. On CTP (n = 17; average 48 hours from TBI) hyperglycolytic lactate was always associated with normal or hyperemic CTP, whilst hypoxic lactate was associated with oligemic CTP (Table 1). Methods Patients with a TBI admitted to a designated trauma center in 2009/10 were screened retrospectively and included according to the IMPACT study criteria. The predictive outcome was calculated for included patients using the full IMPACT-TBI model. To assess coagulopathy and extracranial injury we used the prothrombin time percentage (PT), platelet count (109), and injury severity score (ISS). PT, platelets and ISS were added to the IMPACT model, creating new models. The predictive power of the new models was tested by comparing their area under the receiver operating characteristic curve (AUC) with the original model. Figure 1 (abstract P323). g Results Totally 342 were included. Six-month mortality was 32% and unfavorable outcome 57%. The IMPACT model’s predictive power for mortality and neurological outcome; AUC: 0.85, CI: 0.81 to 0.89; and AUC: 0.81, CI: 0.76 to 0.86. P320 Epidemiology of moderate and severe traumatic brain injury in Cairo University Hospital in 2010 T Montaser1, A Hassan2 1Shobra General Hospital, Cairo, Egypt; 2King Khaled University Hospital, Riyadh, Saudi Arabia Critical Care 2013, 17(Suppl 2):P320 (doi: 10.1186/cc12258) Nonsurvivors and patients with an unfavorable outcome had lower platelets (159; 165 vs. 191; 198), PT (76; 77 vs. 86; 90) and higher ISS (50; 44% ISS >25 vs. 34; 32% ISS >25) compared with survivors and favorable outcome (P <0.05). Addition of PT but not ISS nor platelets resulted in a signifi cant improvement of the IMPACT model’s predictive power for 6-month mortality; AUC: 0.87, CI: 0.81 to 0.89, P = 0.017 (Figure 1). In multivariate analysis PT remained an independent signifi cant predictor of outcome when adjusting for IMPACT prognosis and ISS (OR: 0.985, CI: 0.975 to 0.996). p g Conclusion Addition of PT to the IMPACT model improves the predictive power for 6-month mortality but not neurological outcome when adjusting for ISS. p p y g when adjusting for ISS. References 1. Steyerberg EW, et al.: PLoS Med 2008, 5:e165. 2. Marmarou A, et al.: J Neurotrauma 2007, 24:239-250. P323 Nonischemic endogenous lactate production in humans with severe traumatic brain injury N Sala1, T Suys1, JB Zerlauth1, J Bloch1, P Magistretti2, R Meuli1, M Oddo1 1Lausanne University Hospital, Lausanne, Switzerland; 2EPFL, Lausanne, Switzerland Critical Care 2013, 17(Suppl 2):P323 (doi: 10.1186/cc12261) Introduction Evidence suggest that endogenous lactate, produced by aerobic glycolysis, is an important substrate for neurons, particularly in Figure 1 (abstract P322). P320 Epidemiology of moderate and severe traumatic brain injury in Cairo University Hospital in 2010 T Montaser1, A Hassan2 1Shobra General Hospital, Cairo, Egypt; 2King Khaled University Hospital, Riyadh, Saudi Arabia Critical Care 2013, 17(Suppl 2):P320 (doi: 10.1186/cc12258) Epidemiology of moderate and severe traumatic brain injury in Cairo University Hospital in 2010 T Montaser1, A Hassan2 1Shobra General Hospital, Cairo, Egypt; 2King Khaled University Hospital, Riyadh, Saudi Arabia Critical Care 2013, 17(Suppl 2):P320 (doi: 10.1186/cc12258) T Montaser1, A Hassan2 y Critical Care 2013, 17(Suppl 2):P320 (doi: 10.1186/cc12258) Introduction Traumatic brain injury (TBI) is a contributing factor to approximately one-third of all injury-related deaths in the USA annually. Updated statistical records for TBI in Egypt are lacking. The current research is aiming to estimate the prevalence of TBI in Egypt in order to develop a comprehensive TBI prevention program. p p p p g Methods A 1-year period (one calendar month every quarter of 2010) descriptive epidemiological study of moderate and severe TBI cases admitted to the emergency department, Cairo main university hospital. The data collection sheet included personal data (age, sex and residency), incident-related data (cause, nature and time of injury) and both clinical and radiological fi ndings. y Conclusion Heavily alcohol intoxicated TBI patients are commonly initially transported to a non-neurosurgical trauma center and this may worsen 6-month neurological outcome. References 1. Hartl R, et al.: J Trauma 2006, 60:1250-1256. 1. Hartl R, et al.: J Trauma 2006, 60:1250-1256. 2. Shahin H, et al.: J Trauma 2010, 69:1176-1181. 2. Shahin H, et al.: J Trauma 2010, 69:1176-1181. Table 1 (abstract P320). Causes of TBI in diff erent age groups in Egypt NO = 844/ Abuse/ Violence/ Struck by/ Other causes/ Age (years) 4 months FFH MVC neglect assaults against not known 1 to 5 54 (6%) 20 (37%) 10 (19%) 8 (15%) – 8 (15%) 8 (14%) 6 to 18 91 (11%) 28 (31%) 20 (22%) 5 (6%) 6 (5 %) 22 (25%) 10 (11%) 19 to 29 150 (18%) 49 (33%) 45 (30%) – 14 (9%) 18 (12%) 24 (16%) 30 to 55 381 (45%) 114 (30%) 134 (35%) – 19 (5%) 50 (13%) 64 (17%) Above 55 168 (20%) 47 (28%) 41 (24%) 11 (7%) 5 (3%) 25 (15%) 39 (23%) Table 1 (abstract P320). Causes of TBI in diff erent age groups in Egypt S122 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 P322 conditions of increased energy demand. This study aimed to examine brain lactate metabolism in patients with severe traumatic brain injury (STBI). j References 1. Steyerberg EW, et al.: PLoS Med 2008, 5:e165. 1. Steyerberg EW, et al.: PLoS Med 2008, 5:e165. 2. Marmarou A, et al.: J Neurotrauma 2007, 24:239-250. 2. Marmarou A, et al.: J Neurotrauma 2007, 24:239-250. Figure 1 (abstract P322). Table 1 (abstract P323). Lactate elevations and brain perfusion ( ) p Oligemia (%) Normal (%) Hyperemia (%) HG lactate 0 58 42 HX lactate 100 0 0 HG, hyperglycolytic; HX, hypoxic. Conclusion Our fi ndings suggest predominant nonischemic lactate release after TBI and identify, for the fi rst time, an association between cerebral hyperglycolytic lactate production and normal to supranormal brain perfusion. Our data support the concept that lactate may be used as energy substrate by the injured human brain. gy y j Acknowledgement Supported by the Swiss National Science Foundation. Figure 1 (abstract P322). P323 P323 Nonischemic endogenous lactate production in humans with severe traumatic brain injury N Sala1, T Suys1, JB Zerlauth1, J Bloch1, P Magistretti2, R Meuli1, M Oddo1 1Lausanne University Hospital, Lausanne, Switzerland; 2EPFL, Lausanne, Switzerland Critical Care 2013, 17(Suppl 2):P323 (doi: 10.1186/cc12261) Nonischemic endogenous lactate production in humans with severe traumatic brain injury Yagi, N Saito, Y Hara, H Hisashi Matumoto, K Mashiko Introduction In the prehospital setting, it is diffi cult to use the Glasgow Coma Scale (GCS) to evaluate the consciousness state using in pediatric patients with severe trauma. The Japan Coma Scale (JCS) is a consciousness scale used widely in Japan and, with its four grades, is simpler and quicker to use than the GCS. This study examined whether Introduction In the prehospital setting, it is diffi cult to use the Glasgow Coma Scale (GCS) to evaluate the consciousness state using in pediatric patients with severe trauma. The Japan Coma Scale (JCS) is a consciousness scale used widely in Japan and, with its four grades, is simpler and quicker to use than the GCS. This study examined whether Introduction Evidence suggest that endogenous lactate, produced by aerobic glycolysis, is an important substrate for neurons, particularly in S123 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Methods In this prospective observational study, measurements of OSND were carried out in 21 patients of a polyvalent 10-bed adult ICU before and 2 minutes after changes of PEEP from a baseline of 5 with 1 cmH2O increments, to a maximum of 12. Two measurements were performed with a 7.5 MHz probe (MicroMaxx; Fujifi lm Sonosite Inc., USA) in each eye before and after PEEP changes. Demographic data (APACHE II score, age, sex, diagnosis) Ppeak and Pm were also recorded. Exclusion criteria were intracranial pathology, ocular neuritis and trauma. Normality with P–P plots and the Kolmogorov–Smirnov test were calculated followed by bivariate and single linear regression analysis (IBM SPPS v17, signifi cance P <0.05). the JCS can predict clinical traumatic brain injury (TBI) and outcome in pediatric patients aged 3 to 15 years in the prehospital setting. Methods This retrospective study analyzed data from the Japan Trauma Data Bank. P323 Registered pediatric patients aged 3 to 15 years with severe trauma (maximum Abbreviated Injury Scale score ≥3 or Injury Severity Score ≥9) were divided into four groups according to JCS score in the prehospital setting (Grade 0: alert, Grade 1: possible eye-opening, not lucid, Grade 2: possible eye-opening upon stimulation, Grade 3: no eye-opening and coma). TBI was defi ned as maximum head AIS ≥3. y gi Results A total of 1,562 patients were included (Grade 0: 673, Grade 1: 410, Grade 2: 230, Grade 3: 249). Victims of blunt trauma accounted for 98.1%. Median age was 9 (interquartile range: 7 to 12) years, median ISS was 16 (9 to 21). There was strong agreement between the JCS in prehospital setting and GCS scores on arrival at hospital (r = –0.745, P <0.001). Multivariate analysis adjusted for age and ISS revealed that the JCS was independently associated with TBI (odds ratio (OR): 2.5; 95% CI: 2.1 to 2.8, P <0.001) and hospital mortality (OR: 3.8; 95% CI 2.4 to 6.0, P <0.001). See Table 1. y gi Results Seventy-two measurements were included for further analysis. Mean  ±  SD values were: PEEP (7.9  ±  2.3), Ppeak (24.35  ±  5), Pm (12.03 ± 3.16), OSNDright (4.03 ± 0.79), OSNDleft (4.1 ± 0.79), ΔOSND (0.24 ± 0.2), while values for age and APACHE II score were 62 ± 4 years and 14.4 ± 1.4 respectively. PEEP and OSND seem to have a moderate relation (tendency equation: OSND = 2.454 + 0.581×PEEP) compared with the weaker eff ect of Ppeak and Pm (Table 1). Table 1 (abstract P325). Correlations (r2) between airway pressures and OSND OSNDright OSNDleft PEEP 0.581* 0.571* Ppeak 0.467* 0.448* Pm 0.491** 0.467** *P <0.01, **P <0.05. Table 1 (abstract P324) TBI, n (%) Mortality (%) JCS grade 0 213 (31.6) 0.1 JCS grade 1 225 (62.2) 0.7 JCS grade 2 184 (80.0) 2.6 JCS grade 3 230 (92.4) 15.7 Table 1 (abstract P325). Correlations (r2) between airway pressures and OSND Conclusion Our study identifi ed a moderate relation between PEEP and OSND and a weaker one between Ppeak, Pm and OSND. Thus, in selected cases OSND could serve as a bedside marker of eff ect of airway pressure to ICP. Yet, larger studies are needed to come to a safer conclusion. Conclusion There was strong association between JCS score and clinical outcome in pediatric patients with severe trauma. P325f 1. Geeraerts T, et al.: Int Care Med 2008, 34:2062-2067. P325 Eff ect of positive end-expiratory pressure on ultrasound measurement of optical sheath nerve diameter: preliminary study T Aslanidis, E Anastasiou, E Geka, E Efthimiou, A Myrou, A Kontos, D Papadopoulos, E Boultoukas, M Giannakou-Peftoulidou A.H.E.P.A. University Hospital, Thessaloniki, Greece Critical Care 2013, 17(Suppl 2):P325 (doi: 10.1186/cc12263) P325 Eff ect of positive end-expiratory pressure on ultrasound measurement of optical sheath nerve diameter: preliminary study T Aslanidis, E Anastasiou, E Geka, E Efthimiou, A Myrou, A Kontos, D Papadopoulos, E Boultoukas, M Giannakou-Peftoulidou A.H.E.P.A. University Hospital, Thessaloniki, Greece Critical Care 2013, 17(Suppl 2):P325 (doi: 10.1186/cc12263) P326 P326 National survey of current protocols and management of the traumatic brain injury patients in UK ICUs B Lewinsohn, S Panchatsharam, S Wijayatilake, A Billini, G Delacedra, R Jain, J Khan, R Shetty, A Lewinsohn Queens Hospital, Romford, UK Critical Care 2013, 17(Suppl 2):P326 (doi: 10.1186/cc12264) P323 The results support the use of the JCS in the prehospital transport destination criteria for children. Reference Reference National survey of current protocols and management of the traumatic brain injury patients in UK ICUs j y p B Lewinsohn, S Panchatsharam, S Wijayatilake, A Billini, G Delaced R Jain, J Khan, R Shetty, A Lewinsohn Queens Hospital, Romford, UK Critical Care 2013, 17(Suppl 2):P326 (doi: 10.1186/cc12264) Introduction Non-invasive ocular ultrasonography has recently been proposed to detect elevated intracranial pressure (ICP). On the other hand, the eff ect of positive end-expiratory pressure (PEEP) on ICP is well documented. The aim of the present ongoing study is to record the eff ect of changes of airway pressures on optical sheath nerve diameter (OSND). Introduction Following primary neurological insult, initial manage ment of traumatic brain-injured (TBI) patients has a clearly defi ned pathway [1]. However, after arrival at tertiary centers, further manage ment is not Figure 1 (abstract P326). Figure 1 (abstract P326). S124 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 computed for longer periods. Signifi cantly better outcome (Table  1) was observed in patients for whom optimal CPP derived from LAx was maintained. Table 1 (abstract P327) Nonsurvivors Survivors P value Time within CPPopt-% 18.3 (15.5; 24.4) 24.8 (19.7; 28.7) 0.0004 |CPP-CPPopt| (mmHg) 7.05 (4.98; 9.73) 5.37 (4.49; 6.82) 0.0024 Table 1 (abstract P327) standardized. Intracranial hypertension (ICH), systemic hypotension, hypoxia, hyperpyrexia and hypocapnia have all been shown to independently increase mortality [2]. Despite numerous studies, there is currently no level 1 evidence to support any specifi c management [3]. Our objective was to provide an overview of the current clinical management protocols in the UK. g p Methods Thirty-one ICUs managing patients with severe TBI were identifi ed from the RAIN (Risk Adjustment In Neurocritical care) study, and a telephone survey was conducted. computed for longer periods. Signifi cantly better outcome (Table  1) was observed in patients for whom optimal CPP derived from LAx was maintained. Results A total 97% of units used a cerebral perfusion pressure protocol for the initial management, with 83% targeting pressures of 60 to 70 mmHg and 17% aimed for >70 mmHg. Ninety-one percent of units monitored CO2 routinely with 61% targeting CO2 of 4.5 to 5 kPa (Figure  1). Regarding osmotherapy, mannitol was still the preferred agent, with 48% of units using it as fi rst line; 32% used hypertonic saline, while 20% of units used either depending on clinicians’ preference. g References 1. NICE: Head Injury; Triage, Assessment, Investigation and Early Management of Head Injury. London: National Collaborating Centre for Acute Care; September 2007. g Methods A retrospective analysis of rcSO2 utility in AMS. rcSO2 data were collected every 30 seconds for AMS patients who had a head CT. Patients with a negative head CT were compared with those with an abnormal head CT. ROC analysis was performed to fi nd the AUC for each summary statistic and performance characteristics. Subgroup analysis was done to determine whether rcSO2 predicted injury and location. 2. Wijayatilake et al.: Updates in the management of intracranial pressure in traumatic brain injury. Curr Opin Anaesthesiol 2012, 25:540-547. 3. Guidelines for the Management of Severe Traumatic Brain Injury. 3rd edition. New York: Brain Trauma Foundation; 2007. 3. Guidelines for the Management of Severe Traumatic Brain Injury. 3rd edition. New York: Brain Trauma Foundation; 2007. c 2 p j y Results rcSO2 readings across 5, 15, 20, and 30 minutes were stable (Figure  1). rcSO2 readings with one or both sides <50% or a wide diff erence between L and R cerebrum was predictive of an abnormal CT scan. A mean diff erence of 4.2 was 82% sensitive for detecting a CT lesion with 62% specifi city, 88% PPV, and 52% NPV; a mean diff erence of 12.2 was 100% specifi c for an abnormal head CT. Lower mean rcSO2 readings localized to the CT pathology side, and higher rcSO2 readings trend toward the EDH group. 4. Barochia et al.: Bundled care for septic shock. Crit Care Med 2010, 38:668-678. 4. Barochia et al.: Bundled care for septic shock. Crit Care Med 2010, 38:668-678. 4. Barochia et al.: Bundled care for septic shock. Crit Care Med 2010, 38:668-678. National survey of current protocols and management of the traumatic brain injury patients in UK ICUs Sixteen percent questioned were currently enrolled on the Eurotherm hypothermia trial, while 16% never used hypothermia and one unit used prophylactic hypothermia routinely. The remaining 65% of units used hypothermia only to manage refractory ICH. P327 f Low-frequency autoregulation index for calculation of optimal cerebral perfusion pressure in severe traumatic brain injury F Guiza1, G Meyfroidt1, M Schuhmann2, G Van den Berghe1, I Piper3, B Depreitere1 1University Hospitals Leuven, Belgium; 2University Hospital Tübingen, Germany; 3Southern General Hospital, Glasgow, UK Critical Care 2013, 17(Suppl 2):P327 (doi: 10.1186/cc12265) Cerebral rSO2 monitoring in pediatric altered mental status patients T Abramo, I Kane Vanderbilt School of Medicine, Nashville, TN, USA Critical Care 2013, 17(Suppl 2):P328 (doi: 10.1186/cc12266) Vanderbilt School of Medicine, Nashville, TN, USA Critical Care 2013, 17(Suppl 2):P328 (doi: 10.1186/cc12266) Conclusion There is no clear consensus on the initial targets used. The surviving sepsis campaign showed that protocol-led care can reduce mortality [4]. Perhaps it is time for a similar approach to be adopted, with specialists coming to together to review the evidence and formulate guidelines that can then be tested. Introduction Pediatric patients with altered mental status (AMS) present with poor histories resulting in delayed testing and potential poor outcomes. Non-invasive detection for altered cerebral physiology related to TBI would improve resuscitation and outcome. Cerebral rSO2 (rcSO2) studies demonstrate its utility in certain neurological emergencies. References References 1. Aries M, et al.: Crit Care Med 2012, 40:2456-2463. 2. Piper I, et al.: Acta Neurochirurgica 2003, 145:615-628. 1. Aries M, et al.: Crit Care Med 2012, 40:2456-2463. 2. Piper I, et al.: Acta Neurochirurgica 2003, 145:615-628. 1. Aries M, et al.: Crit Care Med 2012, 40:2456-2463. 1. Aries M, et al.: Crit Care Med 2012, 40:2456 2463. 2. Piper I, et al.: Acta Neurochirurgica 2003, 145:615-628. Low-frequency autoregulation index for calculation of optimal cerebral perfusion pressure in severe traumatic brain injury F Guiza1, G Meyfroidt1, M Schuhmann2, G Van den Berghe1, I Piper3, B Depreitere1 Conclusion Cerebral rcSO2 monitoring can non-invasively detect altered cerebral physiology and pathology related to TBI as the cause for pediatric altered mental status. The utility of rcSO2 monitoring has shown its potential for localizing and characterizing intracranial lesions among these altered children. Further studies utilizing rcSO2 monitoring as an adjunct tool in pediatric altered mental status evaluation and management are ongoing. Introduction Traumatic brain injury (TBI) is a major cause of permanent disability and death in young patients. Controversy exists regarding the optimal cerebral perfusion pressure (CPP) required in TBI management. A tool for monitoring autoregulation and determining an optimal CPP is the pressure reactivity index (PRx), defi ned as a moving correlation coeffi cient between the mean arterial blood pressure (MAP) and intracranial pressure (ICP) at a frequency of at least 60 Hz. This requirement of high frequency has constrained its use to a few academic centers. An association was shown between outcome and continuous optimal CPP based on 4 hours of PRx [1]. We present a novel low-frequency autoregulation index (LAx), based on correlations between ICP and MAP at a standard minute-by-minute time resolution. Methods A total of 182 patients from the Brain-IT [2] multicentre European database had registered outcome and ICP and MAP for the fi rst 48 ICU hours. Twenty-one TBI patients admitted to the university hospitals of Leuven, Belgium and Tubingen, Germany were continuously monitored using ICM+ software (Cambridge Enterprise) allowing for continuous PRx calculation. Autoregulation indices versus CPP plots for PRx and LAx were computed to determine optimal CPP every minute during the fi rst 48 ICU hours [1]. Intravenous paracetamol for fever control in acute brain-injured patients: cerebral and hemodynamic eff ects E Picetti1, I Rossi1, P Ceccarelli1, S Risolo1, P Schiavi2, V Donelli1, A Crocamo1, M Antonini1, M Caspani1 1I Servizio Anestesia e Rianimazione, Azienda Ospedaliero-Universitaria di Parma, Italy; 2Neurochirurgia e Neurotraumatologia, Azienda Ospedaliero- Universitaria di Parma, Italy Critical Care 2013, 17(Suppl 2):P329 (doi: 10.1186/cc12267) Introduction Fever is a dangerous secondary insult for the injured brain [1]. We investigated the cerebral and hemodynamic eff ects of intravenous (i.v.) paracetamol administration for the control of fever in neurointensive care unit (NICU) patients. Methods The i.v. paracetamol (1 g in 15 minutes) was administered to NICU patients with a body temperature (Temp.) >37.5°C. Its eff ects on mean arterial pressure (MAP), heart rate (HR), intracranial pressure (ICP), cerebral perfusion pressure (CPP), jugular venous oxygen saturation (SjVO2) and Temp. were recorded at the start of paracetamol infusion (T0) and after 30 (T30), 60 (T60) and 120 (T120) minutes. Interventions for the maintenance of CPP  >60  mmHg or ICP  <20  mmHg were recorded. i Results On the Brain-IT database, LAx resulted in an optimal CPP for 90% of the fi rst 48 hours. Table 1 shows recommendations with respect to outcome. In the Leuven–Tübingen database, PRx and LAx resulted in 44% and 92% recommendations respectively. The average diff erence between methods was 5.26 mmHg.f Conclusion The diff erences in optimal CPPs derived from PRx and LAx were not clinically signifi cant. LAx allowed for recommendations to be Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 S125 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Results Fifteen NICU patients (nine subarachnoid hemorrhage, fi ve traumatic brain injury, mean age 54.9 ± 16.8, seven (50%) males, median GCS 7) were prospectively studied. We analyzed the administration of one dose of paracetamol for each patient (total 14 cases). After infusion of paracetamol we found a decrease of Temp. (from 37.8 ± 0.3 to 37.4 ± 0.4°C, P <0.001), MAP (from 94.7 ± 9.9 to 86.1 ± 6.7 mmHg, P = 0.008), CPP (from 79.6 ± 13.1 to 70.8 ± 7.6 mmHg, P = 0.011) and HR (from 71.5 ± 14.9 to 63.8 ± 16.3 bpm, P <0.001) with respect to the starting value (ANOVA for repeated measures), whereas ICP and SjVO2 remained unchanged (Figure 1). In fi ve cases norepinephrine infusion was started for CPP <60 mmHg. In another two cases, for the same reason, the norepinephrine dosage was augmented. 1. Greer DM, Funk SE, Reaven NL, Ouzounelli M, Uman GC: Impact of fever on outcome in patients with stroke and neurologic injury. Stroke 2008, 39:3029-3035. Intravenous paracetamol for fever control in acute brain-injured patients: cerebral and hemodynamic eff ects We evaluated sociodemographic and clinical factors associated with readmissions using a logistic regression model.i P330 Retrospective analysis of the hemodynamic eff ects of induction of barbiturate coma in patients with refractory elevated intracranial pressure S D’Hollander1, M Vander Laenen1, JB Gillet2, C De Deyne1, R Heylen1, W Boer1, F Jans1 1ZOL Genk, Belgium; 2UZ Leuven, Belgium Critical Care 2013, 17(Suppl 2):P330 (doi: 10.1186/cc12268) g g g Results Among 18,342 adult patients with TBI identifi ed in the registry, 14,777 patients were included among which 2,363 had severe, 1,106 moderate and 11,308 mild traumatic brain injury. Most patients were young (mean age: 52  ±  23 years) and had no comorbidity (73.6%). Overall, 1,032 patients (7.0%) were readmitted within 30 days, 12.7% within 3 months and 17.6% within 6 months. At 30 days post discharge, 311 (30.1%) were readmitted for a complication. The median length of stay was 8 days (Q1 to Q3: 3 to 20). More than 10% of patients aged ≥75 years with ≥1 comorbidity or with ≥1 admission prior to index hospitalisation were readmitted. The severity of the TBI was not an independent predictor of readmission. Age, highest AIS, number of comorbidities, number of admissions prior to index hospitalization, level of index trauma center and discharge destination were associated with readmissions on multivariate analysis. Introduction Standard clinical practice in patients with elevated intracranial pressure (ICP) is to keep cerebral perfusion pressure (CPP) at (or above) 60  mmHg: we performed this retrospective study to analyze whether induction of barbiturate coma impedes compliance to this target CPP value. It has been repetitively shown that, although proven as an effi cient therapy for refractory elevated ICP, barbiturate coma may cause a decrease in mean arterial pressure (MAP) and CPP [1]. Methods All patients that had received sodium thiopental in our ICU during 2011 were identifi ed and their medical and nursing records were retrospectively analyzed. The eff ect of administration of sodium thiopental (loading dose 3 to 5  mg/kg, followed by a continuous infusion adjusted to obtain an EEG burst suppression ratio between 40 and 80%) on MAP, ICP and CPP was evaluated. Values are reported as mean ± SD. The changes over time in ICP, MAP and CPP are reported as means with their 95% CI. y Conclusion Readmissions in the months following TBI are frequent, but were not found to be associated with the TBI severity. Intravenous paracetamol for fever control in acute brain-injured patients: cerebral and hemodynamic eff ects The proportion of patients who had infusion of norepinephrine increased from 42.8% at T0 to 78.6% at T120 (P = 0.02, chi-square for trends). Conclusion Use of i.v. paracetamol is eff ective in the maintenance of normothermia in acute brain-injured patients. However, adverse hemodynamic eff ects, which could represent a secondary insult for the injured brain, must be rapidly recognized and treated. Reference 1. Greer DM, Funk SE, Reaven NL, Ouzounelli M, Uman GC: Impact of fever on outcome in patients with stroke and neurologic injury. Stroke 2008, 39:3029-3035. Figure 1 (abstract P328). Figure 1 (abstract P329). *P <0.05 versus T0. Figure 1 (abstract P328). Figure 1 (abstract P329). *P <0.05 versus T0. reason, the norepinephrine dosage was augmented. The proportion of patients who had infusion of norepinephrine increased from 42.8% at T0 to 78.6% at T120 (P = 0.02, chi-square for trends).f Results Fifteen NICU patients (nine subarachnoid hemorrhage, fi ve traumatic brain injury, mean age 54.9 ± 16.8, seven (50%) males, median GCS 7) were prospectively studied. We analyzed the administration of one dose of paracetamol for each patient (total 14 cases). After infusion of paracetamol we found a decrease of Temp. (from 37.8 ± 0.3 to 37.4 ± 0.4°C, P <0.001), MAP (from 94.7 ± 9.9 to 86.1 ± 6.7 mmHg, P = 0.008), CPP (from 79.6 ± 13.1 to 70.8 ± 7.6 mmHg, P = 0.011) and HR (from 71.5 ± 14.9 to 63.8 ± 16.3 bpm, P <0.001) with respect to the starting value (ANOVA for repeated measures), whereas ICP and SjVO2 remained unchanged (Figure 1). In fi ve cases norepinephrine infusion was started for CPP <60 mmHg. In another two cases, for the same q Conclusion Use of i.v. paracetamol is eff ective in the maintenance of normothermia in acute brain-injured patients. However, adverse hemodynamic eff ects, which could represent a secondary insult for the injured brain, must be rapidly recognized and treated. Reference S126 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 P330 admission, and readmissions in the 12 following months. Primary outcome was unplanned readmission 30 days, 3 months and 6 months post discharge. Intravenous paracetamol for fever control in acute brain-injured patients: cerebral and hemodynamic eff ects Further studies evaluating reasons for readmission are warranted in order to develop strategies to prevent such events. Results In 2011, a total of 20 patients were treated in our ICU with barbiturate coma for refractory elevated ICP. In fi ve patients, systemic hypothermia was simultaneously induced. These patients were excluded from further analysis. Six patients died during their stay in the ICU and nine patients could be discharged to the neurosurgical ward. The mean peak ICP value before induction of barbiturate coma was 26 ± 3 mmHg, and the ICP value 6 hours later was 20 ± 6 mmHg. The MAP at those same time points was 91 ± 12 mmHg and 81 ± 11 mmHg, respectively. The CPP, calculated from both previous values, was 66 ± 13 mmHg and 61 ± 11 mmHg, respectively. At both time points, the dose of noradrenaline was comparable (10.1 ± 9.6 μg/minute and 11.5  ±  13.2  μg/minute, respectively). Analysis of the change in ICP, MAP and CPP over the 6-hour period showed a signifi cant decrease in ICP of –5.8 mmHg (–9.3; –2.3) (mean, 95% CI). The MAP signifi cantly decreased by –10.4 mmHg (–19.1; –1.7). The decrease in CPP was not signifi cant (–4.6 mmHg (–14.2; 5.0)). P332 Risk factors of pituitary disorders following traumatic brain injury F Lauzier1, O Lachance1, I Cote1, B Senay1, P Archambault1, F Lamontagne2 A Boutin1, M Shemilt1, L Moore1, F Bernard3, C Gagnon1, D Cook4, AF Turgeon1 1Universite Laval, Quebec, Canada; 2Universite de Sherbrooke, Canada; 3Universite de Montreal, Canada; 4McMaster University, Hamilton, Canada Critical Care 2013, 17(Suppl 2):P332 (doi: 10.1186/cc12270) Risk factors of pituitary disorders following traumatic brain injury F Lauzier1, O Lachance1, I Cote1, B Senay1, P Archambault1, F Lamontagne2 A Boutin1, M Shemilt1, L Moore1, F Bernard3, C Gagnon1, D Cook4, AF Turgeon1 g 1Universite Laval, Quebec, Canada; 2Universite de Sherbrooke, Canada; 3Universite de Montreal, Canada; 4McMaster University, Hamilton, Canada Critical Care 2013, 17(Suppl 2):P332 (doi: 10.1186/cc12270) g 1Universite Laval, Quebec, Canada; 2Universite de Sherbrooke, Canada; 3Universite de Montreal, Canada; 4McMaster University, Hamilton, Canada Critical Care 2013, 17(Suppl 2):P332 (doi: 10.1186/cc12270) Introduction Pituitary disorders following traumatic brain injury (TBI) are frequent, but their determinants are poorly understood. We performed a systematic review to assess the risk factors of TBI- associated pituitary disorders. p y Methods We searched MEDLINE, Embase, Scopus, The Cochrane Library, BIOSIS, and Trip Database, and references of narrative reviews for cohort, cross-sectional and case–control studies enrolling at least fi ve adults with TBI in whom ≥1 pituitary axis was tested and one potential predictor reported. Two independent investigators selected citations, extracted data and assessed the risk of bias. We pooled the data from all studies assessing a specifi c predictor, regardless of the pituitary axis being evaluated. When more than one pituitary axis was assessed, we used the data related to hypopituitarism or the data from the most defective axis. When a pituitary axis was evaluated several times, we used assessment farthest from the injury. A meta-analysis was performed using random eff ect models and I2 was used to evaluate heterogeneity. Studies were considered at low risk of bias if the authors defi ned inclusion/exclusion criteria, did not use voluntary sampling, and tested > 90% of patients with proper detailed diagnostic criteria. i Conclusion This retrospective study indicates that inducing a thiopental coma in patients with refractory elevated ICP eff ectively reduces ICP. Although the concomitant reduction in MAP was signifi cant, the resulting decrease in CPP was small and nonsignifi cant. Moreover, the target CPP of 60 mmHg was maintained after induction of barbiturate coma. Reference 1. 1. Haddad et al.: Scand J Trauma Resusc Emerg Med 2012, 20:12-27. P332 Haddad et al.: Scand J Trauma Resusc Emerg Med 2012, 20:12-27. P331 H i S127 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 P333 P333 Vasopressin reduces the cumulative epinephrine dose in hypothermic swine with traumatic brain injury AD Goldberg, MG Kashiouris, L Keenan, A Rabinstein, M Kumar Mayo Clinic, Rochester, MN, USA Critical Care 2013, 17(Suppl 2):P333 (doi: 10.1186/cc12271) Introduction Severe traumatic brain injury (TBI) is a major cause of death and of severe neurologic sequelae. Long-term functional outcome of TBI and its best timing of assessment are not well understood, and may be evaluated too prematurely in clinical studies because of resources required to do so without too much missing data. Hence, we conducted a systematic review of studies in severe TBI patients to evaluate the long-term functional outcome. We hypothesized that functional impact measured by the Glasgow Outcome Scale (GOS), or the extended version (GOSe), may plateau after several months in patients with severe TBI. Introduction Severe traumatic brain injury (TBI) is a major cause of death and of severe neurologic sequelae. Long-term functional outcome of TBI and its best timing of assessment are not well understood, and may be evaluated too prematurely in clinical studies because of resources required to do so without too much missing data. Hence, we conducted a systematic review of studies in severe TBI patients to evaluate the long-term functional outcome. We hypothesized that functional impact measured by the Glasgow Outcome Scale (GOS), or the extended version (GOSe), may plateau after several months in patients with severe TBI. Introduction Prevention of secondary neurologic injuries is paramount for improved neurologic outcomes after traumatic brain injury (TBI). Evidence suggests that although therapeutic hypothermia (TH) lowers intracranial pressure and attenuates secondary cerebral insults after TBI [1], it also induces hypotension. Brief episodes of mild hypotension in brain-injured patients can trigger secondary injuries, which have been associated with increased mortality in patients with TBI [2]. Vasopressin mitigates hypotension in septic shock and improves coronary perfusion in hypothermic cardiac arrest models [3]. We hypothesized that a low- dose vasopressin infusion may reduce the cumulative epinephrine dose in hypothermic, brain-injured swine. Methods We performed a systematic review of randomized controlled trials and cohort studies (prospective and retrospective) in patients with severe TBI. We searched MEDLINE, Embase, Cochrane Central, BIOSIS, CINAHL and Trip Database from their inception to December 2011. References of included studies were searched for additional studies. P331 H i P331 Hospital readmissions following traumatic brain injury A Boutin, K Francisque, L Moore, F Lauzier, X Neveu, A Turgeon Université Laval, Québec, Canada Critical Care 2013, 17(Suppl 2):P331 (doi: 10.1186/cc12269) p p p g Results Among 13,559 citations, we included 26 studies (1,708 patients). Increased age was associated with pituitary disorders (weighted mean diff erence = 3.2, 95% CI = 0.3 to 6.1, 19 studies, 1,057 patients, I2 = 59%). This fi nding was no longer signifi cant when only considering studies with low risk of bias. TBI severity was associated with an increased risk of developing pituitary disorders (RR = 1.49, 1.24 to 1.77, I2 = 17% for moderate/severe vs. mild TBI; RR = 1.78, 1.09 to 2.91, I2 = 80% for severe vs. mild/moderate TBI), while sex was not (RR for male = 1.05, 0.98 to 1.13, 15 studies, 870 patients, I2 = 0%). Among CT scan fi ndings, only skull fractures were associated with pituitary disorders (RR = 1.75, 1.13 to 2.70, six studies, 345 patients, I2 = 47%). An insuffi cient number of studies with low risk of bias assessing the association between GCS, CT scan fi ndings and pituitary disorders was retrieved to perform meta-analysis. Introduction Evaluating resource utilization is paramount in critically ill patients with traumatic brain injury (TBI), but little is known on readmissions after hospital discharge. We evaluated rates and determinants of unplanned readmission following TBI. Methods We conducted a multicenter retrospective cohort study from April 1998 to March 2009. Data were obtained from a Canadian provincial trauma system, based on mandatory contribution from 59 trauma centres, and a hospital discharge database. Patients aged ≥16  years with TBI (ICD-9 or ICD-10 codes of 850-854 and S06, respectively) were included. Patients who died during the index hospitalization, who lived outside the province, who could not be linked with the hospital discharge database were excluded. We collected baseline and trauma characteristics, hospital admissions in the 12 months preceding index Conclusion Age, TBI severity and skull fractures are associated with an increased risk of pituitary disorders. Further studies are necessary to identify additional factors that will help developing targeted screening strategies. References 1. Polderman KH, et al.: Intensive Care Med 2002, 28:1563-1573. 2. Fearnside MR, et al.: Br J Neurosurg 1993, 7:267-279. 2. Fearnside MR, et al.: Br J Neurosurg 199 Results All 31 (100%) units used ICP monitoring and parenchymal ICP bolt was the most widely used method (77%). Thirty (96.7%) units used a cerebral perfusion pressure protocol to guide therapy. Twenty-eight (90.3%) units used continuous capnography. Twelve (38.7%) units used transcranial Doppler. Eight (25.8%) units used partial pressure of oxygen in brain tissue. Four (12.9%) units used microdialysis. Only one unit used jugular bulb oximetry. None of the units used near-infrared spectrometry or optic nerve sheath diameter. Eight units (25.8%) used a cerebral function monitor and seven (22.5%) units used the bispectral index for guiding depth of sedation. 3. Voelckel WG, et al.: Anesth Analg 2000, 91:627-634. Monitoring of severe traumatic brain injury patients in UK ICUs: a national survey Monitoring of severe traumatic brain injury patients in UK ICUs: a national survey S Panchatsharam, B Lewinsohn, G De La Cerda, D Wijayatilake Queen’s Hospital, Romford, UK Critical Care 2013, 17(Suppl 2):P335 (doi: 10.1186/cc12273) S Panchatsharam, B Lewinsohn, G De La Cerda, D Wijayatilake Queen’s Hospital, Romford, UK Introduction Prevention of secondary brain injury is the cornerstone in the management of patients with severe traumatic brain injury (TBI) and raised intracranial pressure (ICP). Although a variety of monitoring methods are available, due to lack of strong evidence their use varies considerably [1]. The objective of this survey was to provide an overview of the current practice in monitoring of patients with severe TBI in all neuro-ICUs across the UK. Results The median cumulative epinephrine dose in the animals that received the vasopressin infusion was 715  mg with a 25th to 75th interquartile range (IQR) of 320 to 930  mg. The median cumulative epinephrine dose in the control group was 2,044  mg (IQR 1,640 to 2,344 mg). This was statistically signifi cant (P = 0.003), based on the Wilcoxon rank-sum test. Conclusion A low-dose infusion of vasopressin can signifi cantly reduce vasopressor requirements and improves hemodynamics in hypothermic, brain-injured swine. This hemodynamic stability may improve neurological outcomes. Methods The ICUs managing adult patients with severe TBI were identifi ed from the RAIN (Risk Adjustment In Neurocritical care) study sites, and the Society of British Neurosurgeons. Thirty-one centers were identifi ed, and a telephonic survey was conducted by the investigators. Data were collected using a single-page questionnaire containing 18 questions. P331 H i Two reviewers independently determined study eligibility and collected data. The primary outcome measure was the proportion of unfavourable functional outcome (GOS 1 to 3 or GOSe 1 to 4) at 6 to 12 months, 12 to 18 months, 18 to 24 months and more than 24 months after severe TBI. We calculated Freeman Tukey-type arcsine square- root transformations and pooled data using random-eff ect models. Heterogeneity was assessed with the I2 test and sensitivity analyses were based on a priori hypotheses. Methods Six domestic cross-bred pigs were subjected to epinephrine infusion after general anesthesia, standardized TBI and transpulmonary hypothermia (32°C for 48 hours). All animals received the same care, aiming for a mean arterial pressure >60 mmHg. At hour 24, animals received additional vasopressin infusion at 0.04 units/minute. We measured the cumulative epinephrine dose for each animal pre and post vasopressin infusion (Figure 1) and performed a two- sample Wilcoxon rank-sum test, comparing the median cumulative epinephrine doses in the two groups. Results In total, 4,432 studies were assessed for eligibility; 209 studies (n = 31,540) were included. In the 188 studies using the GOS, a poor functional outcome was observed in 56.6% (95% CI = 54.0 to 59.1%, I2 = 91%), 51.9% (95% CI = 38.0 to 59.0%, I2 = 84%), 57.0% (95% CI = 48.2 to 55.5%, I2 = 93%) and 56.9% (95% CI = 48.2 to 65.1%, I2 = 93%) of patients at 6 to 12  months, 12 to 18  months, 18 to 24  months and beyond 24  months, respectively. In the 18 studies using GOSe, a poor functional outcome was observed in 62.9% of patients at 6 to 12 months (95% CI = 55.9 to 69.2%, I2 = 90%) and 54.6% at 12 to 18  months (95% CI  =  43.2 to 65.8%, I2  =  90%). Heterogeneity was present in most analyses and was not entirely explained by the planned sensitivity analyses. Figure 1 (abstract P333). Eff ect of vasopressin on cumulative amount of epinephrine administered. Conclusion Considering that the incidence of patients with an unfavourable outcome remained constant at diff erent assessments, a follow-up of severe TBI patients longer than 12  months does not provide incremental information. Functional outcomes measured longer than 12 months after the injury may not be warranted in clinical studies. Survival analysis after head injury: is a normal INR reassuring? K Leclerc-Gagne Hôpital Sacré-Coeur de Montréal, Canada Critical Care 2013, 17(Suppl 2):P336 (doi: 10.1186/cc12274) Critical Care 2013, 17(Suppl 2):P337 (doi: 10.1186/cc12275) Introduction Intracranial bleeding after head injury is an important issue in the emergency department (ED), and elevated INR increases this risk [1]. A retrospective study showed an association between diff erent levels of normal INRs (<1.6) and elevated risk of intracranial bleeding, with a signifi cant elevation of this risk over an INR threshold of 1.3 [2]. The objective of the study was to evaluate clinical impact of an INR within the normal range in patients with head trauma. We compared mortality between patients with INR <1.3 and those with INR ≥1.3 to <1.6. Introduction Traumatic brain injury (TBI) is a leading cause of death and disability worldwide. Because mortality seriously underestimates the impact of TBI, an outcome tool including vegetative state and severe disability should be used. Our purpose was to study the long- term outcome of a small cohort of severe TBI patients in Brazil. Methods This was a retrospective analysis of a prospectively collected database. We included 34 consecutive adult patients admitted with severe TBI to a tertiary private hospital in Rio de Janeiro, Brazil between 2009 and 2011. We analyzed data on demographics, admission, clinical scores, imaging and complications, as well as hospital mortality and 1-year extended Glasgow Outcome Scale (eGOS) of all patients. Methods A post hoc analysis of prospective data collected from 3,356 patients seen in a tertiary-care ED from March 2008 to February 2011. We included patients aged 16 years old and over with an INR <1.6 and a head CT performed within 24 hours of the trauma. We followed these patients until December 2012, performed a chi-square test between mortality of the two groups and calculated the hazard ratio (HR) from survival analysis using Cox regression. y Results We analyzed 33 patients with severe TBI. Mean age was 36 (± 20) years and 79% were male. All patients were mechanically ventilated and 70% underwent ICP monitoring. Overall hospital mortality was 36% and 60% had unfavorable outcome (eGOS <4) after 1  year. In univariate analysis, higher APACHE II scores, the presence of midriasis and intracranial hypertension were more frequent in nonsurvivors compared with survivors. g References 1. Lingsma HF, et al.: Early prognosis in traumatic brain injury: from prophecies to predictions. Lancet Neurol 2010, 9:543-554. 1. Lingsma HF, et al.: Early prognosis in traumatic brain injury: from h i di i L N l 2010 9 543 554 1. Lingsma HF, et al.: Early prognosis in traumatic brain injury: from prophecies to predictions Lancet Neurol 2010 9:543-554 1. Wijayatilake et al.: Updates in the management of intracranial pressure in traumatic brain injury. Curr Opin Anaesthesiol 2012, 25:540-547. tilake et al.: Updates in the management of intracranial pressure in Wijayatilake et al.: Updates in the management of intracranial pres 1. Wijayatilake et al.: Updates in the management of intracranial pressure in traumatic brain injury. Curr Opin Anaesthesiol 2012, 25:540-547. 2. Chauny JM, et al.: Non Elevated INR and Post Traumatic Intracranial Bleeding. Day of Research in Neurotraumatology. Montreal: Hôpital du Sacré-Cœur; 22 June 2012. 2. Chesnut et al.: A trial of intracranial-pressure monitoring in traumatic brain injury. N Engl J Med 2012, 367:2471-2481. 2. Chesnut et al.: A trial of intracranial-pressure monitoring in traumatic brain injury. N Engl J Med 2012, 367:2471-2481. P337 One-year extended Glasgow Outcome Scale and hospital mortality predictors in patients with severe traumatic brain injury in Brazil R Turon1, FR Ferreira1, D Prado1, P Kurtz2, M Damasceno1 1Hospital de Clínicas de Niteroi, Brazil; 2Clinica São Vicente, Rio de Janeiro, Brazil Critical Care 2013, 17(Suppl 2):P337 (doi: 10.1186/cc12275) P338 P338 Early respiratory deterioration predicts poor outcome after aneurysmal subarachnoid hemorrhage E Van Lummel, L Vergouw, M Van der Jagt Erasmus MC, Rotterdam, the Netherlands Critical Care 2013, 17(Suppl 2):P338 (doi: 10.1186/cc12276) One-year extended Glasgow Outcome Scale and hospital mortality predictors in patients with severe traumatic brain injury in Brazil R Turon1, FR Ferreira1, D Prado1, P Kurtz2, M Damasceno1 One-year extended Glasgow Outcome Scale and hospital mortality predictors in patients with severe traumatic brain injury in Brazil R Turon1, FR Ferreira1, D Prado1, P Kurtz2, M Damasceno1 Survival analysis after head injury: is a normal INR reassuring? K Leclerc-Gagne Hôpital Sacré-Coeur de Montréal, Canada Critical Care 2013, 17(Suppl 2):P336 (doi: 10.1186/cc12274) Furthermore, older age, higher APACHE II score and worse GCS on admission were associated with unfavorable outcome after 1 year, as measured by eGOS <4.i y g g Results Patient mean age was 55.1 years (SD ±23), 65% were men and mean follow-up duration was 3.1 years (SD ±0.8). A total of 115 patients (15.9%) died during follow-up: 16 (36.4%) in the group with INR ≥1.3 and 99 (14.6%) in the group with INR <1.3 (P <0.001). Results showed a Figure 1 (abstract P336). Survival analysis of patients with INR <1.3 and with INR ≥1.3. y y Conclusion Our descriptive results suggest a signifi cant burden of severe TBI in a small cohort of young patients. Moreover, severity of the primary disease, age and pupil refl exes, as well as intracranial hypertension seemed to be associated with worse neurological outcome. Long-term functional outcome in adults with severe TBI: a meta-analysis y M Asselin1, Y Lachance1, G Lalonde1, A Boutin1, M Shemilt1, L Moore1, F Lauzier1, R Zarychanski2, P Archambault1, F Lamontagne1, D Fergusson3, A Turgeon1 1Université Laval, Québec, Canada; 2University of Manitoba, Winnipeg, Canada; 3University of Ottawa, Canada Critical Care 2013, 17(Suppl 2):P334 (doi: 10.1186/cc12272) M Asselin1, Y Lachance1, G Lalonde1, A Boutin1, M Shemilt1, L Moore1, F Lauzier1, R Zarychanski2, P Archambault1, F Lamontagne1, D Fergusson3, A Turgeon1 1Université Laval, Québec, Canada; 2University of Manitoba, Winnipeg, Canada; 3University of Ottawa, Canada Critical Care 2013, 17(Suppl 2):P334 (doi: 10.1186/cc12272) S128 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 http://ccforum.com/supplements/17/S2 Conclusion This survey shows that there is no clear consensus on what types of monitoring should be used to guide management of patients with TBI. A CPP protocol, based on measurement of ICP, is the most widely used method, although a recent randomized control trial did not show any benefi t in outcome [2]. Other invasive monitoring methods, although they may help in individualized care, are still not yet popular to due lack of strong evidence. Larger multicentre portfolio studies are needed to establish their benefi ts. References signifi cantly higher risk of death in the group with INR ≥1.3: HR = 2.99 (95% CI = 1.8 to 5.1); P <0.001 (Figure 1). g Conclusion In patients with head injury and normal INR (<1.6), there is an association between an INR ≥1.3 and higher risk of death. Therefore, it would be useful to request an INR in patients presenting with a head injury when bleeding is suspected, even in the absence of anticoagulant. f P339 Evaluation of proximal–distal velocity gradient in spastic middle cerebral artery after aneurismal subarachnoid hemorrhage M Gotti1, F Stretti1, S Piff eri1, V Conte1, M Zara1, N Stocchetti2 1Fondazione IRCCS Ca’ Granda-Ospedale Maggiore Policlinico, Milan, Italy; 2Fondazione IRCCS Ca’ Granda-Ospedale Maggiore Policlinico, Milan University, Milan, Italy Critical Care 2013, 17(Suppl 2):P339 (doi: 10.1186/cc12277) Results Twenty-fi ve patients (age 57  ±  12  years, WFNS 1 to 5) were included in the analysis. Overall increased levels of MPs were observed at early phase after aSAH when compared with controls. Contrarily to our hypothesis, in the early phase no signifi cant diff erences of MP level were observed in WFNS 4 to 5 (n = 9) when compared with WFNS 1 to 2 (n = 16) patients. Interestingly, patients with most severe ECI and DCI showed the highest values of MPs, but this trend did not reach statistical signifi cance, probably because of the small number of patients included in the study. Introduction Delayed cerebral ischemia (DCI) worsens neurological outcome in patients with aneurysmal subarachnoid hemorrhage (aSAH). DCI pathogenesis is multifactorial and not completely understood, but vasospasm plays a central role. Transcranial color- coded duplex sonography (TCCDS) is a non-invasive bedside tool to explore cerebral vessels, but specifi city is still suboptimal. The aim of this study was to evaluate the proximal–distal gradient of mean cerebral blood fl ow velocity (CBF-V) in middle cerebral artery (MCA) as a possible indicator of critical vasospasm. Conclusion Microparticles and microthrombosis may increase the severity of early and delayed ischemic damage after aSAH. p p Methods Consecutive aSAH patients (WFNS 1 to 5, age 18 to 80 years) admitted to NeuroICU between November 2011 and September 2012 were included in this study. TCCDS was used to assess CBF-V in MCA of both sides: we defi ned TCCDS vasospasm as CBF-V >120 cm/second. Magnetic resonance angiography (MRA, 3D TOF HR) was performed to evaluate vasospasm at early (<3 days) and delayed (7 to 10 days) time points. When patients underwent MRA, in the same clinical conditions, we recorded CBF-V with TCCDS at two levels: at the origin (proximal CBF-V) and at the end (distal CBF-V) of the MCA. Then we calculated the absolute value of proximal–distal CBF-V gradient for MCA of both sides. We defi ned clinical vasospasm as the appearance of a new neurological defi cit confi rmed by imaging. P338 Early respiratory deterioration predicts poor outcome after aneurysmal subarachnoid hemorrhage E Van Lummel, L Vergouw, M Van der Jagt Erasmus MC, Rotterdam, the Netherlands Critical Care 2013, 17(Suppl 2):P338 (doi: 10.1186/cc12276) Introduction Pulmonary complications are frequently occurring medical complications after aneurysmal subarachnoid hemorrhage (aSAH) [1]. Early respiratory deterioration (ERD) may be associated with delayed cerebral ischemia (DCI) or outcome and would then be a potential target for therapeutic interventions. We investigated whether respiratory deterioration within the fi rst 72  hours after admission predicted DCI or poor outcome. Methods We conducted a retrospective study in 137 consecutively admitted patients with aSAH, admitted between October 2007 and October 2011 to the ICU of a university hospital. ERD was defi ned as increased need for ventilatory support the second or third day after admission (Table 1). Elective intubation for a surgical procedure was not included as ERD. Inclusion criteria were availability of detailed information on respiratory status and level of support, admission within 48  hours after hemorrhage and age ≥18  years. Multivariable Figure 1 (abstract P336). Survival analysis of patients with INR <1.3 and with INR ≥1.3. Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 S129 Table 1 (abstract P338). Categories of respiratory support Level of respiratory support Category (compared with fi rst admission day) 1 Up to 5 l O2 2 >5 l O2 by nasal canulla or Venturi mask 3 Nonrebreathing mask or non-invasive positive-pressure ventilation 4 Invasive mechanical ventilation When mechanically Decreased PaO2/FiO2 ratio terminated Results We included 26 consecutive aSAH patients (WFNS 1 to 5, age 57 ± 12 years). The absolute value of MCA CBF-V gradient was higher in: (1) vessels aff ected by TCCDS vasospasm when compared with vessels not aff ected (63 cm/second, IR 23 to 85 and 15 cm/second IR 7 to 23 respectively, P <0.001); (2) vessels aff ected by MRA vasospasm when compared with vessels not aff ected (43 cm/second, IR 18 to 78 and 13 cm/second IR 6 to 22, P <0.001); and (3) in patients who developed clinical vasospasm than in patients who did not (56 cm/second IR 11 to 124 and 16 cm/second IR 8 to 30 respectively, P <0.01). Considering only the subset of MCAs aff ected by MRA vasospasm, the CBF-V gradient was higher when clinical vasospasm was also present (75 cm/ second IR 56 to 124 and 23 cm/second IR 17 to 30 respectively, P <0.05). Conclusion Proximal–distal CBF-V gradient of MCA might be a reliable indicator of critical vasospasm but further studies are warranted to defi ne threshold values and specifi city in aSAH patients. P338 Early respiratory deterioration predicts poor outcome after aneurysmal subarachnoid hemorrhage E Van Lummel, L Vergouw, M Van der Jagt Erasmus MC, Rotterdam, the Netherlands Critical Care 2013, 17(Suppl 2):P338 (doi: 10.1186/cc12276) survival analysis was used to investigate associations of DCI, death and Glasgow Outcome Scale (GOS) with ERD adjusted for condition on admission, Hijdra score, treatment of ruptured aneurysm and pulmonary comorbidity. GOS was assessed at 3 to 6 months after the bleed. DCI was defi ned as described recently [2]. P340 P340 Microparticles in aneurismal subarachnoid hemorrhage: role in acute and delayed cerebral ischemia V Conte1, A Artoni1, S Magnoni1, M Leoni1, L Capuano1, V Civelli1, N Stocchetti2 1Fondazione IRCCS Ca’ Granda-Ospedale Maggiore Policlinico, Milan, Italy; 2Fondazione IRCCS Ca’ Granda-Ospedale Maggiore Policlinico, Milan University, Milan, Italy Critical Care 2013, 17(Suppl 2):P340 (doi: 10.1186/cc12278) i y Results Mean age of the patients was 55.9 (± 12.7) and 63.5% was female. A total 46.7% of the patients developed DCI. Mortality was 25.6%. Forty percent of the patients were classifi ed as having ERD. ERD was not associated with DCI (adjusted HR = 1.48; 95% CI = 0.88 to 2.49; P = 0.14). ERD showed a trend towards an association with mortality (adjusted HR = 2.22; 95% CI = 0.96 to 5.14; P = 0.06; additionally adjusted for age, and rebleed). A clear association was found between absence of ERD and functional outcome with ordinal logistic regression analysis (0.98 point increase in GOS score at 3 to 6 months; 95% CI = 0.14 to 1.82; P = 0.02; additionally adjusted for age and rebleed). Introduction Microthrombosis has been demonstrated in early and delayed cerebral ischemia after aneurysmal subarachnoid hemorrhage (aSAH). Markers of coagulation activation as microparticles (MPs) are an established risk factor for thrombosis. Our hypothesis was that levels of microparticles might correlate with aSAH severity, early cerebral ischemia (ECI) and delayed cerebral ischemia (DCI). y j g Conclusion ERD within 72  hours after admission is associated with increased risk of poor functional outcome after aSAH, but not DCI. Further investigations are required to assess whether prevention of ERD may improve outcome. Methods Consecutive aSAH patients (age 18 to 80) admitted to our department between November 2011 and September 2012 were enrolled in the study. Total MPs (anxV+), platelet MPs (PLT-MPs, anx+/ CD41+), tissue factor MPs (TF-MPs, anx+/CD142+), and endothelial MPs (E-MPs, anx+/CD144+) were assessed in venous blood samples. Measurement of MPs levels were obtained at early (<3 days) and delayed phase (7 to 10 days). Multiparameter MRI (T2; FLAIR; T1; AngioRM 3D TOF HR; DWI) was performed to evaluate ischemic damage and vasospasm at the same time points. Qualitative evaluation of severity of ischemic damage was performed on DWI sequences. Levels of MPs were evaluated comparing diff erent groups in terms of SAH severity (World Federation of Neurological Surgeons score or WFNS), ECI severity and occurrence of delayed complications (vasospasm and DCI).i References References 1. Friedman JA, et al.: Neurosurgery 2003, 52:1025-1031. 2. Vergouwen MD, et al.: Stroke 2010, 41:2391-2395. P339 The relationships between the absolute value of TCCDS CBF-V gradient and (1)  TCCDS vasospasm, (2)  MRA vasospasm and (3) clinical vasospasm were explored. P341 P341 Intracranial pressure after subarachnoid hemorrhage b d 1 l 1 2 h 1 S h 1 Intracranial pressure after subarachnoid hemorrhage A Lombardo1, T Zoerle1, ER Zanier2, L Longhi1, N Stocchetti1 1Fondazione IRCCS Ca’ Granda-Ospedale Maggiore Policlinico, University of Milan, Italy; 2Mario Negri Institute, Milan, Italy Critical Care 2013, 17(Suppl 2):P341 (doi: 10.1186/cc12279) Introduction Elevated intracranial pressure (ICP) may have deleterious eff ects on cerebral metabolism and mortality after aneurysmal subarachnoid hemorrhage (SAH) [1,2], but its relevance has not yet been well explored. Aims of this study are to track ICP changes after SAH, to identify clinical factors associated with it and to explore the relationship between ICP and outcome. Introduction Elevated intracranial pressure (ICP) may have deleterious eff ects on cerebral metabolism and mortality after aneurysmal subarachnoid hemorrhage (SAH) [1,2], but its relevance has not yet been well explored. Aims of this study are to track ICP changes after SAH, to identify clinical factors associated with it and to explore the relationship between ICP and outcome. S130 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Methods A total of 116 consecutive SAH patients with ICP monitoring were enrolled. Episodes of ICP >20 mmHg for at least 5 minutes and the mean ICP value for every 12-hour interval were analyzed. The highest mean ICP collected in every patient was identifi ed. ICP values were analyzed in relation to clinical and CT fi ndings; 6-month outcome and ICU mortality were also introduced in multivariable logistic models. with aSAH. Studies including more than 50% of non-aneurismal SAH were excluded. Studies were considered at low risk of bias if the authors defi ned inclusion/exclusion criteria, avoided voluntary sampling, and tested >90% of included patients with proper detailed diagnostic criteria. Studies testing all pituitary axes were considered as evaluating hypopituitarism, which was defi ned as the dysfunction of ≥1 axis. We used a Freeman Tukey-type arcsine square-root transformation and pooled prevalences using the DerSimonian–Laird random-eff ect method. We determined the degree of heterogeneity with I2 values. Results Among 12,363 citations, we included 28 studies (1,628 patients). Patients were mostly female (64%) aged 50.5 ± 5.4. Sixteen studies reported the severity of aSAH, 14 reported the procedure for securing the aneurysm and 13 reported the location of aneurysm. Overall, hypopituitarism was observed in 53.4% of patients at short- term (<3 months), 36.5% at mid-term (3 to 12 months) and 34.2% at long-term (>12 months) (Table 1). Correlation between the timing of endovascular coiling or microsurgical clipping and long-term outcomes of patients after aneurysmal subarachnoid haemorrhage N Ojukwu, R Jain, S Wijayatilake, A Bellini, R Shetty, J Khan, G De la Cerda Queen’s Hospital, London, UK Critical Care 2013, 17(Suppl 2):P343 (doi: 10.1186/cc12281) Correlation between the timing of endovascular coiling or microsurgical clipping and long-term outcomes of patients after aneurysmal subarachnoid haemorrhage N Ojukwu, R Jain, S Wijayatilake, A Bellini, R Shetty, J Khan, G De la Cerda Queen’s Hospital, London, UK Critical Care 2013, 17(Suppl 2):P343 (doi: 10.1186/cc12281) References 1. Heuer GG, et al.: Relationship between intracranial pressure and other clinical variables in patients with aneurismal subarachnoid hemorrhage. J Neurosurg 2004, 101:408-416. 1. Heuer GG, et al.: Relationship between intracranial pressure and other clinical variables in patients with aneurismal subarachnoid hemorrhage. J Neurosurg 2004, 101:408-416. 2. Nagel A, et al.: Relevance of intracranial hypertension for cerebral metabolism in aneurismal subarachnoid hemorrhage. Clinical article. J Neurosurg 2009, 111:94-101. 2. Nagel A, et al.: Relevance of intracranial hypertension for cerebral metabolism in aneurismal subarachnoid hemorrhage. Clinical article. J Neurosurg 2009, 111:94-101. P341 Intracranial pressure after subarachnoid hemorrhage b d 1 l 1 2 h 1 S h 1 There was an insuffi cient number of studies with low risk of bias to perform sensitivity analyses according to study quality. y Results Eighty-one percent of patients had at least one episode of elevated ICP and 36% had a highest mean ICP >20 mmHg. The number of patients with highest mean ICP  >20  mmHg or with episodes of HICP was maximum at day 3 after SAH and decreased only after day 7. Neurological status, aneurysmal rebleeding, amount of blood on CT and CT ischemic lesion occurred within 72 hours from SAH were signifi cantly related to highest mean ICP >20 mmHg in a multivariable model. Patients with highest mean ICP >20 mmHg showed signifi cantly higher mortality in ICU. However, ICP is not an independent predictor of 6 months unfavorable outcome. Conclusion Elevated intracranial pressure is a common complication in the fi rst week after SAH. It is associated with early brain injury severity and ICU mortality. Conclusion The exact prevalence of pituitary disorders following aSAH remains uncertain, mainly due to high heterogeneity and the small number of studies with low risk of bias. However, the prevalence seems to decrease during the recovery phase. The prevalence, risk factors and clinical signifi cance of pituitary disorders in aSAH will require further rigorous evaluation. y References Prevalence of pituitary disorders associated with aneurysmal subarachnoid hemorrhage g F Lauzier, I Cote, A Bureau, M Shemilt, A Boutin, L Moore, C Gagnon, AF Turgeon Universite Laval, Quebec, Canada Critical Care 2013, 17(Suppl 2):P342 (doi: 10.1186/cc12280) Introduction Aneurysmal subarachnoid haemorrhage (aSAH) is associated with a high morbidity and mortality. Although UK Anaesthesia Guidelines advocate early coiling or clipping of the aneurysm within the fi rst 72 hours of admission for all grades of aSAH, the optimal timing of treatment and whether this is linked with better neurological long-term outcome are a subject of debate [1]. We aimed to investigate whether the timing of the occlusion of the aneurysm translates into better outcome. Introduction Pituitary disorders are an often-neglected consequence of aneurysmal subarachnoid hemorrhage (aSAH). We systematically reviewed their prevalence, aiming particularly at studies with low risk of bias. Methods We searched Embase, MEDLINE, The Cochrane Library, Trip Database, references of included studies and narrative reviews. We included cohort studies, cross-sectional studies and RCTs published in any language that tested the integrity of ≥1 pituitary axis in adults Methods A retrospective analysis of prospective collected data in a tertiary neuroscience centre from January to September 2012. Neuroprotective eff ect of a new synthetic aspirin–decursinol adduct in a rat model of ischemic stroke Neuroprotective eff ect of a new synthetic aspirin–decursino adduct in a rat model of ischemic stroke YH Lee, CW Park, JH Cho, MH Won Kangwon National University, Chuncheonsi, South Korea Critical Care 2013, 17(Suppl 2):P345 (doi: 10.1186/cc12283) patients were managed according to the local Guidelines for the management of aSAH. Outcome was assessed at 3 months using the extended Glasgow Outcome Scale (GOSE) defi ning good recovery as a GOSE ≥7 and poor outcome as GOSE ≤6. Introduction Stroke is a major cause of death. This study investigated the preventative eff ect of a new synthetic drug on brain function in experimentally induced ischemic stroke. Introduction Stroke is a major cause of death. This study investigated the preventative eff ect of a new synthetic drug on brain function in experimentally induced ischemic stroke. Results A total of 28 patients were included within the study period. Three patients were not expected to survive the fi rst 24 hours and were not included in the study. Seventeen patients were classifi ed as good grade aSAH (WFNS I to III) and eight as poor grade (IV to V). Twenty-two patients underwent successful coiling while the other three required clipping due to unsuccessful coiling. We did not fi nd any correlation between the timing of coiling/clipping and the 3-month GOSE (Figure 1). A total 44% of the patients had a poor 3-month GOSE while 56% had a good long-term functional outcome. The overall mortality rate was 21%. Methods Male Sprague–Dawley rats were administered aspirin (ASA), decursinol (DA) or ASA-DA before and after ischemic insults. Brain and neuronal damage were examined by TTC staining, PEP-CT, NeuN immunohistochemistry and F-J B histofl uorescence. Gliosis was also observed by GFAP and iba-1 immunohistochemistry. y y Results Pretreatment with 20  mg/kg, but not 10  mg/kg, of ASA-DA protected against ischemic neuronal death and damage, and its neuroprotective eff ect was much more pronounced than that of ASA or DA alone. In addition, treatment with 20 mg/kg ASA-DA reduced the ischemia-induced activation of astrocytes and microglia.i Conclusion Overall mortality in patients with aSAH is low when aneurysm is treated early post rupture of aneurysm. We did not fi nd any correlation between the timing of occlusion of aneurysm and the 3-month functional outcome. Prevalence of pituitary disorders associated with aneurysmal subarachnoid hemorrhage All Table 1 (abstract P342) Hypopituitarism GH ACTH TSH Gonadal ADH Short term All studies 53.4% (20.5 to 84.7) 30.4% (11.5 to 53.2) 13.2% (4.4 to 25.1) 49.2% (0.1 to 17.7) 26.1% (8.8 to 48.0) 6.3% (2.7 to 11.0) n (patients) 5 (235) 8 (346) 13 (537) 7 (327) 8 (351) 3 (481) I2 96.0% 93.5% 90.2% 89.1% 93.0% 61.1% Studies of low risk of bias (patients) 2(56) – – 2(56) 1(26) – Mid-term All studies 36.5% (13.6 to 62.8) 7.3% (0.6 to 18.3) 8.9% (1.3 to 21.0) 8.8% (2.4 to 18.0) 3.9% (0.0 to 11.9) 3.7% (0.6 to 13.5) n (patients) 4 (109) 6 (158) 7 (208) 5 (176) 6 (178) 2(56) I2 85.7% 70.9% 79.4% 59.4% 66.8% 29.0% Studies with low risk of bias (patients) 1(40) – – 1(40) – – Long-term All studies 34.2% (18.7 to 51.4) 17.1% (9.4 to 26.4) 11.2% (4.8 to 19.4) 2.3% (0.4 to 5.3) 3.2% (0.7 to 6.8) 0% (0 to 1.5) n (patients) 12 (486) 13 (531) 13 (531) 12 (486) 13(351) 3 (142) I2 92.5% 82.9% 82.9% 49.0% 62.9% 0% Studies with low risk of bias (patients) 2(40) 2(40) 2(40) 2(40) 1(10) – Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 S131 Results All WFNSpt 1 to 2 patients completed neuropsychological tests at each time point. WFNSpt 3 and WFNS 4 patients were testable in 80% and 50% of the cases respectively at early time points. WFNS 5 patients were not testable at any time point. In all testable patients, cognitive functions were severely impaired at early time points. At 3 months in WFNS 1 to 3 a good recovery of language defi cits while only a partial recovery of attention, memory and executive functions were observed; at the same time point 70% of WFNS 4 patients became testable, but they had a worse recovery of all cognitive functions. At 1 month after SAH less than 30% of patients return to work, at 3 months approximately 50%. Despite a good recovery of everyday life activities at 3 months, for all patients quality of life was lower than a normal population.f Figure 1 (abstract P343). Time of coiling/clipping versus GOSE boxplot. Prevalence of acute critical neurological disease in children: a global epidemiological assessment (PANGEA) Prevalence of acute critical neurological disease in children: a global epidemiological assessment (PANGEA) E Fink1, R Tasker2, J Beca3, MJ Bell1, RS Clark1, J Hutchison4, M Vavilala5, RS Watson1, L Weissfeld6, PM Kochanek1, DC Angus7, PANGEA Investigators1 1Children’s Hospital of Pittsburgh, PA, USA; 2Children’s Hospital Boston, MA, USA; 3Starship Children’s Hospital, Auckland, New Zealand; 4The Hospital for Sick Children, Toronto, Canada; 5Seattle Children’s Hospital, Seattle, WA, USA; 6University of Pittsburgh, PA, USA; 7University of Pittsburgh Medical Center, Pittsburgh, PA, USA Critical Care 2013, 17(Suppl 2):P346 (doi: 10.1186/cc12284) Prevalence of pituitary disorders associated with aneurysmal subarachnoid hemorrhage p q y p p Conclusion Cognitive dysfunction has diff erent time courses after aSAH: signifi cant defi cits in diff erent cognitive domains, worse quality of life and diffi culties in return to work persist in more than 50% of patients at 3 months following SAH. P344 Time course of neuropsychological functioning after aneurysmal subarachnoid hemorrhage B Zarino1, G Bertani1, V Conte1, S Magnoni1, A Di Cristofori1, N Stocchetti2 1Fondazione IRCCS Ca’ Granda-Ospedale Maggiore Policlinico, Milan, Italy; 2Fondazione IRCCS Ca’ Granda-Ospedale Maggiore Policlinico, Milan University, Milan, Italy Critical Care 2013, 17(Suppl 2):P344 (doi: 10.1186/cc12282) Reference 1. van der Schaaf I, et al.: Stroke 2006, 37:572-573. 1. van der Schaaf I, et al.: Stroke 2006, 37:572-573. Neuroprotective eff ect of a new synthetic aspirin–decursinol adduct in a rat model of ischemic stroke Conclusion Our fi ndings indicate that ASA-DA, a new synthetic drug, prevents against transient focal cerebral ischemia, which provides a resource for the development of its clinical application for stroke. Time course of neuropsychological functioning after aneurysmal subarachnoid hemorrhage An aff ordable and reliable bedside test could change practice to an individual tailored dosing of LMWH. The aim of our study was to evaluate whether a prophylactic dose regimen of 40 mg enoxaparine in cardiac surgical patients increases the anti-Xa activity to the level necessary for effi cient prevention of a thromboembolic event [1]. Secondarily we tested whether there was a reliable correlation between a bedside anti-Xa measurement compared with a two-stage chromogenic assay at the laboratory [2]. assay remain controversial in daily clinical practice. One of the major obstacles is the cost of such a test. An aff ordable and reliable bedside test could change practice to an individual tailored dosing of LMWH. The aim of our study was to evaluate whether a prophylactic dose regimen of 40 mg enoxaparine in cardiac surgical patients increases the anti-Xa activity to the level necessary for effi cient prevention of a thromboembolic event [1]. Secondarily we tested whether there was a reliable correlation between a bedside anti-Xa measurement compared with a two-stage chromogenic assay at the laboratory [2]. Results Of 3,113 subjects screened, 1,009 (32%) met enrollment criteria. The mean number of subjects enrolled per site for each study day was 2.4. Most sites were dedicated pediatric ICUs with a mean number of 22 ICU beds (range 3 to 72). ICUs had resources to invasively monitor intracranial pressure (93%), continuous electroencephalography (14%), invasive and non-invasive brain tissue oxygenation (14% and 57%), and somatosensory evoked potentials (39%). There were on average 11 ICU faculty and six fellows per site, and nearly one-half reported a neurocritical care ICU team. Subjects were 58% male and 52% white, and 60% had normal pre-admission PCPC scores (85%). Status epilepticus and cardiac arrest (both 21%) had the highest prevalence. Sixty-one per cent of subjects were mechanically ventilated during ICU admission. ICU length of stay was a mean 29 days (median 43 days) and hospital LOS was a mean 43 days (median 61 days). Survival at hospital discharge was 87% with 58% of subjects discharged home and 17% to inpatient rehabilitation.i y y Methods This was an open, single-centre, prospective, nonrandomized clinical trial at a university hospital. All patients that needed prophylactic dosing of enoxaparine after cardiac surgery were duly informed and after giving written consent we included 44 patients with a mean Euroscore of 1.66. Eff ective dosage of enoxaparin for intensive care patients S Robinson, A Zincuk, U Larsen, P Toft Odense University Hospital, Odense, Denmark Critical Care 2013, 17(Suppl 2):P349 (doi: 10.1186/cc12287) Eff ective dosage of enoxaparin for intensive care patients S Robinson, A Zincuk, U Larsen, P Toft Odense University Hospital, Odense, Denmark Critical Care 2013, 17(Suppl 2):P349 (doi: 10.1186/cc12287) gi Results In total, 943 patients met the inclusion criteria with a mean follow-up period of 1 year. The mean age was 74.3 years and 50.4% were men. A total of 108 deaths (11.5%) were recorded within the follow-up period; eight (25%) for dabigatran compared with 44 (12.6%) for warfarin and 56 (9.9%) for aspirin. The mortality risk for patients on dabigatran was signifi cantly higher than for patients on warfarin: HR = 2.1 (95% CI: 1.0 to 4.5), P = 0.05 after controlling for age. Aspirin had a lower (but not statistically signifi cant) mortality risk compared with warfarin; HR = 0.75 (95% CI: 0.50 to 1.14), P = 0.18 after controlling for age. y p Critical Care 2013, 17(Suppl 2):P349 (doi: 10.1186/cc12287) Introduction We hypothesized that higher doses of enoxaparin would improve thromboprophylaxis without increasing the risk of bleeding. Critically ill patients are predisposed to venous thromboembolism, leading to increased risk of adverse outcome [1]. Peak anti-factor Xa (anti-Xa) levels of 0.1 to 0.4 IU/ml, 4 hours post administration of enoxaparin, refl ect adequate thromboprophylaxis for medico-surgical patients. Conclusion The results showed higher overall mortality in patients who presented to the ED with a bleeding complication and were taking dabigatran compared with warfarin or aspirin. Physicians should be aware of the potential higher mortality with dabigatran over warfarin when treating a bleeding patient. However, this was a single- centre retrospective analysis with a small number of patients taking dabigatran (n = 32), and further studies are needed to corroborate the results. p Methods The sample population consisted of 78 patients, randomized to receive subcutaneous (s.c.) enoxaparin: 40 mg ×1 (control group), versus 30 mg ×2, 40 mg ×2 or 1 mg/kg ×1 (test groups) for a period of 3 days. Anti-Xa activity was measured at baseline, and at 4, 12, 16 and 24 hours post administration on each day. Patients did not diff er signifi cantly between groups. i Results On day 1 of administration, doses of 40  mg ×1 and 40  mg ×2 yielded similar mean peak anti-Xa of 0.20  IU/ml and 0.17  IU/ml respectively, while a dose of 30  mg ×2 resulted in subtherapeutic levels of anti-Xa (0.08 IU/ml). Time course of neuropsychological functioning after aneurysmal subarachnoid hemorrhage B Zarino1, G Bertani1, V Conte1, S Magnoni1, A Di Cristofori1, N Stocchetti2 1Fondazione IRCCS Ca’ Granda-Ospedale Maggiore Policlinico, Milan, Italy; 2Fondazione IRCCS Ca’ Granda-Ospedale Maggiore Policlinico, Milan University, Milan, Italy Critical Care 2013, 17(Suppl 2):P344 (doi: 10.1186/cc12282) Introduction Early and delayed cognitive dysfunctions are an under- studied issue after aneurysmal subarachnoid hemorrhage (aSAH), irrespective of neurological outcome. The aim of this study was to describe early and delayed changes of cognitive functions, activities of everyday life, and quality of life in aSAH patients. Introduction Acute neurological injury is a leading cause of morbidity and mortality in children. Global prevalence and regional disparities of etiology, interventions, and outcomes are unknown. The aim of this point-prevalence study was to measure the burden of pediatric neurological injury and to describe variations in interventions and outcomes in ICUs. Methods Consecutive aSAH patients admitted to our ICU between November 2011 and September 2012 were prospectively studied. Patients underwent neuropsychological evaluation at early (<3 days, 10 days) and delayed time points (1 month, 3 months). Patients were tested for language, verbal fl uency, short-term and long-term memory, attention, executive functions, praxis, and neglect. Impairments in activities of everyday life were assessed using the Activities of Daily Living scale and the Instrumental Activities of Daily Living scale. The SF-36 was used to assess the quality of life at 3  months. Since complications of aneurysm treatment in addition to aSAH severity may signifi cantly aff ect cognitive status, patients were evaluated according to the World Federation of Neurological Surgeons score after treatment (WFNSpt). Methods One hundred and three ICUs on six continents enrolled subjects on 4 specifi c days in a 1-year period. Included subjects were between ages 7 days and 17 years who were diagnosed with acute traumatic brain injury, stroke, cardiac arrest, central nervous system infection or infl ammation, status epilepticus, spinal cord lesion, hydrocephalus, or brain mass. Sites completed a secure web-based case report form that included subject and hospital demographics, details about the neurological disease, interventions, length of stay, and Pediatric Cerebral Performance Category (PCPC) score (good outcome = PCPC 1 to 3) and mortality at hospital discharge. S132 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 assay remain controversial in daily clinical practice. One of the major obstacles is the cost of such a test. P347 Survival analysis of patients taking dabigatran who consulted an emergency department for acute bleeding: a fi rst alarm OA Arlet, EN Notebaert, JP Paquet, RD Daoust, MV Vincent, JC Chauny Hôpital du Sacré-Coeur de Montréal, Université de Montréal, Canada Critical Care 2013, 17(Suppl 2):P347 (doi: 10.1186/cc12285) P347 Survival analysis of patients taking dabigatran who consulted an emergency department for acute bleeding: a fi rst alarm OA Arlet, EN Notebaert, JP Paquet, RD Daoust, MV Vincent, JC Chauny Hôpital du Sacré-Coeur de Montréal, Université de Montréal, Canada Critical Care 2013, 17(Suppl 2):P347 (doi: 10.1186/cc12285) Survival analysis of patients taking dabigatran who consulted an emergency department for acute bleeding: a fi rst alarm OA Arlet, EN Notebaert, JP Paquet, RD Daoust, MV Vincent, JC Chauny Hôpital du Sacré-Coeur de Montréal, Université de Montréal, Canada Critical Care 2013, 17(Suppl 2):P347 (doi: 10.1186/cc12285) Introduction The newly approved oral anticoagulant dabigatran has no eff ective antidote. We therefore suspected an overall increase in mortality in patients presenting to the emergency department (ED) with a bleeding complication on dabigatran compared with warfarin or aspirin. Conclusion The bedside anti-Xa activity assay with a Hemochron device tends to show some correlation with the two-stage chromogenic assay, but insuffi cient to be used as an alternative, in this small but uniform patient population. Use of a standard dosing protocol for enoxaparine administration is prone for underdosage in post-cardiac surgery patients and may increase postoperative morbidity. R f p Methods We conducted a post hoc analysis on a database of all patients admitted to a tertiary-care ED with any kind of bleeding or suspicion of one from March 2011 to August 2012 who were taking dabigatran, warfarin, or aspirin. The primary endpoint was long-term survival. Patients were censored at death or at the end of the study period (7 December 2012). We performed a Cox proportional hazard model, controlled for age, to calculate the hazard ratio (HR) for dabigatran versus warfarin and one for warfarin versus aspirin. Statistical signifi cance was set at α = 0.05 and results are presented with 95% CI. Results In total, 943 patients met the inclusion criteria with a mean follow-up period of 1 year. The mean age was 74.3 years and 50.4% were men. A total of 108 deaths (11.5%) were recorded within the follow-up period; eight (25%) for dabigatran compared with 44 (12.6%) for warfarin and 56 (9.9%) for aspirin. The mortality risk for patients on dabigatran was signifi cantly higher than for patients on warfarin: HR = 2.1 (95% CI: 1.0 to 4.5), P = 0.05 after controlling for age. Aspirin had a lower (but not statistically signifi cant) mortality risk compared with warfarin; HR = 0.75 (95% CI: 0.50 to 1.14), P = 0.18 after controlling for age. Methods We conducted a post hoc analysis on a database of all patients admitted to a tertiary-care ED with any kind of bleeding or suspicion of one from March 2011 to August 2012 who were taking dabigatran, warfarin, or aspirin. The primary endpoint was long-term survival. Eff ective dosage of enoxaparin for intensive care patients S Robinson, A Zincuk, U Larsen, P Toft Odense University Hospital, Odense, Denmark Critical Care 2013, 17(Suppl 2):P349 (doi: 10.1186/cc12287) Patients receiving 1 mg/kg enoxaparin achieved near-steady-state levels from day 1 with mean peak anti-Xa levels of 0.34 IU/ml. Steady-state anti-Xa was achieved for all doses of enoxaparin at day 3. At steady state, mean peak anti-Xa levels of 0.13 IU/ ml and 0.15 IU/ml were achieved with doses of 40 mg ×1 and 30 mg ×2 respectively. This increased signifi cantly to 0.33 IU/ml and 0.40 IU/ml for doses of 40 mg ×2 and 1 mg/kg enoxaparin respectively (P = 0.0000) (Figure 1). A dose of 1 mg/kg enoxaparin yielded therapeutic anti-Xa levels for over 80% of the study period. There were no adverse eff ects. Survival analysis of patients taking dabigatran who consulted an emergency department for acute bleeding: a fi rst alarm Patients were censored at death or at the end of the study period (7 December 2012). We performed a Cox proportional hazard model, controlled for age, to calculate the hazard ratio (HR) for dabigatran versus warfarin and one for warfarin versus aspirin. Statistical signifi cance was set at α = 0.05 and results are presented with 95% CI. 1. Ribic C, et al.: J Crit Care 2009, 24:197-205. 1. Ribic C, et al.: J Crit Care 2009, 24:197-205. 1. Ribic C, et al.: J Crit Care 2009, 24:197-205. 2. Gehrie E, et al.: Am J Hematol 2012, 87:194-196. Time course of neuropsychological functioning after aneurysmal subarachnoid hemorrhage The demographic specifi cations, medical and surgical history of all patients were collected. Anti-Xa activity was measured at three diff erent points in time. We determined baseline, peak and trough anti-Xa activity: preoperatively, and respectively 4 hours after the third dose of enoxaparine and 30 minutes before the fourth dose. Each measurement was done with both techniques, the two-stage chromogenic assay at the laboratory (Biophen®) and the bedside assay (Hemochron® Jr). Conclusion Acute neurological disease is a signifi cant pediatric health issue. These data suggest a vital need for increased research and healthcare resources to assist in the challenge of improving outcomes for these children. Results Our dose regimen of enoxaparine achieved in one-half of the included patients a suffi cient anti-Xa activity for prevention of thromboembolic events. One-half of the patients with insuffi cient anti- Xa activity had a body mass index over 30 kg/m2. Comparison of the bedside assay with the two-stage chromogenic assay by means of the Pearson’s correlation coeffi cient showed correlation of the two tests if no variables were taken into account. In the Bland–Altman analysis we could not confi rm this correlation. P348 P348 Bedside anti-Xa measurement for therapeutic assessment of a prophylactic anticoagulation regimen B Dewulf1, I Herck2, F De Somer2, K Francois2, J Decruyenaere2 1AZ Sint-Jan, Brugge, Belgium; 2University Hospital, Gent, Belgium Critical Care 2013, 17(Suppl 2):P348 (doi: 10.1186/cc12286) P351 The fi rst-in-class hepcidin antagonist NOX-H94, a PEGylated anti-hepcidin L-RNA oligonucleotide, is in development for targeted treatment of anemia of infl ammation. We investigated whether NOX-H94 prevents the infl ammation-induced serum iron decrease during experimental human endotoxemia. Methods A randomized, double-blind, placebo-controlled trial in 24 healthy young men. At T = 0 hours, 2 ng/kg E. coli endotoxin was administered intravenously (i.v.), followed by 1.2 mg/kg NOX-H94 or placebo i.v. at T = 0.5 hours. Blood was drawn serially after endotoxin administration for measurements of infl ammatory parameters, Introduction Anemia is very frequently encountered on the ICU. Increased hepcidin production is one of the cornerstones of the pathophysiology of anemia of infl ammation. The fi rst-in-class hepcidin antagonist NOX-H94, a PEGylated anti-hepcidin L-RNA oligonucleotide, is in development for targeted treatment of anemia of infl ammation. We investigated whether NOX-H94 prevents the infl ammation-induced serum iron decrease during experimental human endotoxemia. Introduction Unfractionated heparin is preferred over LMWH in ICU patients but LMWH is used more frequently in many European ICUs. Thromboprophylaxis with standard doses of nadroparin and enoxaparin has been shown to result in signifi cantly lower anti-Xa in ICU patients when compared with medical patients [1,2]. p p p Methods ICU patients (SAPS 44  ±  16, MV, n  =  44; pressors n  =  32) received 7,500 IU (Group 1, n = 25) or 5,000 IU dalteparin s.c. (Group 2, n  =  29). Twenty-nine medical patients receiving 5,000  IU dalteparin served as controls (Group 3). g p Methods A randomized, double-blind, placebo-controlled trial in 24 healthy young men. At T = 0 hours, 2 ng/kg E. coli endotoxin was administered intravenously (i.v.), followed by 1.2 mg/kg NOX-H94 or placebo i.v. at T = 0.5 hours. Blood was drawn serially after endotoxin administration for measurements of infl ammatory parameters, p Results Group 2 had signifi cantly lower areas under the Xa curve (AUC) compared with Groups 1 and 3 (Table 1). Diff erences were not signifi cant between Groups 1 and 3. Peak anti-Xa activities (Cmax-anti-Xa) Table 1 (abstract P351). g Referencesfl 1. Dörffl er-Melly J, deJonge E, dePont AC, et al.: Bioavailability of subcutaneous low-molecular-weight heparin to patients on vasopressors. Lancet 2002, 359:849-850. 1. Cook D, et al.: N Engl J Med 2011, 364:1305-1314. 2. Priglinger U, Delle Karth G, Geppert A, et al.: Prophylactic anticoagulation with enoxaparin: is the subcoutaneous route appropriate in the critically ill? Crit Care Med 2003, 31:1405-1409. 2. Priglinger U, Delle Karth G, Geppert A, et al.: Prophylactic anticoagulation with enoxaparin: is the subcoutaneous route appropriate in the critically ill? Crit Care Med 2003, 31:1405-1409. P350 Abstract withdrawn P350 Abstract withdrawn P351 Anti-Xa activities following 5,000 IU and 7,500 IU of s.c. dalteparin in critically ill patients and 5,000 IU in medical patients: a prospective randomized study GH Heinz1, U Priglinger2, I Pabinger3 1University Clinic of Internal Medicine II, Vienna, Austria; 2University Clinic of Emergency Medicine, Vienna, Austria; 3University Clinic of Internal Medicine I, Vienna, Austria Critical Care 2013, 17(Suppl 2):P351 (doi: 10.1186/cc12289) Randomized double-blind placebo-controlled PK/PD study on the eff ects of a single intravenous dose of the anti-hepcidin Spiegelmer NOX-H94 on serum iron during experimental human endotoxemia L Van Eijk1, DW Swinkels1, A John1, F Schwoebel2, F Fliegert2, L Summo2, S Vauleon2, JM Laarakkers1, K Riecke2, P Pickkers1 1Radboud University Nijmegen Medical Centre, Nijmegen, the Netherlands; 2NOXXON Pharma AG, Berlin, Germany Critical Care 2013, 17(Suppl 2):P352 (doi: 10.1186/cc12290) Anti-Xa activities following 5,000 IU and 7,500 IU of s.c. dalteparin in critically ill patients and 5,000 IU in medical patients: a prospective randomized study Randomized double-blind placebo-controlled PK/PD study on the eff ects of a single intravenous dose of the anti-hepcidin Spiegelmer NOX-H94 on serum iron during experimental human endotoxemia L Van Eijk1, DW Swinkels1, A John1, F Schwoebel2, F Fliegert2, L Summo2, S Vauleon2, JM Laarakkers1, K Riecke2, P Pickkers1 1Radboud University Nijmegen Medical Centre, Nijmegen, the Netherlands; 2NOXXON Pharma AG, Berlin, Germany Critical Care 2013, 17(Suppl 2):P352 (doi: 10.1186/cc12290) Randomized double-blind placebo-controlled PK/PD study on the eff ects of a single intravenous dose of the anti-hepcidin Spiegelmer NOX-H94 on serum iron during experimental human endotoxemia L Van Eijk1, DW Swinkels1, A John1, F Schwoebel2, F Fliegert2, L Summo2, S Vauleon2, JM Laarakkers1, K Riecke2, P Pickkers1 1Radboud University Nijmegen Medical Centre, Nijmegen, the Netherlands; 2NOXXON Pharma AG, Berlin, Germany Critical Care 2013, 17(Suppl 2):P352 (doi: 10.1186/cc12290) GH Heinz1, U Priglinger2, I Pabinger3 1University Clinic of Internal Medicine II, Vienna, Austria; 2University Clinic of Emergency Medicine, Vienna, Austria; 3University Clinic of Internal Medicine I, Vienna, Austria Critical Care 2013, 17(Suppl 2):P351 (doi: 10.1186/cc12289) GH Heinz1, U Priglinger2, I Pabinger3 1University Clinic of Internal Medicine II, Vienna, Austria; 2University Clinic of Emergency Medicine, Vienna, Austria; 3University Clinic of Internal Medicine I, Vienna, Austria Critical Care 2013, 17(Suppl 2):P351 (doi: 10.1186/cc12289) Introduction Anemia is very frequently encountered on the ICU. Increased hepcidin production is one of the cornerstones of the pathophysiology of anemia of infl ammation. Bedside anti-Xa measurement for therapeutic assessment of a prophylactic anticoagulation regimen Introduction Dose adjustments of low molecular weight heparin (LMWH) based on daily anti-Xa measurement by chromogenic S133 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum com/supplements/17/S2 http://ccforum.com/supplements/17/S2 Figure 1 (abstract P349). Peak anti-Xa over 3 days with varying doses of enoxaparin. Figure 1 (abstract P349). Peak anti-Xa over 3 days with varying doses of enoxaparin. Conclusion Current standard s.c. doses of 40  mg ×1 enoxaparin (Europe) or 30 mg ×2 (North America) yield levels of anti-Xa thought to be subtherapeutic for critically ill patients. A weight-based dose yielded the best anti-Xa levels, allowed the establishment of near-steady-state levels from the fi rst day of enoxaparin administration, and may thus be more appropriate, convenient, and eff ective. A new study using 1 mg/ kg enoxaparin s.c. with clinical endpoints has recently been approved by the Danish Ministry of Health. R f were delayed (tmax-anti-Xa) in Group 2 compared with Groups 1 and 3 (Table 1). Conclusion In ICU patients a s.c. dose of 5,000  IU dalteparin results in signifi cantly lower Xa activities when compared with normal ward patients. A s.c. dose of 7,500 IU dalteparin in ICU patients resulted in kinetics and peak anti-Xa activities comparable with medical patients receiving 5,000 IU dalteparin. P351 Pharmacokinetics Group 1 (n = 25) Group 2 (n = 29) Group 3 (n = 29) ICU, 7,500 IU ICU, 5,000 IU Normal ward, 5,000 IU P overall AUC-anti-Xa0–12 hours (U/l*hour) 2.51 (1.15 to 4.61) 1.27 (1.15 to 4.4) 2.58 (1.45 to 4.87) <0.001* AUC-anti-Xa12_00 (U/l*hour) 1.37 (0.58 to 13) 1.47 (0.65 to 6.3) 0.89 (0.35 to 3.88) 0.003** Cmax-anti-Xa (U/l) 0.29 (0.10 to 0.52) 0.14 (0.1 to 0.43) 0.33 (0.14 to 0.65) <0.001*** tmax-anti-Xa (hours) 3 (3 to 12) 4.5 (1 to 12) 3 (3 to 6) 0.017§ Cmax, peak anti-Xa level; tmax, time of anti-Xa peak. Medians and range. *Group 1 versus 2, P = 0.001; Group 1 versus 3, P = 0.51. **Group 1 versus 2, P = 0.96; Group 1 versus 3, P = 0.045; Group 2 versus 3, P = 0.13. ***Group 1 versus 2, P = 0.001; Group 1 versus 3, P = 0.29. §Group 1 versus 3, P = 0.09; Group 1 versus 2, P = 0.8; Group 2 versus 3, P = 0.018. Table 1 (abstract P351). Pharmacokinetics Cmax, peak anti-Xa level; tmax, time of anti-Xa peak. Medians and range. *Group 1 versus 2, P = 0.001; Group 1 versus 3, P = 0.51. **Group 1 versus 2, P = 0.96; Group 1 versus 3, P = 0.045; Group 2 versus 3, P = 0.13. ***Group 1 versus 2, P = 0.001; Group 1 versus 3, P = 0.29. §Group 1 versus 3, P = 0.09; Group 1 versus 2, P = 0.8; Group 2 versus 3, P = 0.018. S134 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 cytokines, NOX-H94 pharmacokinetics, total hepcidin-25, and iron parameters. The diff erence of serum iron change from baseline at T = 9 hours was defi ned as the primary endpoint.ll Introduction According to many authors, acute necrotizing pancreatitis (ANP) still remains one of the diffi cult problems of abdominal surgery. The complexity of the pathogenesis of the disease, features of the pancreas pathomorphology, abdominal hypertension, and high mortality (30 to 70%) necessitate a search for new ways to treat this disease. i y Results Endotoxin administration led to fl u-like symptoms. Infl am- matory parameters (CRP, body temperature, leucocytes, and plasma levels of TNFα, IL-6, IL-10, and IL-1RA) peaked markedly and similarly in both treatment groups. P351 NOX-H94 was well tolerated. Plasma concen- trations peaked at 0.7 ± 0.4 hours after the start of administration, after which they declined according to a two-compartment model, with a T1/2 of 22.5 ± 4.28 hours. In the placebo group, serum iron increased from 19.0 ± 7.6 μg/l at baseline to a peak at T = 3 hours, returned close to baseline at T = 6 hours and decreased under the baseline concentration at T = 9 hours, reaching its lowest point at T = 12 hours. In the NOX-H94 group, serum iron concentrations rose until T = 6 hours and then slowly declined until T = 24 hours. From 6 to 12 hours post LPS, the serum iron concentrations in NOX-H94-treated subjects were signifi cantly higher than in placebo-treated subjects (P <0.0001, ANCOVA). Methods The study was conducted in 44 patients with ANP, who were divided into two groups according to type of analgesia: epidural or opioids. Patients from the fi rst group (n = 23) had epidural analgesia by ropivacaine 6 to 14 mg/hour during 7 to 10 days, and from the second group (n = 21) opioid analgesia by trimeperidine 20 mg three times a day during the same period. We monitored the level of septic and thrombohemorrhagic complications by clinical and instrumental data, during the month after treatment starting. The hemostatic system was evaluated using indicators of hemoviscoelastography (Mednord-01M analyzer). g p y y Results It was found that all patients with ANP initially have hypercoagulation and fi brinolysis inhibition. Levels of hemostatic disorders correlate with the level of septic complications, treatment in the ICU, and mortality. In the fi rst group we noted a deep vein thrombosis, two pneumonia, seven pseudopancreatic cysts and abscesses, two deaths and time of stay in the ICU as 15.4 days. In the second group: three cases of deep vein thrombosis, four pneumonia, 10 pseudopancreatic cysts and abscesses, two episodes of gastroduodenal bleeding, fi ve deaths and time of stay in the ICU as 27.8 days. Conclusion Experimental human endotoxemia induces a robust infl am matory response and a subsequent decrease in serum iron. Treatment with NOX-H94 had no eff ect on innate immunity, but eff ectively prevented the infl ammation-induced drop in serum iron concentrations. These fi ndings demonstrate the clinical potential of the anti-hepcidin drug NOX-H94 for further development to treat patients with anemia of infl ammation. Hemoviscoelastography: a new method of hemostasis monitoring O Tarabrin, S Galich, D Gavrychenko, G Mazurenko, A Suhanov Odessa National Medical University, Odessa, Ukraine Critical Care 2013, 17(Suppl 2):P355 (doi: 10.1186/cc12293) Introduction The association between haemoglobin concentrations and mortality has been studied in patients with various comorbidities [1,2]. This study aims to determine the association between haemo- globin levels on admission to intensive care and patient length of stay and mortality. Introduction Deep vein thrombosis of lower extremities and pulmonary embolism occupy an important place in the structure of postoperative morbidity and mortality. Introduction Deep vein thrombosis of lower extremities and pulmonary embolism occupy an important place in the structure of postoperative morbidity and mortality. y Methods A retrospective collection of data from patient admissions to a single fi ve-bed ICU over a 15-year period identifi ed 5,826 patients between April 1994 and November 2012. Patients were split into groups according to haemoglobin concentration on admission. The data were analysed to determine whether there was any relationship between haemoglobin concentration at ICU admission and any of our outcome measures (unit and hospital mortality, unit and hospital length of stay). Results Patients with haemoglobin concentrations ≤10  g/dl and >10.1  g/dl were used in mortality comparisons. Patients with a haemo globin concentration ≤10 g/dl had an increase in ICU mortality compared with those with haemoglobin levels >10 g/dl (OR = 1.36, 95% CI = 1.30 to 1.71, P <0.0001). A similar diff erence was seen with hospital mortality (≤10 g/dl 37.7% vs. >10 g/dl 27.3%, P <0.0001). Unit length of stay was signifi cantly longer in patients with admission Hb ≤10 g/ dl (5.34 days) compared with an admission Hb >10 g/dl (4.08 days), P  <0.0001. Hospital length of stay was also signifi cantly longer in patients with Hb ≤10 g/dl versus Hb >10 g/dl (21.6 days vs. 15.5 days, P  <0.0001). There was seen to be an inverse correlation between haemoglobin concentration and patient age (r = –0.174; P <0.0001). Methods After ethics approval and informed consent, we studied the functional state of hemostasis in a group of 57 healthy volunteers, who were not receiving drugs aff ecting coagulation, and 43 patients with postphlebothrombotic syndrome (PPTS). In the PPTS patients we conducted baseline studies of coagulation state and daily monitoring of dynamic changes in the functional state of hemostasis, a comparative evaluation of performance low-frequency piezoelectric vibration hemoviscoelastography (LPVH) and platelet aggregation test (PAT), standard coagulation tests (SCT), and thromboelastogram (TEG). Results We found that LPVH correlated with SCT, PAT and TEG. P355 Hemoviscoelastography: a new method of hemostasis monitoring O Tarabrin, S Galich, D Gavrychenko, G Mazurenko, A Suhanov Odessa National Medical University, Odessa, Ukraine Critical Care 2013, 17(Suppl 2):P355 (doi: 10.1186/cc12293) P353 Haemoglobin concentration on admission to intensive care infl uences hospital mortality rates and length of stay: a retrospective study M Adams, P Dean, P Doherty, S Noble, A Mackay Victoria Infi rmary, Glasgow, UK Critical Care 2013, 17(Suppl 2):P353 (doi: 10.1186/cc12291) P356 P356 Thromboelastographic monitoring of fondaparinux in surgical patients B Martinez1, R Giacomello1, R Paniccia2 1University Hospital of Udine, Italy; 2University of Florence, Italy Critical Care 2013, 17(Suppl 2):P356 (doi: 10.1186/cc12294) P356 Thromboelastographic monitoring of fondaparinux in surgical patients B Martinez1, R Giacomello1, R Paniccia2 1University Hospital of Udine, Italy; 2University of Florence, Italy Critical Care 2013, 17(Suppl 2):P356 (doi: 10.1186/cc12294) Hemoviscoelastography: a new method of hemostasis monitoring O Tarabrin, S Galich, D Gavrychenko, G Mazurenko, A Suhanov Odessa National Medical University, Odessa, Ukraine Critical Care 2013, 17(Suppl 2):P355 (doi: 10.1186/cc12293) However, our proposed method is more voluminous: indexes ICC (the intensity of the contact phase of coagulation), t1 (the time for the contact phase of coagulation), and AO (initial rate of aggregation of blood) are consistent with PAT; indexes ICD (the intensity of coagulation drive), CTA (a constant thrombin activity) and CP (the clot intensity of the polymerization) are consistent with SCT and TEG. In addition, the advantage of this method is to determine the intensity of fi brinolysis – with the indicator IRIS (the intensity of the retraction and clot lysis). Conclusion Haemoglobin concentrations ≤10 g/dl on admission to the ICU are associated with an increase in ICU mortality, hospital mortality, unit length of stay and hospital length of stay when compared with patients admitted with haemoglobin concentrations >10 g/dl. References y y Conclusion LPVH allows one to make a total assessment of all parts of hemostasis: from initial viscosity and platelet aggregation to coagulation and lysis of clots, as well as their interaction. These fi gures are objective and informative, as evidenced by close correlation with the performance of traditional coagulation methods. 1. Zhao Y, et al.: Crit Care 2007, 11:P234. 2. Sekhon MS, et al.: Crit Care 2012, 16:R128. P351 Conclusion Using epidural anesthesia in patients with ANP reduced the number of septic complications on 36.6%, and reduced the mortality rate from 23.8% (second group) to 8.7% (fi rst group). We think that violations of blood coagulation and microcirculation are the basis for ischemia, necrosis in tissues and septic complications. Epidural analgesia is an eff ective method to decrease the level of septic and thrombohemorrhagic complications and mortality in ANP patients. Infl uence of epidural anesthesia on the hemocoagulation disorders and quantity of septic complications in patients with acute necrotizing pancreatitis O Tarabrin, S Shcherbakov, A Tkachenko, O Kushnir, I Grychushenko Odessa National Medical University, Odessa, Ukraine Critical Care 2013, 17(Suppl 2):P354 (doi: 10.1186/cc12292) O Tarabrin, S Shcherbakov, A Tkachenko, O Kushnir, I Grychushenko Odessa National Medical University, Odessa, Ukraine Critical Care 2013, 17(Suppl 2):P354 (doi: 10.1186/cc12292) Introduction Fondaparinux is currently the fi rst therapeutic choice in several clinical settings such as deep venous thrombosis (DVT) S135 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 8.7 ± 3.4 minutes vs. 12.2 ± 1.9 minutes, P <0.001; α-angle 59.7 ± 9.4 vs. 47.2 ± 11.8, P <0.01). This reduction of fi brinolysis was more evident at a urokinase concentration of 160 UI/ml (Figure 1). Conclusion UKIF-TEG could be a feasible point-of-care method to evaluate fi brinolysis in critically ill patients. Reference 1 Gallimore MJ et al : J Thromb Haemost 2005 3:2506-2513 Figure 1 (abstract P357). prophylaxis in orthopedics or in cases of acute coronary syndromes. The main drawback of FOND is that routine monitoring is not currently available. This could be a problem during the management of critical and surgical patients, especially in cases of old patients and renal failure. The aim of this study is to evaluate the ability of thromboelastography (TEG) to determine the level of anticoagulation due to FOND in a surgical population. prophylaxis in orthopedics or in cases of acute coronary syndromes. The main drawback of FOND is that routine monitoring is not currently available. This could be a problem during the management of critical and surgical patients, especially in cases of old patients and renal failure. The aim of this study is to evaluate the ability of thromboelastography (TEG) to determine the level of anticoagulation due to FOND in a surgical population. Figure 1 (abstract P357). Methods We prospectively analyzed all patients to whom elective major orthopedic surgery was consecutively performed in a 2-month period. All the patients received FOND 2.5  mg in the postoperative period according to ACCP 2012 Guidelines. Native and heparinase (hep) TEG (Haemoscope Corporation, Niles, IL, USA) tests activated with kaolin were performed using whole blood citrated samples at four times: T0, before FOND administration; T1, 2  hours after administration; T2, 17 hours after administration (half-life); T3, 24 hours after administration. The following native and hep TEG parameters were analyzed: reaction time (R), α angle, maximum amplitude (MA) and coagulation index (CI). Infl uence of epidural anesthesia on the hemocoagulation disorders and quantity of septic complications in patients with acute necrotizing pancreatitis These parameters were compared with levels of anti-Xa. Unvariate analysis and Spearman’s test were applied to our data. 8.7 ± 3.4 minutes vs. 12.2 ± 1.9 minutes, P <0.001; α-angle 59.7 ± 9.4 vs. 47.2 ± 11.8, P <0.01). This reduction of fi brinolysis was more evident at a urokinase concentration of 160 UI/ml (Figure 1). Conclusion UKIF-TEG could be a feasible point-of-care method to evaluate fi brinolysis in critically ill patients. Reference 1. Gallimore MJ, et al.: J Thromb Haemost 2005, 3:2506-2513. Results Eighteen patients were analyzed. Ten patients met the inclusion criteria. The mean R value increased from T1 to T3. The mean R parameter was in the normal range at any phase of the study and there was no signifi cant diff erences between the R mean value at the diff erent phases. The lowest value of R was at T1, which coincides with plasmatic peak concentration of FOND. This value did not correlate with anti-Xa mean value at T1, which showed the highest value at that time. There was signifi cant diff erence between the mean native and hep R value only at T2 (P <0.05), native and hep α angle at T3, MA and MA hep at T2 (P <0.01) and CI and CI hep at T3 (P <0.02). Only the parameter MA had signifi cant variation over time (P <0.02). 1. Gallimore MJ, et al.: J Thromb Haemost 2005, 3:2506-2513. P358 p References References 1. Falck-Ytter Y, et al.: Chest 2012, 141:2. 1. Falck-Ytter Y, et al.: Chest 2012, 141:2. 2. FUTURA/OASIS Trial Group, Steg PG, Jolly SS, Mehta SR, et al.: Low Methods In this retrospective study, we investigated 1,737 consecutive insertions of central venous catheters on 1,444 patients in a large university hospital during 2009 to 2010. Pre-procedural coagulation status, blood component use, type of catheter, insertion site, and complications during insertion were recorded and compared with bleeding complications documented in electronic charts. 2. FUTURA/OASIS Trial Group, Steg PG, Jolly SS, Mehta SR, et al.: Low-dose vs standard dose unfractioned heparin for percutaneous coronary intervention in acute coronary syndromes treated with fondaparinux: the FUTURA/OASIS randomized trial. JAMA 2010, 304:1339-1349. py TK Kander, US Schött y TK Kander, US Schött TK Kander, US Schött Lund University, Lund, Sweden y Critical Care 2013, 17(Suppl 2):P358 (doi: 10.1186/cc122 Conclusion R represents the time necessary to thrombin formation and in the presence of FOND we hypothesized a prolonged R time. In our population, TEG performed with citrated kaolin-activated whole blood was not able to detect prophylatic doses of FOND in every phase. On the contrary, levels of anti-Xa were able to reveal the exact pharmacokinetics of the drug. Further studies including a large number of patients are necessary. Introduction Bleeding complications in association with insertion of central venous catheters are reported in 0.5 to 1.6% of cases, are rarely fatal, but sometimes require intervention. The aim of this study was to map pre-procedural variables at the insertion of a central venous catheter and to investigate whether any independent variable could be identifi ed as an independent risk factor for bleeding complications associated with insertion of central venous catheters. Urokinase-induced fi brinolysis in thromboelastography (UKIF-TEG) to assess fi brinolysis in critically ill patients Urokinase-induced fi brinolysis in thromboelastography (UKIF-TEG) to assess fi brinolysis in critically ill patients L Zacchetti, M Panigada, M Cressoni, T Marchesi, L Gattinoni Fondazione IRCCS Ca’ Granda Ospedale Policlinico Milano, Milan, Italy Critical Care 2013, 17(Suppl 2):P357 (doi: 10.1186/cc12295) Results No serious bleeding complications were recorded in connection with the insertions of central venous catheters. Sixteen out of 1,769 (0.9%) insertions caused grade 2 bleeding, being defi ned as a bleeding requiring prolonged compression at the insertion site. Insertion of a large-bore central dialysis catheter was found to be an independent risk factor for bleeding complications. Neither conventional coagulation tests nor accidental arterial puncture or the number of needle passes could predict bleeding complications in this study. i y y L Zacchetti, M Panigada, M Cressoni, T Marchesi, L Gattinoni L Zacchetti, M Panigada, M Cressoni, T Marchesi, L Gattinoni Fondazione IRCCS Ca’ Granda Ospedale Policlinico Milano, Milan, Italy Critical Care 2013, 17(Suppl 2):P357 (doi: 10.1186/cc12295) Fondazione IRCCS Ca’ Granda Ospedale Policlinico Milano, Milan, Italy Critical Care 2013, 17(Suppl 2):P357 (doi: 10.1186/cc12295) p itical Care 2013, 17(Suppl 2):P357 (doi: 10.1186/cc12295) Introduction Coagulopathy, particularly a trend toward hyper- coagula bility and hypofi brinolysis, is common in critically ill patients and correlates with worse outcome. Available laboratory coagulation tests to assess fi brinolysis are expensive and time demanding. We investigated whether a modifi ed thromboelastography with the plasminogen activator urokinase (UKIF-TEG) [1] may be able to evaluate fi brinolysis in a population of critically ill patients.i Conclusion This retrospective study shows that serious bleeding complications in association with central line insertion are uncommon and that insertion of dialysis catheter is an independent risk factor for mild bleeding complications. Coagulopathy, measured with conventional coagulation tests, is not an independent risk factor for bleeding complication at insertion of central venous catheters. i y y Methods UKIF-TEG was performed as follows: fi rst urokinase was added to citrate blood to give fi nal concentrations of 160 UI/ml, then thromboelastography (TEG) analysis was started after kaolin activation and recalcifi cation with calcium chloride. Basal TEG (no addition of urokinase) was also performed. Fibrinolysis was determined by the loss of clot strength after the maximal amplitude (MA), and recorded as Ly30 (percentage lysis at 30 minutes after MA) and as Ly60 (percentage lysis at 60 minutes after MA). P359 Effi cacy of tranexamic acid in decreasing blood loss during cesarean section V Zaporozhan, O Tarabrin, D Gavrychenko, G Mazurenko, O Saleh, I Lyoshenko Results UKIF-TEG was performed on 17 healthy volunteers and 18 critically ill patients. Ly60 was predicted by Ly30 according to an exponential function, so we used Ly30 as an indicator of clot lysis. Basal TEG showed increased coagulability and a trend toward less fi brinolysis in critically ill patients compared with healthy volunteers (reaction time Introduction Despite signifi cant progress in obstetric care, the problem of bleeding during labor remains unsolved. Annually in the world, 125,000 women die from obstetric hemorrhage. Introduction Despite signifi cant progress in obstetric care, the problem of bleeding during labor remains unsolved. Annually in the world, 125,000 women die from obstetric hemorrhage. S136 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Methods We performed a randomized, double-blind study in 37 patients who underwent cesarean section. Patients were divided into two groups: the fi rst group (n = 19) received preoperative (30 minutes before operation) tranexamic acid 10 mg/kg; the second group (n = 18) received preoperative placebo. The condition of hemostasis was monitored by haemoviscoelastography. Methods High and low concentrations of both fi brinogen and FXIII were used to reverse coagulopathy induced by 1:1 dilution in vitro with 5% albumin of blood samples from healthy volunteers, monitored by rotational thromboelastometry (ROTEM).i y ( ) Results Haemodilution with albumin signifi cantly attenuated EXTEM maximum clot fi rmness (MCF), α angle (AA), clotting time (CT) and clot formation time (CFT), and FIBTEM MCF (P  <0.001). Following haemodilution, both doses of fi brinogen signifi cantly corrected all ROTEM parameters (P ≤0.02), except the lower dose did not correct AA. Compared with the lower dose, the higher dose of fi brinogen signifi cantly improved FIBTEM MCF and EXTEM MCF, AA and CFT (P <0.001). The lower dose of FXIII did not signifi cantly correct any of the ROTEM parameters, and the high dose only improved EXTEM CT (P = 0.004). All combinations of high/low concentrations of fi brinogen/ FXIII signifi cantly improved all ROTEM parameters examined (P ≤0.001). Fibrinogen concentration generally had a greater eff ect on each parameter than did FXIII concentration; the best correction of ROTEM parameters was achieved with high-dose fi brinogen concentrate and either low-dose or high-dose FXIII. 1Hôpital Jeanne de Flandre, Lille, France; 2Centre hospitalier regional universitaire Lille, France Critical Care 2013, 17(Suppl 2):P362 (doi: 10.1186/cc12300) Critical Care 2013, 17(Suppl 2):P362 (doi: 10.1186/cc12300) Introduction Postpartum hemorrhage (PPH) remains the leading cause of maternal mortality. After IOCS introduction in our north of France tertiary-care obstetric unit, the consecutive cohort of severe PPH was collected prospectively and compared regarding the use of IOCS or not. y Methods Citrated venous blood from 10 healthy volunteers was diluted by 33% using 130/0.42 hydroxyethyl starch or Ringer’s acetate. ROTEM was used to evaluate the eff ect of addition of either fi brinogen concentrate corresponding to 4  g/70  kg, or this fi brinogen dose combined with factor XIII equivalent to 20 IU/kg. Blood was analyzed at 33 or 37°C with ROTEM ExTEM and FibTEM reagents.i Methods A case–control prospective open-label study comparing the management and outcome of a consecutive cohort of 324 severe PPHs in elective and emergency hemorrhagic caesarean sections (CS) over 3 years. Results A signifi cant dilutive response was shown in both groups: hypocoagulation was greater in the starch group. Hypothermia lengthened the following: ExTEM clotting time (CT), clot formation time and α angle; FibTEM maximal clot formation (MCF). Irrespective of temperature, fi brinogen overcorrected Ringer’s acetate’s eff ects on all ROTEM parameters and partially reversed starch’s eff ects on ExTEM CT and FibTEM MCF. FibTEM demonstrated that factor XIII provided an additional procoagulative eff ect in the Ringer’s acetate group at both temperatures but not the starch group. The only ExTEM parameter to be improved by addition of factor XIII was MCF at 33°C.i Results IOCS was used in 70 severe PPHs and 254 severe PPH controls were managed without IOCS. Placenta accreta can be selected as the best indication for RBC restitution. In the 1,500 to 3,000 ml PPH, allogeneic transfusion was decreased in the IOCS group: 17.6 versus 56.3% (P = 0.006); PRBC: 0 (0 to 3) versus 3 (0 to 6) (P = 0.045). IOCS spared 87 blood bank PRBC (17,374 ml); that is, 24.2% of the total transfusion need. No amniotic fl uid embolism has been observed in the group with IOCS whereas one case appeared in the control group without IOCS. Conclusion Regarding the literature [1-4] and our study, IOCS could be used safely in PPH during CS. A leukocyte fi lter for retransfusion has been recommended and Rhesus isoimmunization must be precluded and monitored by repeated fetal RBC testing. In vitro correction of hypothermic and dilutive crystalloid and colloid rotational thromboelastography-monitored coagulopathy with fi brinogen and factor XIII i g D Winstedt, O Thomas, US Schött Intraoperative cell savage for life-threatening haemorrhagic caesarean section Lund University, Lund, Sweden AS Ducloy1, C Barre-Drouard2, E Jaillette2, S Depret-Mosser2, B Wibaut2, B Vallet2, S Susen2 1Hôpital Jeanne de Flandre, Lille, France; 2Centre hospitalier regional universitaire Lille, France References y Conclusion ROTEM shows that fi brinogen concentrate can reverse dilutive coagulation defects induced by colloid and crystalloid at both 33 and 37°C. Some additional reversal was provided by factor XIII: higher doses of both fi brinogen and factor XIII may counteract starch’s eff ects on clot structure. 1. Rainaldi et al.: Br J Anaesth 1998, 80:195-198. P361 2. Bernstein et al.: Anesth Analg 1997, 85:831-833. Dose–response of fi brinogen and factor XIII concentrate for correcting albumin-induced coagulopathy US Schött, DW Winstedt, JH Hanna Lund University, Lund, Sweden Critical Care 2013, 17(Suppl 2):P361 (doi: 10.1186/cc12299) Dose–response of fi brinogen and factor XIII concentrate for correcting albumin-induced coagulopathy US Schött, DW Winstedt, JH Hanna Lund University, Lund, Sweden Critical Care 2013, 17(Suppl 2):P361 (doi: 10.1186/cc12299) 3. Catling et al.: Int J Obstet Anesth 1999, 8:79-84. g 4. Waters et al.: Anesthesiology 2000, 92:1531-1536. P359 Effi cacy of tranexamic acid in decreasing blood loss during cesarean section y g p y Results All patients included in the study before surgery had moderate hypercoagulation and normal fi brinolysis: increasing the intensity of clot formation (ICF) to 11.4% compared with normal rates; the intensity of the retraction and clot lysis (IRCL) was 16.45 ± 1.40 in both groups. At the start of the operation in patients (Group 1), ICF decreased by 9.7% (P <0.05), and IRCL decreased by 27.6% (P <0.05) compared with preoperatively. In Group 2, there was ICF decrease by 8.8% (P <0.05), and IRCL increase by 11.4% (P <0.05) compared with preoperatively. At the end of the operation, the condition of hemostasis in both groups came almost to the same value  – moderate hypocoagulation, depressed fi brinolysis. In both groups there were no thrombotic complications. Intraoperative blood loss in the fi rst group was 300 ± 40.5 and in the second was 500 ± 60.6.i Conclusion Using of tranexamic acid before surgery signifi cantly reduces intraoperative blood loss by 60%, without thrombotic complications. Conclusion Fibrinogen concentrate successfully corrected initiation, propagation and clot fi rmness defi cits induced by haemodilution with albumin, and FXIII synergistically improved fi brin-based clot strength. Reference AS Ducloy1, C Barre-Drouard2, E Jaillette2, S Depret-Mosser2, B Wibaut2, B Vallet2, S Susen2 Introduction Rotational thromboelastography (ROTEM) can detect dilutive and hypothermic eff ects on coagulation and evaluate corrective treatments. The aim of this in vitro study was to study whether fi brinogen concentrate alone or combined with factor XIII could reverse colloid-induced and crystalloid-induced coagulopathies in the presence and absence of hypothermia. 1Hôpital Jeanne de Flandre, Lille, France; 2Centre hospitalier regional universitaire Lille, France In vitro correction of hypothermic and dilutive crystalloid and colloid rotational thromboelastography-monitored coagulopathy with fi brinogen and factor XIII D Winstedt, O Thomas, US Schött Lund University, Lund, Sweden Critical Care 2013, 17(Suppl 2):P360 (doi: 10.1186/cc12298) 1. Anesth Analg 2008, 106:1360-1365. P363 primary composite endpoint  – all-cause mortality, cardiovascular complications, acute respiratory distress syndrome, acute kidney injury requiring renal replacement therapy, septic shock or reoperation at 30 days – occurred in 19.6% of the patients in the liberal strategy group and in 35.6% in the restrictive group (P = 0.012). The liberal strategy group had a signifi cantly lower 30-day mortality rate as compared with the restrictive group (8.2% (95% CI, 4.2 to 15.4%) vs. 22.8% (95% CI, 15.7 to 31.9%), respectively, P  =  0.005). The occurrence of cardiovascular complications was lower in the liberal group than in the restrictive group (5.2% (95% CI, 2.2 to 11.5%) vs. 13.9% (95% CI, 8.4 to 21.9%), respectively, P = 0.038). The restrictive strategy group had a higher 60- day mortality rate as compared with the liberal group (23.8% (95% CI, 16.5 to 32.9%) vs. 11.3% (95% CI, 6.5 to 9.2%), respectively, P = 0.022). Conclusion The liberal RBC transfusion strategy with a hemoglobin trigger of 9 g/dl was associated with fewer major postoperative complications in patients undergoing major cancer surgery compared with the restrictive strategy. Methods Data from 240 cardiac surgery patients were retrospectively analysed. Of these, 157 had routine management with a bolus of protamine to correct the activated clotting time and then expectant management of subsequent bleeding, and 47 had the same but also a protamine infusion of 10 to 80 mg/hour for between 3 and 8 hours postoperatively. Blood loss was measured at 1, 6, 12 and 24 hours. In all, excessive bleeding was investigated using thromboelastography (TEG). Rebound heparinisation was determined by a ratio of R-times (heparinase/plain) <0.8. The Mann–Whitney U test and the chi-squared test were used to assess statistical signifi cance.if p y Conclusion The liberal RBC transfusion strategy with a hemoglobin trigger of 9 g/dl was associated with fewer major postoperative complications in patients undergoing major cancer surgery compared with the restrictive strategy. gi Results There was no signifi cant diff erence in blood loss between the two groups. Blood loss at 1 hour in the infusion and non-infusion group was 145 and 88 ml, respectively (P = 0.06); at 6 hours: 450 and 392 ml (P = 0.5); at 12 hours: 620 and 595 ml (P = 0.62); and at 24 hours: 971 and 872 ml (P = 0.12). P364 P364 Transfusion Requirements in Surgical Oncology Patients (TRISOP): a randomized, controlled trial J Almeida1, F Galas1, E Osawa1, J Fukushima1, S Moulin1, C Park1, E Almeida1, S Vieira1, J Vincent2, A Rhodes3, M Balzan1, J Inacio1, H Palomba1, R Nakamura1, F Bergamin1, A Sandrini1, U Ribeiro Jr1, J Auler Jr1, L Hajjar1 1Instituto do Cancer do Estado de São Paulo, Brazil; 2Erasme Hospital, Université libre de Bruxelles, Brussels, Belgium; 3St George’s Healthcare NHS Trust and St George’s University of London, UK Critical Care 2013, 17(Suppl 2):P364 (doi: 10.1186/cc12302) p Results One hundred and thirty-six patients (66.3%) were exposed to RBC transfusion. In the intraoperative room, 63.4% of patients received at least one RBC unit, and in the ICU, 11.2% of children were transfused. From all patients, 66 (32.1%) presented the combined endpoint. Patients with complications had higher RACHS-1 score, were younger (69 months (0 to 137) vs. 73 months (37 to 138), P <0.001), had a lower weight (13 kg (3 to 23) vs. 20 kg (12 to 36), P <0.001), a longer time of surgery (475 minutes (410 to 540) vs. 353 (275 to 433), P <0.001), a longer duration of cardiopulmonary bypass (205 minutes (175 to 235) vs. 106 minutes (73 to 123), P = 0.003), a lower SVO2 at the end of surgery (59% (IQR 39 to 80) vs. 78% (71 to 83), P <0.001), a higher arterial lactate at the end of surgery (6.9 mmol/l (4.3 to 9.2) vs. 2.7 mmol/l (73 to 123), P = 0.003), a lower intraoperative hematocrit (26.2 ± 5.6% vs. 29.5 ± 6% (P <0.001)) and a lower hematocrit at the end of surgery (33.4 ± 6.7% vs. 36.9 ± 6.9% (P <0.001)) as compared with patients without complications. Patients with complications were more exposed to RBC transfusion in the intraoperative room (75% vs. 57%, P = 0.011) and in the ICU (21% vs. 6.4%, P = 0.002). In an adjusted model of logistic regression, RBC transfusion is an independent risk factor of combined endpoint (OR 4.25 (95% CI, 1.359 to 13.328), P = 0.013). Conclusion Blood transfusion after pediatric cardiac surgery is a risk factor for worse outcome including 30-day mortality. P363 Does protamine infusion reduce postoperative blood loss and heparin rebound in cardiac surgery patients? A retrospective analysis of 240 patients on the cardiac ICU p p p heparin rebound in cardiac surgery patients? A retrospectiv Introduction Natural colloid albumin induces a lesser degree of dilutional coagulopathy than synthetic colloids. Fibrinogen concentrate has emerged as a promising strategy to treat coagulopathy, and factor XIII (FXIII) works synergistically with fi brinogen to correct coagulopathy following haemodilution with crystalloids. Objectives were to examine the ability of fi brinogen and FXIII concentrates to reverse albumin- induced dilutional coagulopathy. analysis of 240 patients on the cardiac ICU E Helme, H Meeran, N Al-Subaie St George’s Hospital, London, UK g p Critical Care 2013, 17(Suppl 2):P363 (doi: 10.1186/cc12301) Introduction Cardiac surgery is associated with signifi cant blood loss. Teoh and colleagues demonstrated a reduction in postoperative S137 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 primary composite endpoint  – all-cause mortality, cardiovascular complications, acute respiratory distress syndrome, acute kidney injury requiring renal replacement therapy, septic shock or reoperation at 30 days – occurred in 19.6% of the patients in the liberal strategy group and in 35.6% in the restrictive group (P = 0.012). The liberal strategy group had a signifi cantly lower 30-day mortality rate as compared with the restrictive group (8.2% (95% CI, 4.2 to 15.4%) vs. 22.8% (95% CI, 15.7 to 31.9%), respectively, P  =  0.005). The occurrence of cardiovascular complications was lower in the liberal group than in the restrictive group (5.2% (95% CI, 2.2 to 11.5%) vs. 13.9% (95% CI, 8.4 to 21.9%), respectively, P = 0.038). The restrictive strategy group had a higher 60- day mortality rate as compared with the liberal group (23.8% (95% CI, 16.5 to 32.9%) vs. 11.3% (95% CI, 6.5 to 9.2%), respectively, P = 0.022). Conclusion The liberal RBC transfusion strategy with a hemoglobin trigger of 9 g/dl was associated with fewer major postoperative complications in patients undergoing major cancer surgery compared with the restrictive strategy. bleeding with the use of a protamine infusion and an abolishment of heparin rebound [1]. The aim of this study was to see whether the use of postoperative protamine infusions in our cardiac ITU was associated with a reduction in heparin rebound and blood loss. 1. Teoh K, et al.: J Thorac Cardiovasc Surg 2004, 128:211-219. 2. Teoh K, et al.: Circulation 1993, 88:420-425. References 1. Teoh K, et al.: J Thorac Cardiovasc Surg 2004, 128:211-219. 2. Teoh K, et al.: Circulation 1993, 88:420-425. y Methods We performed a prospective cohort study of 205 patients undergoing cardiac surgery for congenital heart disease. We recorded baseline characteristics, RACHS-1 score, intraoperative data, transfusion requirement and severe postoperative complications as need for reoperation, acute kidney injury, arrhythmia, severe sepsis, septic shock, bleeding, stroke, and death during 30 days. We performed univariate analysis using baseline, intraoperative and postoperative variables. Selected variables (P  <0.10) were included in a forward stepwise multiple logistic regression model to identify predictive factors of a combined endpoint including 30-day mortality and severe complications. Conclusion Blood transfusion after pediatric cardiac surgery is a risk factor for worse outcome including 30-day mortality. Avoiding blood transfusion must be a goal of best postoperative care. P365 Red blood cell transfusion is a predictor of mortality and morbidity in children undergoing cardiac surgery C Colognesi, R Maia, L Hajjar, F Bergamin, J Fukushima, E Osawa, J Almeida, L Camara, S Zeferino, V Guimaraes, F Galas Heart Institute, São Paulo, Brazil Critical Care 2013, 17(Suppl 2):P365 (doi: 10.1186/cc12303) Introduction Red blood cell (RBC) transfusion is associated with morbidity and mortality in critically ill patients. Congenital cardiac surgeries are associated with high rates of bleeding and consequently with high rates of allogeneic transfusion. We aimed to evaluate the association of transfusion with worse outcomes in children undergoing cardiac surgery. P363 There was also no signifi cant diff erence in those getting heparin rebound with 40% in the infusion group and 47% in the non-infusion group (P = 0.54). gy Acknowledgement ClinicalTrials.gov NCT 01560494. Acknowledgement ClinicalTrials.gov NCT 01560494. g Conclusion Unlike Teoh and colleagues [1], we did not fi nd an advantage in using protamine infusions. That there were still cases of heparin rebound in the infusion group suggests that the infusion was not as eff ective as expected and/or the dose was inadequate. However, previous studies assessed heparin rebound using isolated clotting parameters [1,2]. Here, we used TEG. As TEG measures the thrombodynamic properties of whole blood coagulation, perhaps it is a more reliable indicator of heparin activity? As a retrospective study, there are limitations; namely, the nonstandardised management of the patients and the potential bias in the anaesthetists’ selection of patients for an infusion. This group may be inherently higher risk for bleeding. However, heparin rebound is common and protamine is a simple, relatively safe and low-cost intervention compared with transfusion and so further study is needed. 1. Teoh K, et al.: J Thorac Cardiovasc Surg 2004, 128:211-219. 2 Teoh K et al : Circulation 1993 88:420-425 P364 Avoiding blood transfusion must be a goal of best postoperative care Introduction The purpose of this study was to evaluate whether a restrictive strategy of red blood cell (RBC) transfusion was superior to a liberal one for reducing mortality and severe clinical complications among patients undergoing major cancer surgery. Introduction The purpose of this study was to evaluate whether a restrictive strategy of red blood cell (RBC) transfusion was superior to a liberal one for reducing mortality and severe clinical complications among patients undergoing major cancer surgery. Methods The trial was designed as a phase III, randomized, controlled, parallel-group, superiority trial. The inclusion criteria were adult patients with cancer who were undergoing major abdominal surgery requiring postoperative care in an ICU. The patients were randomly allocated to treatment with either a liberal RBC transfusion strategy (transfusion when hemoglobin levels decreased below 9 g/dl) or a restrictive RBC transfusion strategy (transfusion when hemoglobin levels decreased below 7 g/dl). The primary outcome was a composite endpoint of death or severe complications. The patients were monitored for 30 days. Results A total of 1,521 patients were screened for eligibility and 248 met the inclusion criteria. After exclusions for medical reasons or a lack of consent, 198 patients were included in fi nal analysis, with 101 allocated to the restrictive group and 97 to the liberal group. The Conclusion Blood transfusion after pediatric cardiac surgery is a risk factor for worse outcome including 30-day mortality. Avoiding blood transfusion must be a goal of best postoperative care. S138 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 patients in the MT group (23.7%; 27/114) and 87 in the non-MT group. The MT group was signifi cantly higher in the ratio of females (P <0.001), ISS (P <0.01), PT-INR (P <0.001), APTT (P <0.05), ABCs (P <0.001) and TASHs (P <0.001) than in the on-MT group. On the other hand, the MT group was signifi cantly lower in Ps (P  <0.05) and fi brinogen level (P  <0.001) than the non-MT group. In the receiver operating characteristics (ROC) analysis, the area under the curve (AUC) to distinguish a MT was the highest for TASHs (0.831, P <0.001), followed by fi brinogen (0.758, P <0.001), and ABCs (0.732, P <0.001). P367i i Results During the study period, 913 admissions were documented, 843 of which were included in the study. Among them, 144 (17%) were transfused. When comparing TCs with nontransfused cases (NTCs), the odds ratio (OR) of new or progressive multiple organ dysfunction syndrome (NPMODS) was 2.39 (95% CI = 1.58 to 3.62, P <0.001). This association remained statistically signifi cant in the multivariate analysis for children with admission PRISM score ≤5 (OR = 2.41, 95% CI = 1.08 to 5.37, P = 0.032). TCs were ventilated longer than NTCs (14.1 ± 32.6 days vs. 4.3 ± 9.6 days, P <0.001). This diff erence was still signifi cant after adjustment using a Cox model. Moreover, we observed an adjusted dose–eff ect relationship between RBC transfusions and length of mechanical ventilation. The PICU length of stay was signifi cantly increased for TCs (12.4 ± 26.2 days vs. 4.9 ± 10.2 days, P <0.001), even after multivariate adjustment (hazard ratio of PICU discharge for TCs: 0.61, 95% CI = 0.5 to 0.74, P <0.001). We also observed an adjusted dose–eff ect relationship between RBC transfusions and PICU length of stay. The paired analysis for comparison of pre-transfusion and post- transfusion values showed that the arterial partial pressure in oxygen was signifi cantly reduced after the fi rst transfusion (mean diff erence 42.8 mmHg, 95% CI = 27.2 to 58.3, P <0.001). The paired analysis also showed an increased proportion of renal replacement therapy, while the proportions of sepsis, severe sepsis and septic shock did not diff er. Conclusion RBC transfusions were associated with prolonged mechanical ventilation and prolonged PICU stay. The risk of NPMODS was increased in some transfused children. Moreover, our study questions the ability of stored RBCs to improve oxygenation in critically ill children. These results should help to improve transfusion practice in the PICU. P364 Fibrinogen was only a predictor of a MT without a scoring system such as ABCs and TASHs, and the optimal cutoff value was 205 mg/dl. P366 P366 Red blood cell transfusion results in worse outcomes in patients with left ventricular dysfunction undergoing on-pump cardiac surgery R Miadda, C Colognesi, L Hajjar, M Sundin, L Camara, S Zeferino, F Bergamin, A Leme, V Guimaraes, F Galas Heart Institute, São Paulo, Brazil Critical Care 2013, 17(Suppl 2):P366 (doi: 10.1186/cc12304) Introduction Cardiac surgeries are associated with high rates of bleeding and consequently with high rates of allogeneic transfusion. Patients with left ventricular dysfunction usually present a higher incidence of complications after cardiac surgery, including low output syndrome and organ dysfunction. It is a matter of controversy whether this subgroup of patients need higher hemoglobin levels to optimize oxygen delivery. We aimed to evaluate the association of transfusion with worse outcomes in patients with left ventricular dysfunction. pf g Conclusion We found that the level of fi brinogen was the most valuable predictor of a MT in the coagulation or fi brinolysis markers. It is certain that the level of fi brinogen at admission was not as useful as the TASHs about predicting a MT in this study. Whereas the scoring systems require the assessment of several factors, the measurement of fi brinogen is simple, easy and quick. We strongly suggest that the level of fi brinogen will be a useful predictor of a MT at in severe blunt traumatic patients. p y Methods A prospective cohort study was conducted in 215 patients undergoing cardiac surgery, presenting ejection fraction <35%. We recorded baseline characteristics, intraoperative data, transfusion requirement and severe postoperative complications as need for reoperation, acute kidney injury, arrhythmia, severe sepsis, septic shock, bleeding, stroke, and death during 30  days. We performed univariate analysis using baseline, intraoperative and postoperative variables associated with severe postoperative complications. Selected variables (P <0.10) were included in a forward stepwise multiple logistic regression model to identify predictive factors of a combined endpoint including 30-day mortality and severe complications.i p References 1. Timothy C, et al.: J Trauma 2009, 66:346-352. 2. Yücel N, et al.: J Trauma 2006, 60:1228-1236. 3. Sørensen B, et al.: Semin Thromb Hemost 2012, 38:268-273. 1. Timothy C, et al.: J Trauma 2009, 66:346-352. 1. Timothy C, et al.: J Trauma 2009, 66:346-352. 2. Yücel N, et al.: J Trauma 2006, 60:1228-1236. 3. Sørensen B, et al.: Semin Thromb Hemost 2012, 38:268-273. 1. Timothy C, et al.: J Trauma 2009, 66:346 352. 2. Yücel N, et al.: J Trauma 2006, 60:1228-1236. 3. Sørensen B, et al.: Semin Thromb Hemost 2012, 38:268-273. 2. Yücel N, et al.: J Trauma 2006, 60:1228-1236. 3. Sørensen B, et al.: Semin Thromb Hemost 2012, 38:268-273. Association between red blood cell transfusions and clinical outcome in critically ill children y P Demaret1, M Tucci2, T Ducruet2, H Trottier2, J Lacroix2 Results Two hundred and fi fteen patients were included in the study. The mean ejection fraction was 31%. From all patients, 129 (54.4%) presented the combined endpoint. The incidence of RBC transfusion was higher in the group with complications compared with the group without complications both intraoperative and postoperative, P  <0.001. Patients with complications were older, had lower initial hemoglobin levels and had higher EuroSCORE. At multivariate analysis, age (OR = 1.07, 95% CI = 1.013 to 1.137, P = 0.016) and perioperative RBC transfusion (OR = 7.78, 95% CI = 2.13 to 28.41, P = 0.002) were independent predictors of severe postoperative complications after cardiac surgery. Introduction Red blood cell (RBC) transfusions are frequent in critically ill children. Their benefi ts are clear in several situations. However, issues surrounding their safety have emerged in the past decades. It is important to identify the potential complications associated with RBC transfusions, in order to evaluate their risk–benefi t ratio better. i Methods A single-center prospective observational study of all children admitted to the pediatric intensive care unit (PICU) over a 1-year period. The variables possibly related to RBC transfusions were identifi ed before the study was initiated, and their presence was assessed daily for each child. In transfused cases (TCs), a variable was considered as a possible outcome related to the transfusion only if it was observed after the fi rst transfusion. g y Conclusion In patients with left ventricular dysfunction, red blood transfusion after cardiac surgery is a risk factor for worse outcome including 30-day mortality. Defi ning the need for a massive transfusion in severe blunt traumatic patients T Umemura, H Ishikura, Y Nakamura, K Hoshino, T Nishida, T Kamitani Fukuoka University, Fukuoka, Japan Critical Care 2013, 17(Suppl 2):P367 (doi: 10.1186/cc12305) Introduction We do not have enough criteria to make a judgment of the need for a massive transfusion (MT) in severe blunt traumatic patients. As a scoring system to predict the need for a MT, we usually use the Assessment Blood Consumption score (ABCs) and/or the Trauma-Associated Severe Hemorrhage score (TASHs). However, for these scoring systems, the procedure is slightly complicated. The aim of this study was to establish a predictor of a MT using coagulation or fi brinolysis markers. i Methods A retrospective analysis of MT was conducted in patients with severe blunt traumatic injury, which was defi ned as Injury Severity Score (ISS) of 16 or more admitted to the ICU between 1 June 2009 and 31 December 2010. Blood samples were collected from patients immediately after arriving at our level I trauma center. We defi ned the patients who received more than 10 unit packed red blood cells (PRBCs) within the fi rst 24 hours as a MT group and who received less than 9 units PRBCs as a non-MT group. After the demographic data, number of units of PRBCs and the need for massive transfusions were recorded and analyzed in each groups, we compared data between two groups. Results There were 114 patients who met the inclusion criteria. Fifty patients received blood transfusions (43.9%; 50/114). There were 27 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 S139 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 P369 Microcirculation and blood transfusions during sepsis: leukoreduced (LR) versus non-LR red blood cells A Donati1, E Damiani1, R Domizi1, C Scorcella1, A Carsetti1, S Tondi1, R Castagnani1, N Mininno1, P Pelaia1, C Ince2 1Università Politecnica delle Marche, Ancona, Italy; 2Academic Medical Center, Amsterdam, the Netherlands Critical Care 2013, 17(Suppl 2):P369 (doi: 10.1186/cc12307) P369 density and fl ow were assessed with sidestream dark-fi eld imaging sublingually. Thenar tissue O2 saturation (StO2) was measured using near-infrared spectroscopy and a vascular occlusion test was performed. Results The De Backer score (P = 0.02), total vessel density (P = 0.08), perfused vessel density (P  =  0.04), proportion of perfused vessels (P = 0.01), and microvascular fl ow index (P = 0.04, Figure 1) increased only in Group  2. The StO2 upslope (Figure  2) during reperfusion increased in both groups (P <0.05). Massive blood transfusion for obstetric haemorrhage S Simmons1, WE Pollock1, L Phillips2, S McDonald3 p 1Mercy Hospital for Women, Heidelburg, Australia; 2Monash University, Prahran, Australia; 3La Trobe University, Bundoora, Australia Critical Care 2013, 17(Suppl 2):P370 (doi: 10.1186/cc12308) Figure 1 (abstract P369). Blood transfusion and microvascular fl ow. Prahran, Australia; 3La Trobe University, Bundoora, Australia Critical Care 2013, 17(Suppl 2):P370 (doi: 10.1186/cc12308) Introduction Obstetric haemorrhage remains a leading cause of maternal mortality and severe morbidity. Cardiovascular and haemostatic physiology alters in pregnancy and massive transfusion protocols have been implemented for obstetric haemorrhage based on limited evidence. The objective of this study was to examine the pattern and rate of blood products used in massive transfusion for obstetric haemorrhage in a tertiary obstetric hospital. Methods Massive transfusion was defi ned as 5 or more units of red blood cells within 4 hours in accordance with the Australian Massive Transfusion Registry defi nition. Following ethics approval, all cases fi lling this criterion were identifi ed in the hospital’s birthing and blood bank systems. Data were extracted from the medical histories and analysed using SPSS. P <0.05 was considered statistically signifi cant. i Results Twenty-eight women in three years (2009 to 2011) underwent a massive transfusion for obstetric haemorrhage, with nine receiving more than 10 units of RBCs in 24 hours. Eleven (39%) were admitted to the ICU and 11 underwent a hysterectomy, of which six were admitted to the ICU. The median estimated blood loss was 4,335 ml (IQR 3,000 to 5,000). Median blood product delivery was RBC 8 units (IQR 6 to 13); FFP 4 units (IQR 4 to 8); platelets 4 units (IQR 0 to 8) and cryoprecipitate 3 units (IQR 0 to 10). One-half of the women received the fi rst four units of RBCs in less than 34 minutes. Other blood products were started a median of 49  minutes, 44  minutes and 75  minutes after the RBC transfusion commenced, respectively. Eight women had a fi brinogen level <0.8 g/l on the initial coagulation test during the haemorrhage. The remaining 20 women had a median fi brinogen level of 3.7 g/l (IQR 3.15 to 4.9). There was no diff erence in the transfusion of RBCs (P = 0.20), FFPs (P = 0.96) and platelets (P = 0.48) in women who showed an initial low fi brinogen and those who did not, although there was a diff erence in the number of units of cryoprecipitate (P <0.05). Massive blood transfusion for obstetric haemorrhage The median lowest Hb during the haemorrhage was 66  g/l (IQR 51 to 80) and median discharge Hb was 103 g/l (IQR 96 to 113). No blood product reaction was noted and there was one death. Figure 1 (abstract P369). Blood transfusion and microvascular fl ow. Figure 2 (abstract P369). Blood transfusion and microvascular reactivity. Conclusion Massive transfusion for obstetric haemorrhage involved rapid blood product administration with no consistent pattern in the ratio of products administered. Defi ning the need for a massive transfusion in severe blunt traumatic patients In Group 1 the baseline StO2 and StO2 downslope during ischemia increased, probably refl ecting a lower O2 consumption. Introduction Microcirculatory alterations during sepsis impair tissue oxygenation, which may be further worsened by anemia. Blood transfusions proved not to restore O2 delivery during sepsis [1]. The impact of storage lesions and/or leukocyte-derived mediators in red blood cell (RBC) units has not yet been clarifi ed [2]. We compared the eff ects of leukoreduced (LR) versus nonLR packed RBCs on microcirculation and tissue oxygenation during sepsis. 2 p Conclusion Unlike nonLR RBCs, the transfusion of fresh LR RBCs seems to improve microvascular perfusion and might help to restore tissue oxygenation during sepsis. yg References 1. Marik PE, et al.: JAMA 1993, 269:3024-3029. Methods A prospective randomized study. Twenty patients with either sepsis, severe sepsis or septic shock requiring RBC transfusion randomly received nonLR (Group 1, n = 10) or LR (Group 2, n = 10) fresh RBCs (<10 days old). Before and 1 hour after transfusion, microvascular 2. Sparrow RL: Blood Transfus 2010, 8(Suppl 3):s26-s30. 3 Blood transfusion practices in the ICU of a level 1 trauma centre and tertiary cardiac unit P373 P373 Incidence, risk factors and outcome of TRALI after cardiac surgery A Dijkhuizen, R De Bruin, S Arbous Leiden University Medical Center, Leiden, the Netherlands Critical Care 2013, 17(Suppl 2):P373 (doi: 10.1186/cc12311) Introduction Transfusion-related acute lung injury (TRALI) is a syndrome that presents as a sudden onset of respiratory distress 6 hours after transfusion of blood products. The diagnosis is based on clinical and radiographic fi ndings. Particularly at risk for TRALI are cardiac surgery patients. However, specifi c patient risk factors and data on outcome are largely unknown. The aim of this study was to investigate incidence, risk factors and outcome of TRALI in cardiac surgical patients on cardiopulmonary bypass. g p p Results A total 175  units of PRC were transfused in the ICU, over 105 episodes during the 2-month period (excluding immediately postoperative transfusions). Ninety-four units (53.7%) administered in 64 transfusion episodes (61.0%) occurred contrary to the guidelines. In 89.3% of these cases the recorded reason for transfusion was an apparently low Hb level. The median (IQR (range)) Hb level at which patients were transfused: within guidelines was 6.9 g/dl (6.6 to 7.7 (4.8 to 13.9)); within guidelines, excluding cases of acute blood loss, was 6.6 g/dl (6.5 to 6.8 (5.5 to 6.9)); and outside the guidelines was 7.7 g/dl (7.4 to 8.2 (7.0 to 9.7)). One unit of PRC was transfused in 57 episodes (54.3%), 2 units of PRC were transfused in 36 episodes (34.3%), and 3 to 6 units were transfused in 12 episodes (11.4%), with two-thirds of the latter due to acute haemorrhage. g y y Methods All thoracic surgery patients from a university hospital in the Netherlands of 18 years and older admitted to the ICU from January 2009 until December 2011 were screened. Included patients were observed during surgery and the fi rst 24 hours on the ICU for the onset of possible TRALI. The Canadian Consensus Conference TRALI defi nition was used. Two independent physicians blinded to the predictor variables scored the chest radiographs for the onset of bilateral interstitial abnormalities on 2K monitors. When interpretation diff ered, chest radiographs were reviewed by a third physician to achieve consensus. The European System for Cardiac Operative Risk Evaluation (EURO score) and the American Association of Anesthesiology (ASA) were scored before surgery. 3 Blood transfusion practices in the ICU of a level 1 trauma centre and tertiary cardiac unit Y Mustafa, B Pouchet University Hospitals Birmingham NHS Foundation Trust, Birmingham, UK Critical Care 2013, 17(Suppl 2):P371 (doi: 10.1186/cc12309) Introduction Blood transfusions are associated with longer ICU and hospital inpatient durations, and an increase in mortality [1]. This study was undertaken to investigate whether the practice of packed red cell Figure 2 (abstract P369). Blood transfusion and microvascular reactivity. S140 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 4.9 ± 0.5 (P = 0.09). Cetor® also reduced BALF levels of MIP-2 from 214 (54) to 127 (22) pg/ml (P <0.01). KC and IL-6 levels were not aff ected. Conclusion In a model of antibody-mediated TRALI, C1 inhibitor attenuated pulmonary infl ammation. C1 inhibition may be a potential benefi cial intervention in TRALI. 4.9 ± 0.5 (P = 0.09). Cetor® also reduced BALF levels of MIP-2 from 214 (54) to 127 (22) pg/ml (P <0.01). KC and IL-6 levels were not aff ected. Conclusion In a model of antibody-mediated TRALI, C1 inhibitor attenuated pulmonary infl ammation. C1 inhibition may be a potential benefi cial intervention in TRALI. (PRC) transfusions in the ICU was in accordance with the best clinical evidence. A number of studies, most notably the TRICC study [2], have shown that indications for ICU blood transfusions are a haemoglobin (Hb) level of <7  g/dl or evidence of acute haemorrhage [3]. These criteria were therefore employed. (PRC) transfusions in the ICU was in accordance with the best clinical evidence. A number of studies, most notably the TRICC study [2], have shown that indications for ICU blood transfusions are a haemoglobin (Hb) level of <7  g/dl or evidence of acute haemorrhage [3]. These criteria were therefore employed. y Methods This study prospectively examined episodes of PRC unit transfusions over a 2-month period in the ICU of a large level 1 trauma centre and a tertiary cardiac unit. The number of PRC units transfused in each episode was recorded by nurses, along with the proposed indication and concurrent Hb level. The data were analysed to assess the number of transfusions administered contrary to the guidelines, along with the average Hb level at which a PRC unit was transfused and the average number of units administered per episode. P372 P372 C1 inhibitor attenuates pulmonary infl ammation in an in vivo model of transfusion-related acute lung injury M Müller1, PR Tuinman1, G Jongsma1, AM Tuip-de Boer1, L Boon2, SS Zeerleder1, NP Juff ermans1 1Academic Medical Center, Amsterdam, the Netherlands; 2Bioceros, Utrecht, the Netherlands Critical Care 2013, 17(Suppl 2):P372 (doi: 10.1186/cc12310) 3 Blood transfusion practices in the ICU of a level 1 trauma centre and tertiary cardiac unit By calculating the Acute Physiology and Chronic Health Evaluation (APACHE) II and IV scores the severity of illness was determined on arrival in the ICU. Conclusion Our results indicate a liberal transfusion threshold currently exists in the ICU. Patients are frequently receiving excessive PRC transfusions for Hb levels above the recommended concentration. In the 2-month study period, these were associated with a cost of approximately £12,220. We recommend increased staff awareness of the guidelines to reduce the number of unnecessary transfusions. This would decrease exposure of ICU patients to unnecessary risks of blood transfusion, reduce cost of treatment and help to preserve a valuable resource. f 1Academic Medical Center, Amsterdam, the Netherlands; 2Bioceros, Utrecht, the Netherlands 1Academic Medical Center, Amsterdam, the Netherlands; 2Bioceros, Utrecht, the Netherlands 1Academic Medical Center, Amsterdam, the Netherlands; 2Bioceros, Utrecht, the Netherlands Critical Care 2013, 17(Suppl 2):P372 (doi: 10.1186/cc12310) Introduction Transfusion-related acute lung injury (TRALI) has a high incidence in critically ill and surgical patients and contributes to adverse outcome, while specifi c therapy is absent. Recently it was demonstrated that complement activation plays a pivotal role in TRALI. We aimed to determine whether a C1 inhibitor is benefi cial in a two-hit mouse model of antibody-mediated TRALI. 1. Vlaar APJ, et al.: Crit Care Med 2010, 38:771-778. References Results In total, 1,787 cardiac surgical patients were included. A total of 19 (1.1%) patients developed TRALI within 24 hours following surgery. Patients developing TRALI were older compared with patients not developing TRALI, mean age respectively 71 and 65 years (P = 0.035). Furthermore, patients developing TRALI had higher APACHE II, APACHE IV, EURO and ASA score (P = 0.000, P = 0.000, P = 0.000 and P = 0.37). Compared with patients not developing TRALI, patients developing TRALI had a longer cardiopulmonary bypass time. Of the 19 patients developing TRALI, four patients (21.1%) died during ICU stay, in total six patients (31.6%) died during hospital stay. Patients developing TRALI were ventilated longer and had a longer ICU and hospital stay. 1. Corwin HL, et al.: Crit Care Med 2004, 32:39-52. 1. Corwin HL, et al.: Crit Care Med 2004, 32:39-52. 1. Corwin HL, et al.: Crit Care Med 2004, 32:39-52. 2. Hebert PC: Br J Anaesth 1998, 81(Suppl 1):25-33. 3. Napolitano et al.: Crit Care Med 2009, 37:3124-3157. 2. Hebert PC: Br J Anaesth 1998, 81(Suppl 1):25-33. 3. Napolitano et al.: Crit Care Med 2009, 37:3124-3157. g j y M Müller1, PR Tuinman1, G Jongsma1, AM Tuip-de Boer1, L Boon2, SS Zeerleder1, NP Juff ermans1 g g p y Conclusion Older age, higher APACHE II, IV, EURO and ASA scores and a longer time on cardiopulmonary bypass and ventilation time are risk factors for the development of TRALI. The ICU and hospital stay are longer, and the prognosis is bad for patients developing TRALI. Reference Albumin-induced coagulopathy is less severe and more eff ectively reversed with fi brinogen concentrate than is synthetic colloid-induced coagulopathy y Methods BALB/c mice were primed with lipopolysaccharide (LPS, from E. coli 0111:B4) that was administered intraperitoneally in a dose of 0.1 mg/kg, after which TRALI was induced by injecting MHC-I antibody against H2Kd (IgG2a,k) at a dose of 2 mg/kg. Mice infused with PBS or LPS served as controls. Concomitantly, mice infused with the MHC-I antibody were treated with C1 inhibitor (Cetor®; Sanquin, Amsterdam, the Netherlands) in a dose of 400 IU/kg intravenously. After infusion, mice were mechanically ventilated with a lung-protective pressure- controlled mode for 2  hours and then sacrifi ced, after which a bronchoalveolar lavage (BAL) was done. Statistics were analyzed by one-way ANOVA, values expressed as mean and standard deviation. Results Injection of LPS and MHC-I antibodies resulted in TRALI, indicated by increased levels of protein in the BAL fl uid, wet/dry ratios and levels of KC, MIP-2 and IL-6. C1 inhibitor Cetor® signifi cantly reduced total protein in BAL fl uid from 792 (169) to 421 (230) μg/ml (P <0.001) and tended to reduce the wet/dry ratio from 5.3 ± 0.3 to DW Winstedt, JH Hanna, US Schött Lund University, Lund, Sweden Critical Care 2013, 17(Suppl 2):P374 (doi: 10.1186/cc12312) Introduction Volume resuscitation is essential to restore normovolemia during hemorrhagic shock, burns and sepsis. However, synthetic colloids cause dilutional coagulopathy. The aims were to determine whether the natural colloid albumin induces a lesser degree of coagulopathy compared with synthetic colloids, and the comparative eff ectiveness of fi brinogen concentrate to reverse coagulopathy following dilution with these solutions. Methods Rotational thromboelastometry-based tests were used to examine coagulation parameters in samples from 10 healthy volunteers, in undiluted blood and samples diluted 1:1 with saline, S141 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Ringer’s acetate, hydroxyethyl starch (HES) 130/0.4, buff ered HES 130/0.4, 3% dextran 60, 6% dextran 70 or 5% albumin. Samples were analyzed before and after addition of 2 mg/ml fi brinogen concentrate. Results EXTEM maximum clot fi rmness (MCF), clot formation time (CFT) and α angle (AA) decreased signifi cantly upon dilution with all colloid solutions (P  <0.001), although a signifi cantly greater coagulopathic eff ect was seen for samples diluted with synthetic colloid solutions versus albumin (P ≤0.001). Albumin-induced coagulopathy is less severe and more eff ectively reversed with fi brinogen concentrate than is synthetic colloid-induced coagulopathy A signifi cant reduction in the platelet component of clot strength (EXTEM MCF – FIBTEM MCF) was seen for samples diluted with synthetic colloids (P  <0.001) but not albumin (P = 0.10). Following addition of fi brinogen, FIBTEM MCF, EXTEM MCF and EXTEM AA were signifi cantly higher, and EXTEM CFT was signifi cantly shorter in samples diluted with albumin versus those treated with HES or dextran (P ≤0.001). P375 P375 Network meta-analysis of clinical trials of fl uid treatments in sepsis demonstrates improved survival with albumin compared with crystalloid and hydroxyethyl starch M Bansal1, A Farrugia2, G Martin3 1Plasma Protein Therapeutics Association, Annapolis, MD, USA; 2University of Western Australia, Crawley, Australia; 3Emory University School of Medicine, Atlanta, GA, USA Critical Care 2013, 17(Suppl 2):P375 (doi: 10.1186/cc12313) Critical Care 2013, 17(Suppl 2):P375 (doi: 10.1186/cc12313) Critical Care 2013, 17(Suppl 2):P375 (doi: 10.1186/cc12313) Introduction Fluid resuscitation is widely practiced in treating sepsis. Comparative assessment of the diff erent fl uid modalities is hampered by a paucity of direct trials. We present a network meta-analysis for assessing the relative eff ectiveness of two fl uid treatments in sepsis when they have not been compared directly in a randomized trial but have each been compared with a common treatment. p g Results The economic model shows the potential cost saving by switching from open to closed albumin administration systems. It is measured in occurrence of BSI in the ICU, estimated occurrence of BSI in the albumin population and incidence of overall mortality in the ICU and cost of BSI per albumin patient. Finally, the model describes the potential cost savings by switching to a closed system. Using closed albumin delivery systems may overall provide a cost saving for the hospital and healthcare sector. Assuming 800 patients treated in an ICU per year with all in all 4,800 ml albumin, the total annual cost saving would be £24,000 switching from open to closed albumin infusions. Methods A systematic review of trials sepsis yielded 13 trials for assessment in network meta-analysis. The indirect comparison between albumin, HES and crystalloid was conducted using Bayesian methods for binomial likelihood, fi xed-eff ects network meta-analysis with a Monte Carlo Gibbs sampling method. Studies in septic patients with crystalloid as a reference treatment compared with any formulation of the colloid treatments albumin or HES were included, as were direct head-to-head trials between the two colloids.f g Conclusion Switching from an open to a closed albumin infusion system may result in a reduction in the risk of external contamination, resulting in less infection and may provide a cost saving for the healthcare system. Conclusion Switching from an open to a closed albumin infusion system may result in a reduction in the risk of external contamination, resulting in less infection and may provide a cost saving for the healthcare system. P376 Assessing the economic impact of catheter-related bloodstream infections when switching from open to closed system delivery of human albumin solutions in ICUs F Axelsen1, P Riss2 1Baxter Healthcare, Zurich, Switzerland; 2Medical University of Vienna, Austria Critical Care 2013, 17(Suppl 2):P376 (doi: 10.1186/cc12314) y References Results Odds ratios between the diff erent treatments were obtained (Figure 1). Ranking the interventions [1] demonstrated that albumin ranked highest in lowering mortality at a 96.4% probability compared with 3.6% and 0.01% for crystalloid and HES, respectively.l 1. Maki et al.: Infect Control Hosp Epidemiol 2011, 32:50-58. 2. Tacconelli et al.: J Hosp Infection 2009, 72:97-103. 3. Franzetti et al.: Epidemiol Infect 2009, 137:1041-1048. 1. Maki et al.: Infect Control Hosp Epidemiol 2011, 32:50-58. 1. Maki et al.: Infect Control Hosp Epidemiol 2011, 32:50-58. 2 Tacconelli et al : J Hosp Infection 2009 72:97-103 1. Maki et al.: Infect Control Hosp Epidemiol 2011, 32:50-58. 2. Tacconelli et al.: J Hosp Infection 2009, 72:97-103. 3. Franzetti et al.: Epidemiol Infect 2009, 137:1041-1048. 2. Tacconelli et al.: J Hosp Infection 2009, 72:97-103. Conclusion Albumin as a fl uid therapy in sepsis is associated with the lowest mortality of the three modalities studied. Reference . Salanti G, Ades AE, Ioannidis JP: Graphical methods and numerical summaries for presenting results from multiple-treatment meta-analysis: an overview and tutorial. J Clin Epidemiol 2011, 64:163-171. Assessing the economic impact of catheter-related bloodstream infections when switching from open to closed system delivery of human albumin solutions in ICUs , 1Baxter Healthcare, Zurich, Switzerland; 2Medical University of Vienna, Austria Critical Care 2013, 17(Suppl 2):P376 (doi: 10.1186/cc12314) Introduction Hospital-acquired infections create a signifi cant burden to the healthcare system. To understand the economic impact, a model was developed to assess the cost of diff erences in catheter-related bloodstream infections (BSI) in ICUs based on either closed or open albumin systems. Conclusion Hemodilution using albumin induced a lesser degree of coagulopathy compared with the synthetic colloids HES and dextran. In addition, albumin-induced coagulopathy was more eff ectively reversed following addition of fi brinogen concentrate compared with coagulopathy induced by synthetic colloids. y Methods A model was developed with the aim of showing the economic consequences of diff erences between intravenous (i.v.) infusions of albumin via open or closed infusion containers. The model took a healthcare perspective in the UK. The impact of central venous catheter-related BSI was evaluated. The model took diff erences in BSI rates and the associated cost consequences for the hospital into account. The model bases on the evidence that closed system delivery helps to reduce the risk of external contamination, which decreases the BSI rate and reduces overall mortality [1-3]. We assessed the total costs using public list prices and literature. The model assumes that infection rates for using open and closed albumin delivery systems are similar to any other closed or open nonalbumin i.v. fl uid delivery system used in the ICU. The model looks at reduction in central-line-associated BSI and the potential cost saving of this reduction. P375 Procedural sedation in the emergency department Methods An electronic-dedicated database was created to retro- spectively collect volume, type of fl uids infused and plasmatic acid– base balance variations in postoperative ICU patients from admission to 9:00 am of the day after. SIDin was calculated as the average SID of all fl uids infused during the whole study period (crystalloids, colloids and blood products). Arterial base excess variation (ΔBEa) was computed as the diff erence between values at 9:00 am on the day after and those at entry. We report data from all patients admitted in 2006 and 2007 (650 patients). MS Shah1, FS Shah2, KP Pope3, AS Abbas4 1Luton and Dunstable Hospital, London, UK; 2Birmingham Hospitals NHS Trust, Birmingham, UK; 3Chesterfi eld NHS Trust, Chesterfi eld, UK; 4Colchester Hospital, Colchester, UK Critical Care 2013, 17(Suppl 2):P380 (doi: 10.1186/cc12318) MS Shah , FS Shah , KP Pope , AS Abbas 1Luton and Dunstable Hospital, London, UK; 2Birmingham Hospitals NHS Trust, Birmingham, UK; 3Chesterfi eld NHS Trust, Chesterfi eld, UK; 4Colchester Hospital, Colchester, UK Critical Care 2013 17(Suppl 2):P380 (doi: 10 1186/cc12318) p , , Critical Care 2013, 17(Suppl 2):P380 (doi: 10.1186/cc12318) Introduction Are safety guidelines being followed when administering procedural sedation in the emergency department? Between November 2004 and November 2008, the NPSA received 498 alerts of patients being given the wrong dose of midazolam for procedural sedation [1]. In the fi rst 5 years of midazolam use there were 86 deaths, most related to procedural sedation [2]. Introduction Are safety guidelines being followed when administering procedural sedation in the emergency department? Between November 2004 and November 2008, the NPSA received 498 alerts of patients being given the wrong dose of midazolam for procedural sedation [1]. In the fi rst 5 years of midazolam use there were 86 deaths, most related to procedural sedation [2]. p Results Nine patients not receiving intravenous infusions were excluded. The remaining population was divided into three groups according to SIDin distribution (Group  1, 18  ±  12; Group  2, 47  ±  6; Group  3, 55  ±  0  mEq/l). We observed a progressive increment in ΔBEa between the groups (1.1 ± 2.0 vs. 2.8 ± 2.9 vs. 3.0 ± 2.8 mmol/l, P <0.001). We further subdivided each group by the median value of baseline HCO3-a (24.3 (22.3 to 26.1) mmol/l) and we analyzed the ΔBEa: we observed a greater increase in patients with lower baseline HCO3-a (Group 1, 1.8 ± 2.9 vs. P379 (SAP), SV and CO were recorded directly before the administration of any colloid (T0) and every 5 minutes for the next 1 hour (T1 to T12). Kolmogorov–Smirnov was used to test normal distribution of data and ANOVA was used for the statistical analysis. P <0.05 was considered statistically signifi cant. P379 Microcirculatory response to fl uid challenge: should we prefer balanced colloids to rebalance tissue perfusion? A Donati, E Damiani, R Domizi, C Scorcella, A Carsetti, S Tondi, R Castagnani, N Mininno, V Monaldi, P Pelaia Università Politecnica delle Marche, Ancona, Italy Critical Care 2013, 17(Suppl 2):P379 (doi: 10.1186/cc12317) y gi Results Demographic data and ASA classifi cation did not diff er statistically signifi cant among the six groups of the study. CO, SV, HR and SAP did not show any statistically signifi cant evolution compared with their baseline value during the study period. Moreover, there were no statistically signifi cant diff erences among the six study groups with regard to any of the recorded parameters. Introduction Fluid resuscitation should improve tissue oxygenation in hypovolemia, besides restoring macrohemodynamic stability [1]. We evaluated the microvascular response to fl uid challenge with diff erent colloid solutions and its relation to macrohemodynamics.l y Conclusion According to our results, volume replacement with the six colloids tested in our study did not result in any hemodynamic response. Within comparison of these six colloids did not reveal any statistically signifi cant diff erence in any of the parameters recorded according to our protocol. Methods An observational study of patients receiving a fl uid challenge (500 ml colloids in 30 minutes) according to the attending physician’s decision. Before and after the infusion, sublingual microcirculation was evaluated with sidestream dark-fi eld imaging (Microscan; Microvision Medical, Amsterdam, the Netherlands). Microvascular fl ow and density were assessed for small vessels [2]. The cardiac index (CI), intrathoracic blood volume index (ITBVI) and extravascular lung water index (ELWI) were measured in seven patients with PiCCO2 (Pulsion Medical System, Munich, Germany). Results Ten patients (two sepsis, four trauma, three intracranial bleeding, one post surgery) received either saline-based hydroxyethyl starch (HES) 130/0.4 (Amidolite®; B.BraunSpA; n = 5) or balanced HES 130/0.42 (Tetraspan®; B.BraunSpA; n = 5). The CI (P = 0.02) and ITBVI (P = 0.07) tended to increase, the EVLWI did not change. Microvascular fl ow and density improved in the whole sample. No correlation was found between macro-circulatory and micro-circulatory parameters. P378 Strong ion diff erence and arterial bicarbonate concentration as cornerstones of the impact of fl uid therapy on acid–base balance D Ottolina, M Ferrari, L Zazzeron, E Scotti, M Stanziano, C Rovati, C Marenghi, L Gattinoni, P Caironi Fondazione IRCCS Ca’ Granda – Ospedale Maggiore Policlinico Università degli Studi di Milano, Milan, Italy Critical Care 2013, 17(Suppl 2):P378 (doi: 10.1186/cc12316) g Conclusion Balanced HES may be more effi cacious than saline-based HES in recruiting the microcirculation, thereby improving tissue O2 delivery. Introduction The biochemical characteristics of infused fl uids may be important in regulating acid–base balance, by modifying plasmatic volume and strong ion diff erence. In vitro and animal studies [1,2] have shown that volume and strong ion diff erence of infused fl uids (SIDin) as well as the arterial baseline bicarbonate concentration (HCO3-a) infl uence acid–base variations. Our aim was to verify these changes in critically ill patients after surgery. Procedural sedation in the emergency department 0.2 ± 2.6, mmol/l, P <0.001; Group 2, 4.0 ± 2.7 vs. 1.5 ± 2.6, mmol/l, P <0.001; Group 3, 4.4 ± 2.8 vs. 1.7 ± 2.0 mmol/l, P <0.001), as compared with those with higher baseline levels. When the study population was divided into quartiles of the diff erence between SIDin and HCO3-a, ΔBEa appeared to increase with the rise of such diff erence (P <0.001).f Methods We searched through the controlled drugs book in resuscitation over a 2-month period and found a list of patients who had received midazolam or fentanyl. From this, we could make a search for the relevant A and E notes for these patients. From these notes, we looked for (see shorthand in Table 1): verbal consent documentation (consent), past medical history recorded (pmhx), safe initial dose of midazolam (midaz), pre-procedure monitoring (pre), post-procedure monitoring (post), and monitoring for 1 hour before discharge (1hr). Following introduction of a reminder in the controlled drugs book/ sedation room and staff education, the case notes were analysed over another 2-month period (24 sets of notes) to assess practise against safety guidelines. f Conclusion SIDin aff ects the acid–base status per se and in relation- ship with HCO3-a. We verifi ed this hypothesis in critically ill patients, highlighting the importance of the diff erence between SIDin and HCO3-a, which better describes and predicts the acid–base modifi cations to fl uid therapy. y g Results See Table 1 (key for shorthand in Methods). Conclusion The re-audit notices within the procedural sedation room and controlled drug book front cover served as a reminder of good practise. The visibility of this reminder (within the CD book) helped ensure better adherence to the audit standard. This reminder will now be kept within the CD book. i References il References 1. Carlesso E, et al.: The rule regulating pH changes during crystalloid infusion. Intensive Care Med 2011, 37:461-468. Carlesso E, et al.: The rule regulating pH changes during crystalloid infusion. Intensive Care Med 2011, 37:461-468. P377 Figure 1 (abstract P375). Forest plot of results of Bayesian fi xed-eff ect network meta-analysis of mortality. Comparing the hemodynamic eff ects of six diff erent colloids N Kteniadakis, V Grosomanidis, E Oloktsidou, B Fyntanidou, C Nouris, K Kotzampasi, D Vasilakos Aristotle Medical School, Thessaloniki, Greece Critical Care 2013, 17(Suppl 2):P377 (doi: 10.1186/cc12315) Comparing the hemodynamic eff ects of six diff erent colloids N Kteniadakis, V Grosomanidis, E Oloktsidou, B Fyntanidou, C Nouris, K Kotzampasi, D Vasilakos Aristotle Medical School, Thessaloniki, Greece Critical Care 2013, 17(Suppl 2):P377 (doi: 10.1186/cc12315) Introduction Despite the fact that colloids are widely used, doubt still remains as to which colloid is the best and what is the ideal volume of administration. The aim of our study was to compare the hemodynamic eff ects of six diff erent colloids. Introduction Despite the fact that colloids are widely used, doubt still remains as to which colloid is the best and what is the ideal volume of administration. The aim of our study was to compare the hemodynamic eff ects of six diff erent colloids. ff Methods A total of 180 patients were enrolled in our study undergoing general surgery procedures, who were assigned to six groups (A, B, C, D, E, F). After fasting fl uid replacement and induction of anesthesia, 500 ml Gelofusine, Haemaccel, Voluven, HES 6%, HES 10% and Hemohes were administered to each group respectively. An oesophageal Doppler monitor probe was inserted into the patients for measuring stroke volume (SV) and cardiac output (CO). Standard monitoring included ECG, IBP, ETCO2 and SpO2, and heart rate (HR). Systemic arterial pressure Figure 1 (abstract P375). Forest plot of results of Bayesian fi xed-eff ect network meta-analysis of mortality. S142 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 References 1. Futier et al.: Crit Care 2011, 15:R214. 2. De Backer et al.: Crit Care 2007, 11:R101. P379 Balanced HES led to a greater increase in capillary density than NaCl HES (Figure 1).fi p References 2. Langer T, et al.: In vivo conditioning of acid–base equilibrium by crystalloid solutions: an experimental study on pigs. Intensive Care Med 2012, 38:686-693. [http://www.npsa.nhs.uk/corporate/news/ reducing-risk-of-midazolam-overdose-in-adults] [http://www.npsa.nhs.uk/corporate/news/ reducing-risk-of-midazolam-overdose-in-adults] S143 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http //ccfor m com/s pplements/17/S2 http://ccforum.com/supplements/17/S2 Figure 1 (abstract P379). Microvascular response to fl uid challenge: eff ects of diff erent colloids. Figure 1 (abstract P379). Microvascular response to fl uid challenge: eff ects of diff erent colloids. Figure 1 (abstract P379). Microvascular response to fl uid challenge: eff ects of diff erent colloids. 2. Epstein B: Data Retrieval Unit HFD-737. Department of Health and Human Services, Offi ce of Epidemiology and Biostatistics, Center for Drug Evaluation and Research; 27 June 1989. P381 Impact of an age, kidney and liver function adjusted sedation protocol in critically ill patients S Haddad, H Tamim, C Gonzales, A Rishu, A Deeb, Y Arabi King Abdulaziz Medical City, Riyadh, Saudi Arabia Critical Care 2013, 17(Suppl 2):P381 (doi: 10.1186/cc12319) Introduction Daily sedation interruption and protocol implementation have been recommended to reduce excessive sedation; however, their use has been inconsistent. We hypothesized that the use of an age, kidney and liver function adjusted sedation protocol would be Table 1 (abstract P380). Before and after education programme and reminder in the controlled drugs book Before (%) After (%) Yes No Yes No Consent 35 65 50 50 Pmhx 55 45 67 33 Midaz 58 42 88 12 Pre 25 75 54 46 Post 10 90 71 29 1hr 55 45 54 46 Table 1 (abstract P380). Before and after education programme and reminder in the controlled drugs book associated with reduced doses and improved outcomes compared with a standard protocol. Table 1 (abstract P380). Before and after education programme and reminder in the controlled drugs book Before (%) After (%) Yes No Yes No Consent 35 65 50 50 Pmhx 55 45 67 33 Midaz 58 42 88 12 Pre 25 75 54 46 Post 10 90 71 29 1hr 55 45 54 46 Methods This was a prospective cohort study comparing 3 months of a standard protocol (control group) with 3 months of an adjusted protocol (intervention group). Benzodiazepines induce hyperglycemia in rats by aff ecting peripheral disposal of glucose VS Salice, FV Valenza, MP Pizzocri, LV Valenti, GC Chevallard, MU Umbrello, SG Gatti, SF Fargion, GI Iapichno, LG Gattinoni Università degli Studi Milano, Milan, Italy Critical Care 2013, 17(Suppl 2):P385 (doi: 10.1186/cc12323) Results We retrieved 902 citations. Among them, nine RCTs fulfi lled inclusion criteria. PS was compared with the standard practice in one RCT, DIS with the standard practice in four, PS with DIS in two, and PS with PS plus DIS (PS/DIS) in two. PS or DIS reduced the duration of mechanical ventilation by 28.9% and ICU stay by 27.8% compared with the standard practice. Neither the duration of mechanical ventilation nor ICU stay diff ered among DIS, PS and PS/DIS. Mortality did not diff er among the three strategies. DIS increased the daily dose of sedatives and the workload of nurses in one recent RCT. Introduction In light of the interest in the relationship between glycemia control in critically ill subjects and outcome, we set up a study to investigate whether benzodiazepine, commonly used in anesthesia and ICUs, interferes with glucose metabolism and to explore the mechanism. Introduction In light of the interest in the relationship between glycemia control in critically ill subjects and outcome, we set up a study to investigate whether benzodiazepine, commonly used in anesthesia and ICUs, interferes with glucose metabolism and to explore the mechanism. Methods A total of 40 sedated and paralyzed Sprague–Dawley rats (301  ±  55  g) were investigated in four consecutive studies. (1)  To investigate the eff ects of diazepam on blood glucose, 15 rats were randomly assigned to intraperitoneal anesthesia with tiopenthal 80 mg/kg (DZP0), tiopenthal 40 mg/kg + diazepam 5 mg/kg (DPZ5) or tiopenthal 40 mg/kg + diazepam 15 mg/kg (DZP15). Blood levels of glucose (GEM premier 3000; IL) were measured at time intervals over 2 hours. (2) Ten animals randomized to DZP0 or DZP5 underwent an intravenous glucose tolerance test with glucose bolus (0.5 g/kg). Acute insulin response, the mean value of blood insulin (Insulin ELISA kit; Millipore) from 2 to 10 minutes after glucose bolus, was measured as index of insulin secretion. (3)  A hyperinsulinemic euglycemic clamp obtained by a continuous intravenous infusion of insulin (130  mUI/ kg/minute) was run in 10 animals randomized to DZP0 or DZP5 and the glucose infusion rate (GIR, mg/kg/minute) was assessed [1]. Benzodiazepines induce hyperglycemia in rats by aff ecting peripheral disposal of glucose (4) The eff ect of midazolam on blood glucose was tested in fi ve additional animals (M5: tiophental 40  mg/kg + midazolam 5  mg/kg). Data are presented as mean  ±  SEM. Statistical analysis (ANOVA, t test) was conducted with Sigma Stat 3.1 (Systat Software). Conclusion PS or DIS decreased the duration of mechanical ventilation and ICU stay. PS seems to be superior to DIS based on the doses of sedatives and the workload of nurses. P383 Chemical and physical compatibility of continuous intravenous drug infusion combinations used in paediatric intensive care C Cole1, A Fox1, M Van Der Merwe2, L Dickson2, K Ball2, A Bevan1, A Hocking1, J Pappachan1 1University Hospitals Southampton, UK; 2University of Portsmouth, UK Critical Care 2013, 17(Suppl 2):P383 (doi: 10.1186/cc12321) Introduction The aim of this research was to provide clinically relevant evidence for Y-site compatibility of drug infusion combinations used in the PICU. Pharmacists and clinicians regularly have to interpret limited published data, particularly when more than two drugs are Y-sited. The risk of potential incompatibility must be balanced against that of additional line insertion. Results (1)  Diazepam was associated with higher levels of blood glucose in a dose–response fashion: DZP0 128  ±  7  mg/dl, DZP5 166 ± 7.3, DZP15 197 ± 7 (P <0.05). (2) The acute insulin response to intravenous glucose tolerance test was similar in DZP0 and DZP5 (DZP0 3.97  ±  0.42 ng/ml, DZP5 3.68  ±  0.44, P  =  0.68), while blood glucose levels were diff erent (DZP0 192  ±  5  mg/dl, DZP5 217  ±  5, P  <0.05). (3)  During hyperinsulinemic euglycemic clamp, blood glucose levels were similar (109  ±  3  mg/dl, P  =  0.2), but the DZP5 group showed a trend through lower values of GIR (DZP0 30.8 ± 2 mg/ kg/minute, DZP5 24.7 ± 2, P = 0.08). (4) Infusion of midazolam was associated with higher blood glucose levels (DZP0 128 ± 5 mg/dl, M5 151 ± 6, P <0.05).i Methods A full 28-factorial design (total 256 combinations) was used to investigate chemical and physical compatibility of fi ve drugs (clonidine, morphine, ketamine, midazolam and furosemide). The drugs were studied at their highest commonly infused concentrations and exposed to three variations in environmental conditions (diluent: sodium chloride 0.9% or glucose 10%; temperature 25 or 37°C; and normal room lighting or blue light phototherapy). Chemical stability was assessed using HPLC; >10% reduction in concentration indicated incompatibility. Benzodiazepines induce hyperglycemia in rats by aff ecting peripheral disposal of glucose Physical incompatibility was confi rmed by precipitation, pH or colour change.f Conclusion Both diazepam and midazolam signifi cantly alter plasma glucose levels in rats. Peripheral disposal of glucose rather than altered pancreas secretion of insulin explains the benzodiazepine-associated hyperglycemia. Results Environmental conditions had no eff ect on the drug mixtures. The precipitation observed in incompatible combinations was due to either a change in pH, or with ketamine the presence of benzethonium chloride. Of 31 possible drug combinations, 12 were incompatible. A further three combinations were incompatible at extreme pH, or were of concern and so should be avoided. The incompatible formulations p References We hypothesized that the use of an age, kidney and liver function adjusted sedation protocol would be S144 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 adjusted protocol group (18% vs. 36%, P = 0.004) with no signifi cant diff erences in MVD, ICU and hospital LOS, and hospital mortality. Conclusion The use of an age, kidney and liver function adjusted sedation protocol is associated with lower doses of analgesics and sedatives, lower risk of agitation and lower ICU mortality. all contained furosemide. All combinations of the sedative agents studied were chemically and physically compatible. adjusted protocol group (18% vs. 36%, P = 0.004) with no signifi cant diff erences in MVD, ICU and hospital LOS, and hospital mortality. y y y Conclusion This work provides evidence for Y-site compatibility of morphine, midazolam, clonidine and ketamine in any combination, which will potentially reduce the need for extra intravenous lines. Furosemide is incompatible with any of these sedative drugs and must be infused via a separate line. These results will aid clinical decision- making and help satisfy the requirements of recent UK Department of Health legislation relating to the mixing of medicines. Reference Conclusion The use of an age, kidney and liver function adjusted sedation protocol is associated with lower doses of analgesics and sedatives, lower risk of agitation and lower ICU mortality. P382 Risk and benefi t of protocol sedation and daily interruption of sedation for mechanically ventilated patients: a systematic review K Hosokawa1, M Egi2, M Nishimura3 1Kyoto Prefectural Yosanoumi Hospital, Kyoto, Japan; 2Okayama University Hospital, Okayama, Japan; 3Tokushima University Hospital, Tokushima, Japan Critical Care 2013, 17(Suppl 2):P382 (doi: 10.1186/cc12320) DeFronzo: Am J Physiol 1979, 273:E214-E223. p References In the adjusted protocol, patients were divided into three categories: category 1 (age <60 years, and normal kidney and liver function), category 2 (age = 60 to 70 years, or moderate kidney or liver function impairment), and category 3 (age >70 years, or severe kidney or liver function impairment). The upper limits of analgesics and sedatives doses were determined by age, and kidney and liver function, being lowest in category 3, and lower in category 2 than category 1. All consecutive adults mechanically ventilated patients who required infusion of analgesics and/or sedatives for >24  hours were included in the study. We compared the main outcomes of both groups including average daily doses of analgesics and sedatives; average Sedation–Agitation Scale (SAS), pain and GCS scores; mechanical ventilation duration (MVD); sedation-related complications during ICU stay; ICU and hospital length of stay (LOS), and ICU and hospital mortality. y y Results Two hundred and four patients were included in the study (control group  =  105; adjusted protocol group  =  99). There was no diff erence in baseline characteristics between the two groups. The adjusted protocol group, compared with the control group, received signifi cantly lower average daily doses of fentanyl (2,162 ± 2,110 μg vs. 3,650 ± 3,253 μg, P = 0.0001), nonsignifi cant lower average daily doses of midazolam and dexmedetomidine, and a trend toward higher average daily doses of propofol. Pain score was higher in the adjusted protocol group (0.98 ± 0.72 vs. 0.16 ± 0.35, P <0.0001) with no diff erence in SAS or GCS scores. Sedation-related complications during ICU stay were not diff erent between the two groups; however, agitation (SAS = 5) was less frequent in the adjusted protocol group (3% vs. 30%, P <0.0001). ICU mortality was signifi cantly lower in the P381 Impact of an age, kidney and liver function adjusted sedation protocol in critically ill patients S Haddad, H Tamim, C Gonzales, A Rishu, A Deeb, Y Arabi King Abdulaziz Medical City, Riyadh, Saudi Arabia Critical Care 2013, 17(Suppl 2):P381 (doi: 10.1186/cc12319) Introduction Daily sedation interruption and protocol implementation have been recommended to reduce excessive sedation; however, their use has been inconsistent. Reference 1. Mixing of Medicines Prior to Administration in Clinical Practice: Medical and Non-medical Prescribing [http://www.dh.gov.uk/en/ Publicationsandstatistics/Publications/PublicationsPolicyAndGuidance/ DH_116361] 1. Mixing of Medicines Prior to Administration in Clinical Practice: Medical and Non-medical Prescribing [http://www.dh.gov.uk/en/ Publicationsandstatistics/Publications/PublicationsPolicyAndGuidance/ DH_116361] 1. Mixing of Medicines Prior to Administration in Clinical Practice: Medical and Non-medical Prescribing [http://www.dh.gov.uk/en/ Publicationsandstatistics/Publications/PublicationsPolicyAndGuidance/ DH_116361] g 1Kyoto Prefectural Yosanoumi Hospital, Kyoto, Japan; 2Okayama University Hospital, Okayama, Japan; 3Tokushima University Hospital, Tokushima, Japan Critical Care 2013, 17(Suppl 2):P382 (doi: 10.1186/cc12320) Introduction Protocol-directed sedation (PS) and daily interruption of sedation (DIS) are two major strategies to optimize sedation depth for mechanically ventilated patients. To clarify the clinical risk and benefi ts of PS and DIS, we performed a systematic review of randomized controlled trials (RCTs) in previous literature. Introduction Protocol-directed sedation (PS) and daily interruption of sedation (DIS) are two major strategies to optimize sedation depth for mechanically ventilated patients. To clarify the clinical risk and benefi ts of PS and DIS, we performed a systematic review of randomized controlled trials (RCTs) in previous literature. P384 Abstract withdrawn P384 Abstract withdrawn P384 P384 Abstract withdrawn P384 Abstract withdrawn Methods We searched the MEDLINE database from January 1990 to October 2012 for studies on PS and DIS in critically ill patients. English-language manuscripts were included when they assessed the impact of PS or DIS on outcomes in critically ill patients requiring mechanical ventilation for >48  hours. PS is defi ned as sedation practices implemented by ICU staff using a sedation scale and a written protocol. DIS is defi ned as strategies with cessation of the continuous sedatives. P387 Involvement of mitochondrial ATP-sensitive K+ channels in fentanyl- induced mitochondrial dysfunction of cultured human hepatocytes S Djafarzadeh, M Vuda, J Takala, SM Jakob Bern University Hospital (Inselspital) and University of Bern, Switzerland Critical Care 2013, 17(Suppl 2):P387 (doi: 10.1186/cc12325) Introduction Pharmacological agents used to treat critically ill patients may alter mitochondrial function. The aim of the present study was to investigate whether fentanyl, a commonly used analgesic drug, interacts with hepatic mitochondrial function. Introduction We report our experience in the use of isofl urane for prolonged sedation in severe ARDS patients. Prolonged sedation in the ICU may be diffi cult because of tolerance, drug dependence and withdrawal, drug interactions and side eff ects. Inhaled anesthetics have been proposed for sedation in ventilator-dependent ICU patients. AnaConDa is a device that allows a safety and easy administration of inhaled anesthetics in the ICU. Methods The human hepatoma cell line HepG2 was exposed to fentanyl at 0.5, 2 or 10 ng/ml for 1 hour, or pretreated with naloxone (an opioid receptor antagonist) at 200 ng/ml or 5-hydroxydecanoate (5-HD; a specifi c inhibitor of mitochondrial ATP-sensitive K+ (KATP) channels) at 50  μM for 30  minutes, followed by incubation with fentanyl at 2 ng/ml for an additional hour. The mitochondrial complex I-dependent, II-dependent and IV-dependent oxygen consumption rates of the permeabilized cells were measured using a high-resolution oxygraph (Oxygraph-2k; Oroboros Instruments, Innsbruck, Austria). The respiratory electron transfer capacity of intact cells was evaluated using FCCP (carbonyl cyanide p-trifl uoromethoxyphenylhydrazone) to obtain the maximum fl ux. Methods From January 2009 to June 2012, 15 patients were sedated with isofl urane by means of the AnaConDa device. We consider administration of isofl urane as a washout period from common sedative drugs in patients with (at least one of): high sedative drug dosage (propofol ≥300 mg/hour or midazolam ≥8 mg/hour) to reach the target Richmond Agitation Sedation Score (RASS) or inadequate paralysis; two or more hypnotic drugs to reach the target RASS (propofol, midazolam, hydroxyzine, haloperidol, diazepam, quetiapine); and hypertriglyceridemia. During isofl urane administration previous hypnotic drugs were interrupted. We retrospectively collected data before, during and after administration of isofl urane: hemodynamic parameters, renal and hepatic function, level of sedation (RASS) and sedative drug dosage. All data are reported as mean ± standard deviation, otherwise as median (minimum to maximum). P387 l Results Incubation of HepG2 cells with fentanyl (1  hour, 2  ng/ml) induced a reduction in complex II-dependent and IV-dependent respiration (Figure 1). Cells pretreated with 5-HD before the addition of fentanyl exhibited no signifi cant changes in complex activities in comparison with controls. Pretreatment with naloxone tended to abolish the fentanyl-induced mitochondrial dysfunction. Treatment with fentanyl led to a reduction in cellular ATP content (0.24 ± 0.06 in controls vs. 0.17 ± 0.14 μmol/mg cellular protein in stimulated cells; P = 0.02). We did not observe any diff erence in basal or FCCP-uncoupled respiration rates of cells treated with fentanyl at 2  ng/ml compared with controls (data not shown). Results Mean age was 43 ± 12 years and SAPS II was 35.7 ± 11; 13 patients were treated with ECMO for severe ARDS and four had a history of drug abuse; median ICU length of stay was 41 (15 to 127) days and they were ventilated for 41 (12 to 114) days. Due to severe critical illness, target RASS was –4 for all patients, most of which were also paralysed. Isofl urane was administered in nine patients because of a high level of common sedative drugs, in fi ve patients due to the use of two or more hypnotic drugs and in one patient because of hypertrigliceridemia. Isofl urane administration lasted 5.6  ±  1.8  days. During isofl urane administration no alteration in renal function or hemodynamic instability was recorded. After the isofl urane washout period we observed a reduction in sedative drug dosage in 10 patients while two patients were quickly weaned from mechanical ventilation and the target RASS raised to 0. In two patients isofl urane was precautionarily interrupted because of concomitant alteration of liver function and suspected seizures respectively. Conclusion Fentanyl reduces cultured human hepatocyte mito- chondrial respiration by a mechanism that is blocked by a KATP channel antagonist. In contrast, antagonism with naloxone does not seem to completely abolish the eff ect of fentanyl. Acknowledgement This study was supported by the Swiss National Science Foundation (grant number 32003B_127619). P388f Reference S145 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 P386 P386 Prolonged sedation in ARDS patients with inhaled anesthetics: our experience S Redaelli, P Mangili, V Ormas, S Sosio, L Peluso, F Ponzoni, N Patroniti, A Pesenti University of Milan-Bicocca, Monza, Italy Critical Care 2013, 17(Suppl 2):P386 (doi: 10.1186/cc12324) P389 Conjunctival administration of S+ketamine in rabbits L Hess1, J Malek2, A Kurzova2, Z Simunkova1, M Votava2 1Institute for Clinical and Experimental Medicine, Prague, Czech Republic; 23rd Medical Faculty of Charles University, Prague, Czech Republic Critical Care 2013, 17(Suppl 2):P389 (doi: 10.1186/cc12327) Results During the study period 75 patients underwent procedural sedation and were discharged from the ED. The median age was 40 years and 57% were male. The commonest procedure performed was shoulder reduction (52%). In the propofol group (n = 20) the mean LOS was 100 minutes compared with 165 minutes in those receiving midazolam (n = 40) and 141 minutes in those receiving a combination (n = 15), P = 0.004. There was no diff erence in adverse events between groups. See Figure 1. Introduction Delivering analgesics via conjunctival application could provide rapid and convenient pain relief in disaster medicine. There are sporadic reports from the USA concerning inhalation administration of aerosol with various drugs producing a wide variety of eff ects from anxiolysis, sedation, and loss of aggressiveness to immobilisation. We attempted to determine in an animal experiment whether conjunctival administration of S+ketamine could produce signifi cant eff ect without side eff ects. g p g Conclusion Propofol is increasingly used in EDs for procedural sedation due to its short duration of action. This study suggests that a shorter duration of action and faster recovery may result in a reduced LOS in the ED. f Methods After ethic committee approval, 10 rabbits were administered conjunctival S+ketamine 2.5 mg/kg. Measured parameters were SpO2, blood pressure (BP) and heart rate (HR) before administration and in 1-minute intervals and immobilisation time (loss of righting refl ex). The measurements were performed for 20 minutes. Conjunctival irritation was measured 1, 10 and 20 minutes after administration according to modifi ed technical standard EN IS O 10993-10. ANOVA test was used for statistical analysis of hemodynamic parameters. Figure 1 (abstract P390). Length of stay (minutes) in the ED. Figure 1 (abstract P390). Length of stay (minutes) in the ED. Results The immobilisation time was 207  ±  60  seconds. P388 Eff ects of clonidine on microcirculatory alterations during endotoxemia K S h idt C Phili b A Zi k i S H f P388 Eff ects of clonidine on microcirculatory alterations during endotoxemia K Schmidt, C Philipsenburg, A Zivkovic, S Hofer Universitätsklinikum Heidelberg, Germany Critical Care 2013, 17(Suppl 2):P388 (doi: 10.1186/cc12326) Conclusion Inhaled anesthetics could be successfully used in the ICU especially in case of an inadequate sedation plan; for example, in patients with a history of drug abuse or young severe ARDS patients that required deep sedation and paralysis for a long period. Introduction Endothelial dysfunction during endotoxemia is responsible for the functional breakdown of microvascular perfusion and microvessel permeability. The cholinergic anti-infl ammatory pathway (CAP) is a neurophysiological mechanism that regulates the infl ammatory response by inhibiting proinfl ammatory cytokine synthesis, thereby preventing tissue damage. Endotoxemia-induced Figure 1 (abstract P387). Cellular oxygen consumption after incubation with fentanyl, naloxone or 5-HD. S146 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 eff ect of ketamine or due to the method of administration. There were no signs of conjunctival irritation in any animal (S+ketamine is a preservative-free solution). microcirculatory dysfunction can be reduced by cholinergic CAP activation. Clonidine improves survival in experimental sepsis [1] by reducing the sympathetic tone, resulting in the parasympathetic- mediated CAP activation. The aim of this study was to determine the eff ects of clonidine on microcirculatory alterations during endotoxemia. Methods Using fl uorescent intravital microscopy, we determined the venular wall shear rate, macromolecular effl ux and leukocyte adhesion in mesenteric postcapillary venules of male Wistar rats. Endotoxemia was induced over 120 minutes by intravenous infusion of lipopolysaccharide (LPS). Control groups received an equivalent volume of saline. Clonidine 10  μg/kg was applied as i.v. bolus in treatment groups. Animals received either (i) saline alone, (ii) clonidine 10  minutes prior to saline administration, (iii)  clonidine 45  minutes prior to LPS administration, (iv)  clonidine 10  minutes prior to LPS administration, (v)  clonidine 30  minutes after LPS administration or (vi) LPS alone. p Conclusion Conjunctival S+ketamine 2.5 mg/kg in rabbits produced rapid onset without changes in cardiorespiratory parameters and without signs of irritation of the eye. The results of our project warrant further research to increase the variety of drugs and methods of their administration for anxiolysis, sedation and analgesia in disaster medicine. Acknowledgement The study was supported by scientifi c grant IGA NT 11284. Reference 1. Malek J, et al.: Conjunctival administration of sufentanil in rabbits. P388 Eff ects of clonidine on microcirculatory alterations during endotoxemia K S h idt C Phili b A Zi k i S H f Eur J Pain 2011, Suppl 5:212. 1. Malek J, et al.: Conjunctival administration of sufentanil in rabbits. Eur J Pain 2011, Suppl 5:212. Results All LPS groups (iii to vi) showed a signifi cantly reduced venular wall shear rate compared with the saline group after 120 minutes. There were no signifi cant diff erences between the numbers of adhering leukocytes in the clonidine/LPS groups (iii, iv, v) and the LPS group after 120 minutes. Macromolecular effl ux signifi cantly increased in all groups over the time period of 120 minutes. After 120 minutes there was no diff erence between the LPS group and the clonidine 10 minutes prior to LPS administration group (iv) whereas all other groups (i, ii, iii, v) showed a signifi cantly reduced macromolecular effl ux compared with the LPS group.f Use of propofol for procedural sedation reduces length of stay in the emergency department J Millar, F Adamson, P O’Connor, R Wilson, E Ferrie, R McLaughlin Royal Victoria Hospital, Belfast, UK Critical Care 2013, 17(Suppl 2):P390 (doi: 10.1186/cc12328) Use of propofol for procedural sedation reduces length of stay in the emergency department J Millar, F Adamson, P O’Connor, R Wilson, E Ferrie, R McLaughlin Royal Victoria Hospital, Belfast, UK Critical Care 2013, 17(Suppl 2):P390 (doi: 10.1186/cc12328) Introduction Procedural sedation is used in the emergency department (ED) to facilitate short but painful interventions. Many patients are suitable for discharge after completion. Ideally, the agent used to achieve sedation should not have a prolonged eff ect, allowing safe discharge in the shortest time frame. We hypothesised that propofol, with its short onset and off set, may reduce length of stay (LOS) in comparison with traditional benzodiazepines. Conclusion Clonidine has no positive eff ect on microhemodynamic alterations and leukocyte–endothelial interaction during endotoxemia. The reduction of capillary leakage in clonidine-treated groups depends on the time interval relative to the initiation of endotoxemia. Endothelial permeability and leukocyte activation are regulated by diff erent pathways when stimulated by clonidine during endotoxemia. We conclude that clonidine might have an important time-dependent anti-infl ammatory and protective eff ect on endothelial activation during infl ammation. Methods Data from a prospective registry were analysed for the period 1 August 2011 to 31 January 2012. Patients who underwent procedural sedation and who were discharged from the ED were identifi ed. Individuals were grouped as having received propofol, midazolam or a combination of the two. All were discharged when fully alert and able to eat and drink. Demographic details and the type of procedure undertaken were extracted. ANOVA was performed to identify diff erences in the length of stay between groups, in addition to descriptive analysis. Reference Reference 1. Hofer S, et al.: Crit Care 2009, 13:R11. 1. Hofer S, et al.: Crit Care 2009, 13:R11. 1. Hofer S, et al.: Crit Care 2009, 13:R11. P393 Use of physical restraint in Dutch ICUs: prevalence and motives RJ Raijmakers1, RL Vroegop1, H Tekatli1, M Van den Boogaard2, AW Van der Kooi1, AJ Slooter1 1University Medical Center Utrecht, the Netherlands; 2Radboud University Nijmegen Medical Centre, Nijmegen, the Netherlands Critical Care 2013, 17(Suppl 2):P393 (doi: 10.1186/cc12331) Use of physical restraint in Dutch ICUs: prevalence and motives RJ Raijmakers1, RL Vroegop1, H Tekatli1, M Van den Boogaard2, AW Van der Kooi1, AJ Slooter1 1University Medical Center Utrecht, the Netherlands; 2Radboud University Nijmegen Medical Centre, Nijmegen, the Netherlands Critical Care 2013, 17(Suppl 2):P393 (doi: 10.1186/cc12331) Results Data were collected from 54 adults; 50 received an infusion for ≥24 hours. We noted a signifi cant CK rise in 11 (22%) of our patients that could be attributed to propofol alone. They all had raised TG (≥1.7  IU/l); six (12%) were markedly raised (≥3.4  IU/l). No patients became signifi cantly acidotic, developed cardiac arrhythmias or renal failure as a result of propofol. In the 11 patients who met our criteria, highest daily propofol dose varied from 1.5 to 4 mg/kg/hour. Pre-PRIS developed from between 2 and 6 days of infusion. The mean propofol dose in patients displaying Pre-PRIS was 2.2 mg/kg/hour whilst in those not meeting our criteria it was 2.0 mg/kg/hour. No patients developed sequelae of full PRIS. See Table 1. Introduction Physical restraints are used to facilitate essential care and prevent secondary injuries. However, physical restraint may be regarded as humiliating. It may lead to local injury and increase the risk of delirium and post-traumatic stress syndrome. Research on physical restraint is scarce. The aim of this study is to investigate the scope of physical restraint use. Methods Twenty-one ICUs ranging from local hospitals to academic centres were each visited twice and 327 patients were included. We recorded characteristics of restrained patients, motives and awareness of nurses and physicians. Table 1 (abstract P391) Total Trauma Nontrauma ≥24 hours infusion 50 34 16 CK + TG ≥3.4 6 6 0 CK + TG ≥1.7 5 3 2 Results Physical restraint was applied in 74 (23%) patients, ranging from 0 to 54% in diff erent hospitals. Frequent motives for restraint use were ‘possible threat to airway’ (36%) and ‘pulling lines/probes’ (31%). Restrained subjects more often had a positive CAM-ICU (34% vs. 16%, P <0.001), could less frequently verbally communicate (14% vs. 49%, P <0.001), and received more often antipsychotics (49% vs. P394 Factors infl uencing the use of physical restraints in Canadian ICUs B Sneyers1, E Luk2, M Perreault3, D Williamson3, L Rose2, S Mehta4, L Burry4 1Université Catholique de Louvain, Brussels, Belgium; 2Lawrence S. Bloomberg Faculty of Nursing, University of Toronto, Ontario, Canada; 3Université de Montréal, Canada; 4Mount Sinai Hospital, University of Toronto, Ontario, Canada Critical Care 2013, 17(Suppl 2):P394 (doi: 10.1186/cc12332) Factors infl uencing the use of physical restraints in Canadian ICUs B Sneyers1, E Luk2, M Perreault3, D Williamson3, L Rose2, S Mehta4, L Burry4 1Université Catholique de Louvain, Brussels, Belgium; 2Lawrence S. Bloomberg Faculty of Nursing, University of Toronto, Ontario, Canada; 3Université de Montréal, Canada; 4Mount Sinai Hospital, University of Toronto, Ontario, Canada Critical Care 2013, 17(Suppl 2):P394 (doi: 10.1186/cc12332) P389 There were no changes in cardiorespiratory parameters (initial, 10 and 20 minutes after administration: HR 255.5 ± 24.7, 265.8 ± 26.0 and 267.8 ± 20.4, systolic BP 114.3 ± 8.6, 108.2 ± 10.7 and 113.9 ± 11.9 mmHg, diastolic BP 66.8 ± 11.6, 68.4 ± 10.6 and 69.7 ± 9.3 mmHg and SpO2 99.2 ± 1.0, 98.2 ± 0.6 and 98.7 ± 0.5). These results are in contrast to conjunctival administration of sufentanil 5 μg/kg that caused a signifi cant decrease of SpO2 and HR [1]. We can speculate that the reason for stability of cardiorespiratory parameters was due to the sympathoadrenergic S147 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 P391 Pre-PRIS? Prospective monitoring for early markers of propofol infusion syndrome M Stovell, S Smith, M Udberg, W Loh, P Nair Walton Centre Neurology & Neurosurgery, Liverpool, UK Critical Care 2013, 17(Suppl 2):P391 (doi: 10.1186/cc12329) P391 Conclusion The study shows a 1% adverse event rate. This supports use of propofol sedation by emergency physicians but within the limits of a strict governance framework. Our safety analysis using the World SIVA adverse events tool provides a reference point for further studies. R f P391 Pre-PRIS? Prospective monitoring for early markers of propofol infusion syndrome 1. Green SM, Yealy DM: Procedural sedation goes Utstein: the Quebec guidelines. Ann Emerg Med 2009, 53:436-438. 1. Green SM, Yealy DM: Procedural sedation goes Utstein: the Quebec guidelines. Ann Emerg Med 2009, 53:436-438. 1. Green SM, Yealy DM: Procedural sedation goes Utstein: the Quebec guidelines. Ann Emerg Med 2009, 53:436-438. Introduction The use of propofol for sedation in intensive care has been associated with the propofol infusion syndrome (PRIS) characterised by cardiac dysfunction, metabolic acidosis, renal failure, rhabdomyolysis and hyperlipidaemia. We prospectively monitor biochemical markers that we believe demonstrate early signs of this dangerous, often fatal syndrome. When this Pre-PRIS state is identifi ed, propofol is withdrawn whilst the syndrome is still reversible. 2. Green SM, Krauss B: Barriers to propofol use in emergency medicine. Ann Emerg Med 2008, 52:392-398. 3. Mason KP, Green SM, Piacevoli Q; International Sedation Task Force: Adverse event reporting tool to standardize the reporting and tracking of adverse events during procedural sedation: a consensus document from the World SIVA International Sedation Task Force. Br J Anaesth 2012, 108:13-20. 3. Mason KP, Green SM, Piacevoli Q; International Sedation Task Force: Adverse event reporting tool to standardize the reporting and tracking of adverse events during procedural sedation: a consensus document from the World SIVA International Sedation Task Force. Br J Anaesth 2012, 108:13-20. y Methods We prospectively audited our monitoring of these markers over a 4-month period in propofol-sedated patients: propofol infusion rate, creatine kinase (CK), triglycerides (TG), creatinine, lactate, pH and base defi cit. We defi ned the criteria for Pre-PRIS as requiring a CK ≥320 mmol/l that had doubled from its base level and a rise in TG ≥1.7 IU/l; both that followed a trend with propofol dose. P393 28%, P <0.001), or benzodiazepines (55% vs. 36%, P = 0.003). The use of physical restraint was registered in the patient’s fi les in 48% of cases. Of the 310 interviewed nurses, 258 (83%) were familiar with a physical restraint protocol and 89 (29%) used it in any situation. Thirty percent of the 60 interviewed physicians were aware of the physical restraint status of their patients. Conclusion We propose that a paired rise in CK and TG that can be attributed to propofol alone represents a Pre-PRIS state that is at risk of developing into full PRIS. We noted this in 22% of our patients, all on modest doses of propofol. It is unclear what proportion of patients will develop the full syndrome as it is not ethically possible to continue propofol in this situation. We advocate daily monitoring of CK and TG to identify Pre-PRIS so that propofol can be reduced or substituted to avoid the morbidity and mortality of the full syndrome. Conclusion Physical restraint is frequently used in Dutch ICUs, but the frequency diff ers strongly between diff erent ICUs. Attending physicians are often not aware of physical restraint use. P392 Safety profi le of 1,008 cases of propofol sedation in the emergency department Safety profi le of 1,008 cases of propofol sedation in the emergency department S Bradburn, G Lloyd, B Newstead Royal Devon and Exeter Hospital, Exeter, UK Critical Care 2013, 17(Suppl 2):P392 (doi: 10.1186/cc12330) Introduction Until recently there were no guidelines for the reporting of adverse events (AEs) during procedural sedation [1,2]. A consensus document released in 2012 by the world SIVA International Sedation Task Force proposed a benchmark for defi ning AEs [3]. We analysed 1,008 cases of procedural sedation in the emergency department. Introduction Physical restraint (PR) use in critically ill patients has been associated with delirium, unplanned extubation, prolonged ICU length of stay, and post-traumatic stress disorder. Our objectives were to defi ne prevalence of PR use, and to examine patient, treatment, or institutional factors associated with their use in Canadian ICUs. Introduction Physical restraint (PR) use in critically ill patients has been associated with delirium, unplanned extubation, prolonged ICU length of stay, and post-traumatic stress disorder. Our objectives were to defi ne prevalence of PR use, and to examine patient, treatment, or institutional factors associated with their use in Canadian ICUs. Methods The study is based on 1,008 patients who received procedural sedation with propofol in the emergency department between December 2006 and March 2012. Patients were selected and sedated to a strict protocol by ED consultant staff . We applied the AE tool by performing a search through patient records, discussion with consultants performing the sedation and consensus opinion.i Methods We conducted an analysis of a database of sedative, analgesic, antipsychotic and neuromuscular blocker prescribing practices and PR use in 711 mechanically ventilated (MV) adults in 51 Canadian ICUs (3,619 patient-days). Data were collected during a 2-week period (2008 to 2009). Logistic and Poisson regression analysis were used to identify factors associated with PR use and number of days of use respectively. Results PR was used on 1 or more days in 52.6% (374/711) of patients. For patients restrained, the mean number of days restrained was Results From 1,008 cases we identifi ed 11 sentinel (six of hypotension, fi ve cases of hypoxia), 34 moderate, 25 minor and three minimal risk adverse events. S148 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 4.1 ± 4.0 and the mean number of restraint applications per patient was 1.2 ± 0.5. Current perspectives, beliefs and practices concerning delirium in critically ill patients: a multicenter survey among Dutch healthcare professionals Z Trogrlic, M Van der Jagt, P Van der Voort, J Bakker, E Ista, on behalf of iDECePTIvE Study Group Results We assessed 1,576 patients, corresponding with 8,150 ICU treatment days with a median length of stay of 3 days (IQR 2 to 5). The mean age of the patients was 62 years (SD = 16) and 58% were male. Delirium occurred in 23% (356/1,576) of patients with a median duration of 3 days (IQR 2 to 7) and ranged from 11 to 40% for each ICU. Delirium assessment with the CAM-ICU at any point during ICU stay was performed in 38% of all patients. Screening with CAM-ICU was applied in three ICUs, in 29 to 96% of the patients in these centers. Of 3,564 documented screening days with the CAM-ICU, it was positive in 1,459 (41%); in only 120 (8%) of these CAM-ICU-positive days there was a documented action or treatment started for delirium. However, patients still received haloperidol on 52% (n = 766) of all CAM-ICU- positive days. Patients received benzodiazepines in 49% (n  =  1,141) of patient sedation days. Delirium preventive interventions were physiotherapy (19% of 8,150 ICU days), mobilization (10%), glasses use (2.6%) and hearing aid use (0.3%). Presence of hearing or visual impairment at admission was not documented in 65% of patients. Erasmus MC University Medical Center, Rotterdam, the Netherlands Critical Care 2013, 17(Suppl 2):P397 (doi: 10.1186/cc12335) Introduction The beliefs, knowledge and practices regarding ICU delirium among ICU professionals may vary. This may interfere with the implementation of the Dutch ICU Delirium guideline. We aimed to get insight into potential barriers and facilitators for delirium guideline implementation that may help to fi nd an eff ective implementation strategy. Methods An online survey was sent to healthcare professionals from the six participating ICUs. Respondents included ICU physicians, nurses and delirium experts (psychiatrists, neurologists, geriatricians, nurse experts). The survey consisted of statements on beliefs, knowledge and practices towards ICU delirium. Agreement with statements by more than 75% of respondents were regarded as facilitating items and agreement lower than 50% as barriers for implementing protocolled care. Conclusion Daily screening for ICU delirium with a validated screening instrument is applied in less than one-half of the time in critically ill patients and management of delirium is often not guided by this screening. Haloperidol was used as the fi rst-choice medication. P395 Current practices in ICU delirium management: a prospective multicenter study in the Netherlands Z Trogrlic, E Ista, A Slooter, J Bakker, M Van der Jagt, on behalf of iDECePTIvE Study Group Erasmus MC University Medical Center, Rotterdam, the Netherlands Critical Care 2013, 17(Suppl 2):P395 (doi: 10.1186/cc12333) Results Of 768 eligible patients, complete data on delirium could be collected in 738 patients. The incidence of delirium was 14.2% in the dexamethasone group and 14.9% in the placebo group (odds ratio = 0.95, 95% CI = 0.63 to 1.43). No signifi cant diff erence was found on the duration of delirium between the intervention (median = 2 days, interquartile range 1 to 3  days) and placebo (median  =  2 days, interquartile range 1 to 2 days) group (P = 0.45). The use of restrictive measures and administration of sedatives, haloperidol, benzodiazepine and opiates were comparable between both groups. Introduction As part of a multicenter prospective study on barriers and facilitators for implementation of protocolled care for delirious critically ill patients, we aimed to describe current practices in delirium management in a representative cohort of ICU patients. p p g p Conclusion Intraoperative injection of dexamethasone seems not to aff ect the incidence or duration of delirium in the fi rst 4 days after cardiac surgery, suggesting this regimen is safe to use in the operative setting with respect to psychiatric adverse events. Reference Methods We included consecutively admitted patients during a 4-month period in a prospective multicenter study in six ICUs (one academic, teaching and nonteaching hospitals) in the southwest of the Netherlands. We assessed: percentage of patients screened for delirium with validated screening tools; pharmacological treatment with haloperidol or other antipsychotic drugs; psychohygiene (use of hearing aids and or glasses, preventing sleep disturbances); and access to early mobilization and physiotherapy. Delirium was pragmatically defi ned as administration of haloperidol and/or delirium reported by physicians or ICU nurses in patient records, as assessed by a designated research nurse. Diff erences between centers were tested with non- parametric tests. 1. Dieleman JM, et al.: JAMA 2012, 308:1761-1767. 1. Dieleman JM, et al.: JAMA 2012, 308:1761-1767. Intraoperative dexamethasone and delirium after cardiac surgery: a randomized clinical trial AC Sauër, AJ Slooter, DS Veldhuijzen, MM Van Eijk, D Van Dijk University Medical Center Utrecht, the Netherlands Critical Care 2013, 17(Suppl 2):P396 (doi: 10.1186/cc12334) Introduction The objective of this study is to investigate the eff ect of intraoperative administration of dexamethasone versus placebo on the incidence of delirium in the fi rst four postoperative days after cardiac surgery. Introduction The objective of this study is to investigate the eff ect of intraoperative administration of dexamethasone versus placebo on the incidence of delirium in the fi rst four postoperative days after cardiac surgery. Methods Within the context of the large multicenter Dexamethasone for Cardiac Surgery (DECS) trial [1] for which patients were randomized to 1 mg/kg dexamethasone or placebo at induction of anesthesia, a monocenter substudy was conducted. The primary outcome of this study was the incidence of delirium in the fi rst four postoperative days. Secondary outcomes were duration of delirium, use of restrictive measures and sedative, antipsychotic and analgesic requirements. Delirium was assessed daily by trained research personnel, using the Richmond Agitation Sedation Scale and the Confusion Assessment Method. Medical, nursing and medication charts were evaluated for signs of delirium and use of prespecifi ed medication. Analysis was by intention to treat. Conclusion We identifi ed multiple factors associated with PR use and number of days of PR use. Particularly among those related to treatment, strategies such as daily sedation interruption or use of antipsychotics are associated with PR use and increasing the number of days of use. P392 Safety profi le of 1,008 cases of propofol sedation in the emergency department Treatment characteristics associated with PR use were: daily benzodiazepine dose (OR = 1.01, 95% CI: 1.00 to 1.01), daily opiate dose (OR = 1.00, 95% CI: 1.00 to 1.01), daily sedation interruption (OR = 1.84, 95% CI: 1.24 to 2.72), use of antipsychotics (OR = 3.42, 95% CI: 1.98 to 5.91) and agitation (Sedation–Agitation Scale (SAS) >4, OR = 2.55, 95% CI: 1.20 to 5.44). Additionally, number of days of PR use was increased according to the following: daily sedation interruption (IRR  =  4.52, 95% CI: 2.09 to 9.75), use of antipsychotics (IRR = 18.65, 95% CI: 7.95 to 43.77), the presence of a heavily sedated score (SAS <3, IRR = 2.57, 95% CI: 1.06 to 6.24) or the presence of an adverse event, such as self- extubation (IRR = 10.46, 95% CI: 2.55 to 42.92). Hospital characteristics associated with PR use included the proportion of MV patients in the ICU. University hospitals were associated with reduced PR use, compared with community hospitals (OR = 0.272, 95% CI: 0.15 to 0.50). Nonmodifi able patient characteristics (age, sex, APACHE score, patient category, prior substance abuse, prior psychotropic medication, pre- existing psychiatric condition or dementia) were not associated with PR use. mobilization) were carried only in a small minority or were not documented. To implement protocolled delirium care in the region at study, a multifaceted tailored implementation program is needed. P396 Intraoperative dexamethasone and delirium after cardiac surgery: a randomized clinical trial AC Sauër, AJ Slooter, DS Veldhuijzen, MM Van Eijk, D Van Dijk University Medical Center Utrecht, the Netherlands Critical Care 2013, 17(Suppl 2):P396 (doi: 10.1186/cc12334) International validation of the delirium prediction model for ICU patients (PREDELIRIC): a multicenter observati onal study Methods We included patients in whom days with delirium could be compared with days without delirium, based on the Confusion Assessment Method for the ICU and inspection of medical records. Patients with conditions aff ecting thermal regulation, including infectious diseases, and those receiving therapies aff ecting body temperature were excluded. Twenty-four ICU patients were included after screening 334 delirious ICU patients. Daily temperature variability was determined by computing the mean absolute second derivative of the temperature signal. Per patient, temperature variability during delirious days was compared with nondelirium days using a Wilcoxon signed-rank test. With a linear mixed model, diff erences between delirium and nondelirium days with regard to temperature variability were analysed adjusted for daily mean Richmond Agitation and Sedation Scale scores, daily maximum Sequential Organ Failure Assessment score, and within-patient correlation. M van den Boogaard1, L Schoonhoven2, E Maseda3, C Plowright4, C Jones5, A Luetz6, PV Sackey7, P Jorens8, LM Aitken9, F van Haren10, JG van der Hoeven1, P Pickkers1 M van den Boogaard1, L Schoonhoven2, E Maseda3, C Plowright4, C Jones5, A Luetz6, PV Sackey7, P Jorens8, LM Aitken9, F van Haren10, JG van der Hoeven1, P Pickkers1 1Radboud University Nijmegen Medical Centre, Nijmegen, the Netherlands; 2Radboud University Nijmegen Medical Centre, Scientifi c Institute for Quality of Healthcare, Nijmegen, the Netherlands; 3Hospital Universitario La Paz, Madrid, Spain; 4Medway Maritime Hospital, Kent, UK; 5Whiston Hospital, Prescot, UK; 6Charité – Universitaetsmedizin Berlin, Germany; 7Karolinska University Hospital, Stockholm, Sweden; 8University of Antwerpen, Belgium; 9Princess Alexandra Hospital, Woolloongabba, Australia; 10Canberra Hospital, Canberra, Australia Critical Care 2013, 17(Suppl 2):P398 (doi: 10.1186/cc12336) Critical Care 2013, 17(Suppl 2):P398 (doi: 10.1186/cc12336) Critical Care 2013, 17(Suppl 2):P398 (doi: 10.1186/cc12336) Introduction Delirium is a serious and frequent disorder in ICU patients. The aim of this study was to internationally validate the existing Prediction of Delirium ICU (PREDELIRIC) model to predict delirium in ICU patients. Results Temperature variability was increased during delirium days compared with days without delirium (mean diff erence = –0.007, 95% CI = –0.004; –0.011, P <0.001). Adjusting for confounders did not alter our fi ndings (adjusted mean diff erence  =  –0.005, 95% CI  =  –0.008; –0.002, P <0.001). Methods A prospective multicenter cohort study was performed in eight ICUs in six countries (Table 1). The 10 predictors (age, APACHE II score, urgent and admission category, infection, coma, sedation, morphine use, urea level, metabolic acidosis) were collected within 24  hours after ICU admission. P398 International validation of the delirium prediction model for ICU patients (PREDELIRIC): a multicenter observati onal study M van den Boogaard1, L Schoonhoven2, E Maseda3, C Plowright4, C Jones5, A Luetz6, PV Sackey7, P Jorens8, LM Aitken9, F van Haren10, JG van der Hoeven1, P Pickkers1 Current perspectives, beliefs and practices concerning delirium in critically ill patients: a multicenter survey among Dutch healthcare professionals Measures aimed at delirium prevention (psychohygiene and early Results Of the 565 surveys distributed, 360 were completed (63.7%). The majority of respondents were ICU nurses (79%). Delirium was considered a major problem (83%) that requires adequate treatment (99%) and is underdiagnosed (81%). Respondents considered that S149 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 routine screening of delirium can improve prognosis (95%). However, only a minority (20%) answered that delirium is preventable. Only 39% of the respondents had received any training about delirium in the previous 3 years and 77% of them found training useful. The mean delirium knowledge score was 6.6 out of 10 (SD = 1.54). When all groups were mutually compared, nurses scored lower than delirium experts (ANOVA, P = 0.013). The respondents (58%; n = 210) from three ICUs indicated that CAM-ICU assessment was department policy. However, 50% (n = 106) of these respondents felt unfamiliar with CAM- ICU and only 47% (n = 99) of them indicated that a positive CAM-ICU was used for treatment decisions. Haloperidol was the fi rst-choice pharmacological treatment. Only 21% of all respondents knew that a national ICU delirium guideline existed, but in-depth knowledge was generally low. was 82 ± 16% and inter-rater reliability 0.87 ± 0.17. Overall delirium incidence was 19.9% (range 27.2%). Despite signifi cant diff erences between centres on all 10 predictors, the overall area under the receiver operating characteristic curve (AUROC) of the eight centers was 0.77 (95% CI: 0.74 to 0.79). was 82 ± 16% and inter-rater reliability 0.87 ± 0.17. Overall delirium incidence was 19.9% (range 27.2%). Despite signifi cant diff erences between centres on all 10 predictors, the overall area under the receiver operating characteristic curve (AUROC) of the eight centers was 0.77 (95% CI: 0.74 to 0.79). Conclusion The overall predictive value of the PREDELIRIC model was good, indicating that the prediction model can now be used internationally. P399 Delirium in ICU patients is associated with increased temperature variability AW Van der Kooi, TH Kappen, RJ Raijmakers, IJ Zaal, AJ Slooter University Medical Center Utrecht, the Netherlands Critical Care 2013, 17(Suppl 2):P399 (doi: 10.1186/cc12337) Delirium in ICU patients is associated with increased temperature variability Delirium in ICU patients is associated with increased temperature variability y AW Van der Kooi, TH Kappen, RJ Raijmakers, IJ Zaal, AJ Slooter University Medical Center Utrecht, the Netherlands Critical Care 2013, 17(Suppl 2):P399 (doi: 10.1186/cc12337) y AW Van der Kooi, TH Kappen, RJ Raijmakers, IJ Zaal, AJ Slooter University Medical Center Utrecht, the Netherlands Critical Care 2013, 17(Suppl 2):P399 (doi: 10.1186/cc12337) y Conclusion Our survey showed that healthcare professionals considered delirium an important but underdiagnosed form of organ failure. In contrast, screening tools for delirium are scarcely used, knowledge can be improved and protocolled treatment based on positive screening is often lacking. These results suggest that the focus of implementation of ICU delirium management should not be on motivational aspects, but on knowledge improvements, training in screening tools and implementation of treatment and prevention protocols. Introduction Delirium is an acute disturbance of consciousness and cognition. It is a common disorder in the ICU and associated with impaired long-term outcome [1,2]. Despite its frequency and impact, delirium is poorly recognized by ICU physicians and nurses using delirium screening tools [3]. A completely new approach to detect delirium is to use monitoring of physiological alterations. Temperature variability, a measure for temperature regulation, could be an interesting parameter for monitoring of ICU delirium, but this has never been investigated before. The aim of this study was to investigate whether temperature variability is aff ected during ICU delirium. International validation of the delirium prediction model for ICU patients (PREDELIRIC): a multicenter observati onal study CAM-ICU was used to identify ICU delirium. Conclusion Temperature variability is increased during delirium in ICU patients, which refl ects the encephalopathy that underlies delirium. Opportunities for delirium monitoring using temperature variability should be further explored. Particularly, in combination with electroencephalography it could provide the input for an objective tool to monitor delirium. Results A total of 2,852 adult ICU patients were screened and 1,824 (64%) were included. Main exclusion reasons were length of stay <1 day (19.1%) and sustained coma (4.1%). Mean  ±  CAM-ICU compliance Gastrointestinal symptoms in the ICU: a comparison of primary and secondary acute gastrointestinal injury A Reintam Blaser, J Starkopf , p University of Tartu, Tartu University Hospital, Tartu, Estonia y y Critical Care 2013, 17(Suppl 2):P401 (doi: 10.1186/cc12339) Introduction We aimed to clarify the diff erences between primary and secondary acute GI injury. Methods A total of 2,690 consecutive adult patients were retro- spectively studied during their fi rst week in the ICU. Pathology in the GI system or laparotomy defi ned the primary GI insult. If GI symptoms developed without primary GI insult it was considered secondary GI injury. Absent bowel sounds (BS), vomiting/regurgitation, diarrhoea, bowel distension, GI bleeding, and high gastric residuals (GRV >1,000 ml/24 hours) were recorded daily. ventilation with relative risk of 9 to 27% and with mortality of 25 to 50% [1,2]. One of the promoting factors of VAP is the increased pH of the gastric acid, which occurs when H2-receptor antagonists (H2RA) or proton pump inhibitors (PPI) are used for stress ulcer prophylaxis. The aim of this study was to investigate eff ects of PPI versus H2RA on the occurrence of VAP and, as second endpoint, the occurrence of gastrointestinal bleeding (GIB). y Results In total, 2,690 patients (60.4% male), median age 59 years (range 16 to 92), were studied. Eighty-four per cent of them were ventilated, 72% received vasopressor/inotrope. Median (IQR) APACHE II score was 14 (9 to 21) and SOFA on the fi rst day was 6 (3 to 10). A total 35.5% had primary GI pathology. During the fi rst week 34% of patients had absent BS, 38% vomiting/regurgitation, 9% diarrhoea, 7% bowel distension, 6% high GRV and 5% GI bleeding. All symptoms except diarrhoea occurred more often (<0.001) in patients with primary GI insult. Eighty-fi ve per cent of patients with primary GI insult versus 46% without developed at least one GI symptom. The incidence of GI symptoms was signifi cantly higher in nonsurvivors. ICU mortality was lower in patients with primary than secondary GI injury (43.6% vs. 61.2%, P = 0.046). Nonsurvivors without primary GI insult developed GI symptoms later (Figure 1).f Methods After ethics committee approval mechanically ventilated (>48  hours) adults were recruited in a prospective randomized controlled, double-blind clinical trial and randomised into PPI or H2RA groups. Patients with admission diagnosis of pneumonia or other acute infl ammatory pulmonary disease, severe sepsis/septic shock, and/or regular PPI/H2RA users were excluded. References 1. Pisani MA, et al.: Am J Respir Crit Care Med 2009, 180:1092-1097. 2. Ely EW, et al.: JAMA 2004, 291:1753-1762. 1. Pisani MA, et al.: Am J Respir Crit Care Med 2009, 180:1092-1097. 2 El EW t l JAMA 2004 291 1753 1762 Table 1 (abstract P398). PREDELIRIC AUROC of diff erent centers Table 1 (abstract P398). PREDELIRIC AUROC of diff erent centers Table 1 (abstract P398). PREDELIRIC AUROC of diff erent centers Delirium (%) AUROC 95% CI Belgium (%) 86 (15.2) 0.79 0.74 to 0.84 Germany (%) 60 (26.9) 0.85 0.80 to 0.91 Spain (%) 23 (18.0) 0.88 0.81 to 0.94 Sweden (%) 30 (39.0) 0.71 0.60 to 0.83 Australia Brisbane (%) 42 (12.8) 0.81 0.75 to 0.88 Australia Canberra (%) 23 (11.8) 0.80 0.72 to 0.89 UK_Prescot (%) 73 (30.8) 0.65 0.57 to 0.73 UK_Kent (%) 26 (36.6) 0.76 0.65 to 0.88 PREDELIRIC overall 363 (19.9) 0.77 0.74 to 0.79 y 3. van Eijk MM, et al.: Am J Respir Crit Care Med 2011, 184:340-344. P400 Long-term outcome of delirium in critically ill patients A Wolters, D Van Dijk, O Cremer, D De Lange, A Slooter UMC Utrecht, the Netherlands Critical Care 2013, 17(Suppl 2):P400 (doi: 10.1186/cc12338) Long-term outcome of delirium in critically ill patients A Wolters, D Van Dijk, O Cremer, D De Lange, A Slooter UMC Utrecht, the Netherlands Critical Care 2013, 17(Suppl 2):P400 (doi: 10.1186/cc12338) Introduction In ICU patients, little research has been performed on the relationship between delirium and long-term outcome, including health-related quality of life (HRQoL), cognitive functioning and mortality. In addition, results seem to be inconsistent. Furthermore, in studies that reported increased mortality in delirious patients, no proper adjustments were made for severity of illness during ICU admission. This study was conducted to investigate the association S150 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Figure 1 (abstract P401). Daily dynamics of the number of GI symptoms. P <0.001 between survivors and nonsurvivors in all time points. between ICU delirium and long-term HRQoL, cognitive functioning and mortality. The hypothesis was that delirious patients have worse long-term outcome in comparison with nondelirious patients. between ICU delirium and long-term HRQoL, cognitive functioning and mortality. The hypothesis was that delirious patients have worse long-term outcome in comparison with nondelirious patients. Methods A prospective observational cohort study was conducted. Gastrointestinal symptoms in the ICU: a comparison of primary and secondary acute gastrointestinal injury The diagnostic criteria for VAP were: leukocytosis, increased PCT level, fever, purulent tracheal secretion, positive microbiological result of tracheal sample, and new/ increased infi ltrate on chest X-ray. Data are presented as median (interquartile range, IQR). For statistical analysis Mann–Whitney U test and chi-square tests were used. q Results Out of 94 patients recruited 14 were excluded due to early weaning/death (n  =  9) or protocol violation (n  =  6). The remaining 79 patients were analysed (PPI, n  =  38; H2RA, n  =  41). There was no signifi cant diff erence (P  >0.05) between the groups regarding demographics: age 67 (56 to 77) versus 72 (58 to 79)  years; male/ female: 23/15 versus 25/16; length of mechanical ventilation: 5 (3 to 9) versus 5 (2 to 8) days; APACHE II score: 28 (22 to 32) versus 26 (21 to 36) (PPI vs. H2RA, respectively). There was no signifi cant diff erence in the number of cases with VAP in the PPI versus H2RA groups: 9 (24%) versus 10 (24%). None of the patients developed GI bleeding during their stay on the ICU. Conclusion Primary and secondary acute GI injury have diff erent incidence, dynamics and outcome. References A median of 12 months after ICU discharge, questionnaires were sent to all survivors. HRQoL and cognitive functioning were measured with the EuroQol-6D. Age, gender and severity of illness were considered relevant covariates. Severity of illness was estimated using the APACHE IV score and the maximal SOFA score during admission. HRQoL was investigated with linear regression analysis, cognitive functioning using logistic regression and mortality with Cox regression analysis. y y Results The patient population consisted of 690 patients admitted to the ICU, subdivided into delirious (n = 257) and nondelirious patients (n = 433). During follow-up, 181 (26%) patients died. The response rate of the questionnaire was 70.6%. After adjusting for the predefi ned covariates, delirium was signifi cantly associated with a lower HRQoL (adjusted β: –0.137; 95% CI  =  –0.140 to –0.005) and more mild and severe cognitive impairment (adjusted odds ratio: respectively: 2.3; 95% CI = 1.3 to 4.2 and 5.8; 95% CI = 1.3 to 15.2). No signifi cant association between delirium and long-term mortality was found (adjusted hazard ratio: 1.0; 95% CI = 0.7 to1.4). Conclusion Delirium during ICU admission was associated with lower HRQoL and worse cognitive functioning, 1 year after discharge. Furthermore, delirium on the ICU was not associated with long-term mortality after adjusting for relevant covariates, including severity of illness during ICU admission. Early diagnosis of liver failure in septic patients using the maximal liver function capacity test (LiMAx test): comparison with conventional methods M Kaff arnik, M Stockmann, J Lock Charite Berlin Campus Virchow, Berlin, Germany Critical Care 2013, 17(Suppl 2):P403 (doi: 10.1186/cc12341) Introduction Patients with bacterial sepsis often suff er from multiorgan failure. No reliable parameter exists for the exact estimation of the liver function. The indocyaningreen test (ICG test) showed a correlation between liver function and mortality rate. The new maximal liver function capacity test (LiMAx test) is a real-time method to investigate the liver function. g p pp Results HVPG increased in Group 1 from 3.7 to 5.4 mmHg (P <0.05) and decreased in Group 2 (4.8 to 4.3 mmHg, P = NS) (Table 1). Liver function as assessed by ICG-PDR decreased in Group 1 by day 1 (18.5 to 15.3, P <0.05) and remained stable in Group 2 (25.5 to 26.8, P = NS). Renin, aldosterone, ADH, adrenaline, noradrenaline and dopamine increased signifi cantly (P <0.05) in Group 1 during operation. Group 2 showed a signifi cant rise only in ADH and dopamine. Acute kidney injury occurred in fi ve of 13 patients in Group 1 (P <0.05). Methods Thirty septic patients were prospectively included in the study. The LiMAx test was performed on days 0, 2, 5 and 10 after sepsis onset and was compared with ICG test and liver-specifi c laboratory parameters. Primary endpoint was the mortality rate after 90 days. Secondary endpoint was the comparison with the ICG test. Results The LiMAx test showed low results initially with increasing values on days 5 and 10. The 90-day mortality rate in patients with a low LiMAx test on day 2 (<100 μg/kg/hour) was signifi cantly higher than in patients with a LiMAx test >100 μg/kg/hour. The LiMAx test was comparable with the ICG test. Patients with indication for renal replacement therapy during hospital treatment showed signifi cantly lower LiMAx values than patients without dialysis. See Figure 1. Table 1 (abstract P404) Group 1 Group 2 Renin 36.4 7.9 PostOP 189.7* 30.5 Day 1 258 31.3 ADH 3.49 2.59 PostOP 16.11* 10.8* Day 1 10.8 2.39 Creatine 0.8 0.83 Day 1 1.25* 0.91 *P <0.05 compared with preOP value. p y g Conclusion With the LiMAx test we can detect a liver dysfunction in septic patients early on days 0 to 2. P403 P403 Early diagnosis of liver failure in septic patients using the maximal liver function capacity test (LiMAx test): comparison with conventional methods M Kaff arnik, M Stockmann, J Lock Charite Berlin Campus Virchow, Berlin, Germany Critical Care 2013, 17(Suppl 2):P403 (doi: 10.1186/cc12341) Early diagnosis of liver failure in septic patients using the maximal liver function capacity test (LiMAx test): comparison with conventional methods The LiMAx test is equal with the ICG test and a result of <100  μg/kg/hour on day 2 suggests a low probability of survival in septic patients. 1. Stockmann et al.: New liver function test for prediction of postoperative outcome in liver surgery. HPB 2010, 12:139. 2. Lock et al.: Early diagnosis of primary nonfunction and indication for reoperation after liver transplantation. Liver Transpl 2010, 16:172. 2. Lock et al.: Early diagnosis of primary nonfunction and indication for reoperation after liver transplantation. Liver Transpl 2010, 16:172. Figure 1 (abstract P403). Kaplan–Meier survival curve of patients with high and low LiMAx results. Conclusion Depending on resection size liver resection acutely increases portal venous pressure and induces neurohumoral activation resulting in compromised renal function and increased risk of developing AKI. P402f Eff ects of proton pump inhibitor versus H2-receptor antagonist stress ulcer prophylaxis on ventilator-associated pneumonia: a pilot study JF Fogas, KK Kiss, FG Gyura, ZT Tóbiás, ZM Molnár University of Szeged, Hungary Critical Care 2013, 17(Suppl 2):P402 (doi: 10.1186/cc12340) Eff ects of proton pump inhibitor versus H2-receptor antagonist stress ulcer prophylaxis on ventilator-associated pneumonia: a pilot study JF Fogas, KK Kiss, FG Gyura, ZT Tóbiás, ZM Molnár University of Szeged, Hungary Critical Care 2013, 17(Suppl 2):P402 (doi: 10.1186/cc12340) University of Szeged, Hungary Critical Care 2013, 17(Suppl 2):P402 (doi: 10.1186/cc12340) y Conclusion The results of this pilot study suggest that there may be no diff erence in the incidence of VAP and GI bleeding if stress ulcer prophylaxis is performed by H2RA or PPI. As the latter is more expensive, its use as fi rst choice in critical care should be questioned. Introduction Ventilator-associated pneumonia (VAP) is the most frequent ICU-acquired infection among patients receiving mechanical S151 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 However, the completion of the study on the planned 198 patients is required to come to the fi nal conclusions. References 1. Efrati S, et al.: J Clin Monit Comput 2010, 24:161-168. 2. Torres A, et al.: Intensive Care Med 2009, 35:9-29. However, the completion of the study on the planned 198 patients is required to come to the fi nal conclusions. References P Biesenbach, T Grünberger, B Payer, A Ferlitsch, W Druml, E Fleischmann, F Luf, J Mühlbacher Medical University of Vienna, Austria Critical Care 2013, 17(Suppl 2):P404 (doi: 10.1186/cc12342) Introduction Liver dysfunction  – in correlation to the severity of functional impairment – but also any increase in portal pressure per se (hepatorenal refl ex) can induce alterations in renal function and ultimately result in hepatorenal syndrome (HRS). In this prospective investigation we determined the impact of liver resection on portal venous pressure by measuring the hepatic venous pressure gradient (HVPG), on concentrations of vasoactive peptides and on renal function. Methods Twenty patients (mean age 66.3 years) undergoing elective liver resection surgery because of malignant tumor were assessed and grouped according to resection size: (Group 1) hemihepatectomy, n = 13 versus (Group 2) segmentectomy, n = 7. HPVG was measured before and after resection by canulation of a hepatic vein under fl uoroscopic guidance, liver function was assessed by the indocyanine green plasma disappearance rate (ICG-PDR). However, the completion of the study on the planned 198 patients is required to come to the fi nal conclusions. References 1. Efrati S, et al.: J Clin Monit Comput 2010, 24:161-168. 2. Torres A, et al.: Intensive Care Med 2009, 35:9-29. Impact of liver resection on portal venous pressure and renal function p p p function P Biesenbach, T Grünberger, B Payer, A Ferlitsch, W Druml, E Fleischmann, F Luf, J Mühlbacher Medical University of Vienna, Austria Critical Care 2013, 17(Suppl 2):P404 (doi: 10.1186/cc12342) 1. Efrati S, et al.: J Clin Monit Comput 2010, 24:161-168. 2. Torres A, et al.: Intensive Care Med 2009, 35:9-29. Does just fungal evidence increase the mortality of crit patients with liver cirrhosis? A retrospective analysis patients with liver cirrhosis? A retrospective analysis T Lahmer, U Mayr, M Messer, B Saugel, C Schultheiss, R Schmid, W Huber University Hospital Munich, Germany Introduction Patients with liver cirrhosis (LC) are susceptible to a variety of complications. Especially, infection disorders can reduce their life expectancy markedly. The aim of this retrospective analysis was to detect the incidence and prognosis in patients with LC and fungal evidence (FE). Table 1 (abstract P407) Variable AUC Standard error 95% CI APACHE II score 0.934 0.023 0.889 to 0.980 SOFA score 0.908 0.026 0.857 to 0.960 Number OF 0.901 0.026 0.849 to 0.952 AUC, areas under the curves. Methods A 3-year retrospective analysis of ICU patients with LC. Patients with LC with/without FE were compared with a matched control group based on comparable age and APACHE II score. Specimens were collected based on clinical and/or laboratory (infection signs) reasons and were analyzed by microbiological standard methods. Results A total of 101 patients with LC were enrolled in this analysis. Basic data for both groups: Child–Pugh Score: 4*A, 18*B, 79*C; reason for ICU admission: 28*hepatorenal syndrome, 18*SBP, 20*GI bleeding, 19*sepsis, 16*pneumonia; etiology of LC: 83*alcoholic, 8*NASH, 8*viral (hepatitis B/C), 2*PSC. Fifty-fi ve (54%) patients with LC had FE (mean age: 57.6 ± 14 years, sex M/F: 40/15, APACHE II score 27 ± 2). Compared with the 46 patients with LC but without FE (mean age: 59.2 ± 12 years, sex M/F: 35/11, APACHE II score 23 ± 4), these patients have a signifi cantly higher APACHE II score (27 ± 2 vs. 23 ± 4; P <0.001) and signifi cantly higher mortality (43/55 (78%) vs. 15/46 (33%); P <0.001). Multivariate analysis showed that FE is associated with a higher mortality (P <0.001) but is not associated with age, sex or CHILD score. Seventeen (31%) of the 55 patients in the control group had FE (screening patients n = 147, mean age 65.8 ± 12 years, sex M/F: 35/20, APACHE II score 26.5 ± 3, reason for ICU admission: 19*pneumonia, 16*sepsis, 9*intracerebral haemorrhage, 7*acute abdomen, 4*lymphatic disorder). The mortality rate was signifi cantly lower (27/55 (49%) vs. 43/55 (78%); P = 0.003) as compared with patients with LC and FE. The main localisation of fungal was in both groups the lung (46 vs. 14), followed by urine (18 vs. 2), blood culture (6 vs. 1) and ascites (4 vs. 0). P405 P405 Value of the preoperative Model for End-stage Liver Disease score in predicting postoperative outcome in living donor liver transplantation recipients Y Nassar, K Omar, A Battah, A Mwafy Cairo University, Giza, Egypt Critical Care 2013, 17(Suppl 2):P405 (doi: 10.1186/cc12343) P405 Value of the preoperative Model for End-stage Liver Disease score in predicting postoperative outcome in living donor liver transplantation recipients Y Nassar, K Omar, A Battah, A Mwafy Cairo University, Giza, Egypt Critical Care 2013, 17(Suppl 2):P405 (doi: 10.1186/cc12343) P405 Value of the preoperative Model for End-stage Liver Disease score in predicting postoperative outcome in living donor liver transplantation recipients Cairo University, Giza, Egypt Cairo University, Giza, Egypt y gyp Critical Care 2013, 17(Suppl 2):P405 (doi: 10.1186/cc12343) Introduction Partial liver grafting predisposes recipients to a unique set of potential technical and anatomic complications that are not prevalent in whole deceased donor grafts. We aimed to assess the preoperative Model for End-stage Liver Disease (MELD) score, postoperative 30-day clinical and laboratory follow-up, and detection of the value of MELD score in predicting 30-day postoperative outcome of LDLT recipients. Figure 1 (abstract P403). Kaplan–Meier survival curve of patients with high and low LiMAx results. Methods A prospective multicenter registry involving 142 patients who underwent LDLT from October 2004 to December 2010. Preoperative S152 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 and long lengths of stay [1]. Survival among patients with predicted SAP at admission has been shown to correlate with the duration of organ failure (OF) [2]. The systemic determinant of severity in a new classifi cation of acute pancreatitis (AP) is also based on identifi cation of patients with transient or persistent OF [3]. MELD score was calculated: (0.378×log (bilirubin) (mg/dl) + 1.120×log (INR)  + 0.957×log (creatinine) (mg/dl)  + 0.643)×10 then rounded to the nearest integer. Postoperative daily recording of all the clinical and laboratory data was done for a period of 30 days. Results The survival rate recorded in our study was 86.62%. The most frequent complications were renal complications (86.6%), pulmonary complications (73.9%), neurological complications (14%), cardiovascular complications (12.6%), infectious complications (13.3%), intra-abdominal infections (10.5%) and immunosuppressant toxicity (7.7%). MELD score proved to be a good predictor of perioperative outcome, patients with MELD score (>12) was associated with less favorable outcome, accuracy was (80.3%). P407 0 Predictors of persistent organ failure in ICU patients with severe acute pancreatitis P408 Increased plasma levels of thrombopoietin in patients with severe acute pancreatitis 1Città della Salute e della Scienza Hospital, Torino, Italy; 2University of Torino, Italy y Critical Care 2013, 17(Suppl 2):P408 (doi: 10.1186/cc12346) Introduction The aim of this study was to evaluate the accuracy of thrombopoietin (TPO) plasma levels as a biomarker of clinical severity in patients with acute pancreatitis (AP). TPO is a humoral growth factor that stimulates megakaryocyte proliferation and diff erentiation [1]. Furthermore, it favors platelet aggregation and polymorphonuclear leukocyte activation [2]. Elevated plasmatic concentrations of TPO have been shown in patients with critical diseases, including ACS, burn injury and sepsis [2]. In particular, clinical severity is the major determinant of elevated TPO levels in patients with sepsis [3]. AP is a relatively common disease whose diagnosis and treatment are often diffi cult, especially in the clinical setting of the emergency department (ED). About 20% of patients with AP develop a severe form of the disease. In order to early identify those patients aff ected by severe AP, several biomarkers have been studied. No data regarding TPO plasma levels in patients with AP are currently available. y p Conclusion ICU patients with LC have a high incidence of FE. The mortality rate is therefore signifi cantly higher as compared with patients with LC without FE and also as compared with a control group without LC. Therefore early antimycotic treatment should be considered. P405 Methods The aim of the study was to retrospectively determine the predictors of early persistent OF in ICU patients with SAP. The analysis involved 152 patients. The median time interval between the onset of AP and admission was 24 (9; 48) hours. The patients were divided into two groups: the fi rst group (n = 46) had transient OF and the second group (n = 106) had persistent OF. The ability of the APACHE II score, total SOFA score and number of organ/system failure to discriminate transient from persistent OF was explored with receiver operating characteristic (ROC) curves.i Conclusion MELD score >12 was associated with less favorable outcome with an accuracy of 80.3%. Results Hospital mortality was signifi cantly higher in the second group as compared with the fi rst group (45% vs. 7%, P = 0.000); while infectious complications were 39% versus 11% (P = 0.001) and median lengths of ICU stay were 8 (5; 18) days for the second group and 6 (4; 7) days for the fi rst group (P = 0.001). Optimum cutoff levels (by ROC curve analysis) were APACHE II score ≥12 (sensitivity 0.790; 1  – specifi city 0.119), total SOFA score ≥4 (sensitivity 0.829; 1 – specifi city 0.190), and failure ≥2 organs/systems (sensitivity 0.819; 1 – specifi city 0.214). See Table 1. Does just fungal evidence increase the mortality of crit patients with liver cirrhosis? A retrospective analysis Predominantly candida species were detected (especially C. albicans/glabrata), fi ve patients in the LC group had aspergillus fumigatus. In LC patients, FE could be detected in many cases in several compartments. Conclusion Persistent OF in ICU patients with SAP is associated with the highest risk of hospital mortality. Early identifi cation of patients with a high likelihood of persistent OF (APACHE II score ≥12, total SOFA score ≥4, failure ≥2 organs/systems) is an important goal in determining optimal management of ICU patients with SAP. p References References 1. Harrison DA, et al.: Crit Care 2007, 11(Suppl 1):S1. 2. Johnson CD, et al.: Gut 2004, 53:1340-1344. 3 D lli EP t l A S 2012 [E b h d f i t] 3. Dellinger EP, et al.: Ann Surg 2012. [Epub ahead of print] P411 Urinary neutrophil gelatinase-associated lipocalin as an early predictor of acute kidney injury in cardiac surgery patients T García Rodríguez San Miguel Hospital Santa Creu i Sant Pau, Barcelona, Spain Critical Care 2013, 17(Suppl 2):P411 (doi: 10.1186/cc12349) Urinary neutrophil gelatinase-associated lipocalin as an early predictor of acute kidney injury in cardiac surgery patients T García Rodríguez San Miguel Hospital Santa Creu i Sant Pau, Barcelona, Spain Critical Care 2013, 17(Suppl 2):P411 (doi: 10.1186/cc12349) Introduction To evaluate whether urinary neutrophil gelatinase- associated lipocalin (uNGAL) detects acute kidney injury (AKI) earlier than the estimated glomerular fi ltration rate (eGFR) in cardiac surgery patients. Introduction The objective was to evaluate the eff ect of the renal dysfunction measured by pRIFLE on clinical outcome in children admitted to the pediatric intensive care unit (PICU). Introduction The objective was to evaluate the eff ect of the renal dysfunction measured by pRIFLE on clinical outcome in children admitted to the pediatric intensive care unit (PICU). p Methods Two-hundred and seventy-four adult patients undergoing cardiac surgery were consecutively included from February to December 2011. Exclusion criteria were absence of diuresis due to end-stage renal disease or chronic renal failure and a previous cardiac catheterism with i.v. contrast use the week before surgery. Four serial blood and urine samples immediately before (PRE) and after (POST) surgery, and 1 day (1d) and 2 days (2d) after surgery were obtained. uNGAL was measured in an Architect 6200 (Abbott Diagnostics). AKIN criteria were used to diagnose AKI. The study was approved by the local ethics committee and all patients gave informed consent. Delta uNGAL was defi ned as the diff erence between the PRE and the POSTs concentrations. Methods A retrospective cohort involving all children admitted between August 2009 and July 2010 to a Brazilian PICU located in a university-affi liated hospital. Patients were classifi ed according to the pRIFLE at admission as well the maximum pRIFLE during the PICU stay. The endpoints were: length of stay (LOS) in the PICU, LOS on mechanical ventilation, mortality rate quoted by predicted mortality of the Pediatric Index of Mortality (PIM2). y Results In total, 375 children were included in the study. A total 169 (45%) of them presented renal dysfunction according to the pRIFLE. Renal injury and renal failure were associated with higher LOS in the PICU and length of mechanical ventilation. The observed and predicted mortality by PIM2 did not show diff erences. However, the more severe renal presentation (Injury and Failure) was associated with higher mortality rate and was not predicted by PIM2 (8% vs. 11%, P <0.01). Thorough evaluation for the new acute kidney injury criteria by Kidney Disease Improving Global Outcomes Thorough evaluation for the new acute kidney injury criteria by Kidney Disease Improving Global Outcomes Thorough evaluation for the new acute kidney injury criteria by Kidney Disease Improving Global Outcomes y J Izawa, S Uchino, T Fujii, T Arii, T Fukushima, S Kawano Jikei University School of Medicine, Tokyo, Japan Critical Care 2013, 17(Suppl 2):P410 (doi: 10.1186/cc12348) J Izawa, S Uchino, T Fujii, T Arii, T Fukushima, S Kawano Jikei University School of Medicine, Tokyo, Japan Critical Care 2013, 17(Suppl 2):P410 (doi: 10.1186/cc12348) Introduction Two previous classifi cations of acute kidney injury (AKI) that are known as RIFLE criteria and AKIN criteria have shown that AKI is associated with increased morbidity and mortality. Diff erences in predicting ability for prognosis, however, have been reported. In 2012, Kidney Disease Improving Global Outcomes (KDIGO) created the new AKI criteria, combining RIFLE and AKIN criteria. However, such a combination might cause inconsistency among each defi nition in the criteria. We have investigated all of the defi nitions in the new KDIGO criteria in detail. Methods This is a retrospective historical cohort study including adult patients admitted to the ICU (Jikei University, Tokyo, Japan) between January 2010 and October 2011. Patients undergoing chronic dialysis were excluded. KDIGO criteria were applied to all patients to diagnose AKI. Hospital mortality of patients with AKI diagnosed by the 10 defi nitions in the criteria was compared. Results We studied 41 patients with AP (17 severe and 24 mild pancreatitis). No diff erences for gender and age were detected between patients with mild and severe disease. TPO plasma levels were signifi cantly higher in patients with severe AP (99.33 ± 23.68 pg/ ml) than in those with mild AP (50.81  ±  6.73  pg/ml) (Figure  1). The ROC curve led us to calculate a cutoff value of 51.55 pg/ml. This value identifi ed correctly 12 out of the 17 patients aff ected by severe AP. i Results A total of 2,399 patients were evaluated. AKI occurred in 26.6% with standard defi nition of KDIGO; 18.0% with creatinine criteria alone; 15.5% with urine output alone. By multivariable analysis, each AKI stage was associated with hospital mortality: 5.6%, odds ratio 2.95, for Stage 1; 10.1%, odds ratio 5.52, for Stage 2; 30.2%, odds ratio 21.30, for Stage  3. Crude hospital mortality stratifi ed by the 10 defi nitions showed increasing trends with stage progression. P411 Urinary neutrophil gelatinase-associated lipocalin as an early predictor of acute kidney injury in cardiac surgery patients T García Rodríguez San Miguel Hospital Santa Creu i Sant Pau, Barcelona, Spain Critical Care 2013, 17(Suppl 2):P411 (doi: 10.1186/cc12349) Results One-hundred and eighty-one patients (66.1%) were men; mean age was 68.2  ±  12.2 years. Valve replacement was performed in 123, coronary artery bypass graft (CABG) in 81, valve surgery + CABG in 48, cardiac transplant in fi ve, aorta aneurism surgery in nine, and other procedures in eight patients. ICU and hospital stays were 6.7 ± 8.1 and 15.7 ± 13.9 days, respectively. Renal replacement therapy (RRT) was required in 16 patients (5.8%) within 48 hours of ICU stay and in 28 patients (10.2%) within 4 weeks. Mortality at 28 days was 2.9%. Eighty-six patients (31.4%) were diagnosed with AKI within 48 hours of surgery. Area under the ROC curve of POST uNGAL for AKI diagnosis was 0.72 (0.66 to 0.79) (P <0.0001) at an optimal cutoff value of 180 μg/l, Conclusion Renal dysfunction measured by pRIFLE is a very common fi nding (45%) among pediatric patients admitted to the PICU. The more severe renal aff ection is associated with higher mortality, length of MV and length of PICU stay. Thorough evaluation for the new acute kidney injury criteria by Kidney Disease Improving Global Outcomes Mortality of the three defi nitions in Stage 1 was from 4.2% to 8.8%. Stage 2 had two defi nitions and their mortality was 12.1% and 12.9%. Stage 3 had fi ve defi nitions and their mortality was from 20.5% to 55.6%.i i yf y Conclusion TPO may be proposed as biomarker of clinical severity in patients with AP at the time of fi rst evaluation in the ED. Further studies, involving larger numbers of patients, are needed in order to validate these preliminary data. References 1. Kaushansky K: J Thromb Haemost 2003, 1:1587-1592. 2. Lupia E, et al.: Mediators Infl amm 2012, in press. 3. Zakynthinos SG, et al.: Crit Care Med 2004, 32:1004-1010. i Conclusion AKI defi ned by the new KDIGO criteria was associated with increased hospital mortality. In addition, defi nitions in the KDIGO criteria seem to be appropriate because of clear relations between mortality and stage progression. P409 P409 Severe renal dysfunction measured by pRIFLE in children is an independent factor associated with death and not predicted by PIM2 J Piva1, P Lago2, G Pedro3, F Cabral3 1UFRGS, Porto Alegre, Brazil; 2Hospital de Clinicas de Porto Alegre, Brazil; 3PUCRS, Porto Alegre, Brazil Critical Care 2013, 17(Suppl 2):P409 (doi: 10.1186/cc12347) Predictors of persistent organ failure in ICU patients with severe acute pancreatitis Predictors of persistent organ failure in ICU patients with severe acute pancreatitis I Aleksandrova, M Ilynskiy, S Rei, V Kiselev, G Berdnikov, L Marchenkova Hospital Research Institute for Emergency Medicine N.V. Sklifosovsky, Moscow, Russia Critical Care 2013, 17(Suppl 2):P407 (doi: 10.1186/cc12345) I Aleksandrova, M Ilynskiy, S Rei, V Kiselev, G Berdnikov, L Marchenkova Hospital Research Institute for Emergency Medicine N.V. Sklifosovsky, Moscow, Russia Critical Care 2013, 17(Suppl 2):P407 (doi: 10.1186/cc12345) Methods We enrolled patients with AP at the moment of the fi rst clinical evaluation in the ED. AP patients were classifi ed as having mild or severe forms of AP on the basis of the APACHE II score (≥8). TPO concentrations were determined by ELISA. Introduction Severe acute pancreatitis (SAP) requiring admission to an ICU is associated with high mortality (hospital mortality reached 42%) S153 Critical Care 2013, Volume 17 Suppl 2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Figure 1 (abstract P408). Plasma TPO levels in patients with AP. 2. Hassinger AB, Becker CL: Predictive power of serum cistatin C. Pediatr Crit Care Med 2012, 13:435-440. 1. Picca S, Picardo S: Acute kidney injury in an infant after cardiopulmonary bypass. Semin Nephrol 2008, 28:470-476. P413 Perioperative sodium bicarbonate to prevent acute kidney injury after cardiac surgery: a multicenter double-blind randomized controlled trial A Haase-Fielitz1, M Haase1, M Plass2, P Murray3, M Bailey4, R Bellomo5, S Bagshaw6 Perioperative sodium bicarbonate to prevent acute kidney injury after cardiac surgery: a multicenter double-blind randomized controlled trial Atrial natriuretic peptide attenuates metabolic acidosis and infl ammation of the kidney, lung and heart in a rat model of renal ischemia–reperfusion injury Reference 1. Vollmar AM: The role of atrial natriuretic peptide in the immune system. Peptide 2005, 26:1086-1094. Introduction Renal ischemia–reperfusion injury (IRI) is a common cause of acute kidney injury and occurs in various clinical conditions including shock and cardiovascular surgery. Renal IRI releases proinfl ammatory cytokines within the kidney. Atrial natriuretic peptide (ANP) has natriuretic, diuretic and anti-infl ammatory eff ects [1] and plays an important role of regulating blood pressure and volume homeostasis. The hypothesis was that renal IRI induces infl ammation not only in the kidney but also in remote organs such as the lung and heart and ANP attenuates renal injury and infl ammation in the kidney, lung and heart. References 1. Picca S, Picardo S: Acute kidney injury in an infant after cardiopulmonary bypass. Semin Nephrol 2008, 28:470-476. 1. Picca S, Picardo S: Acute kidney injury in an infant after cardiopulmonary bypass. Semin Nephrol 2008, 28:470-476. 2. Hassinger AB, Becker CL: Predictive power of serum cistatin C. Pediatr Crit Care Med 2012, 13:435-440. S154 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 with 78.7% specifi city, 64% sensitivity and 74.1% accuracy. uNGAL advanced diagnosis of AKI in 44 patients (51.2%), whereas diagnosis was achieved at the same time as AKI criteria in 11 patients; AKI criteria outperformed uNGAL in only 36% of cases. Accordingly, uNGAL was useful to diagnose postoperative AKI in 63.9% of cases. Median delta uNGAL was 12.5 (from –1.9 to 71.1) and 154.5 (from 16.6 to 484.5) μg/l in non-AKI and AKI patients, respectively (P <0.0001) and its area under the ROC curve for AKI prediction was 0.70 (0.63 to 0.77) (P <0.0001). measured. The kidney, lung and heart were immunostained to examine the localization of IL-6 and NF-κB and assigned an expression score. The wet/dry ratio of the lung was also measured. Results Renal IRI induced metabolic acidosis, pulmonary edema, mRNA expression of IL-6 in the kidney, lung and heart. Renal IRI increased immunohistochemical localization of IL-6 in the proximal convoluted tubule of the left kidney and NF-κB in the bronchial epithelial cells of the lung. ANP attenuated metabolic acidosis, pulmonary edema and expression of IL-6 mRNA in the kidney, heart, and lung. ANP decreased immunohistochemical localization of IL-6 in the left kidney and NF-κB in the lung. p Conclusion Compared with AKIN criteria, a urinary NGAL concentration >180 μg/l anticipates AKI diagnosis in more than 50% of cardiac surgery patients in the fi rst 24 to 48 hours after intervention. Conclusion These fi ndings suggested that infl ammation within the kidney after renal IRI was extended into the lung and heart. ANP attenuated metabolic acidosis and infl ammation in the kidney, lung and heart in a rat model of renal IRI. ANP may attenuate organ cross- talk between the kidney, lung and heart. P412 P412 Atrial natriuretic peptide attenuates metabolic acidosis and infl ammation of the kidney, lung and heart in a rat model of renal ischemia–reperfusion injury M Khin Hnin Si, C Mitaka, M Tulafu, S Abe, S Ikeda Tokyo Medical and Dental University Graduate School, Tokyo, Japan Critical Care 2013, 17(Suppl 2):P412 (doi: 10.1186/cc12350) P414 p Methods We connected the urinary catheter of 39 ICU patients to a quasi-continuous urine analyzer (Kidney INstant monitorinG®), allowing measurement of pH (pHU), sodium, chloride, potassium and ammonium concentrations (Na+U, Cl–U, K+U, NH4+U) every 10 minutes. The study period lasted 3 hours after a single intravenous bolus of furosemide (time 0). In 13 patients receiving two or more administrations over a longer period (46 (26 to 49) hours), according to clinical needs, we reviewed data on fl uid therapy, hemodynamics and acid–base balance from the beginning to the end of the observation. Introduction Here, we compare the value of acute tubular damage markers measured early in the course after cardiac surgery for prediction of postoperative AKI compared with conventional markers used in clinical routine for risk assessment of acute renal function loss. Methods One hundred adult patients undergoing cardiac surgery in the control arm of a RCT (NCT00672334) were analyzed. We quantifi ed the following biomarkers in urine: NGAL, hepcidin, midkine, IL-6, α1- microglobulin; in plasma: NGAL, hepcidin, haptoglobin, CK, CKMB, CRP, leukocytes, lactate, urea and creatinine. Introduction Here, we compare the value of acute tubular damage markers measured early in the course after cardiac surgery for prediction of postoperative AKI compared with conventional markers used in clinical routine for risk assessment of acute renal function loss. Results Ten minutes after furosemide administration, Na+U and Cl–U rose from 65 ± 6 to 140 ± 5 and from 109 ± 6 to 150 ± 5 mEq/l respectively, while K+U fell from 60 ± 5 to 39 ± 4 mEq/l (P <0.001 for all electrolytes vs. time 0) with a consequent increase in urinary anion gap (AGU = Na+U + Cl–U – K+U). Urinary output increased from 10 (5 to 19) to 53 (29 to 71) ml/10 minutes (P <0.05). After the fi rst hour Cl–U remained higher than Na+U, which progressively decreased, leading to a reduction in AGU and pHU over time. In parallel, a progressive increment in NH4+U was observed. In patients receiving more than one administration we observed an increase in arterial base excess (1.8  ±  0.8 vs. 5.0  ±  0.6  mmol/l, P  <0.001) and plasmatic strong ion diff erence (SIDpl) (31 (30 to 33) vs. 35 (34 to 36) mEq/l, P = 0.01) during the study period. These changes were due to a decrease in plasmatic Cl– concentration (109.0 ± 1.1 vs. P414 P414 Tubular damage biomarkers linked to infl ammation or iron metabolism predict acute kidney injury A Haase-Fielitz1, S Westphal1, R Bellomo2, P Devarajan3, M Westerman4, P Mertens1, M Haase1 1Otto von-Guericke University, Magdeburg, Germany; 2Austin Hospital, Melbourne, Australia; 3Cincinnati Children’s Hospital, Cincinnati, OH, USA; 4Intrinsic LifeSciences, La Jolla, CA, USA Critical Care 2013, 17(Suppl 2):P414 (doi: 10.1186/cc12352) balance in response to the administration of furosemide. Methods We connected the urinary catheter of 39 ICU patients to a quasi-continuous urine analyzer (Kidney INstant monitorinG®), allowing measurement of pH (pHU), sodium, chloride, potassium and ammonium concentrations (Na+U, Cl–U, K+U, NH4+U) every 10 minutes. The study period lasted 3 hours after a single intravenous bolus of furosemide (time 0). In 13 patients receiving two or more administrations over a longer period (46 (26 to 49) hours), according to clinical needs, we reviewed data on fl uid therapy, hemodynamics and acid–base balance from the beginning to the end of the observation. Results Ten minutes after furosemide administration, Na+U and Cl–U rose from 65 ± 6 to 140 ± 5 and from 109 ± 6 to 150 ± 5 mEq/l respectively, while K+U fell from 60 ± 5 to 39 ± 4 mEq/l (P <0.001 for all electrolytes vs. time 0) with a consequent increase in urinary anion gap (AGU = Na+U + Cl–U – K+U). Urinary output increased from 10 (5 to 19) to 53 (29 to 71) ml/10 minutes (P <0.05). After the fi rst hour Cl–U remained higher than Na+U, which progressively decreased, leading to a reduction in AGU and pHU over time. In parallel, a progressive increment in NH4+U was observed. In patients receiving more than one administration we observed an increase in arterial base excess (1.8  ±  0.8 vs. 5.0  ±  0.6  mmol/l, P  <0.001) and plasmatic strong ion diff erence (SIDpl) (31 (30 to 33) vs. 35 (34 to 36) mEq/l, P = 0.01) during the study period. These changes were due to a decrease in plasmatic Cl– concentration (109.0 ± 1.1 vs. 106.6 ± 0.9 mEq/l, P = 0.009). Plasmatic sodium and potassium concentrations did not change. In these patients, considering the total amount of administered fl uids and urine, a negative water and chloride balance was observed (–460 ± 403 ml and –48 ± 48 mEq, respectively). P415 increase in urinary NGAL in patients receiving bicarbonate infusion was observed compared with control (P  =  0.011). The incidence of postoperative RRT was similar but hospital mortality was increased in patients treated with bicarbonate compared with chloride (11/174 (6.3%) vs. 3/176 (1.7%), OR 3.89 (1.07 to 14.2), P = 0.031). See Figure 1. Conclusion On this basis of our fi ndings we do not recommend the use of perioperative infusions of sodium bicarbonate to reduce the incidence or severity of AKI in this patient group. Renal response and acid–base balance alterations during furosemide administration Renal response and acid–base balance alterations during furosemide administration L Zazzeron, D Ottolina, E Scotti, M Ferrari, M Stanziano, C Rovati, F Zadek, C Marenghi, L Gattinoni, P Caironi Fondazione IRCCS Ca’ Granda – Ospedale Maggiore Policlinico, Milan, Italy Critical Care 2013, 17(Suppl 2):P415 (doi: 10.1186/cc12353) Renal response and acid–base balance alterations during furosemide administration L Zazzeron, D Ottolina, E Scotti, M Ferrari, M Stanziano, C Rovati, F Zadek, C Marenghi, L Gattinoni, P Caironi Fondazione IRCCS Ca’ Granda – Ospedale Maggiore Policlinico, Milan, Italy Critical Care 2013, 17(Suppl 2):P415 (doi: 10.1186/cc12353) Introduction Furosemide is one of the most employed diuretics in the ICU for its ability to induce negative water balance. However, one common side eff ect is metabolic alkalosis [1]. We aimed to describe the time course of urinary excretion and changes in plasmatic acid–base balance in response to the administration of furosemide. P414 106.6 ± 0.9 mEq/l, P = 0.009). Plasmatic sodium and potassium concentrations did not change. In these patients, considering the total amount of administered fl uids and urine, a negative water and chloride balance was observed (–460 ± 403 ml and –48 ± 48 mEq, respectively). Methods One hundred adult patients undergoing cardiac surgery in the control arm of a RCT (NCT00672334) were analyzed. We quantifi ed the following biomarkers in urine: NGAL, hepcidin, midkine, IL-6, α1- microglobulin; in plasma: NGAL, hepcidin, haptoglobin, CK, CKMB, CRP, leukocytes, lactate, urea and creatinine. y Results Preoperatively, no biomarker predicted AKI. At ICU arrival, four urinary tubular damage markers showed good discriminatory ability (AUC ≥0.80) for subsequent development or absence of AKI (Figure 1). An excellent predictive value was found for uNGAL/uhepcidin ratio (AUC 0.90, Figure  1). This ratio combines an AKI prediction marker (NGAL) and a marker of protection from AKI (hepcidin), potentiating their individual discriminatory values. Contrarily, at ICU admission, none of the plasma biomarkers was a good early AKI predictor with AUC-ROC ≥0.80. Conclusion Several urinary markers of acute tubular damage predict AKI after cardiac surgery and the biologically plausible combination of NGAL and hepcidin provides excellent AKI prediction. Conclusion Furosemide acts immediately after administration, causing a rise in urinary output, Na+U and Cl–U concentrations. Loop-diuretic- induced metabolic alkalosis may be due to an increased urinary chloride loss and the associated increase in SIDpl. Figure 1 (abstract P414). 1. Wilcox CS: Metabolic and adverse eff ects of diuretics. Semin Nephrol 1999, 19:557-568. Perioperative sodium bicarbonate to prevent acute kidney injury after cardiac surgery: a multicenter double-blind randomized controlled trial A Haase-Fielitz1, M Haase1, M Plass2, P Murray3, M Bailey4, R Bellomo5, S Bagshaw6 g 1Otto von-Guericke University, Magdeburg, Germany; 2German Heart Center, Berlin, Germany; 3University College, Dublin, Ireland; 4ANZIC, Melbourne, Australia; 5Austin Hospital, Melbourne, Australia; 6University of Alberta, Canada Critical Care 2013, 17(Suppl 2):P413 (doi: 10.1186/cc12351) g Methods Male Sprague–Dawley rats were anesthetized with pento- barbital. Tracheostomy was performed and rats were ventilated at VT 10 ml/kg with 5 cmH2O PEEP. The right carotid artery was catheterized for blood sampling and continuous blood pressure measurements. The right femoral vein was catheterized for infusion of saline or ANP. Rats were divided into three groups; IRI group (n = 10), left renal pedicle was clamped for 30 minutes; IRI+ANP group (n = 10), left renal pedicle was clamped for 30 minutes, ANP (0.2 μg/kg/minute, for 3 hours 25 minutes) was started 5 minutes after clamp; and Sham group (n = 6), the sham- operated rats. Hemodynamics, arterial blood gas, and plasma lactate levels were measured at baseline and at 1 hour, 2 hours and 3 hours after declamp. The mRNA expression of IL-6 in the kidney, lung, and heart were Introduction Evidence suggests a nephroprotective eff ect of urinary alkalinization in patients at risk of acute kidney injury (AKI). y y Methods In a multicenter, double-blind, RCT we enrolled 350 adult cardiac surgery patients. At induction of anesthesia, patients received either 24  hours of intravenous infusion of sodium bicarbonate (5.1 mmol/kg) or sodium chloride (5.1 mmol/kg). The primary endpoint was the proportion of patients developing AKI. Results Sodium bicarbonate increased urinary pH (from 6.0 to 7.5, P  <0.001). More patients in the bicarbonate group (83/174 (47.7%)) developed AKI compared with control (64/176 (36.4%), OR  =  1.60 (95% CI, 1.04 to 2.45); unadjusted P = 0.032). A greater postoperative Figure 1 (abstract P413). Figure 1 (abstract P413). Figure 1 (abstract P413). S155 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 P416 Red cell distribution width is not a predictor of mortality in acute kidney injury P Purvis1, J Kinsella1, R Docking2 1University of Glasgow, UK; 2Glasgow Royal Infi rmary, Glasgow, UK Critical Care 2013, 17(Suppl 2):P416 (doi: 10.1186/cc12354) Value of Tc-99m DMSA renal scan in assessing the prognosis and outcome of acute renal failure due to sepsis Methods A retrospective observational study of renal function in all patients admitted to a London teaching hospital ICU for ≥5 days and surviving to hospital discharge in 2011. We excluded cases with current or new diagnosis of end-stage renal disease or renal transplant. We assessed AKI in ICU by KDIGO 1 criteria and hospital discharge eGFR by the CKD-EPI equation. For comparison we assumed a normal GFR in a healthy individual as 120 ml/minute/1.73 m2 at age 20 decreasing by 0.8 per year over age 20. Value of Tc-99m DMSA renal scan in assessing the prognosis and outcome of acute renal failure due to sepsis S Fawzi, K Mashour, A Amin, H Gamal Cairo University, Cairo – Manial, Egypt y gyp Critical Care 2013, 17(Suppl 2):P417 (doi: 10.1186/cc12355) Introduction Acute renal failure (ARF) is a common complication in patients admitted to the ICU. Sepsis is also a well-known risk factor for the development of ARF. The combination of ARF and severe sepsis was reported to carry a mortality up to 70% whereas the mortality of ARF alone is 40 to 45%. The aim of the study is to evaluate the role of renal perfusion scanning in detecting the prognosis and outcome of patients with acute renal failure due to sepsis. Results We identifi ed 282 patients, 180 of whom had AKI. Median age was 50 and 68% were male. Median hospital discharge serum creatinine was 57 μmol/l (range 16 to 654), median eGFR was signifi cantly higher than predicted normal GFR for age at 115 versus predicted 95 (P <0.001, median diff erence 16). In patients who had not had AKI discharge the eGFR was 119 versus normal predicted 98 (P <0.001, median diff erence 19), suggesting that eGFR could be overestimating true GFR in our population by at least a factor of 1.23 (Figure 1). Applying this correction factor to eGFRs of patients who had recovered from AKI resulted in 44% more diagnoses of CKD (eGFR <60) at hospital discharge (36 vs. 25). p Methods Forty patients with acute renal failure due to sepsis, aged between 15 and 74  years, were enrolled in the study. They were monitored for their ICU prognosis and outcome after doing renal perfusion scanning. All patients were subjected to routine ICU and laboratory investigations including APACHE II and SOFA score. Results Thirty patients had normal renal scan and 10 patients had abnormal renal scan. Red cell distribution width is not a predictor of mortality in acute kidney injury Red cell distribution width is not a predictor of mortality in acute kidney injury P Purvis1, J Kinsella1, R Docking2 1University of Glasgow, UK; 2Glasgow Royal Infi rmary, Glasgow, UK Critical Care 2013, 17(Suppl 2):P416 (doi: 10.1186/cc12354) Introduction Given the signifi cant morbidity and mortality associated with acute kidney injury (AKI), there is a need to fi nd factors to help aid decision-making regarding levels of therapeutic support. As a prognostic biomarker, the red cell distribution width (RDW) has attracted interest in the setting of critical care when added to existing scoring systems [1]. By examining RDW in a previously studied AKI cohort, we aimed to evaluate the utility of this routine blood test. Methods A cohort of 209 mixed critical care patients who received renal replacement therapy for AKI had their demographic and biochemical data retrieved from electronic databases. Outcomes were gathered for ICU and hospital mortality. Incomplete datasets were discarded, leading to 153 complete sets. RDW data were taken from the fi rst sample after admission to the ICU, as were all other biochemical values apart from pre-RRT creatinine and potassium. Overall cohort characteristics were gathered, and two groups were created: those with a RDW value within normal range (≤14.5%) and those with a greater than normal value Figure 1 (abstract P414). S156 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 (>14.5%). We then further subgrouped RDW to assess the correlation between rising levels and ICU mortality. Figure 1 (abstract P418). eGFR at hospital discharge versus expected normal GFR for age in 280 ICU survivors. Results A total 77.1% of our cohort had a RDW greater than the normal laboratory range at time of ICU admission. Key baseline characteristics (age, APACHE II score, length of stay, ICU mortality) did not diff er signifi cantly between patients with normal and abnormal RDW. When subgroup analysis was performed, no statistically signifi cant correlation between rising RDW and ICU mortality was found (Spearman correlation = 0.426, P = 0.233). Conclusion In this cohort of critically ill patients with AKI, RDW was not found to be a predictor of mortality. Our results contradict those of recent studies [1,2]. However, both groups of RDW patients in our study suff ered a higher ICU mortality than in other studies. Red cell distribution width is not a predictor of mortality in acute kidney injury To further explain these fi ndings, we intend to perform multivariate logistic regression analysis and assess the eff ect of social deprivation on RDW. R f 1. Hunziker et al.: Red cell distribution width improves the simplifi ed acute physiology score for risk prediction in unselected critically ill patients. Crit Care 2012, 16:R89. 1. Hunziker et al.: Red cell distribution width improves the simplifi ed acute physiology score for risk prediction in unselected critically ill patients. Crit Care 2012, 16:R89. Figure 1 (abstract P418). eGFR at hospital discharge versus expected normal GFR for age in 280 ICU survivors. 2. Oh et al.: Red blood cell distribution width is an independent predictor of mortality in acute kidney injury patients treated with continuous renal replacement therapy. Nephrol Dial Transplant 2012, 27:589-594. 2. Oh et al.: Red blood cell distribution width is an independent predictor of mortality in acute kidney injury patients treated with continuous renal replacement therapy. Nephrol Dial Transplant 2012, 27:589-594. to survivors of critical illness who may have reduced muscle mass. We hypothesized that eGFR may underestimate rates and severity of CKD in ICU survivors. Varying models of intra-abdominal hypertension and their eff ect on renal function in a porcine model Varying models of intra-abdominal hypertension and their eff ect on renal function in a porcine model R Wise1, R Rodseth1, I De Laet2, L Luis3, L Correa-Martin4, M Garcia4, G Castellanos5, M Malbrain2 1University of Kwazulu Natal, Durban, South Africa; 2ZNA Stuivenberg, Antwerp, Belgium; 3Neuron NPh, S.A., Granada, Spain; 4Jesús Usón Minimally Invasive Surgery Centre, Cacères, Spain; 5‘Virgen de la Arrixaca’ University Hospital, Murcia, Spain Critical Care 2013, 17(Suppl 2):P419 (doi: 10.1186/cc12357) , 1University of Kwazulu Natal, Durban, South Africa; 2ZNA Stuivenberg, Antwerp, Belgium; 3Neuron NPh, S.A., Granada, Spain; 4Jesús Usón Minimally Invasive Surgery Centre, Cacères, Spain; 5‘Virgen de la Arrixaca’ University Hospital, Murcia, Spain Critical Care 2013, 17(Suppl 2):P419 (doi: 10.1186/cc12357) 1. Ympa YP, et al.: Am J Med 2005, 118:827-832. Introduction Intra-abdominal hypertension (IAH) is an independent predictor of renal impairment and mortality [1]. Organ dysfunction caused by the pressure eff ect of IAH is well understood, but how this is modifi ed in the presence of bowel obstruction is unclear. The aim of this study was to determine how diff erent IAH models cause renal dysfunction in a pig model. 1. Ympa YP, et al.: Am J Med 2005, 118:827-832. Value of Tc-99m DMSA renal scan in assessing the prognosis and outcome of acute renal failure due to sepsis The mortality percentage was higher among abnormal renal scan cases (three out of 10, 30%) compared with cases with normal renal scan (seven out of 30, 23.3%) with nonsignifi cant P value: 0.6. The median length of stay/day in ICU was longer among nonsurvivors than survivors 15.5  ±  10, 11.5  ±  8, P value: 0.058 (approaching signifi cance). APACHE II score was higher in nonsurvivors than survivors 23.9± 3.2, 19.6± 4.2, P value: 0.0001. The percentage of mortality among cases that needed mechanical ventilation was higher (nine out of 16, 56.3%) compared with mortality cases that did not need mechanical ventilation (one out of 24, 4.2% with P value: 0.0001). Conclusion ARF may exert an independent adverse eff ect on outcome in septic and septic shock patients. It is also a risk factor for mortality. Tc-99m DMSA scanning is useful for detecting renal dysfunction and help to predict the outcome and prognosis. R f Conclusion eGFR may overestimate renal function in survivors of critical illness confounding identifi cation of CKD in this at-risk population. Prospective studies with measurement of actual GFR are required to assess the burden of CKD in survivors of critical illness. Use, type and duration of cardiopulmonary bypass are related to acute kidney injury occurrence and urinary NGAL concentrations T García Rodríguez San Miguel Use, type and duration of cardiopulmonary bypass are related to acute kidney injury occurrence and urinary NGAL concentrations T García Rodríguez San Miguel Use, type and duration of cardiopulmonary bypass are related to acute kidney injury occurrence and urinary NGAL concentrations T García Rodríguez San Miguel Hospital Santa Creu i Sant Pau, Barcelona, Spain Critical Care 2013, 17(Suppl 2):P422 (doi: 10.1186/cc12360) Methods We included medical ICU patients with sepsis and oliguria (urine output <1 ml/kg/hour) who were prescribed i.v. furose mide. Patients with known chronic renal failure, hypovolemia (CVP  <10  mmHg) or severe hypotension (MAP <80 mmHg) were excluded. Resistive index (1 − (end diastolic velocity / maximum systolic velocity)×100) was measured in at least three segmental arteries of both kidneys, the average of all measurements was reported as the result. Repeated assessments were viewed as independent if separated by more than 24 hours. Furosemide was given intravenously in the dose of 40  mg after RI measurement. Positive response to furosemide was defi ned as doubling of hourly dieresis or achieving urine output >1.5 ml/kg/hour after drug administration. Introduction To analyze whether variables related to cardiopulmonary bypass (CPB) infl uence acute kidney injury (AKI) occurrence and urinary neutrophil gelatinase-associated lipocalin (uNGAL) in cardiac surgery patients. Introduction To analyze whether variables related to cardiopulmonary bypass (CPB) infl uence acute kidney injury (AKI) occurrence and urinary neutrophil gelatinase-associated lipocalin (uNGAL) in cardiac surgery patients. Methods A total of 274 adult cardiac surgery patients were consecu- tively included from February to December 2011. Exclusion criteria were absence of diuresis due to end-stage renal disease or chronic renal failure and cardiac catheterism with i.v. contrast in the week before surgery. CPB, when performed, was used as standard CPB (SCPB) or MiniCPB. We obtained four serial blood and urine samples, immediately before (PRE) and after (POST) surgery, and 1  day (1d) and 2 days (2d) after surgery. uNGAL was measured by Architect 6200 (Abbott Diagnostics). AKIN criteria were used to diagnose AKI. The study was approved by the local ethics committee and all patients gave informed consent. g g g Results We included 47 patients with a total of 59 measurements. In 28 cases patients had positive response to furosemide. Median RI in responders was 0.67 (range 0.55 to 0.78) and in nonresponders 0.79 (range 0.58 to 0.81); P  =  0.027. Estimated glomerular fi ltration rate based on hospital discharge creatinine may signifi cantly overestimate renal function and underestimate chronic kidney disease in survivors of critical illness l d ll l Methods Twenty-four pigs were divided into three groups; a control group (n = 5), a pneumoperitoneum (Pn) (n = 10), and an intestinal occlusion (Oc) model (n  =  10). IAP was maintained for 3  hours at 20 mmHg during which time creatinine, urea, urine output, potassium, and glomerular fi ltration pressure (GFP) were measured. Statistical analysis was performed using repeated-measures ANOVA.ii Introduction Acute kidney injury (AKI) complicates over 50% of ICU admissions. Episodes of AKI are a major risk factor for development or progression of chronic kidney disease (CKD); however, methods of estimated glomerular fi ltration rate (eGFR) may be poorly calibrated Results Over the fi rst 3  hours there was a statistically signifi cant diff erence between the control group and both IAH models for S157 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 potassium (P  =  0.002), urea (P  =  0.045), creatinine (P  =  0.012), GFP (P <0.001), and urine output (P = 0.003). Over the full 5-hour period there was a statistically signifi cant diff erence in the potassium measurement between the Pn and the Oc model (P = 0.01). The other parameters did not show a signifi cant diff erence: urea (P = 0.171), creatinine (P = 0.074), GFP (P = 0.141), and urine output (P = 0.242).i potassium (P  =  0.002), urea (P  =  0.045), creatinine (P  =  0.012), GFP (P <0.001), and urine output (P = 0.003). Over the full 5-hour period there was a statistically signifi cant diff erence in the potassium measurement between the Pn and the Oc model (P = 0.01). The other parameters did not show a signifi cant diff erence: urea (P = 0.171), creatinine (P = 0.074), GFP (P = 0.141), and urine output (P = 0.242).i Methods A prospective single-center cohort study started in May 2012 in an academic cardiac surgery ICU. Blood and urine were collected preoperatively (baseline), at ICU admission and at several time points until 48 hours after ICU admission. Patients of age <18  years were excluded, as well as patients with AKI based on the KDIGO sCr and urine output criteria, patients with chronic kidney disease stage 5 and/or with recent renal transplantation (≤3 months before cardiac surgery). AKI was defi ned on KDIGO sCr criteria only. Estimated glomerular fi ltration rate based on hospital discharge creatinine may signifi cantly overestimate renal function and underestimate chronic kidney disease in survivors of critical illness l d ll l Baseline sCr was assessed as the sCr concentration measured before start of cardiac surgery. Urinary CHI3L1 was normalized to urinary creatinine. For both serum and urine CHI3L1, we measured changes from baseline. Data are expressed as median (interquartile range), and number (%). Conclusion As expected the IAH models resulted in signifi cantly worse renal function after 3 hours. This early renal dysfunction may be as a result of an early infl ammatory process that has been associated with the pathophysiology of acute kidney injury. Potassium was signifi cantly elevated in the Pn group as compared with the Oc group. Early changes in potassium levels with IAH may be a marker of early renal dysfunction and the usefulness of other renal biomarkers, such as NGAL, prompts further investigation. Results Data for 17 patients (nine males, eight females) were generated for interim analysis. Five patients (29.4%), of which four were females, developed AKI KDIGO class 1. AKI patients were older compared with non-AKI patients (82.0 years (77.5 to 83.5) vs. 66.5 (60.3 to 75.8); P  =  0.002). The baseline sCr did not diff er signifi cantly between the AKI (1.12 mg/dl (0.80 to 1.15)) and non-AKI (0.90 mg/dl (0.86 to 1.01); P = 0.383) group. Baseline serum CHI3L1 was highly variable in both AKI and non-AKI patients. Moreover, no trend towards a higher serum CHI3L1 level in AKI versus non-AKI patients was observed either at baseline or during the study follow-up. In contrast, the increase in the urinary CHI3L1 to urinary creatinine ratio from baseline was higher in the AKI versus non-AKI group at time points 4 hours and 6 hours after admission to the ICU (2.00 ng/mg Cr (1.38 to 3.03) vs. 0.26 (0.05 to 0.88); P = 0.002, resp. 2.47 (1.09 to 7.26) vs. 0.23 (–0.18 to 0.74); P = 0.006) and, more importantly, occurred before the diagnosis of AKI by sCr (>16 hours after ICU admission). Use, type and duration of cardiopulmonary bypass are related to acute kidney injury occurrence and urinary NGAL concentrations T García Rodríguez San Miguel Construction of receiver operating characteristic curve showed 83% sensitivity and 81% specifi city for the cutoff RI 0.73. No other measured patient characteristic was found to be predictive of response to diuretic treatment. Conclusion Our results show that the RI could be used to guide diuretic treatment in nonhypovolemic, nonhypotensive septic patients. Further studies are needed to confi rm those preliminary results. Results One hundred and eighty-one patients (66.1%) were men; mean age was 68.2 ± 12.2 years. ICU and hospital stays were 6.7 ± 8.1 and 15.7 ± 13.9 days, respectively. Twenty-eight-day mortality was 2.9%. Eighty-six patients (31.4%) were diagnosed with AKI within 48 hours after surgery. In total, 219 patients required CPB (195 SCPB, 24 MiniCPB) and 55 did not (no-CPB). Seven no-CPB patients (12.7%) developed AKI and their median uNGAL POST was 330 (42.6 to 489.9) μg/l compared with 13.6 (6.9 to 38.3) μg/l in the 48 patients without AKI (P <0.0001). Of the 195 patients undergoing SCPB, 76 (38.9%) developed AKI and 119 did not; POST uNGAL was 204 (34.8 to 575.7) μg/l and 44.5 (13.2 to 175.8) μg/l (P <0.0001), respectively. In the 24 patients under MiniCPB, POST uNGAL was 113 (58.8 to 211.8) μg/l in those (three patients, 12.5%) developing AKI and 19.1 (9.2 to 41.8) μg/l in those without AKI (P = 0.01). Aortic clamp time (r = 0.31, P <0.0001) and cardiopulmonary bypass time (r = 0.30, P <0.0001) correlated with POST uNGAL concentrations. Reference 1. Malbrain ML, et al.: Incidence and prognosis of intraabdominal hypertension in a mixed population of critically ill patients: a multiple- center epidemiological study. Crit Care Med 2005, 33:315-322. 1. Malbrain ML, et al.: Incidence and prognosis of intraabdominal hypertension in a mixed population of critically ill patients: a multiple- center epidemiological study. Crit Care Med 2005, 33:315-322. Renal resistive index can predict response to furosemide in septic patients with oliguria I Gornik, A Godan, V Gasparovic y p g , Critical Care 2013, 17(Suppl 2):P420 (doi: 10.1186/cc12358) Introduction Oliguria is common in septic patients and is frequently therapeutically addressed with loop diuretics; that is, furosemide. Diuretic treatment in shock and hypovolemia is not rational, but can be tried in oliguric patients with normovolemia or hypervolemia and without hypotension. In such patients it still does not always increase dieresis and can also be harmful. The resistive index is a measure of pulsatile blood fl ow that refl ects the resistance to blood fl ow caused by the microvascular bed distal to the site of measurement. It can refl ect functional status of the tissue distal to the point of measurement. We investigated whether measuring the renal resistive index (RI) could be helpful in determining which patients will respond to furosemide treatment. Conclusion This interim analysis of an ongoing pilot study, with 300 ICU patients in three cohorts (general ICU, pediatric and adult cardiac surgery ICU), shows promising results for urinary CHI3L1 (ng/mg Cr) as an early diagnostic biomarker for AKI in adult ICU patients that undergo elective cardiac surgery. P422 P421 P421 Urinary chitinase 3-like 1, a novel biomarker for acute kidney injury in adult cardiac intensive care patients: a pilot study J De Loor1, E Hoste2, I Herck2, K Francois2, L De Crop2, C Clauwaert2, K Demeyere1, E Meyer1 1Faculteit diergeneeskunde, Merelbeke, Belgium; 2UZ Gent, Belgium Critical Care 2013, 17(Suppl 2):P421 (doi: 10.1186/cc12359) P423 Symmetrical dimethylarginine is a more sensitive biomarker of renal dysfunction than creatinine t e apy ( ). Stat st cs: OC UC. S SS 0. Results There were 91 patients (34 female, 57 male), 65  ±  14 years, APACHE II score 20.6 ± 8.3, SOFA score 9.3 ± 4.2. Etiology: 10 ARDS, 36 sepsis, 22 cirrhosis, six pancreatitis, eight cardiogenic shock, two CNS-aff ections, seven various. Mortality was best predicted by SOFA (ROC-AUC: 0.747; P  <0.001) and APACHE II (AUC: 0.705; P  <0.001). Among admission parameters of renal function only NGAL signifi cantly predicted mortality (AUC: 0.647; P  =  0.023), whereas creatinine (P  =  0.290) and BUN (P  =  0.067) were not predictive. ROC-AUCs for NGAL further increased after 12 hours (0.659), 24 hours (0.691; P = 0.01) and 48  hours (0.728; P  =  0.004). NGAL on admission also predicted requirement of RRT during the ICU stay (AUC: 0.678; P = 0.015), which was also predicted by creatinine (AUC 0.688; P  =  0.010) and BUN (AUC: 0.649; P  =  0.043). Among baseline TPTD parameters, only the pulmonary vascular permeability index (PVPI) signifi cantly predicted mortality (AUC: 0.700; P  =  0.007). The EVLWI (AUC 0.628; P  =  0.083) slightly failed signifi cance. Cardiac index, global end-diastolic volume index and heart rate were not predictive. Furthermore, PVPI was the only TPTD-derived parameter to predict requirement of RRT (AUC: 0.693; P = 0.009).i Symmetrical dimethylarginine is a more sensitive biomarker of renal dysfunction than creatinine JJ Dixon1, K Lane1, RN Dalton2, IA MacPhee1, BJ Philips1 1Acute Kidney Injury Research Group, St George’s Hospital and University of London, UK; 2King’s College, University of London, UK Critical Care 2013, 17(Suppl 2):P423 (doi: 10.1186/cc12361) Symmetrical dimethylarginine is a more sensitive biomarker of renal dysfunction than creatinine y JJ Dixon1, K Lane1, RN Dalton2, IA MacPhee1, BJ Philips1 1Acute Kidney Injury Research Group, St George’s Hospital and University of London, UK; 2King’s College, University of London, UK Critical Care 2013, 17(Suppl 2):P423 (doi: 10.1186/cc12361) y JJ Dixon1, K Lane1, RN Dalton2, IA MacPhee1, BJ Philips1 1Acute Kidney Injury Research Group, St George’s Hospital and University of London, UK; 2King’s College, University of London, UK Critical Care 2013, 17(Suppl 2):P423 (doi: 10.1186/cc12361) Introduction Current defi nitions of acute kidney injury (AKI) are based upon changes in serum creatinine (SCr) concentration and urine output: both have limitations in patients with AKI. Diagnosis may be delayed if using these criteria alone. Assessment of neutrophil gelatinase-associated lipocalin as a biomarker of acute kidney injury in acute heart failure patients Results Mean GFR measured by single injection was 78.7 ± 28.5 ml/ minute/1.73 m2, and 78.9 ± 28.6 ml/minute/1.73 m2 when measured by CIVLDI (P = 0.82). Mean SDMA concentration was similar on both occasions (641 ± 38 vs. 623 ± 22 nmol/l; P = 0.68). Tubular reabsorption of SDMA was lower in subjects with GFR <60  ml/minute/1.73  m2 (12  ±  8% vs. 18  ±  8% in subjects with GFR >60  ml/minute/1.73  m2; P = 0.0002), and tubular secretion of SCR was higher in subjects with GFR <60 ml/minute/1.73 m2 (29% vs. 23%; P <0.0001). The third-order polynomial equation (r = 0.93) estimated GFR better than quadratic (r  =  0.92) and linear (r  =  0.87) equations. Bland–Altman comparison revealed no bias when the third-order equation was used (precision: ±20 ml/minute/1.73 m2). p Critical Care 2013, 17(Suppl 2):P425 (doi: 10.1186/cc12363) Introduction Growing evidence hints that bidirectional interaction between heart failure and kidney disease and renal insuffi ciency is a strong predictor of mortality as well as causally linked to the progression of heart failure. Neutrophil gelatinase-associated lipocalin (NGAL) is an early predictor of acute kidney injury (AKI). We evaluated the impact of NGAL on morbidity and mortality in patients with acute heart failure. Methods Seventy-six patients presenting with symptoms consistent with acute heart failure (median age 72 years, 56% male) were enrolled. Plasma NGAL levels were measured by an ELISA at admission and compared with the glomerular fi ltration rate (eGFR) and B-natriuretic peptide (BNP) levels. The primary outcome was AKI development defi ned by RIFLE criteria (fall in GFR >25% or creatinine rise ≥50% from baseline, or a fall in urine output <0.5 ml/kg/hour) and secondary outcomes were duration of hospital stay and in-hospital mortality. Conclusion SDMA appears to be an accurate and precise estimate of GFR and a more sensitive biomarker of renal dysfunction than SCr. We predict SDMA will perform better than SCr as a biomarker of AKI. This forms the basis of a future study. P425 Assessment of neutrophil gelatinase-associated lipocalin as a biomarker of acute kidney injury in acute heart failure patients H Michalopoulou, H Michalopoulou, P Stamatis, J Xenogiannis, D Stamatis Metaxa Hospital, Athens, Greece Critical Care 2013, 17(Suppl 2):P425 (doi: 10.1186/cc12363) Urinary chitinase 3-like 1, a novel biomarker for acute kidney injury in adult cardiac intensive care patients: a pilot study J D L 1 E H 2 I H k2 K F 2 L D C 2 C Cl 2 Introduction As a proof of concept, the potential added value of chitinase 3-like 1 (CHI3L1) as a more early and specifi c diagnostic parameter for acute kidney injury (AKI) was investigated in adult ICU patients that underwent elective cardiac surgery. Introduction As a proof of concept, the potential added value of chitinase 3-like 1 (CHI3L1) as a more early and specifi c diagnostic parameter for acute kidney injury (AKI) was investigated in adult ICU patients that underwent elective cardiac surgery. S158 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Incidence of AKI in patients without CPB or with MiniCPB was identical and signifi cantly lower (P <0.0001) than in SCPB patients. Conclusion The incidence of AKI was higher in patients undergoing SCPB. Aortic clamp and cardiopulmonary bypass time correlated with the POST uNGAL concentration. The higher values found in patients without AKI undergoing SCPB suggest that a subclinical AKI, only detectable by uNGAL, could exist in this group. with mortality. Regarding a delayed increase in creatinine, other biomarkers of AKI including neutrophil gelatinase-associated lipocalin (NGAL) have been suggested. It was the aim of our study to compare the predictive capabilities of NGAL and transpulmonary thermodilution (TPTD)-derived parameters within 48 hours after ICU admission. Incidence of AKI in patients without CPB or with MiniCPB was identical and signifi cantly lower (P <0.0001) than in SCPB patients. i Conclusion The incidence of AKI was higher in patients undergoing SCPB. Aortic clamp and cardiopulmonary bypass time correlated with the POST uNGAL concentration. The higher values found in patients without AKI undergoing SCPB suggest that a subclinical AKI, only detectable by uNGAL, could exist in this group. p Methods Urinary NGAL, serum creatinine and BUN as well as TPTD- derived parameters were measured 0 hours, 12 hours, 24 hours and 48  hours after ICU admission. Primary endpoint: prediction of ICU mortality. Secondary endpoint: requirement of renal replacement therapy (RRT). Statistics: ROC-AUC. IBM SPSS 20. P423 Symmetrical dimethylarginine is a more sensitive biomarker of renal dysfunction than creatinine We have previously validated a method of measuring the glomerular fi ltration rate (GFR) intended for use in patients with AKI (a continuous infusion of very low dose iohexol; CIVLDI). In this study we compare the performance of symmetrical dimethylarginine (SDMA) against SCr and CIVILDI. SDMA is the structural isomer of the endogenous nitric oxide inhibitor asymmetrical dimethylarginine. SDMA increases in parallel with SCr. Despite this, no formula exists to estimate GFR from SDMA concentration. Tubular secretion of SCr may be as high as 40%. SDMA may be a more sensitive biomarker of renal dysfunction than SCr; however, the tubular reabsorption of SDMA is unknown. The aims were to compare the performance of SDMA against SCr and accurately measured GFR, and to derive a formula to estimate GFR from SDMA concentration. Conclusion Urinary NGAL on admission signifi cantly predicts mortality, whereas creatinine and BUN were not predictive. The predictive capabilities of NGAL further increased after 12  hours, 24  hours and 48 hours, with the ROC-AUC of NGAL 48 hours exceeding the AUC of the APACHE II score. NGAL on admission also predicts requirement of RRT. Among TPTD-derived parameters, PVPI on admission is signifi cantly associated with mortality and requirement of RRT. Methods Seventeen volunteers had GFR measured twice via measuring the clearance of a 5 ml i.v. bolus of iohexol, or CIVLDI. Serum and urine iohexol, Cr and SDMA were measured by high-performance liquid chromatography/tandem mass spectrometry (HPLC-ms/ms) at 10 time points. Fractional excretion of SDMA and SCr were calculated using iohexol as the denominator. SDMA was plotted against measured GFR, and estimated GFR equations were derived from linear, quadratic and third-order polynomial plots. P424 Prognostic value of neutrophil gelatinase-associated lipocalin and transpulmonary thermodilution-derived parameters within 48 hours after admission W Huber, J Rauch, B Saugel, S Mair, M Messer, T Lahmer, C Schultheiss, P Luppa, RM Schmid Klinikum Rechts der Isar, Technical University of Munich, II. Medizinische Klinik, Munich, Germany Critical Care 2013, 17(Suppl 2):P424 (doi: 10.1186/cc12362) Prognostic value of neutrophil gelatinase-associated lipocalin and transpulmonary thermodilution-derived parameters within 48 hours after admission W Huber, J Rauch, B Saugel, S Mair, M Messer, T Lahmer, C Schultheiss, P Luppa, RM Schmid Klinikum Rechts der Isar, Technical University of Munich, II. Medizinische Klinik, Munich, Germany Critical Care 2013, 17(Suppl 2):P424 (doi: 10.1186/cc12362) Results AKI developed in 11 patients (14.4%). The subgroup that during hospitalization developed renal dysfunction had increased NGAL values with respect to patients with preserved renal function (146 ± 42 vs. 84 ± 24 ng/ml, P <0.05). NGAL signifi cantly associated with admission eGFR (r = 0.72, P <0.01) and BNP levels (r = 0.80, P <0.01). For prediction of AKI, NGAL >140 ng/ml has been related to renal insuffi ciency with sensitivity of 83% and specifi city of 74% with area under the receiver operator curve of 0.70 (0.58 to 0.82). In the follow-up setting, NGAL was associated with recurrent hospital admission for heart failure (HR: 3.1, CI: 1.5 to 6.2) and cardiac death (HR: 2.2, CI: 1.8 to 4.5). Neutrophil gelatinase-associated lipocalin predicts postoperative fl uid overload after cardiac surgery Survivors of acute kidney injury requiring renal replacement therapy rarely receive follow-up: identifi cation of an unmet need CJ Kirwan, R Taylor, JR Prowle Survivors of acute kidney injury requiring renal replacement therapy rarely receive follow-up: identifi cation of an unmet need CJ Kirwan, R Taylor, JR Prowle The Royal London Hospital, Barts Health NHS Trust, London, UK Critical Care 2013, 17(Suppl 2):P427 (doi: 10.1186/cc12365) l g y M Haase1, P Devarajan2, P Michael3, R Bellomo4, A Haase-Fielitz1 1Otto von-Guericke University, Magdeburg, Germany; 2Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA; 3German Heart Center, Berlin, Germany; 4Austin Hospital, Melbourne, Australia Critical Care 2013, 17(Suppl 2):P426 (doi: 10.1186/cc12364) Introduction Acute kidney injury (AKI) occurs in more than 50% of ICU admissions, requiring renal replacement therapy (RRT) in around 10% of cases. There is now increasing evidence that AKI is a risk factor for the development and progression of chronic kidney disease (CKD); however, when AKI occurs as a complication of critical illness appropriate follow-up may be neglected. Accordingly, we reviewed the follow-up of renal function in all patients who received RRT on our ICU and survived to hospital discharge. Introduction Neutrophil gelatinase-associated lipocalin (NGAL), measured early after cardiac surgery, has been demonstrated to predict postoperative acute kidney injury (AKI). Fluid overload potentially masks a subsequent acute renal function loss through dilution of serum creatinine and maintenance of urine output just above AKI- defi ning criteria. g Methods A retrospective audit of patients who received RRT in a central London adult critical care unit during 2011. i Methods We investigated the early postoperative value of NGAL versus that of simultaneously measured serum creatinine to predict subsequent fl uid overload. We studied 100 adult cardiac surgery patients in the control arm of a RCT (NCT00672334). Severe postoperative fl uid overload was defi ned as positive fl uid balance >10% of preoperative body weight within 48 hours after surgery.l Results Of 921 patients admitted, 203 received RRT with 109 surviving to hospital discharge. We excluded 52 patients who had end-stage renal disease, renal transplant or known glomerular disease. Of the remaining 57 AKI patients, median age was 60 (range: 18 to 77) and 37 (65%) were male. Median discharge creatinine was 74.5  μmol/l (27 to 662). Forty-two (74%) were off ered follow-up, but in only six cases (11%) was this to nephrology services. Neutrophil gelatinase-associated lipocalin predicts postoperative fl uid overload after cardiac surgery Twenty-eight attended follow-up (fi ve to nephrology) at a median time of 6 weeks; however, creatinine was measured at in only 14 and in six of these it had risen (by median 16.5 μmol/l). In addition, 14 patients had creatinine measured 3 to 6 months post discharge and in eight it had risen (by median 31.5 μmol/l). y y Results Severe postoperative fl uid overload was present in 5% of patients with a mean positive fl uid balance of 15.8  ±  9.5  l. At ICU admission, urine NGAL predicted severe fl uid overload (AUC-ROC 0.82 (95% CI = 0.70 to 0.94)) (Figure 1) and mortality (AUC 0.88 (0.78 to 0.97)). Serum creatinine measured at the same time did not predict severe fl uid overload (AUC 0.52 (0.26 to 0.79)) or mortality (AUC 0.61 (0.16 to 0.99)).l Conclusion Early NGAL-guided adjustments to fl uid management may reduce organ edema after cardiac surgery. Findings should be validated in larger cohorts. Conclusion Follow-up of patients who received RRT for AKI in the ICU was poor and they were rarely referred to nephrologists. Where renal function was measured after discharge, there was evidence of progressive renal dysfunction; however, renal function was often not assessed. We propose an algorithm for clinicians to guide follow-up. See Figure 1. Figure 1 (abstract P426). NGAL at ICU admission predicts fl uid overload of >10% of body weight. Figure 1 (abstract P426). NGAL at ICU admission predicts fl uid overload of >10% of body weight. P427 P426 Neutrophil gelatinase-associated lipocalin predicts postoperative fl uid overload after cardiac surgery M Haase1, P Devarajan2, P Michael3, R Bellomo4, A Haase-Fielitz1 1Otto von-Guericke University, Magdeburg, Germany; 2Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA; 3German Heart Center, Berlin, Germany; 4Austin Hospital, Melbourne, Australia Critical Care 2013, 17(Suppl 2):P426 (doi: 10.1186/cc12364) Prognostic value of neutrophil gelatinase-associated lipocalin and transpulmonary thermodilution-derived parameters within 48 hours after admission W Huber, J Rauch, B Saugel, S Mair, M Messer, T Lahmer, C Schultheiss, P Luppa, RM Schmid Klinikum Rechts der Isar, Technical University of Munich, II. Medizinische Klinik, Munich, Germany Critical Care 2013, 17(Suppl 2):P424 (doi: 10.1186/cc12362) Introduction Outcome of ICU patients is predicted by multifactorial scores such as APACHE II and SOFA. Furthermore, markers of single organ failure such as the extravascular lung water index (EVLWI) and several biomarkers of acute kidney injury (AKI) have been associated Conclusion NGAL is emerging as a promising biomarker of AKI in the setting of acute heart failure and elevated NGAL levels indicate a poor prognosis in this population regarding morbidity and mortality. S159 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 P426 Eff ect of continuous high-volume hemofi ltration on patients with acute respiratory distress syndrome Eff ect of continuous high-volume hemofi ltration on patients with acute respiratory distress syndrome A Kuzovlev1, E Tishkov2, O Bukaev2 1V.A. Negovsky Scientifi c Research Insitute of General Reanimatology RAMS, Moscow, Russia; 2A.I. Evdokimov Moscow State University of Medicine and Dentistry, Moscow, Russia Critical Care 2013, 17(Suppl 2):P431 (doi: 10.1186/cc12369) Eff ect of continuous high-volume hemofi ltration on patients with acute respiratory distress syndrome A Kuzovlev1, E Tishkov2, O Bukaev2 1V.A. Negovsky Scientifi c Research Insitute of General Reanimatology RAMS, Moscow, Russia; 2A.I. Evdokimov Moscow State University of Medicine and Dentistry, Moscow, Russia Critical Care 2013, 17(Suppl 2):P431 (doi: 10.1186/cc12369) A Kuzovlev1, E Tishkov2, O Bukaev2i , , 1V.A. Negovsky Scientifi c Research Insitute of General Reanimatology RAMS, Moscow, Russia; 2A.I. Evdokimov Moscow State University of Medicine and Dentistry, Moscow, Russia , , 1V.A. Negovsky Scientifi c Research Insitute of General Reanimatology RAMS, Moscow, Russia; 2A.I. Evdokimov Moscow State University of Medicine and Dentistry, Moscow, Russia Critical Care 2013, 17(Suppl 2):P431 (doi: 10.1186/cc12369) Introduction The aim of this study was to investigate the eff ects of intermittent venovenous high-volume hemofi ltration (IHVHF) on extravascular lung water, respiratory function and cytokine removal in patients with acute respiratory distress syndrome (ARDS) associated with postoperative multiple organ dysfunction syndrome in severe abdominal infection patients. Results The average quantity of the substitutive renal therapy procedures in the CG was 13.4 ± 0.7, in the IG it was 8 ± 0.6 (P <0.05). The recuperation of the renal excretory functions was on 19 ± 1 day in 12 patients of the CG, and on 11 ± 1.3 day in the IG, from the moment of substitutive renal therapy start (P <0.05). Lethality in the CG was 43% (nine patients), and in the IG it was 29% (six patients, P <0.05). The duration of the MLV in the CG and IG was 21 ± 1.2 days and 16 ± 1.2, respectively (P <0.05). In the IG the duration of the ICU was lower by 23%, hospitality duration was lower by 17% (P <0.05).fi Methods Thirty patients with severe abdominal infection and ARDS were randomized into the IHVHF group (n  =  15) and control group (n = 15). All patients were mechanically ventilated. The IHVHF technique with a rate of 100 to 120 ml/kg/hour during 6 to 8 hours was used. Can treatment with the molecular adsorbent recirculation system be the solution for type-1 hepatorenal syndrome? L Lavayssiere University Hospital, Toulouse, France Critical Care 2013, 17(Suppl 2):P430 (doi: 10.1186/cc12368) Can treatment with the molecular adsorbent recirculation system be the solution for type-1 hepatorenal syndrome? L Lavayssiere University Hospital, Toulouse, France Critical Care 2013, 17(Suppl 2):P430 (doi: 10.1186/cc12368) University Hospital, Toulouse, France Introduction The aim of this retrospective study was to determine whether the molecular adsorbent recirculation system (MARS) can improve renal function in HSR1 patients. Methods Thirty-two patients with chronic liver disease and HRS1 were treated by MARS sessions that were performed every other day. The endpoint was renal function improvement by 28 days after diagnosis of HRS1 that was defi ned as a serum creatinine level <133  μmol/l. Partial renal recovery was defi ned as a 10% decrease in baseline serum creatinine level. y Conclusion Individuals who develop dialysis-dependent AKI in the ICU setting in general terms either die or recover. Sepsis is the most common association with death. The need for mechanical ventilation and inotropic therapy are both associated with increased incidence of death. Results The mean number of MARS sessions required by each patient was 3.5  ±  1.5. The median time between admission and the start of MARS therapy was 3 (0 to 15) days. Of the total patients, 13 (40%) had improved renal function. Among these, nine (28%) had complete renal recovery. Among the patients that survived, only 40% (6/15) had improved renal function. When MARS was started, 53% of patients had failed renal failure according to RIFLE criteria. Seven patients received a liver transplant after diagnosis of HRS. Of these, four had complete or partial recovery after transplantation (57%) versus nine of the 25 patients who did not undergo liver transplantation (36%), P = NS. The 28-day survival rate was 47%. P429fi P429 Effi ciency of substitutive renal therapy in the complex intensive care of multiple organ failure in patients with polytrauma S Kravtsov, A Shatalin, V Agadzhanyan, D Skopintsev Federal State Budgetary Medical Prophylactic Institution ‘Scientifi c Clinical Center of the Miners’ Health Protection’, Leninsk-Kuznetsky, Russia Critical Care 2013, 17(Suppl 2):P429 (doi: 10.1186/cc12367) Introduction This study was to evaluate the effi ciency of the early start of intermittent substitutive renal therapy in patients with polytrauma complicated by multiple organ failure syndrome. 1. Nadim MK, Genyk YS, Tokin C, et al.: Impact of etiology of acute kidney injury on outcomes following liver transplantation: acute tubular necrosis versus hepatorenal syndrome. Liver Transpl 2012, 18:539-548. 2. Cholongitas E, Senzolo M, Patch D, Shaw S, O’Beirne J, Burroughs AK: Cirrhotics admitted to intensive care unit: the impact of acute renal failure on mortality. Eur J Gastroenterol Hepatol 2009, 21:744-750. 1. Nadim MK, Genyk YS, Tokin C, et al.: Impact of etiology of acute kidney injury on outcomes following liver transplantation: acute tubular necrosis versus hepatorenal syndrome. Liver Transpl 2012, 18:539-548. p y p g y Methods Forty-two patients with polytrauma complicated by multiple organ failure syndrome were included in the study. The age of the patients was from 20 to 60 years (38.3 ± 1.6 years average). All patients were divided into two equal groups. In the control group (CG) the criteria for the start of the substitutive renal therapy were: hyperkalemia ≥6  mmol/l, plasma creatinine ≥280  μmol/l, diuresis ≤20  ml/hour. In the investigation group (IG) there were subtests to carry out the substitutive renal therapy, allowing one to start it in the earlier period of the multiple organ failure progression. These are increase of Na+ >150  mmol/l, osmolarity >300  mOsm/l, elevation of the plasma toxicity according to the average molecule concentration ≥1.0, diuresis decrease ≤40 ml/hour. These were examined: lethality, quantity of the substitutive renal therapy procedures, mechanical lung ventilation duration (MLV), intensive care and hospital duration. The substitutive renal therapy was carried out by AK-200-Ultra apparatus (Gambro, Sweden). The statistical analysis was realized using Statistica 6.1 and the Mann–Whitney U test. 2. Cholongitas E, Senzolo M, Patch D, Shaw S, O’Beirne J, Burroughs AK: Cirrhotics admitted to intensive care unit: the impact of acute renal failure on mortality. Eur J Gastroenterol Hepatol 2009, 21:744-750. 2. References 1. Metnitz PG, et al.: Eff ect of acute renal failure requiring renal replacement therapy on outcome in critically ill patients. Crit Care Med 2002, 30:2051-2058. 2. Uchino S, et al.: Acute renal failure in critically ill patients: a multinational, multicenter study. JAMA 2005, 294:813-818. 2. Uchino S, et al.: Acute renal failure in critically ill patients: a multinational, multicenter study. JAMA 2005, 294:813-818. y Conclusion MARS therapy improved renal function in only very few patients with HRS1. Even if liver transplantation is the best option, MARS could be used as a bridge until transplantation. Some authors argue that prolonged duration of dialysis (>1 month) before transplantation may result in chronic renal failure probably because there are two etiologies of AKI in end-stage liver disease: acute tubular necrosis (ATN) and real functional renal failure. ATN before liver transplantation has been identifi ed as a risk factor for patients’ mortality and chronic renal failure at 1 and 5 years post liver transplantation. Further prospective controlled studies including large number of patients are required. References P429fi Cholongitas E, Senzolo M, Patch D, Shaw S, O’Beirne J, Burroughs AK: Cirrhotics admitted to intensive care unit: the impact of acute renal failure on mortality. Eur J Gastroenterol Hepatol 2009, 21:744-750. Aetiology and outcomes for dialysis-dependent acute kidney injury patients on the ICU M Hameed1, P Carmichael2 1Salford Royal NHS Foundation Trust, Salford, UK; 2The Royal Wolverhampton Hopsitals NHS Trust, Wolverhampton, UK Critical Care 2013, 17(Suppl 2):P428 (doi: 10.1186/cc12366) Introduction AKI is a common occurrence in sick hospitalized patients, in particular those admitted to intensive care. Published data suggest that 4 to 5% of all critically ill patients develop severe AKI and require initiation of renal replacement therapy (RRT) [1,2]. Such patients have high mortality rates often exceeding 60% [2]. We aimed to review the outcomes of patients admitted to the ICU and required renal replacement therapy for AKI. We examined whether aetiology of AKI, comorbidity burden, hospital length of stay and treatment in ICU had any signifi cant association with survival in the study cohort.i i Methods During 2009, 56 patients were identifi ed to have received RRT with AKI who were admitted to the ICU at the Royal Wolverhampton Hospitals NHS Trust. Computerised and paper-based case records were examined for these patients to collect the data. AKIN classifi cation was used to classify the severity of AKI. Figure 1 (abstract P426). NGAL at ICU admission predicts fl uid overload of >10% of body weight. Figure 1 (abstract P426). NGAL at ICU admission predicts fl uid overload of >10% of body weight. Figure 1 (abstract P427). A suggested follow-up pathway for AKI survivors. S160 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Results Median age at admission was 66 years (27 to 85) with 29 males and 27 females. Thirty-one (55.4%) patients had sepsis and 20 (35.7%) patients had ATN as the main cause of AKI. Thirty-two patients (57%) had three organ failures at the time of commencement of RRT. Forty- six patients (82.1%) received haemofi ltration only. Thirty-two (57%) patients died, with more than 80% of these occurring in the ITU. There was no signifi cant diff erence in survival when compared with duration of haemofi ltration, length of stay, number of organs failed and number of comorbidities. However, signifi cantly more patients that died had AKI due to sepsis (P = 0.003) or if they received mechanical ventilation (P = 0.48) or inotropes (0.04). Of the 27 patients who survived until discharge from hospital, 18 (66.7%) had normal renal function, eight (29.6%) had AKIN stage I and only one patient required maintenance haemodialysis. Dialyzer clearance of myoglobin with middle molecule fi lter and low blood fl ow CVVHD in patients with rhabdomyolysis-associated acute kidney injury y j y I Leppanen1, T Ahonen1, J Tenhunen2 1Tampere University Hospital, Tampere, Finland; 2Uppsala University Hospital, Uppsala, Sweden Critical Care 2013, 17(Suppl 2):P432 (doi: 10.1186/cc12370) y j y I Leppanen1, T Ahonen1, J Tenhunen2 1Tampere University Hospital, Tampere, Finland; 2Uppsala University Hospital, Uppsala, Sweden Critical Care 2013, 17(Suppl 2):P432 (doi: 10.1186/cc12370) Figure 1 (abstract P433). Daily fl uid balance and 95% CIs among survivors and nonsurvivors. Introduction Rhabdomyolysis-associated acute kidney injury can be treated with various modes of renal replacement therapy (RRT) [1,2]. We have used continuous venoveno hemodialysis (CVVHD) with middle molecule fi lter and low extracorporeal blood fl ow (100 to 180 ml/minute) to avoid albumin loss and hemodynamic instability.f y y Methods We treated nine patients suff ering from rhabdomyolysis and acute kidney injury with various causes. We used a CVVHD mode with low (100 to 180 ml/minute) blood fl ow and a middle molecule fi lter (Ultrafl ux EMiC2; 1.8 m2 surface area, polysulfone membrane, cutoff at 40 kDa, blood fi lling volume 130 ml, blood fl ow range 100 to 350 ml/ minute, maximum dialysate fl ow 1,000  ml/minute). Myoglobin and albumin concentrations were measured from prefi lter and postfi lter samples and dialyzer clearances were calculated (CLdial  =  blood fl ow×Cpre – Cpost/Cpre + UF×Cpre/Cpost). Measurements were taken at 0, 15 minutes, 30 minutes, 4 hours, 12 hours, 24 hours, 36 hours and 48 hours from the start of the CRRT or until CRRT was no longer needed. Results The baseline and prefi lter plasma albumin concentrations varied from 8 to 29 g/l. Postfi lter and post-treatment albumin concentrations remained comparable. Prefi lter concentrations of myoglobin (17.8 kDa) varied from 96,109 to 747 μg/l and the dialyzer clearance of myoglobin from 50.6 to 0 ml/minute. The mean dialyzer clearance was 23.1 ml/ minute and the median clearance 22.2 ml/minute. Maximal changes between corresponding prefi lter and postfi lter samples were: absolute concentration change 27,091 μg/l, percentage 34%, dialyzer clearance 50.6  ml/minute. Clearances were achieved with low extracorporeal blood fl ow between 100 and 180 ml/minute, most commonly 120 ml/ minute. Ultrafi ltration (UF) was used only at fi ve of the 56 time points calculated, because of hemodynamic instability. All patients required either vasopressor or vasopressor and inotrope support. P432 P432 Dialyzer clearance of myoglobin with middle molecule fi lter and low blood fl ow CVVHD in patients with rhabdomyolysis-associated acute kidney injury I Leppanen1, T Ahonen1, J Tenhunen2 1Tampere University Hospital, Tampere, Finland; 2Uppsala University Hospital, Uppsala, Sweden Critical Care 2013, 17(Suppl 2):P432 (doi: 10.1186/cc12370) Dialyzer clearance of myoglobin with middle molecule fi lter and low blood fl ow CVVHD in patients with rhabdomyolysis-associated acute kidney injury The highest dialyzer clearance of myoglobin (50.6 ml/minute) was measured at the prefi lter myoglobin of 29,266 μg/l, blood fl ow 180 ml/ml and no UF. Figure 1 (abstract P433). Daily fl uid balance and 95% CIs among survivors and nonsurvivors. Conclusion Among critically ill patients who are receiving CRRT, daily fl uid balance is an independent predictor of mortality, even after adjusting for the daily severity of illness and comorbidities. References Conclusion Among critically ill patients who are receiving CRRT, daily fl uid balance is an independent predictor of mortality, even after adjusting for the daily severity of illness and comorbidities. References 1. Bagshaw SM, et al.: Crit Care 2008, 12:169. 2. Bellomo R, et al.: N Engl J Med 2009, 361:1627. 3. Bellomo R, et al.: Crit Care Med 2012, 40:1753-1760. Eff ect of continuous high-volume hemofi ltration on patients with acute respiratory distress syndrome The PiCCOplus system was used to monitor cardiac output (CO), extravascular lung water index (EVLWI) and intrathoracic blood volume index (ITBVI). Arterial partial pressure of oxygen (PaO2), arterial partial pressure of carbon dioxide (PaCO2), oxygenation index (PaO2/FiO2) and dynamic lung compliance (Cdyn), oxygen delivery and oxygen consumption were measured. Serum concentrations of TNFα, IL-6, IL-8 Conclusion The effi ciency of the substitutive renal therapy depends directly on the hydroelectrolytic and metabolic changes and toxicosis degrees in the polytrauma complicated by multiple organ failure syndrome. The early start of the dialysis methods treatment allows one to achieve the earlier recuperation of the renal functions and to decrease the lethality level by 14%. S161 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 P433l and lactate were measured by ELISA method. The APACHE II scores before and after IHVHF therapy were detected.i Daily fl uid balance predicts hospital mortality in critically ill patients receiving continuous renal replacement therapy M Kashiouris, A Akhoundi, S Chaudhary, V Velagapudi, A Goldberg, K Kashani Mayo Clinic, Rochester, MN, USA Critical Care 2013, 17(Suppl 2):P433 (doi: 10.1186/cc12371) Results All indexes in the control group did not show any signifi cant improvement before and after treatment (P  <0.05). There was a signifi cant decrease of serum concentrations of TNFα, IL-6, IL-8 and lactate, increase of CO and decrease of EVLWI 24  hours after IHVHF (P <0.05). The indexes of oxygen delivery, consumption, PaO2, and Cdyn improved signifi cantly, PaO2/FiO2 increased markedly compared with those before IHVHF and the control group. Signifi cant diff erences were detected in all of the hemodynamics and oxygen indexes (P <0.05). Introduction It has been suggested that fl uid balance is a biomarker in critically ill patients [1]. There is a paucity of randomized trials examining the eff ect of daily fl uid balance on outcomes in patients on continuous renal replacement therapy (CRRT). The RENAL trial did not fi nd mortality diff erence with higher CRRT dose [2], but did not investigate the eff ect of daily fl uid balance on patient outcomes. A post hoc analysis suggested survival benefi t in patients with negative fl uid balance [3]. In this study, we hypothesize that daily fl uid balance is an independent predictor of mortality in critically ill patients. y yg Conclusion IHVHF presents with a signifi cant benefi cial eff ect on respiratory function in patients with ARDS as a result of removal of cytokines, decrease in EVLWI, improvement of hemodynamic parameters, and correction of disturbances in oxygen balance. IHVHF adjuvant treatment for ARDS reduces pulmonary edema, improves PaO2/FiO2 and Cdyn and mechanical ventilation parameters. IHVHF may be a promising treatment for ARDS in postoperative multiple organ dysfunction syndrome in severe abdominal infection patients. Reference Methods We conducted a retrospective cohort study in eight ICUs of a tertiary academic center. We constructed a robust clustered linear regression model of daily fl uid balance and all-cause hospital mortality among 595 critically ill patients receiving CRRT. We adjusted the model for the Charlson comorbidity score, the daily SOFA scores in the fi rst week after initiation of CRRT as well the type of ICU. 1. 1. Honoré P, et al.: Anestesiol Reanimatol 2008, 6:4-11. 2. Sorrentino SA, et al.: Crit Care Med 2011, 39:184-186. 1. Albert C, et al.: Crit Care Resusc 2012, 14:159-162. P433l Honoré P, et al.: Anestesiol Reanimatol 2008, 6:4-11. Results After adjusting for the type of ICU and the daily severity of illness, patients who died had on average 779  ml higher daily fl uid balance compared with patients who survived (P  <0.001, 95% CI = 385 to 1,173 ml, Figure 1). Severity of illness predicted daily fl uid accumulation; each additional point of the SOFA score predicted an additional 57 ml of extra daily fl uid (P = 0.002). P432 Fluid balance and renal outcomes in patients requiring renal replacement therapy in the ICU prefi lter myoglobin of 29,266 μg/l, blood fl ow 180 ml/ml and no UF. Conclusion In a rhabdomyolysis-associated kidney injury a middle molecule fi lter and a low blood fl ow CVVHD off er a safe and eff ective treatment choice for patients requiring vasopressor or vasopressor and inotrope for hemodynamic support. Introduction Fluid overload is associated with mortality in critically ill patients with acute kidney injury (AKI) [1-3]. We explored this relationship in patients with AKI who received renal replacement therapy (RRT) in the ICU to investigate the relationship between fl uid 1. Albert C, et al.: Crit Care Resusc 2012, 14:159-162. 2 Sorrentino SA et al : Crit Care Med 2011 39:184-186 Pharmacokinetics of gentamicin and vancomycin during continuous venovenous hemofi ltration in critically ill septic patients with acute kidney injury p Results A total of 522 patients were included (319 male, mean age 64 years); 264 (50.6%) died in hospital. Survivors and those who died were similar with respect to age, weight and incidence of heart failure, liver cirrhosis and primary renal diagnoses. Independent risk factors for hospital mortality were higher Sequential Organ Failure Assessment score at RRT initiation (odds ratio (OR)  =  1.18, 95% confi dence interval = 1.11 to 1.25), higher Charlson comorbidity index (OR = 1.22, 1.09 to 1.37), lower baseline creatinine (OR  =  0.984 per 10  μmol/l, 0.970 to 0.998), lower minimum mean arterial pressure (MAP) on day 1 (OR = 0.81 per 10 mmHg, 0.66 to 0.95) and more positive fl uid balance for the fi rst 7 days (OR 1.03 per 100 ml, 1.02 to 1.05). Of 172 hospital survivors, 57 (33.1%) were RRT dependent at discharge. Although univariable analysis suggested lower SOFA scores and higher baseline serum creatinine levels in those who remained RRT dependent, no factor was independently associated with RRT dependence at discharge in a multivariate model. y j y N Petejova1, J Duricova1, A Martinek1, J Zahalkova2 1University Hospital, Ostrava, Czech Republic; 2Hospital, Sternberk, Czech Republic Introduction Acute kidney injury (AKI) is a common complication of critical illness and sepsis [1]. Dosing of antibacterial agents in septic patients is complicated by altered pharmacokinetics due to both acute renal failure and critical illness [2]. Current dosing regimens for administration of gentamicin and vancomycin to septic patients with AKI on continuous venovenous hemofi ltration (CVVH) at a fi ltration rate of 45 ml/kg/hour are missing. Methods Seventeen septic patients with AKI treated with vancomycin and seven patients with gentamicin on CVVH were included. In the vancomycin group, patients received the fi rst dose of 1.0 g intravenously followed by 1.0 g/12 hours if not adjusted. In the gentamicin group, patients received a loading dose of 240 mg followed by a maintenance dose every 24 hours. The vancomycin maintenance dose was optimized to achieve AUC0–24/MIC ≥400 (Cmin >10 mg/l), gentamicin target was Cmax/MIC of 8 to 10. Maintenance doses were adjusted according to drug level simulation using a pharmacokinetic programme. g Conclusion In this cohort of patients with AKI requiring RRT, a more positive fl uid balance over 1 week and lower initial minimum MAP were associated with mortality. References 1. Uchino S, et al.: JAMA 2005, 294:813-818. 2. Roberts JA, et al.: Crit Care Med 2009, 37:840-851. Methods Esomeprazole 40  mg was administered intravenously once daily in nine intensive care patients and ranitidine 50  mg was administered intravenously three times daily in nine intensive care patients with acute renal failure undergoing CVVHDF who required stress ulcer prophylaxis. The concentration of esomeprazole and ranitidine in serum and ultradiafi ltrate was determined by high- performance liquid chromatography. The intragastric pH was obtained via intragastric pH-metry.i Pharmacokinetics and pharmacodynamics of esomeprazole and ranitidine during continuous venovenous hemodiafi ltration pi Conclusion CVVH at a fi ltration rate of 45  ml/kg/hour leads to high removal of both antibiotics. Due to rapid change in patient’s clinical status it was impossible to predict a fi xed dosage regimen. We recommend administration of unreduced loading dose and: blood sampling as early as 6  hours after fi rst vancomycin dose; blood sampling 30 to 60 minutes after gentamicin administration and before the next dose; and the maintenance dose should be based on drug- level monitoring. Introduction Esomeprazole, a proton pump inhibitor, and ranitidine, a histamine-2 receptor antagonist are frequently used in intensive care patients requiring stress ulcer prophylaxis. Continuous venovenous hemodiafi ltration (CVVHDF) is an important extracorporeal renal replacement therapy in critically ill patients suff ering from multiple organ failure. This study investigates the pharmacokinetics and pharmacodynamics of esomeprazole and ranitidine in anuric critically ill patients undergoing CVVHDF. p References S162 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 balance and intradialytic hypotension with mortality and recovery of renal function. balance and intradialytic hypotension with mortality and recovery of renal function. intragastric pH >5 and median intragastric pH decreased signifi cantly in the ranitidine group over time (P <0.05) but not in the esomeprazole group. Methods We conducted a retrospective cohort study among patients aged ≥16 years who had RRT initiated and continued for ≥2 days in a level 2 or 3 ICU at two academic centres, and had fl uid balance data available. Patients with end-stage kidney disease, within 1  year of a renal transplant or who had RRT initiated to treat a toxic ingestion were excluded. We used multivariable logistic regression to determine the relationship between mean daily fl uid balance over the fi rst 7 days following RRT initiation and the outcomes of mortality and RRT dependence in survivors. g Conclusion Although pharmacokinetics of esomeprazole and raniti- dine are only little infl uenced by CVVHDF, we found a signifi cant decrease of intragastric pH in patients treated with ranitidine. Esome- prazole may be superior for long-term stress ulcer prophylaxis in critically ill patients undergoing CVVHDF. P436 P436 P436 Pharmacokinetics of gentamicin and vancomycin during continuous venovenous hemofi ltration in critically ill septic patients with acute kidney injury N Petejova1, J Duricova1, A Martinek1, J Zahalkova2 1University Hospital, Ostrava, Czech Republic; 2Hospital, Sternberk, Czech Republic Critical Care 2013, 17(Suppl 2):P436 (doi: 10.1186/cc12374) P435 P435 Pharmacokinetics and pharmacodynamics of esomeprazole and ranitidine during continuous venovenous hemodiafi ltration V Fuhrmann1, P Schenk2, W Jaeger3, M Miksits3, R Kitzberger4, U Holzinger1, C Madl5 1Medical University Vienna, Austria; 2Landesklinikum Thermenregion Hochegg, Grimmenstein, Austria; 3University Vienna, Austria; 4Kaiser Franz Josef Krankenhaus, Vienna, Austria; 5Krankenanstalt Rudolfstiftung Wien, Vienna, Austria Critical Care 2013, 17(Suppl 2):P435 (doi: 10.1186/cc12373) Pharmacokinetics and pharmacodynamics of esomeprazole and ranitidine during continuous venovenous hemodiafi ltration V Fuhrmann1, P Schenk2, W Jaeger3, M Miksits3, R Kitzberger4, U Holzinger1, C Madl5 Pharmacokinetics of gentamicin and vancomycin during continuous venovenous hemofi ltration in critically ill septic patients with acute kidney injury Among survivors, a less positive fl uid balance was not associated with increased risk of RRT dependence at discharge, suggesting that conservative fl uid management does not signifi cantly attenuate renal recovery. gi y References References 1. Bouchard J, et al.: Kidney Int 2009, 76:422-427. 2. Grams ME, et al.: Clin J Am Soc Nephrol 2011, 6:966-973. 3. Payen D, et al.: Crit Care 2008, 12:R74. Results The median vancomycin total clearance (Cltot) was 0.89 and 0.55  ml/minute/kg on the fi rst and second day of the study. CRRT clearance accounted for about 50 to 60% of vancomycin Cltot found in a population with normal renal function (0.97  ml/minute/kg). Vancomycin serum concentrations after the fi rst dose were below the required target of 10 mg/l as early as 6 hours in 10 patients. AUC0–24/ MIC ≥400 ratio was achieved in 67% of patients on the fi rst day. The median gentamicin Cltot was 0.68 and 0.79 ml/minute/kg on the fi rst and second day of the study. CRRT clearance accounted for about 50% of gentamicin Cltot found in a population without renal impairment (0.73 ml/minute/kg). The target Cmax/MIC ratio was achieved in 78% of patients after the fi rst dose. Muscle wasting in the ICU can be reliably monitored using ultrasound H Jørgensen1, B Nedergaard2, T Gilsaa1 1Lillebaelt Hospital, Kolding, Denmark; 2Odense University Hospital, Svendborg, Denmark Critical Care 2013, 17(Suppl 2):P439 (doi: 10.1186/cc12377) g Critical Care 2013, 17(Suppl 2):P439 (doi: 10.1186/cc12377) Introduction The aim of this study was to establish the intraobserver and interobserver variation of ultrasonographic measurements of the rectus femoris muscle cross-section area (RF-CSA). Muscle wasting is frequent in the ICU, aff ecting more than one-half of the patients with severe sepsis [1]. Muscle mass reduces rapidly, and 15 to 20% is lost within the fi rst week [1]. To monitor muscle mass, ultrasound has the benefi ts of being both readily available in the ICU and non-invasive. Ultrasonographic measurement of RF-CSA has an almost perfect correlation with MRI (mean interclass correlation (ICC) = 0.999) [2] and RF-CSA is linearly related to maximum voluntary contraction strength in both healthy subjects and COPD patients (r = 0.78) [3]. Conclusion Adipogenesis and accumulation of M2-macrophages are hallmarks of critical illness, irrespective of nutritional management in humans and mice. Critical illness evokes macrophage polarization to the M2-state not only in adipose tissue but also in liver and lungs, which is further accentuated by fasting. P440 Critical illness induces nutrient-independent adipogenesis and accumulation of alternatively activated tissue macrophages M Marques, S Vander Perre, A Aertgeerts, S Derde, F Guiza, M Casaer, G Hermans, G Van den Berghe, L Langouche KU Leuven, Belgium Critical Care 2013, 17(Suppl 2):P440 (doi: 10.1186/cc12378) P437 ff P437 Eff ects of two diff erent regional citrate anticoagulation CVVH protocols on acid–base status and phosphate supplementation S Morabito1, V Pistolesi1, V Pistolesi1, L Tritapepe2, L Tritapepe2, E Vitaliano3, E Vitaliano3, L Zeppilli1, L Zeppilli1, F Polistena1, F Polistena1, E Fiaccadori4, E Fiaccadori4, A Pierucci1, A Pierucci1 1Umberto I, Policlinico di Roma, ‘Sapienza’ University, Rome, Italy; 2Umberto I, Policlinico di Roma, Rome, Italy; 3Pertini Hospital, Rome, Italy; 4University of Parma Medical School, Parma, Italy Critical Care 2013, 17(Suppl 2):P437 (doi: 10.1186/cc12375) Results The mean peak prefi lter concentration, AUC0–24, volume of distribution, half-life, total clearance and hemodiafi ltration clearance of esomeprazole were 6.9 μmol/l, 30.6 μmol.hour/l, 53.3 l, 2.0 hours, 1.4 l/ hour and 0.2 l/hour, respectively. In the ranitidine group the mean peak prefi lter concentration, AUC0–8, volume of distribution, half-life, total clearance and hemodiafi ltration clearance were 5.5 μmol/l, 12.0 μmol. hour/l, 91.4 l, 14.4 hours, 5.8 l and 1.3 l, respectively. Median time of Introduction Regional citrate anticoagulation (RCA) is increasingly used in high bleeding risk patients undergoing CRRT. Regardless of anticoagulation protocol, hypophosphatemia occurs frequently in Introduction Regional citrate anticoagulation (RCA) is increasingly used in high bleeding risk patients undergoing CRRT. Regardless of anticoagulation protocol, hypophosphatemia occurs frequently in S163 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 distance from the anterior superior iliac spine to the superior patellar border [1]. RF-CSA was calculated by planimetry. At each scan, three measurements were made. For intraobserver variation, the 3×3 scans were analyzed using the interclass correlation coeffi cient. For interobserver variation, the three measurements from each observer were averaged and compared using Bland–Altman statistics. Results Intraobserver variation: 15 healthy adults, age 39.6 ± 2.4 years, weight 66.8 ± 2.3 kg, sex three male/12 female. ICC: 0.996 (95% CI: 0.990 to 0.998). Interobserver variation: 15 ICU patients, age: 77 ± 8.3 years, weight: 71.3 ± 9.1 kg, sex nine male/six female. Bland–Altman: bias: –0.07 cm2, 95% limits of agreement –0.188 to 0.048 cm2. Conclusion Ultrasonographic measurement of RF-CSA is easily distance from the anterior superior iliac spine to the superior patellar border [1]. RF-CSA was calculated by planimetry. At each scan, three measurements were made. For intraobserver variation, the 3×3 scans were analyzed using the interclass correlation coeffi cient. References 1. Langouche L, et al.: Am J Respir Crit Care Med 2010, 182:507-516. 2. Casaer MP, et al.: N Engl J Med 2011, 365:506-517. References 1. Parry SM, et al.: BMJ Open 2012, e001891. doi:10.1136/bmjopen 2. Reeves ND, et al.: Eur J Appl Physiol 2004, 91:116-118. 3. Seymour JM, et al.: Thorax 2009, 64:418-423. g p Results In 30 cardiac surgery patients with AKI, acid–base status and electrolytes have been evaluated comparing protocol A (20 patients, running time 5,283  hours) versus protocol B (10 patients, 1,170  hours) (median (IQR)): pH 7.40 (7.36 to 7.44) versus 7.43 (7.41 to 7.47) (P <0.0001), bicarbonate 22.1 (20.9 to 23.5) versus 24.4 (23.2 to 25.6) mmol/l (P <0.0001), base excess –3.1 (–4.6 to –1.15) versus 0 (–1.5 to 1.1) (P <0.0001), systemic Ca2+ 1.16 (1.1 to 1.23) versus 1.14 (1.08 to 1.19)  mmol/l (P  <0.0001), phosphate 0.7 (0.5 to 1) versus 1.1 (0.9 to 1.4)  mmol/l (P <0.0001). Protocol A required bicarbonate infusion in 90% of patients (6 ± 6.4 mmol/hour) and sodium phosphate supplementation in all cases (4.1  ±  2.4  g/day). A lower amount of phosphate supplementation (0.9  ±  2  g/day) was needed in 30% of patients undergoing protocol B while bicarbonate infusion was never required. Filter life was comparable (51.8 ± 36.5 vs. 46.8 ± 30.3 hours, P = NS).f P437 ff For interobserver variation, the three measurements from each observer were averaged and compared using Bland–Altman statistics. CRRT. The aim was to evaluate the eff ects on electrolyte and acid–base status of a new RCA-CVVH protocol using an 18 mmol/l citrate solution combined with a phosphate-containing replacement fl uid, compared with a previously adopted RCA-CVVH protocol combining a 12 mmol/l citrate solution with a conventional replacement fl uid. CRRT. The aim was to evaluate the eff ects on electrolyte and acid–base status of a new RCA-CVVH protocol using an 18 mmol/l citrate solution combined with a phosphate-containing replacement fl uid, compared with a previously adopted RCA-CVVH protocol combining a 12 mmol/l citrate solution with a conventional replacement fl uid. distance from the anterior superior iliac spine to the superior patellar border [1]. RF-CSA was calculated by planimetry. At each scan, three measurements were made. For intraobserver variation, the 3×3 scans were analyzed using the interclass correlation coeffi cient. For interobserver variation, the three measurements from each observer were averaged and compared using Bland–Altman statistics. pl Methods Until September 2011, RCA-CVVH was routinely performed in our centre with a 12  mmol/l citrate solution and a postdilution replacement fl uid with bicarbonate (HCO3 – 32, Ca2+ 1.75, Mg2+ 0.5, K+ 2 mmol/l) (protocol A). In cases of metabolic acidosis, not related to inappropriate citrate metabolism and persisting after optimization of RCA-CVVH parameter setting, bicarbonate infusion was scheduled. Starting from September 2011, in order to optimize buff er balance and to reduce the need for phosphate supplementation, a new RCA-CVVH protocol has been designed using an 18  mmol/l citrate solution combined with a recently introduced phosphate-containing replacement fl uid with bicarbonate (HCO3 – 30, phosphate 1.2, Ca2+ 1.25, Mg2+ 0.6, K+ 4 mmol/l) (protocol B). g g Results Intraobserver variation: 15 healthy adults, age 39.6 ± 2.4 years, weight 66.8 ± 2.3 kg, sex three male/12 female. ICC: 0.996 (95% CI: 0.990 to 0.998). Interobserver variation: 15 ICU patients, age: 77 ± 8.3 years, weight: 71.3 ± 9.1 kg, sex nine male/six female. Bland–Altman: bias: –0.07 cm2, 95% limits of agreement –0.188 to 0.048 cm2. g Conclusion Ultrasonographic measurement of RF-CSA is easily learned and quickly performed. It has a very low intraobserver and interobserver variation and can be recommended as a reliable method for monitoring muscle wasting in the ICU. R f Critical illness induces nutrient-independent adipogenesis and accumulation of alternatively activated tissue macrophages M Marques, S Vander Perre, A Aertgeerts, S Derde, F Guiza, M Casaer, G Hermans, G Van den Berghe, L Langouche KU Leuven, Belgium Critical Care 2013, 17(Suppl 2):P440 (doi: 10.1186/cc12378) Critical Care 2013, 17(Suppl 2):P440 (doi: 10.1186/cc12378) Introduction In artifi cially fed critically ill patients, adipose tissue reveals an increased number of small adipocytes and accumulation of M2-type macrophages [1]. We hypothesized that nutrient-independent factors of critical illness explain these fi ndings, and also that M2- macrophage accumulation during critical illness may not be limited to adipose tissue. Conclusion Protocol B provided a buff er balance more positive than protocol A and allowed one to adequately control acid–base status without additional bicarbonate infusion and in the absence of alkalosis, despite the use of a standard bicarbonate concentration replacement solution. Furthermore, the combination of a phosphate-containing replacement fl uid appeared eff ective to prevent hypophosphatemia. p Methods We performed a randomized investigation in a septic mouse model of critical illness and a study of ICU patient biopsies. In the critically ill mouse, we compared the eff ect of parenteral nutrition (n  =  13) with fasting (n  =  11) on body composition, adipocyte cell size, and macrophage accumulation in adipose tissue, liver and lung. Fed healthy control mice (n  =  11) were studied for comparison. In vivo adipose tissue was harvested after 1 week of illness from human patients (n = 40) who participated in a RCT on early parenteral nutrition versus tolerating nutrient restriction [2], adipose tissue morphology was characterized and compared with healthy controls (n = 13). P438 Abstract withdrawn P438 Abstract withdrawn P438 Results Irrespective of nutritional intake, critically ill mice lost body weight, total fat and fat-free mass. Part of the fat loss was explained by reduced ectopic fat accumulation. Adipocyte cell number and the adipogenic markers peroxisome proliferator-activated receptor γ and CCAT/enhancer binding-protein β increased with illness, again irrespective of nutritional intake. Macrophage accumulation with predominant M2-phenotype was observed in adipose tissue, liver and lungs of critically ill mice, further accentuated by fasting in visceral tissues. Macrophage M2-markers correlated with chemoattractant factor expression in all studied tissues. In human subcutaneous adipose tissue biopsies of critically ill patients, increased adipogenic markers and M2 macrophage accumulation were present irrespective of nutritional intake. P439 Muscle wasting in the ICU can be reliably monitored using ultrasound H Jørgensen1, B Nedergaard2, T Gilsaa1 1Lillebaelt Hospital, Kolding, Denmark; 2Odense University Hospital, Svendborg, Denmark Critical Care 2013, 17(Suppl 2):P439 (doi: 10.1186/cc12377) P444 Strong ion gap can be accurately estimated with a simple bedside equation g y y Methods Obstetric patients were identifi ed from the ICU admissions database and divided into two groups – those treated with magnesium (for suspected pre-eclampsia) and those admitted for other obstetric indications (postpartum hemorrhage, infection, etc.). The baseline calcium values were compared, as well as the lowest and discharge values. Albumin and magnesium values were also compared. All comparisons used Student’s t test. Introduction The anion gap (AG) is used routinely in the assessment of metabolic acidosis, but can be misleading in patients with hypoalbuminemia and other disorders commonly encountered in intensive care. This approach to acid–base analysis relies on assessment of pH, pCO2, sodium, bicarbonate and chloride, and can lead to underestimation or overestimation of the true electrochemical status of a patient, as it does not include important ions such as lactate, calcium, magnesium, and albumin. The strong ion gap (SIG) is an alternative to the AG and is based upon Stewart’s physical chemistry approach. However, the SIG is cumbersome to calculate. As such, a number of shortcut equations have been developed in an eff ort to approximate the SIG. We sought to compare three such equations, the Kellum corrected anion gap (KellAGc), the Moviat equation, and EZSIG, in an eff ort to evaluate precision and accuracy [1-3]. Results Data were collected on 88 parturients admitted over 2 years including 40 (45%) who received magnesium and 48 (55%) who did not. Magnesium-treated women were younger (age: 31 ± 7 vs. 36 ± 5 years, P  =  0.02). The baseline calcium concentrations were similar for the two groups (2.2 ± 0.2 vs. 2.2 ± 0.1 mmol/l, P = 0.85). Patients receiving magnesium had signifi cantly higher magnesium concentrations (2.1  ±  0.4 vs. 0.7  ±  0.2  mmol/l, P  <0.001), and signifi cantly lower calcium concentrations during therapy (1.6 ± 0.3 vs. 1.9 ± 0.3 mmol/l, P  <0.001). At discharge, the calcium levels were closer (magnesium treated 1.9 ± 0.2 vs. untreated 2.1 ± 0.1 mmol/l, P = 0.02). The albumin concentrations did not diff er between the two groups (magnesium treated 27 ± 13 vs. nontreated 33 ± 23 g/l, P = 0.134). Normal values: calcium 2.15 to 2.55 mmol/l, magnesium 0.7 to 0.95 mmol/l, albumin 35 to 50 g/l. f y Methods We conducted a retrospective chart review of consecutive patients admitted to the ICU of George Washington University Medical Center from September 2010 to March 2011. References 1. Kellum JA: Crit Care 2005, 9:500-507. 2. Moviat M, et al: Crit Care 2003, 7:R41-R45. 3. Busse L, et al.: Chest J 2011, 140(4_MeetingAbstracts):1012A-1012A. 3. Busse L, et al.: Chest J 2011, 140(4_MeetingAbstracts):1012A-1012A. P444 Strong ion gap can be accurately estimated with a simple bedside equation Of the 1,516 patients screened, 200 met inclusion criteria, which included availability of all laboratory components to calculate the SIG, obtained within 1 hour of each other. Demographic data and serum values for pH, pCO2, albumin, lactate, sodium, potassium, chloride, bicarbonate, magnesium, phosphate, and calcium were collected. The AG, SIG, KellAGc, EZSIG, and Moviat equations were subsequently calculated and compared using Pearson correlation and Bland–Altman analysis. Conclusion Magnesium therapy was associated with hypocalcemia. Potential causative mechanisms include a renal excretion interaction and magnesium-induced suppression of parathyroid hormone secretion. Physicians should be aware of the potential for symptomatic hypocalcemia during magnesium therapy. g y Results The mean SIG was 3.25 ± 3.5. Mean values for KellAGc, Moviat, and EZSIG were 4.5 ± 5.0, 1.77 ± 2.2, and 3.6 ± 3.7, respectively. Pearson correlation coeffi cients for KellAGc, Moviat, and EZSIG when compared with the SIG were r = 0.77, P = 0.0001; r = 0.88, P = 0.001; and r = 0.89, P = 0.001, respectively. In Bland–Altman analysis, the mean bias for the test equations versus the SIG were: KellAGc (1.25), Moviat (–1.48), and EZSIG (0.40). References Methods The study had two purposes: to determine the intraobserver variation for RF-CSA by one observer scanning 15 healthy adult volunteers three times each at 2-day intervals; and to determine the interobserver variation for RF-CSA by two observers each scanning 15 adult ICU patients on the same day. Patients were in a supine position, legs in passive extension. The transducer was placed perpendicular to the long axis of the right thigh over the RF, two-thirds of the 1. Langouche L, et al.: Am J Respir Crit Care Med 2010, 182:507-516. 2. Casaer MP, et al.: N Engl J Med 2011, 365:506-517. P441 Abstract withdrawn P441 Abstract withdrawn P441 S164 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 P442 of low serum sodium levels, or if hyponatremia is simply a marker for ‘sicker’ patients, is not known. R f of low serum sodium levels, or if hyponatremia is simply a marker for ‘sicker’ patients, is not known. Reference P442 Hypocalcemia during magnesium therapy for obstetric ICU admissions PD Levin, A Szalat, M Vizana, CL Sprung, R Zaguri, Y Haviv Hadassah Hebrew University Medical Center, Jerusalem, Israel Critical Care 2013, 17(Suppl 2):P442 (doi: 10.1186/cc12380) 1. Asadollahi et al.: Hyponatraemia as a risk factor for hospital mortality. QJM 2006, 99:877-880. Introduction Intravenous magnesium sulfate is commonly used in obstetric patients with pre-eclampsia. Following a case of acute symptomatic hypocalcemia we retrospectively examined a cohort of patients to investigate the frequency of hypocalcemia. P445 Relative adrenal insuffi ciency in burns A Mokline, L Gharsallah, I Rahmani, H Oueslati, B Gasri, S Tlaili, R Hammouda, A Ksontini, A Ghanem, AA Messadi Burn and Trauma Center, Tunis, Tunisia Critical Care 2013, 17(Suppl 2):P445 (doi: 10.1186/cc12383) Relative adrenal insuffi ciency in burns Hyponatremia and intensive care outcome Hyponatremia and intensive care outcome P Dean, R Docking, D Govenden, A Davidson, A Mackay Victoria Infi rmary, Glasgow, UK Critical Care 2013, 17(Suppl 2):P443 (doi: 10.1186/cc12381) i y g Critical Care 2013, 17(Suppl 2):P443 (doi: 10.1186/cc12381) Introduction Disorders of sodium (Na+) and water homeostasis are common in hospitalised patients. Hyponatremia in particular has been associated with worse hospital outcome and length of stay [1]. We aimed to defi ne the incidence of hyponatremia (serum Na+ ≤134  mmol/l) in our intensive care population and to determine whether it was associated with ICU outcome or length of stay. Conclusion While all three equations correlated highly with the SIG, the EZSIG and Moviat outperformed the KellAGc in Pearson and Bland– Altman analysis. The EZSIG had a smaller bias than the Moviat equation and a slightly better correlation (0.89 vs. 0.88). In the assessment of critically ill patients, EZSIG is a candidate scanning equation for the measurement of the SIG when all SIG components are not available. f Methods Demographics, APACHE II score, outcome data and admission sodium were retrieved from the Ward Watcher system in the Victoria Infi rmary ICU for 2,440 consecutive admissions from January 2005 to present. We divided patients into three groups depending on serum Na+ (≤134 mmol/l, 135 to 144 mmol/l, ≥145 mmol/l) and compared APACHE II score, length of stay and ICU outcome between patients with a low versus a normal serum Na+. Data were analysed using the chi-squared test, Student’s t test and the Mann–Whitney test where appropriate. P444 P444 Strong ion gap can be accurately estimated with a simple bedside equation L Busse, L Chawla, R Panchamia, D Choi, E Nobakht, E Brasha-Mitchell, M Seneff George Washington University Medical Center, Washington, DC, USA Critical Care 2013, 17(Suppl 2):P444 (doi: 10.1186/cc12382) Etiology of low testosterone levels in mal sepsis requiring mechanical ventilation h A Bech, H Van Leeuwen, H De Boer A Bech, H Van Leeuwen, H De Boer Rijnstate Hospital, Arnhem, the Netherlands yp Results Data from 1,038 patients were included. Admission iCa was available for 976 (94%) patients. A total of 539 (55%) were hypocalcaemic (iCa <1.1 mmol/l). adjCa was available for 1,031 (99%), with 602 (58%) classifi ed as hypocalcaemic (adjCa <2.2 mmol/l). adjCa <2.2 mmol/l had sensitivity of 78% and specifi city of 63% for detecting iCa <1.1 mmol/l. The best previously published formula had an AUC of 0.81 (95% CI = 0.78 to 0.83), compared with an AUC of 0.78 (0.75 to 0.81) for the locally validated formula [1]. It was hypothesized that albumin, pH or phosphate derangements could aff ect the proportion of albumin- bound calcium and the performance of adjustment formulae. Albumin concentration was not correlated with the diff erence between adjCa and iCa. Weak but statistically signifi cant correlations were found for pH (r = 0.164; P <0.001) and phosphate (r = 0.135; P <0.001). The sensitivity and specifi city of adjCa <2.2 mmol/l for detecting hypocalcaemia did not improve when looking at patients with normal pH (sensitivity 60.4%; specifi city 69%) or phosphate (32.2%; 78.9%). j p Critical Care 2013, 17(Suppl 2):P448 (doi: 10.1186/cc12386) Introduction Low testosterone levels are frequently found in critically ill male patients. The etiology and clinical signifi cance is still poorly understood. In the present study we have investigated the kinetics and pathophysiology of altered gonadal hormone synthesis in male patients with severe sepsis and respiratory failure. Methods All male patients with severe sepsis and respiratory failure who were admitted to the ICU of a large teaching hospital in the Netherlands between September 2011 and June 2012 were included. Steroid hormone levels were measured on days 1, 3 and 7. y Results In total, 18 patients were included. The mean age was 69 ± 2 years, mean weight 76 ± 2 kg, APACHE II score 23 ± 2 and most patients suff ered from pneumosepsis. On the fi rst day of intubation, total and free testosterone levels were extremely low in most patients and remained low during the fi rst week (Figure 1). 17β-Estradiol levels were elevated on day 1 and decreased during the fi rst week. LH and i Conclusion adjCa is a relatively poor predictor of iCa in critically ill patients. Table 1 (abstract P447) Table 1 (abstract P447) Low Intermediate High Tertile (n = 216) (n = 219) (n = 220) P value 25(OH)D (ng/ml) 10.4 (± 3.6) 17.6 (± 5.1) 30.7 (± 11.4) <0.001 SAPS 2 32 (± 15) 28 (± 16) 28 (± 16) 0.007 Hospital mortality 58 (26.9%) 48 (21.2%) 29 (13.2%) 0.002 Lethal sepsis 11 (5.1%) 9 (4.1%) 0 (0.0%) 0.005 Conclusion Low 25(OH)D status is predictive of all-cause and sepsis mortality in the critically ill. Interventional studies are needed to investigate the eff ect of vitamin D on mortality and sepsis incidence and outcomes. Low Intermediate High Tertile (n = 216) (n = 219) (n = 220) P value 25(OH)D (ng/ml) 10.4 (± 3.6) 17.6 (± 5.1) 30.7 (± 11.4) <0.001 SAPS 2 32 (± 15) 28 (± 16) 28 (± 16) 0.007 Hospital mortality 58 (26.9%) 48 (21.2%) 29 (13.2%) 0.002 Lethal sepsis 11 (5.1%) 9 (4.1%) 0 (0.0%) 0.005 T Steele , R Kolamunnage-Dona , C Downey , C Toh , I Welters 1University of Liverpool, UK; 2Royal Liverpool University Hospital, Liverpool, UK Critical Care 2013, 17(Suppl 2):P446 (doi: 10.1186/cc12384) Introduction Hypocalcaemia is common in critical illness and accurate assessment is crucial. Small studies have shown that albumin-adjusted calcium (adjCa) does not accurately predict the ionised calcium (iCa) concentration in critically ill patients, yet adjCa continues to be widely used [1]. We investigated the reliability of using adjCa to identify hypocalcaemia in a large, diverse population requiring intensive care. Introduction Hypocalcaemia is common in critical illness and accurate assessment is crucial. Small studies have shown that albumin-adjusted calcium (adjCa) does not accurately predict the ionised calcium (iCa) concentration in critically ill patients, yet adjCa continues to be widely used [1]. We investigated the reliability of using adjCa to identify hypocalcaemia in a large, diverse population requiring intensive care. Methods In a retrospective study of patients admitted to the ICUs of a tertiary care hospital between January 2008 and 2012, iCa and pH were extracted from routine blood gas results and total calcium, albumin and phosphate from routine biochemistry results. adjCa was calculated using a formula derived from and validated on the local population [2]. Sensitivity, specifi city, positive and negative predictive values (PPV and NPV) and area under the curve (AUC) of adjCa for predicting hypocalcaemia (iCa <1.1 mmol/l) were calculated. Vitamin D status and overall and sepsis mortality in critically ill patients Table 1 (abstract P445). Characteristics of the two groups G1 G2 P value Age 38 ± 13 34 ± 12 NS TBSA 57.8 ± 30 26 ± 22 0.019 UBS 158 ± 100 70 ± 99 NS ABSI 8 ± 3 6 ± 3 NS CS T0 37 ± 11 19 ± 14 0.01 D CS 2.2 ± 1.8 19 ± 6 <0.001 Shock 6 (86%) 4 (36%) 0.04 Table 1 (abstract P445). Characteristics of the two groups y , Critical Care 2013, 17(Suppl 2):P447 (doi: 10.1186/cc123 Introduction Vitamin D status may aff ect hospital mortality and infectious complications in critical illness. Methods A total of 655 mixed critically ill patients with available 25(OH)D levels hospitalized between 2008 and 2010 were included. Cox regression analysis adjusted for SAPS 2, age and gender was performed. 25(OH)D levels were categorized by month-specifi c tertiles (high, intermediate, low) to refl ect seasonal variation. Primary endpoint was the correlation between 25(OH)D and hospital mortality. Secondary endpoints were sepsis mortality and blood culture positivity.i y y Results Hospital mortality was signifi cantly higher in patients in the low (HR = 1.98) and the mid-range tertile (HR = 1.88) compared with the highest tertile. Vitamin D levels were signifi cantly lower in patients with lethal sepsis compared with other causes of death (12.3  ±  5.0 vs. 18.2 ± 11.2, P = 0.02). Blood culture positivity rates did not diff er between the groups (23.0% vs. 26.8% vs. 17.3%, P = 0.361). See Table 1. Conclusion RAI is common in severely burned patients during the acute phase, and is associated with shock. Further prospective controlled studies will be necessary to establish risk factors of RAI in severely burned patients and its impact on their prognosis. Relative adrenal insuffi ciency in burns With direct iCa measurements now readily available on ward-based analysers, adjCa should not be used to determine calcium status in critical illness. phosphate or pH. With direct iCa measurements now readily available on ward-based analysers, adjCa should not be used to determine calcium status in critical illness. university-affi liated teaching hospital in Tunis. Patients admitted within the fi rst 24 hours post burn with greater than 10% total body surface area (TBSA) burned were enrolled in this study from 1 January 2009 to 30 June 2010. Exclusion criteria were pregnancy, history of adrenal insuffi ciency, or steroid therapy within 6  months prior to burns. A short corticotrophin test (250 μg) was performed, and cortisol levels were measured at baseline (CS T0) and 60 minutes post test. Adrenal insuffi ciency was defi ned by a response ≤9 μg/dl. Relative adrenal insuffi ciency was further defi ned by a baseline cortisol >20 μg/dl. Results Patients were assigned into two groups: G1 (RAI, n = 7) and G2 (absence AI, n = 11). Comparative study of the two groups shows the results presented in Table 1. 1. Slomp J, et al.: Albumin-adjusted calcium is not suitable for diagnosis of hyper- and hypocalcemia in the critically ill. Crit Care Med 2003, 31:1389-1393. 1. Slomp J, et al.: Albumin-adjusted calcium is not suitable for diagnosis of hyper- and hypocalcemia in the critically ill. Crit Care Med 2003, 31:1389-1393. 2. James M, et al.: Derivation and internal validation of an equation for albumin-adjusted calcium. BMC Clin Pathol 2008, 8:12. Relative adrenal insuffi ciency in burns Relative adrenal insuffi ciency in burns pp p Results Of the 2,440 patients studied, 1,993 had APACHE II data and serum Na+ recorded and so were included for analysis. In total, 453 patients (22.7%) had a serum Na+ ≤134  mmol/l and 1,388 patients (67.1%) had a serum Na+ of 135 to 144 mmol/l. Patients with a low Na+ had a higher mortality (OR = 1.48, 95% CI = 1.16 to 1.90, P <0.001), a higher APACHE II score (22 vs. 19, P  <0.001) and higher mean age (60 years vs. 58 years, P <0.001) than patients with a normal serum Na+. Mean length of stay of patients with low serum Na+ was also longer (5.1  days vs. 4.6  days) although this was not statistically signifi cant (P = 0.09). Introduction Relative adrenal insuffi ciency (RAI) is an uncommon disorder among burn patients, which can often go unrecognized. However, there are no or almost no data about the incidence of this disease in burns. The goal of the current study is to evaluate the incidence of RAI in burn patients during the acute phase. Methods A prospective study, approved by our Institutional Ethics Committee, was conducted in a 20-bed adult burn ICU at a Conclusion In summary, hyponatremia is a useful index of severity of illness in our ICU population. Whether this is a direct adverse eff ect S165 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 university-affi liated teaching hospital in Tunis. Patients admitted within the fi rst 24 hours post burn with greater than 10% total body surface area (TBSA) burned were enrolled in this study from 1 January 2009 to 30 June 2010. Exclusion criteria were pregnancy, history of adrenal insuffi ciency, or steroid therapy within 6  months prior to burns. A short corticotrophin test (250 μg) was performed, and cortisol levels were measured at baseline (CS T0) and 60 minutes post test. Adrenal insuffi ciency was defi ned by a response ≤9 μg/dl. Relative adrenal insuffi ciency was further defi ned by a baseline cortisol >20 μg/dl. Results Patients were assigned into two groups: G1 (RAI, n = 7) and G2 (absence AI, n = 11). Comparative study of the two groups shows the results presented in Table 1. phosphate or pH. Table 1 (abstract P447) Methods In a retrospective study of patients admitted to the ICUs of a tertiary care hospital between January 2008 and 2012, iCa and pH were extracted from routine blood gas results and total calcium, albumin and phosphate from routine biochemistry results. adjCa was calculated using a formula derived from and validated on the local population [2]. Sensitivity, specifi city, positive and negative predictive values (PPV and NPV) and area under the curve (AUC) of adjCa for predicting hypocalcaemia (iCa <1.1 mmol/l) were calculated. Etiology of low testosterone levels in mal sepsis requiring mechanical ventilation h Unpaired t test showed a P value of 0.07. Conclusion Calcium replacement with 40 ml of 10% calcium gluconate is eff ective when the adjusted calcium is <2.0  mmol/l. Replacement with 20 ml of 10% calcium gluconate when the adjusted calcium is between 2.0 and 2.15 mmol/l is not much more eff ective than no replacement. This suggests that either replacement is not needed or there is under-replacement in mild hypocalcaemia. Adjusted calcium may be giving falsely low calcium results in mild hypocalcaemia. The measurement of ionised calcium is a possible solution [2,3]. References 1. Zaloga GP: Crit Care Med 1992, 20:251-262. 2. Slomp J, et al.: Crit Care Med 2003, 31:1389-1393. 3. Byrnes MC, et al.: Am J Surg 2005, 189:310-314. P450 Endogenous melatonin in critically ill patients VS Salice, IG Galluccio, BS Salihovic, IP Piva, FM Marazzo, CV Villa, MT Taverna, MU Umbrello, GM Mistraletti, GI Iapichino Figure 1 (abstract P448). Figure 2 (abstract P448). Figure 1 (abstract P448). the adjusted calcium result and the calcium replacement were documented. Adequate replacement is defi ned as 40 ml of 10% calcium gluconate if the adjusted calcium is <2.0 mmol/l and 20 ml of 10% calcium gluconate if the adjusted calcium result is between 2.0 and 2.15 mmol/l. Figure 2 (abstract P448). Figure 2 (abstract P448). Results Eighty-eight adjusted calcium results were <2.0 mmol/l. In 74 results adequate replacement was given and the average increase in calcium was 0.2 mmol/l. This led to an increase in the adjusted calcium to >2.15 mmol/l in 36% of results and improvement to >2.0 mmol/l in 89% of results. In 14 results no replacement was given and the average increase in calcium was 0.07 mmol/l, which equated to 57% of calcium results remaining <2.0 mmol/l. Unpaired t test showed a P value of 0.0006. A total of 150 adjusted calcium results were between 2.0 and 2.15 mmol/l. In 111 results adequate replacement was given and the average increase was 0.006  mmol/l. The adjusted calcium fell to <2.0 mmol/l in 26% of results and normalised to >2.15 mmol/l in 23%. Where the adjusted calcium fell despite replacement, 51% of results were below 2.05 mmol/l. In 39 results no replacement was given, leading to an average fall in calcium of 0.03  mmol/l. Thirty- three percent of calcium results fell to <2.0 mmol/l and 20% of results normalised. Unpaired t test showed a P value of 0.07. References 1. Zaloga GP: Crit Care Med 1992, 20:251-262. 2. Slomp J, et al.: Crit Care Med 2003, 31:1389-1393. 3. Byrnes MC, et al.: Am J Surg 2005, 189:310-314. Etiology of low testosterone levels in mal sepsis requiring mechanical ventilation h FSH levels were inappropriately low. All lipoprotein fractions and their apo-proteins were reduced as well as 17-OH-progesterone, DHEA and DHEAS. In contrast, androstenedione (adione) levels were elevated. This suggests preferential and stimulated synthesis of androstenedione (Figure 2). The high 17β-estradiol levels indicate that androstenedione is shunted into the estrogen pathway, a process that requires high aromatase activity. The high estradiol/total testosterone ratio supports this conclusion. p Conclusion Calcium replacement with 40 ml of 10% calcium gluconate is eff ective when the adjusted calcium is <2.0  mmol/l. Replacement with 20 ml of 10% calcium gluconate when the adjusted calcium is between 2.0 and 2.15 mmol/l is not much more eff ective than no replacement. This suggests that either replacement is not needed or there is under-replacement in mild hypocalcaemia. Adjusted calcium may be giving falsely low calcium results in mild hypocalcaemia. The measurement of ionised calcium is a possible solution [2,3]. Conclusion Hyperestrogenic hypotestosteronemia is a frequent fi nding in the acute phase of severe sepsis in male patients with respiratory failure. It is suggested to be caused by decreased androgen production and shunting of androgen to estrogen synthesis as a result of increased aromatase activity. The clinical relevance of gonadal hormone substitution needs further study. Effi cacy of calcium replacement in hypocalcaemia H Chatha, K Sim Whiston Hospital, Liverpool, UK Critical Care 2013, 17(Suppl 2):P449 (doi: 10.1186/cc12387 Effi cacy of calcium replacement in hypocalcaemia H Chatha, K Sim Whiston Hospital, Liverpool, UK Critical Care 2013, 17(Suppl 2):P449 (doi: 10.1186/cc12387) Etiology of low testosterone levels in mal sepsis requiring mechanical ventilation h This cannot be explained by abnormalities of albumin, S166 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum com/supplements/17/S2 http://ccforum.com/supplements/17/S2 FSH levels were inappropriately low. All lipoprotein fractions and their apo-proteins were reduced as well as 17-OH-progesterone, DHEA and DHEAS. In contrast, androstenedione (adione) levels were elevated. This suggests preferential and stimulated synthesis of androstenedione (Figure 2). The high 17β-estradiol levels indicate that androstenedione is shunted into the estrogen pathway, a process that requires high aromatase activity. The high estradiol/total testosterone ratio supports this conclusion. Conclusion Hyperestrogenic hypotestosteronemia is a frequent fi nding in the acute phase of severe sepsis in male patients with respiratory failure. It is suggested to be caused by decreased androgen production and shunting of androgen to estrogen synthesis as a result of increased aromatase activity. The clinical relevance of gonadal hormone substitution needs further study. P449 Effi cacy of calcium replacement in hypocalcaemia H Chatha, K Sim Whiston Hospital, Liverpool, UK Critical Care 2013, 17(Suppl 2):P449 (doi: 10.1186/cc12387) the adjusted calcium result and the calcium replacement were documented. Adequate replacement is defi ned as 40 ml of 10% calcium gluconate if the adjusted calcium is <2.0 mmol/l and 20 ml of 10% calcium gluconate if the adjusted calcium result is between 2.0 and 2.15 mmol/l. Results Eighty-eight adjusted calcium results were <2.0 mmol/l. In 74 results adequate replacement was given and the average increase in calcium was 0.2 mmol/l. This led to an increase in the adjusted calcium to >2.15 mmol/l in 36% of results and improvement to >2.0 mmol/l in 89% of results. In 14 results no replacement was given and the average increase in calcium was 0.07 mmol/l, which equated to 57% of calcium results remaining <2.0 mmol/l. Unpaired t test showed a P value of 0.0006. A total of 150 adjusted calcium results were between 2.0 and 2.15 mmol/l. In 111 results adequate replacement was given and the average increase was 0.006  mmol/l. The adjusted calcium fell to <2.0 mmol/l in 26% of results and normalised to >2.15 mmol/l in 23%. Where the adjusted calcium fell despite replacement, 51% of results were below 2.05 mmol/l. In 39 results no replacement was given, leading to an average fall in calcium of 0.03  mmol/l. Thirty- three percent of calcium results fell to <2.0 mmol/l and 20% of results normalised. 50 Endogenous melatonin in critically ill patients The higher melatonin peak in renal failure may be due to an increased distribution volume; greater AUC in patients with liver failure could be due to a less effi cient removal of the hormone from the systemic circulation. The fi nding of increased peak and AUC in nonsurvivor patients could be due to a hormonal response increased by the body stress reaction, potentially similar to cortisol [1], or to a higher production of a physiological antioxidant [2] with a decreased ability to use it. References 1. Venkatesh B, et al.: Best Practice Res Clin End Met 2011, 25:719-733. 2. Flaring UB, et al.: Intensive Care Med 2005, 31:1072-1078. P451 Succinate ameliorates mitochondrial oxygen consumption of metformin-intoxicated human platelets A Protti1, M Monti2, A Lecchi1, A Artoni1, N Greppi1, L Gattinoni1 1Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy; 2Università degli Studi di Milano, Milan, Italy Methods Platelet-rich-plasma was incubated for 72  hours with metformin at a fi nal concentration of 0  mg/l (control), 1.66  mg/l (therapeutic dose) or 166  mg/l (toxic dose). Platelet VO2 was then measured with a Clark-type electrode, in the presence of glutamate plus malate (complex I electron donors) (fi nal concentration: 20 mmol/l for both) or succinate (complex II electron donor) (30 mmol/l), before and after adding cyanide (40  mmol/l). Mitochondrial (cyanide-sensitive) and extra-mitochondrial (cyanide-insensitive) VO2 were corrected for platelet count. Results The main results, from four preliminary experiments, are shown in Figure  1. In the presence of glutamate plus malate, only platelets incubated with a high dose of metformin had a mitochondrial VO significantly lower than controls. In the presence of succinate, Figure 1 (abstract P450). Figure 1 (abstract P451). Mitochondrial oxygen use of human platelets incubated with metformin. Figure 1 (abstract P450). this study is to describe the endogenous blood melatonin values in ICU patients and their correlation with clinical parameters. Figure 1 (abstract P451). Mitochondrial oxygen use of human platelets incubated with metformin. Methods Seventy-three high-risk critically ill patients mechanically ventilated for >48 hours were enrolled. Blood samples for melatonin assay were collected between the 3rd and the 8th day of the ICU stay. Melatonin was determined by radioimmunoassay and ELISA. The peak and the area under the curve (AUC) calculated for each patient were correlated with the clinical parameters using the regression for quantiles test. 50 Endogenous melatonin in critically ill patients Endogenous melatonin in critically ill patients VS Salice, IG Galluccio, BS Salihovic, IP Piva, FM Marazzo, CV Villa, MT Taverna, MU Umbrello, GM Mistraletti, GI Iapichino Università degli Studi Milano, Milan, Italy Critical Care 2013, 17(Suppl 2):P450 (doi: 10.1186/cc12388) Introduction The aim of the study is to examine the effi cacy of calcium replacement in patients with hypocalcaemia in the ICU. Hypocalcaemia aff ects between 15 and 50% of admissions to the ICU [1]. Introduction The aim of the study is to examine the effi cacy of calcium replacement in patients with hypocalcaemia in the ICU. Hypocalcaemia aff ects between 15 and 50% of admissions to the ICU [1]. Introduction Melatonin could have a meaningful role in critically ill patients, because of its immunomodulatory, antioxidant and sleep regulation properties; it is reduced in critical illness. The purpose of Introduction Melatonin could have a meaningful role in critically ill patients, because of its immunomodulatory, antioxidant and sleep regulation properties; it is reduced in critical illness. The purpose of f Methods Adjusted calcium results <2.0  mmol/l were followed up from admission for 5 days or until discharge or death. On each day S167 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 this study is to describe the endogenous blood melatonin values in ICU patients and their correlation with clinical parameters. Methods Seventy-three high-risk critically ill patients mechanically ventilated for >48 hours were enrolled. Blood samples for melatonin assay were collected between the 3rd and the 8th day of the ICU stay. Melatonin was determined by radioimmunoassay and ELISA. The peak and the area under the curve (AUC) calculated for each patient were correlated with the clinical parameters using the regression for quantiles test. Results Endogenous melatonin was found lower in critically ill patients compared with healthy subjects (Figure  1), although it showed a great individual variability and it generally maintained a night-time increase. In the univariate analysis the peak was found related to: blood creatinine (P = 0.034); patients in coma (P = 0.024); hospital mortality (P = 0.016). The AUC was found related to: SAPS II (P = 0.047); creatinine (P <0.001); AST (P <0.001); ALT (P <0.001); hospital mortality (P <0.022). Peak and AUC were found higher in nonsurvivor patients. Conclusion In accord with previous studies, the endogenous blood melatonin was found reduced in ICU patients. References 1. Venkatesh B, et al.: Best Practice Res Clin End Met 2011, 25:719-733. 2. Flaring UB, et al.: Intensive Care Med 2005, 31:1072-1078. 50 Endogenous melatonin in critically ill patients Results Endogenous melatonin was found lower in critically ill patients compared with healthy subjects (Figure  1), although it showed a great individual variability and it generally maintained a night-time increase. In the univariate analysis the peak was found related to: blood creatinine (P = 0.034); patients in coma (P = 0.024); hospital mortality (P = 0.016). The AUC was found related to: SAPS II (P = 0.047); creatinine (P <0.001); AST (P <0.001); ALT (P <0.001); hospital mortality (P <0.022). Peak and AUC were found higher in nonsurvivor patients. g p Conclusion In accord with previous studies, the endogenous blood melatonin was found reduced in ICU patients. The higher melatonin peak in renal failure may be due to an increased distribution volume; greater AUC in patients with liver failure could be due to a less effi cient removal of the hormone from the systemic circulation. The fi nding of increased peak and AUC in nonsurvivor patients could be due to a hormonal response increased by the body stress reaction, potentially similar to cortisol [1], or to a higher production of a physiological antioxidant [2] with a decreased ability to use it. Figure 1 (abstract P451). Mitochondrial oxygen use of human platelets incubated with metformin. Methods Platelet-rich-plasma was incubated for 72  hours with metformin at a fi nal concentration of 0  mg/l (control), 1.66  mg/l (therapeutic dose) or 166  mg/l (toxic dose). Platelet VO2 was then measured with a Clark-type electrode, in the presence of glutamate plus malate (complex I electron donors) (fi nal concentration: 20 mmol/l for both) or succinate (complex II electron donor) (30 mmol/l), before and after adding cyanide (40  mmol/l). Mitochondrial (cyanide-sensitive) and extra-mitochondrial (cyanide-insensitive) VO2 were corrected for platelet count. 1. Venkatesh B, et al.: Best Practice Res Clin End Met 2011, 25:719-733. 2. Flaring UB, et al.: Intensive Care Med 2005, 31:1072-1078. Does chronic metformin therapy off er cardiomyoprotection in coronary artery bypass grafting surgery patients? A case–control study A Delaporte, JP Hacquebard, P Origer, I Pastijn, M Maatouk, O Germay, E Stevens, P Kapessidou, A Roman CHU Saint-Pierre, Brussels, Belgium Critical Care 2013, 17(Suppl 2):P452 (doi: 10.1186/cc12390) g Results Ten patients were selected, fi ve males and fi ve females, with a mean age of 72.2. On admission, nine of 10 patients had a framework of general illness and acute renal failure; one patient appeared with a probable acute abdomen not subsequently confi rmed on CT. Eight patients had a state of dehydration resulting from gastroenteritis, diarrhea, fever; two patients had taken NSAIDs in the  days prior to hospitalization. No patient, in these conditions, has discontinued treatment with metformin. All patients showed a critical clinical framework with a SAPS II mean score of 77.5. Cardiovascular and respiratory support was required in all cases. The hemogas analysis showed that patients had a severe metabolic acidosis (mean pH = 6.96) with increased plasma lactate (mean lactate  =  15.93  mmol/l). Prothrombin activity was normal in eight of 10 patients. The overall mortality was 70%. Introduction Metformin, widely used as an antidiabetic drug, activates the AMP activated protein kinase, a key regulator of the metabolism providing protection under fuel defi ciency. Chronic metformin therapy has been shown in long-term follow-up clinical studies to reduce cardiovascular mortality [1]. In animal experiments, acute metformin pretreatment has been shown to reduce ischemia–reperfusion injury on cardiomyocytes [2]. We want to evaluate whether outcomes are aff ected in coronary artery bypass grafting (CABG) surgery. f Methods We performed a retrospective case–control study on 341 patients admitted exclusively for CABG surgery with cardiopulmonary bypass between January 2006 and April 2012. Eighty-one patients chronically on metformin were matched 1:3 with nonmetformin- treated ones using gender, age, body mass index and number of arterial bypass grafts. Global mortality, ICU-free 28 postoperative days and creatinine kinase MB (CKMB) at postoperative baseline, 8 and 18 hours post baseline were compared.i Conclusion Patient education on correct use of metformin is essential to prevent MALA, specially in those clinical conditions of increased risk (for example, acute renal failure), as recommended by the AIFA 2011 guidelines [4]. In our study, a higher plasma concentration of lactate represents the main negative prognostic factor, as pointed out by other studies [5]. P452 Does chronic metformin therapy off er cardiomyoprotection in coronary artery bypass grafting surgery patients? A case–control study A Delaporte, JP Hacquebard, P Origer, I Pastijn, M Maatouk, O Germay, E Stevens, P Kapessidou, A Roman CHU Saint-Pierre, Brussels, Belgium Critical Care 2013, 17(Suppl 2):P452 (doi: 10.1186/cc12390) Multiscale modeling of acute insulin resistance in critical care Critical Care 2013, 17(Suppl 2):P454 (doi: 10.1186/cc12392) Critical Care 2013, 17(Suppl 2):P454 (doi: 10.1186/cc12392) Introduction Stress hyperglycemia in the critically ill is a complex process in which insulin signaling is systematically hijacked to provide energy substrate for metabolic priorities such as cell healing or infection containment. Fluctuating levels of plasma glucose are associated with increased mortality in the ICU [1]. We develop a multiscale mathematical model that can characterize the severity of stress hyperglycemia based on a fundamental understanding of the signaling molecules involved. Conclusion These data suggest that chronic preoperative treatment with metformin is associated with lower CKMB levels at postsurgery baseline, 8 and 18 hours post baseline with a shorter ICU stay in patients receiving CABG surgery under extracorporeal circulation. References g g Methods Insulin resistance following insult has been shown to be driven primarily by the immune response via the cytokine IL-6 [2]. We created a multiscale mathematical model that links circulating glucose and insulin concentration dynamics from the extended minimal model [3] to a cellular insulin response model [4] that captures insulin- mediated glucose uptake in an insulin-responsive cell. p References References . UK Prospective Diabetes Study Group: Lancet 1998, 352:854-865. 2. Lalau JD: Drug Saf 2010, 33:727-740. 3. Le Gall JR, et al.: JAMA 1993, 270:2957-2963. 4. Italian Medicines Agency 2011 [http://www.agenziafarmaco.gov.it/it/content/ raccomandazioni-sull%E2%80%99utilizzo-dei-medicinali-base-di-metformina- nella-gestione-del-diabete-m] Table 1 (abstract P452). Case–control analysis Table 1 (abstract P452). Case–control analysis Table 1 (abstract P452). Case–control analysis Table 1 (abstract P452). Case–control analysis Statistics, Metformin Nonmetformin P value Mortality 1/81 8/260 0.24 28 days free ICU (days) 23.4 22.9 <0.04 CKMB T 0 hours (ng/ml) 21 26 <0.003 CKMB T+8 hours (ng/ml) 22 53 <0.004 CKMB T+18 hours (ng/ml) 21.5 27.5 <0.012 5. Dell’Aglio DM, et al.: Ann Emerg Med 2009, 54:818-823. 6. Peters N, et al.: Crit Care 2008, 12:R149. Succinate ameliorates mitochondrial oxygen consumption of metformin-intoxicated human platelets Succinate ameliorates mitochondrial oxygen consumption of metformin-intoxicated human platelets A Protti1, M Monti2, A Lecchi1, A Artoni1, N Greppi1, L Gattinoni1 1Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy; 2Università degli Studi di Milano, Milan, Italy Critical Care 2013, 17(Suppl 2):P451 (doi: 10.1186/cc12389) p Results The main results, from four preliminary experiments, are shown in Figure  1. In the presence of glutamate plus malate, only platelets incubated with a high dose of metformin had a mitochondrial VO2 signifi cantly lower than controls. In the presence of succinate, mitochondrial VO2 of controls did not change signifi cantly whereas that of platelets incubated with metformin did. The eff ect of succinate tended to become larger as the dose of metformin was increased from 0 up to 166 mg/l (0.3 ± 0.2 vs. 0.6 ± 0.3 vs. 1.0 ± 0.3 nmol/minute*106 cells) (P = 0.068). Even so, mitochondrial VO2 of platelets incubated with the highest dose of metformin did not return to the levels of controls. Extra-mitochondrial VO2 was always the same. A Protti1, M Monti2, A Lecchi1, A Artoni1, N Greppi1, L Gattinoni1 1Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy; 2Università degli Studi di Milano, Milan, Italy Critical Care 2013, 17(Suppl 2):P451 (doi: 10.1186/cc12389) Introduction Metformin intoxication inhibits mitochondrial complex I and oxygen consumption (VO2). Succinate bypasses complex I by donating electrons to complex II. The aim of this study was to clarify whether succinate ameliorates mitochondrial VO2 of metformin- intoxicated human platelets. S168 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Conclusion Succinate ameliorates (but does not return to normal) mitochondrial VO2 of human platelets incubated with a toxic dose of metformin. predisposing conditions, the use of metformin may result in metformin- associated lactic acidosis (MALA), a rare adverse event associated with a high mortality rate [2]. The aim of this study is to assess risk factors and prognostic factors in patients with MALA. Methods We conducted a retrospective study of patients with MALA admitted to the ICU of ASL 2 Chieti between 1 January 2008 and 30 September 2012. The eligibility criteria were: diagnosis of diabetes mellitus type 2, treatment with oral hypoglycemic drugs containing metformin, increased anion gap metabolic acidosis (pH <7.35, HCO3 – <22 mmol/l, lactate >5 mmol/l). For each patient we evaluated: sex, age, home care, SAPS II score [3], blood tests. Does chronic metformin therapy off er cardiomyoprotection in coronary artery bypass grafting surgery patients? A case–control study The prothrombin activity, which is considered to be a decisive prognostic factor in the study of Peters and colleagues [6], was not impaired in patients with poor outcome. Results Metformin-treated patient mortality was not signifi cantly lower than in nonmetformin (1.2% vs. 3.1%, P = 0.24, Fischer exact test). ICU-free 28 postoperative days were higher in the metformin group (median 23.4 days; 95% CI = 22.5 to 24.3) than in the nonmetformin group (median 22.9 days; 95% CI 22.3 to 23.5, P <0.04, Wilcoxon test). Median CKMB levels at postoperative baseline were 21 versus 26 ng/ml (P <0.003), at 8 hours were 22 versus 53 ng/ml (P <0.004) and 18 hours post baseline were 21.5 versus 27.5 ng/ml (P <0.012). See Table 1. Impact of a real-time electronic persistent hyperglycemia alert on glucose control in the ICU g K Colpaert, S Oeyen, T van den Driessche, C Danneels, J Decruyenaere Ghent University Hospital, Ghent, Belgium Critical Care 2013, 17(Suppl 2):P455 (doi: 10.1186/cc12393) Introduction Hyperglycemia is frequently encountered in critically ill patients, and associated with adverse outcome. Improvement of glucose protocol adherence may be accomplished using electronic alerts. We confi gured a non-intrusive real-time electronic alert, called a GLYC sniff er, as part of our Intensive Care Information System (ICIS) that continuously evaluates the occurrence of persistent hyperglycemia and hypoglycemia. The GLYC sniff er is confi gured in such a way to give an alert: if two consecutive glucose values are >150 mg/dl, with a minimum interval of 60 minutes; or if a glucose value is <80 mg/dl. We wanted to evaluate whether the GLYC sniff er would improve the glucose control. p p Conclusion The MPC–MHE algorithm achieves targeted glucose control in response to changing patient dynamics and multiple measured disturbances for a pilot population of 10 VPs. Furthermore, the MHE scheme updates patient parameters in real time in response to changing patient dynamics. P457 Methods A single-center, prospective intervention study during a 6-month period in our 22-bed surgical ICU. Two study phases were compared: a 3-month pre-alert phase with no alerting to the nurses, and a 3-month intervention phase where the GLYC sniff er was alerting through the Clinical Notifi cation System of our ICIS.f Hyperglycemia in intensive care identifi es patients at high risk of developing diabetes: preliminary data of a multicentre study E Lukic, I Gornik, M Grgic-Medic, D Milicic, V Vegar, V Ivancan, M Peric, V Gasparovic, I Pavlic Renar University Hospital Centre Zagreb, Croatia Critical Care 2013, 17(Suppl 2):P457 (doi: 10.1186/cc12395) i y Results A total of 652 diff erent patients having a total of 699 admission episodes was recorded during the study period. There were no signifi cant diff erences between the two study groups regarding baseline demographic data, fi rst glucose value upon admission. A total of 2,335 GLYC sniff er alerts were recorded during the whole study period: 84.3% persistent hyperglycemia alerts (1,021 in the pre-alert group, 948 in the alert group), and 15.7% hypoglycemia alerts (139 vs. 227). A signifi cantly lower percentage of glucose values in the alert group were hyperglycemic (19.5% vs. 26.5%, P <0.001). The proportion of persistent hyperglycemic values (i.e. P455 Results MPC controller performance on one VP is shown in Figure 1. Across a population of 10 VPs, the average [Gbl] under MPC is 6.31  mmol/l, the average minimum is 4.62  mmol/l, the population individual minimum is 3.49 mmol/l and the average absolute average residual error is 0.83 mmol/l from a 5.6 mmol/l target. With standard intervention, the 10 VPs have an average [Gbl] of 9.32  mmol/l, an average minimum [Gbl] of 3.77 mmol/l, and a population minimum [Gbl] of 2.78 mmol/l. Algorithm performance deteriorates signifi cantly if the imputed sampling time exceeds 30 minutes, underlining the importance of dynamic variations in insulin sensitivity in this population. y References References 1. Van den Berghe G: N Engl J Med 2001, 345:1359-1367. 2. Thorell A: Clin Nutr 1996, 15:75-79. 3. Roy A: Diabetes Technol Ther 2006, 8:617-626. 4. Sedaghat A: Am J Physiol Endocrinol Metab 2002, 283:E1084-E1101. References 1. Van den Berghe G: N Engl J Med 2001, 345:1359-1367. 2. Thorell A: Clin Nutr 1996, 15:75-79. 3. Roy A: Diabetes Technol Ther 2006, 8:617-626. 4. Sedaghat A: Am J Physiol Endocrinol Metab 2002, 283:E1084-E1101. References 1. Van den Berghe G: N Engl J Med 2001, 345:1359-1367. 2. Thorell A: Clin Nutr 1996, 15:75-79. 3. Roy A: Diabetes Technol Ther 2006, 8:617-626. 4. Sedaghat A: Am J Physiol Endocrinol Metab 2002, 283:E1084-E1101. 3. Roy A: Diabetes Technol Ther 2006, 8:617-626. 4. Sedaghat A: Am J Physiol Endocrinol Metab 2002, 283:E1084-E1101. P453 Metformin-associated lactic acidosis: risk factors and prognostic factors Metformin-associated lactic acidosis: risk factors and prognostic factors V Di Falco, A Milano, M Battilana, F Araosta, A Grosso, D Albanese, F Petrini, L Di Liberato ASL Chieti SS. Annunziata, Chieti, Italy Critical Care 2013, 17(Suppl 2):P453 (doi: 10.1186/cc12391) V Di Falco, A Milano, M Battilana, F Araosta, A Grosso, D Albanese, F Petrini, L Di Liberato ASL Chieti SS. Annunziata, Chieti, Italy Critical Care 2013, 17(Suppl 2):P453 (doi: 10.1186/cc12391) Results Inhibitory dynamics driven by IL-6 were incorporated into the cellular model to attenuate an insulin signaling intermediate (insulin receptor substrate 1) according to the proposed biological mechanisms. The percentage reduction in glucose uptake as a function of IL-6 concentration was fi t to data from patients who underwent elective abdominal surgery [2], shown in Figure 1. The overall multiscale model captures decreased insulin signaling as a result of increased IL-6 levels and the subsequent hyperglycemia that may ensue. y Critical Care 2013, 17(Suppl 2):P453 (doi: 10.1186/cc12391) Introduction Metformin, an oral hypoglycemic drug, belongs to the biguanide class and is now generally accepted as fi rst-line treatment in type 2 diabetes mellitus, especially in overweight patients [1]. In some Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 S169 Critical Care 2013, Volume 17 Suppl 2 http://ccforum com/supplements/17/S2 http://ccforum.com/supplements/17/S2 Figure 1 (abstract P454). Reduced glucose uptake driven by increased plasma IL-6 levels. group (9.9% vs. 15.4%, P <0.001). The patients in the alert group spend signifi cantly more time within the set target glucose interval of 80 to 150 mg/dl (82.3% vs. 75.0%, P = 0.009). A signifi cantly lower proportion of patients experienced a new-onset hypoglycemic event (<80 mg/dl) in the alert group (19.3% vs. 26.2%, P = 0.04). g p Conclusion A real-time electronic persistent glycemia sniff er resulted in a signifi cantly higher proportion of normoglycemia, without increasing the variability. Furthermore, hypoglycemic events occurred less frequently, and were resolved more timely. Smart alerting is able to improve quality of care, while diminishing the problem of alert fatigue. P456 P456 Individualized targeted glucose control to avoid hypoglycemia SP Gawel1, G Clermont2, T Ho1, BM Newman3, B Yegneswaran2, RS Parker1 1University of Pittsburgh, PA, USA; 2University of Pittsburgh Medical Center, Pittsburgh, PA, USA; 3Iowa State University, Ames, IA, USA Critical Care 2013, 17(Suppl 2):P456 (doi: 10.1186/cc12394) Introduction Hyperglycemia and hypoglycemia have been linked to worse outcomes in critically ill patients. While there is controversy as to the optimal tightness of glucose control in critically ill patients, there is agreement that an upper limit to safe glucose levels exists and that avoiding hypoglycemic episodes should be prioritized. Our algorithm can assist clinicians in maintaining blood glucose ([Gbl]) within a desired target range while avoiding hypoglycemia. Figure 1 (abstract P454). Reduced glucose uptake driven by increased plasma IL-6 levels. Conclusion A multiscale model has been developed to describe the inhibitory eff ects of IL-6 on insulin-mediated glucose uptake. Cellular inhibitory dynamics were shown to capture reduced insulin sensitivity on the macroscale, which could then be used to characterize insulin sensitivity and to provide insulin treatment advice to reduce glucose variability. Methods Our model predictive control (MPC) algorithm uses insulin and glucose as control inputs and a linearized model of glucose– insulin–fatty acid interactions. To allow the controller model to learn from data, a moving horizon estimation (MHE) technique tailored the tissue sensitivity to insulin to individual responses. Patient data ([Gbl] measurements, insulin and nutritional infusion rates) were from the HIDENIC database at the University of Pittsburgh Medical Center. [Gbl] measurements, typically  hourly, were interpolated to impute a measurement every 5 minutes. The model captured patient [Gbl] via nonlinear least squares by adjusting insulin sensitivity (SI) and endogenous glucose production (EGP0). The resulting virtual patient (VP) is used to evaluate the performance of the MPC–MHE algorithm. Impact of a real-time electronic persistent hyperglycemia alert on glucose control in the ICU consecutive glucose values exceeding the limit of 150 mg/dl) was signifi cantly lower in the alert Introduction A recent study showed that hyperglycaemia (blood glucose ≥7.8 mmol/l) in nondiabetic patients hospitalised in a medical ICU is associated with increased risk of diabetes [1]. We investigated a large mixed ICU population to confi rm these results. Methods This study retrospectively included patients with negative history of diabetes admitted to ICUs during the year 2007. We excluded patients receiving steroids, with newly diagnosed diabetes and those with end-stage disease. Patients were followed-up 5 years after index S170 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Figure 1 (abstract P456). Figure 1 (abstract P456). Figure 1 (abstract P456). Figure 1 (abstract P456). admission. Diagnosis of diabetes within 6  months from the index admission was presumed as revealing DM at inclusion, which excluded the patient. Patients who were taking glucocorticoids during the follow- up period were excluded. The rest of the patients, if consented, were assessed for their diabetic status with a single HbA1c measurement. Introduction The objective of the study was to determine the association between average glucose level (AVG) and hospital outcomes in critically ill patients. Methods We performed a chart review of the MIMICII and HIDENIC databases, prospective cohorts of over 32,000 and 46,000 patients admitted to the ICUs at Beth Israel Deaconess Medical Center between 2001 and 2007 and the ICUs at University of Pittsburgh Medical Center between 1991 and 2008, respectively. All admissions of patients ≥18  years without the diagnosis of DKA and NKHS were included. Diabetics were identifi ed from ICD-9 documentation. Propensity score for death (pDead) was computed from either SAP1 (MIMICII) or APACHE III (HIDENIC) to assess the risk of death. Hypoglycemia was defi ned as AVG ≤60  mg/dl. AVG was computed as the area under the glucose curve throughout ICU admission. Mortality was examined within bins (each bin is categorized by a 10 mg/dl increase in AVG) and was compared between adjacent categories using a chi-square test. The same method was repeated among diabetics, nondiabetics, patients with lower (pDead greater than median) and higher (pDead lower than Results A total of 2,696 patients were included in the study: 1,441 medical and 1,255 surgical, hyperglycaemia (≥7.8  mmol/l) was registered in 1,419 (52%). Impact of a real-time electronic persistent hyperglycemia alert on glucose control in the ICU During the 5 years from index admission 409 died and 38 were taking glucocorticoids, and 10 refused participation. Diabetes was diagnosed in 189 patients during the 6 months after index admission. The remaining cohort of 2,320 patients had 1,169 patients who were hyperglycaemic during hospitalisation. Diabetes was already diagnosed in 132 patients, of whom 112 were hyperglycaemic in the ICU; prediabetes was already diagnosed in 121 patients, of whom 83 had ICU hyperglycaemia. Since 125 patients (55 hyperglycaemic) refused HbA1c testing, it was performed on 1,852 patients and revealed diabetes in 136 patients (107 had hyperglycaemia) and prediabetes in 103 patients (75 were hyperglycaemic in ICU). Overall, patients who had hyperglycaemia had 19.8% cumulative 5-year incidence of diabetes and 14.3% of prediabetes. Nonhyperglycaemic ICU patients had cumulative incidences of 4.5% and 6.6%, respectively. Relative risk for developing diabetes during 5 years after ICU admission is 4.2 (95% CI = 3.2 to 5.9) for patients who did have hyperglycaemia.i Figure 1 (abstract P458). Figure 1 (abstract P458). p yp g y Conclusion The results of this study confi rm in a large mixed medical/ surgical ICU population that hyperglycaemia occurring in severe illness or the perioperative period is associated with increased risk of developing diabetes. Suggesting lifestyle changes to reduce the risk and regular follow-up should be implemented at least for these patients. p Reference p Reference 1. Gornik I, et al.: A prospective observational study of the relationship of critical illness associated hyperglycaemia in medical ICU patients and subsequent development of type 2 diabetes. Crit Care 14:R130. Association between average glucose levels and hospital mortality among critically ill patients B Yegneswaran1, R Parket2, S Gawel2, A Pritchard-Bell2, T Ho2, G Clermont2 1UPMC Hamot, Erie, PA, USA; 2University of Pittsburgh, PA, USA Critical Care 2013, 17(Suppl 2):P458 (doi: 10.1186/cc12396) Figure 1 (abstract P458). Figure 1 (abstract P458). S171 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 with increased risk of mortality (M). Heightened attention to identifi ed risk factors (RF) and causes of hypoglycemic events (HE) may lead to improvement in glycemic control. A limited literature describes RF for H but a detailed root cause analysis (RCA) of individual HE has not been published. median) risk of death. Statistical signifi cance was noted following a Bonferroni correction. Results MIMICII database: 21,209 admissions met inclusion criteria. Mean age: 62.5  ±  16.4  years; female: 9,070 (42.8%); diabetes: 5,496 (25.9%); mean admission SAP1 score: 13.8 ± 5.2; mean LOS: 9 days (IQR 6.15); hospital mortality: 10.3%. HIDENIC database: 38,872 admissions met inclusion criteria. Mean age: 59.4  ±  17.3  years; female: 16,675 (42.9%); diabetes: 11,326 (29.1%); mean admission APACHE III score: 80.2 ± 14; mean LOS: 11 days (IQR 7.22); hospital mortality: 14.3%. In both databases, association between average glucose and hospital mortality followed a bathtub shape with mortality nadir between 80 and 130  mg/dl. No mortality diff erence was observed between adjacent AVG glucose bins in the bottom of the bathtub (Figure  1). This relationship was preserved in nondiabetics, and for patients with higher risk of death. p Methods This is a retrospective analysis of prospectively collected data including 835 patients admitted consecutively between 1 February 2012 and 31 October 2012 to the 16-bed medical–surgical ICU of a university-affi liated teaching hospital. The blood glucose (BG, mg/dl) target was 90 to 120 and the monitoring frequency was every 3 hours, or every 1 hour when the patient received i.v. IN infusion. HE data were collected by the bedside nurse, using a cutoff of BG <60, and information was abstracted from the unit’s database. RF included: shock (SH), renal insuffi ciency (RI), hepatic failure (HF), mechanical ventilation (MV), and diabetes mellitus (DM). Attributable causes included: IN treatment, improper monitoring frequency (MF), interruption of feeding (FE), and spontaneous (SP). P461 Continuous arterial and venous glucose monitoring by quenched chemical fl uorescence in ICU patients after cardiac surgery S Bird1, L Macken1, O Flower1, F Bass1, N Hammond1, S Webb1, N Kennedy1, A Baker1, E Yarad1, C Chau2, M Librande2, P Strasma2, S Finfer1 1Royal North Shore Hospital, Sydney, Australia; 2Glumetrics, Irvine, CA, USA Critical Care 2013, 17(Suppl 2):P461 (doi: 10.1186/cc12399) Results Two groups had similar characteristics at baseline. A total of 1,060 CGM/ABG pairs were analyzed and reference glucose ranged from 4.3 to 18.8 mmol/l. Median (IQR) absolute relative deviation was lower in the FC arm (7.0% (3.5, 13.0) vs. 12.8% (6.3, 21.8), FC vs. MC, P <0.001). Similarly, the percentage of points in the Clarke error grid zone A points meeting ISO criteria were higher with FC (87.8% vs. 70.2%). Sensor bias (median (IQR)) was signifi cantly lower in the FC arm (–0.1 (–0.7, 0.4) mmol/l vs. –1.1 (–2.3, –0.1) mmol/l, P <0.001). The median (IQR) interval between calibrations in FC arm was 169 (122, 213) minutes. Introduction Continuous glucose monitoring (CGM) in ICUs has the potential to improve patient safety outcomes. The GluCath Intravascular CGM System uses a novel quenched chemical fl uorescence sensing mechanism to measure glucose concentration in venous or arterial blood (BG). This is the fi rst report of this system deployed for 48 hours in both arteries and veins of ICU patients. Methods This ongoing clinical study evaluates up to two sensors per subject in 20 patients undergoing cardiac surgery. An arterial sensor is deployed via a standard 20 G radial artery catheter inserted for routine care and an optional venous sensor is deployed percutaneously in an upper arm vein. Data are presented from the fi rst fi ve patients. Outcome measures are qualitative (ease of use, workfl ow fi t) and quantitative (accuracy vs. reference analyzer). Sensors were inserted shortly after ICU admission, with ultrasound guidance for venous sensor insertion. In vivo calibration was performed at 1 and 2 hours, then each morning. Glucose values were recorded every 10 seconds by the system. Hourly arterial reference samples were analyzed via Radiometer ABL 800 Flex Blood Gas Analyzer (BGA).i Conclusion CGM accuracy in the MC arm was comparable with accuracy in subjects with type 1 diabetes. Further signifi cant improvements to CGM accuracy in critical care are possible by increasing the frequency of calibrations. References References 1. Weinstein RL, et al.: Diabetes Care 2007, 30:1125-1130. 2. Kovatchev B, et al.: Diabetes Care 2008, 31:1160-1164. Results Arterial sensors were successfully deployed in all fi ve patients and did not interfere with clinical care, blood pressure monitoring or sampling. One arterial catheter failed resulting in sensor removal at 36  hours. The venous sensor was deployed in three patients, but removed from two patients due to thrombosis identifi ed during surveillance ultrasound examinations. A total of 202 reference BG samples ranging from 5.3 to 11.3  mmol/l were collected. Precision between arterial and venous sensors (n  =  2) was 9.3% CV. Arterial sensor accuracy compared with BGA was 5.5% MARD. One hundred Association between average glucose levels and hospital mortality among critically ill patients We measured rates of severe (<40) and mild (40 to 69) H, and compared them as well as percentage of values in the optimum range (70 to 139) and hyperglycemic range (>139) with data from the preceding 3 months (PRE). Conclusion Low and high average glucose are associated with mortality. P461 Such accurate CGM may provide valuable information to guide insulin therapy in critically ill subjects. f Continuous glucose monitoring in critically ill adults: comparison of two diff erent calibration protocols p g Results Sixty-three (7.5%) patients sustained a total of 79 HE. They were older, more likely to be diabetic, and had higher APACHE IV predicted (37.3% vs. 15.4%, P <0.0001) and observed (25.4% vs. 8.6%, P <0.0001) M than those without H. RF: SH 24.1%; RI 24.1%; HF 8.9%; MV 46.8%; DM 36.5%. Attributable causes: IN 58.2%; MF 8.9%; FE 6.3%; SP 36.7%. BG <40, 40 to 69 and >139 decreased 78.6%, 39.0% and 9.3% (P = 0.0006, P  <0.0001, P  <0.0001). BG 70 to 139 increased 5.5% (P  <0.0001). In total, 4.2% and 22.4% of PRE patients sustained severe and mild H. In total, 0.7% and 14.1% of RCA patients sustained severe and mild H (P = 0.0029, P = 0.0003). two diff erent calibration protocols L Leelarathna1, S English2, H Thabit1, K Caldwell1, J Allen1, K Kumareswaran1, M Wilinska1, M Nodale1, J Mangat1, M Evans1, R Burnstein2, R Hovorka1 1University of Cambridge, UK; 2Addenbrookes Hospital, Cambridge, UK Critical Care 2013, 17(Suppl 2):P459 (doi: 10.1186/cc12397) f p L Leelarathna1, S English2, H Thabit1, K Caldwell1, J Allen1, Introduction We evaluated the clinical and numerical accuracy of the Freestyle Navigator continuous glucose monitoring (CGM) system, in critically ill adults using two diff erent methods of calibration. Studies looking at intensive glucose control have yielded confl icting results with increased rates of iatrogenic hypoglycemia. Availability of accurate real-time glucose information may improve safety and effi cacy of glucose control in the critical care unit. Conclusion IN treatment was associated with barely more than one-half of HE in this population of intensively monitored patients. Implementation of an initiative mandating RCA of every HE, including identifi cation of RF and attributable causes, was associated with a signifi cant improvement in glycemic control. g Methods In a randomized prospective trial, paired CGM and reference glucose (hourly arterial blood gas (ABG)) were collected from 24 adults with critical illness (age 60 ± 14 years, BMI 29.6 ± 9.3 kg/m2, APACHE score range 6 to 19) and hyperglycemia (glucose ≥10 mmol/l or treated with intravenous insulin), over 48 hours. In 12 subjects, CGM was force- calibrated at variable 1 to 6  hourly intervals using ABG glucose (FC arm). In the other 12 subjects, the sensor was calibrated according to the manufacturer’s instructions (1, 2, 10, 24 hours after insertion) using arterial blood and built-in glucometer (MC arm). P461 Performance of the Medtronic Sentrino® continuous glucose management system in the cardiac ICU Performance of the Medtronic Sentrino® continuous glucose management system in the cardiac ICU M Kosiborod1, R Gottlieb2, J Sekella2, D Peterman1, A Grodzinsky1, P Kennedy1, M Borkon1 1Saint Luke’s Mid America Heart Institute, Kansas City, MO, USA; 2Medtronic Minimed, Northridge, CA, USA Critical Care 2013, 17(Suppl 2):P462 (doi: 10.1186/cc12400) 1. Lazar HL, et al.: Ann Thorac Surg 2009, 87:663-669. P460 P460 Root cause analysis of hypoglycemic events in critically ill patients A McDonald, J Krinsley Stamford Hospital, Stamford, CT, USA Critical Care 2013, 17(Suppl 2):P460 (doi: 10.1186/cc12398) Introduction Hypoglycemia (H) is a complication of intensive insulin (IN) therapy of critically ill patients and is independently associated S172 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 htt // f / l t /17/S2 http://ccforum.com/supplements/17/S2 Figure 1 (abstract P461). Arterial sensor ISO-modifi ed Bland–Altman plot. Figure 1 (abstract P461). Arterial sensor ISO-modifi ed Bland–Altman plot. Figure 1 (abstract P461). Arterial sensor ISO-modifi ed Bland–Altman plot. percent (202/202) of arterial sensor measurements met ISO 15197 criteria (within =/–20% of reference measurements if BG ≥4.2 mmol/l; Figure 1). was 12.2%. No diff erences in CGM system accuracy were seen within subgroups of low versus high Society of Thoracic Surgeons (STS) score (MARD 12.1% and 10.6% for STS >8% vs. ≤8%, respectively) or hemodynamic status (MARD 12.0% and 12.4% for compromised vs. stable hemodynamics). Consensus grid analysis showed >99% of SG values within A/B zones, and 0% in D/E zones. No device or study-related adverse events were reported. In total, 80% and 100% of clinicians found Sentrino® easy to use after one and two patients, respectively. Conclusion The GluCath System measured glucose concentration continuously in cardiac surgery ICU patients. Arterial catheter deployment did not appear to compromise line function or patient care. Percutaneous venous deployment was feasible, but may be associated with risk of local venous thrombosis. p y Conclusion The Sentrino® CGM system demonstrated good analytic and clinically relevant accuracy, excellent reliability and safety in critically ill cardiac patients; and was easy to use and integrate in the cardiac ICU. Future studies are needed to determine whether CGM can improve BG control and reduce hypoglycemia in this patient group. Reference P463 Intensive insulin therapy in critically ill children: impact on blood glucose dynamics and its relation with mortality M Van Tornout, M Gielen, T Van Herpe, D Vlasselaers, L Desmet, I Vanhorebeek, G Van den Berghe, D Mesotten University Hospitals Leuven, Belgium Critical Care 2013, 17(Suppl 2):P463 (doi: 10.1186/cc12401) Introduction Blood glucose (BG) control reduces morbidity and length of stay, and is standard practice in patients undergoing cardiac surgery [1]. However, maintaining BG in the target range, while avoiding hypoglycemia, is challenging. Continuous glucose monitoring (CGM) is a promising technology that may help address these challenges. We investigated the performance and safety of Medtronic Sentrino®, a newly developed CGM for critically ill adults, in the cardiac ICU. P464 P464 Implementation of evidence-based protocols improves survival: a 15-year surgical ICU experience in 10,172 patients SA Nasraway Tufts Medical Center, Boston, MA, USA Critical Care 2013, 17(Suppl 2):P464 (doi: 10.1186/cc12402) Tufts Medical Center, Boston, MA, USA Table 1 (abstract P465) Admitted Admitted with without decubitus decubitus P Variable (n = 180) (n = 2,543) value Age 65 or older 106 (58.9%) 1,062 (41.8%) <0.001 APACHE IV score on admission 80.2 (28.2%) 58.7 (30.7) <0.001 Diabetes 53 (29.4%) 420 (16.5%) <0.001 Dialysis 16 (8.9%) 58 (2.3%) <0.001 Hepatic failure 3 (1.7%) 56 (2.2%) 1.000 Mechanically ventilated during 123 (68.3%) 1,457 (57.3%) hospitalization Admission source to the ICU Direct admit 10 (5.6%) 154 (6.1%) ER 84 (46.7%) 1,206 (47.4%) Floor 25 (13.9%) 214 (8.4%) 0.017 OR 19 (10.6%) 459 (18.1%) Other location 42 (23.3%) 510 (20.1%) Outcomes ICU LOS (days) 4.9 (4.9) 3.3 (4.7) <0.001 Hospital LOS (days) 15.6 (20.7) 10.5 (11.1) <0.001 ICU mortality 30 (16.7%) 308 (12.1%) 0.073 Hospital mortality 58 (32.2%) 464 (18.3%) <0.001 Readmission rate for 20 out of 112 173 out of 2,079 0.002 survivors (16.4%) (8.3%) Conclusion The presence of decubiti on admission to the ICU is associated with longer hospitalizations even after adjusting for age, acuity, and organ supportive therapies. DU on admission to ICU provide a unique, unambiguous marker of increased resource utilization. Introduction There has been enormous interest in measuring ICU performance in terms of mortality and resource use, owing to increased public and health insurer scrutiny. We elected to describe the performance of our surgical ICU, using a standardized mortality ratio (SMR = observed / predicted mortality). y Methods The primary cohort was all patients admitted to the surgical ICU from March 2010 through February 2012 and related outcomes. The change in SMR was longitudinally determined from the latest 15- year study period (1997 to 2011) comprised of 10,172 patients. y y Results There were 1,799 ICU admissions in the primary cohort. Hospital mortality, observed and predicted by APACHE IV, was measured. Crude hospital mortality was 8.4%. The hospital SMR (observed / predicted mortality) was 0.58 (95% CI: 0.49 to 0.65). The SMR decreased by 20% from 0.73 to 0.58 over the 15-year study period, an absolute 1% per year decrease (P = 0.039; 95% CI: –0.02 to –0.002), as shown in Figure 1. ( ; ), g Conclusion Mortality was less than predicted and steadily declined during the previous 15 years. Intensive insulin therapy in critically ill children: impact on blood glucose dynamics and its relation with mortality Introduction A large RCT showed that tight glucose control (TGC), targeting age-adjusted normal fasting blood glucose levels with insulin infusion, decreased morbidity and mortality in critically children [1]. However, the incidence of hypoglycemia increased substantially in the TGC group. We aimed to assess the eff ect of TGC on the three domains of blood glucose dynamics (hyperglycemia, hypoglycemia and blood glucose variability) and their independent association with mortality in the pediatric ICU. Methods Adult patients with actual or planned cardiac ICU admission at a single tertiary center were approached for participation and signed consent. Other inclusion criteria were treatment with i.v. insulin (target BG <140 mg/dl) and life expectancy >96 hours. After initiation of i.v. insulin, Sentrino® subcutaneous glucose sensors were inserted into patients’ anterior thighs with planned study participation of 72 to 96  hours. Reference BG was collected according to ICU protocol, obtained from central venous catheter and analyzed with bedside blood gas analyzer (BGA; i-STAT®, Abbott, USA). Sensor glucose (SG) results were displayed, and its predictive alerts and alarms fully enabled. Additional reference BGs were obtained during alarms and calibration. All treatment decisions were based on BGA data, not on SG values. p Methods This is a preplanned substudy of a published RCT in one 10-bed pediatric ICU. Seven hundred patients (age 1 to 16  years), admitted to the PICU between October 2004 and December 2007, were randomized to either TGC (50 to 80 mg/dl in infants, 70 to 100 mg/dl in children) or to the usual care tolerating hyperglycemia up to 215 mg/ dl (UC). Patients with at least two arterial blood glucose measurements were included (UC n = 349; TGC n = 348). Results A total of 21 patients were enrolled; all successfully completed the study. Mean age was 65 years, 38% were women, 67% had diabetes. Types of surgery were CABG (38%), valve replacement (29%), combined CABG and valve (19%) and cardiac transplant (14%). SG was displayed 95% of the time during the study, and 864 paired BG–SG points were used for analysis. Overall mean absolute relative diff erence (MARD) Results Mean blood glucose levels were lowered from 143 ± 34 mg/dl in the UC group to 104 ± 24 mg/dl (P <0.0001). The median number of samples per patient did not diff er between UC (21 (13 to 48)) and TGC (22 (15 to 49)). Decubitus ulcers are associated with prolonged length of stay in critically ill patients Decubitus ulcers are associated with prolonged length of stay in critically ill patients Decubitus ulcers are associated with prolonged length of sta critically ill patients WT McGee1, BH Nathanson2, E Lederman1, TL Higgins1 1Baystate Medical Center, Springfi eld, MA, USA; 2OptiStatim LLC, Longmeadow, MA, USA Critical Care 2013, 17(Suppl 2):P465 (doi: 10.1186/cc12403) y WT McGee1, BH Nathanson2, E Lederman1, TL Higgins1 1Baystate Medical Center, Springfi eld, MA, USA; 2OptiStatim LLC, Longmeadow, MA, USA Critical Care 2013, 17(Suppl 2):P465 (doi: 10.1186/cc12403) WT McGee1, BH Nathanson2, E Lederman1, TL Higgins1 1Baystate Medical Center, Springfi eld, MA, USA; 2OptiStatim LLC, Longmeadow, MA, USA Introduction How decubitus ulcers (DU) diagnosed on admission to the ICU aff ect resource use is unknown. We hypothesize that this easily identifi able clinical fi nding would be associated with longer hospitalizations. Methods Clinical assessment was used to identify patients with DU on admission to the ICU. The association of DU on hospital and ICU length of stay (LOS) was assessed with generalized linear models that adjusted for ICU admission source, age, APACHE IV score, diabetes, sepsis, hepatic failure, dialysis and mechanical ventilation. i Conclusion TGC substantially reduced the hyperglycemic index, at the expense of increased hypoglycemia incidence, but without aff ecting blood glucose variability. Statistical analysis suggested strictly avoiding hyperglycemia in order to bring about outcome benefi t with insulin in critically ill children. p y Results DU was present on admission in 180 of 2,723 (6.6%) during a 19-month period in 2011 to 2012 at a single teaching hospital in New England. Patients with decubiti were sicker on admission (mean (SD) APACHE IV 80.2 (28.2) vs. 58.7 (30.7), P <0.001), more likely to be age 65 or older (58.9% vs. 41.8%, P <0.001) and require more organ supportive therapies (Table 1). DU patients had a higher in-hospital mortality rate (32.2% vs. 18.3%, P  <0.001) and longer mean ICU and hospital LOS (4.9 vs. 3.3 days and 15.6 vs. 10.5 days, respectively (P <0.001)). After multivariate adjustment, DU patients stayed 3.2  days, 95% CI (1.6, 4.8) longer in the hospital than non-DU patients and stayed 2.5 days longer, 95% CI (0.7, 0.43) among survivors. DU patients had longer ICU stays (1.3 days, 95% CI (0.3, 1.3)) in general but were statistically similar among survivors. Reference 1. Vlasselaers D, et al.: Lancet 2009, 373:547-556. 1. Vlasselaers D, et al.: Lancet 2009, 373:547-556. Intensive insulin therapy in critically ill children: impact on blood glucose dynamics and its relation with mortality TGC lowered the hyperglycemic index, a marker of S173 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 hyperglycemia over time, from median 44 (IQR 29 to 66) mg/dl in UC to 12 (7 to 23) mg/dl, as well as the glycemic penalty index, an aggregate measure of dysglycemia, from median 44 (32 to 58) in UC to 22 (16 to 30) (both P  <0.0001). Despite frequent hypoglycemia <40  mg/dl (1.4% in UC, 25.0% in TGC, P <0.0001), TGC did not result in increased blood glucose variability, as refl ected by the standard deviation of the blood glucose time series (TGC: 36 (24 to 50)  mg/dl, UC 34 (26 to 48)  mg/dl, P  =  0.82). In multivariable logistic regression analysis, adjusting for baseline risk factors (age, severity of illness, malignancy, type of admission), hypoglycemia (OR = 1.6, 95% CI = 0.19 to 2.22) and blood glucose variability (OR = 1.0, 95% CI = 0.97 to 1.02, per mg/dl) were not associated with PICU mortality. In contrast, hyperglycemic index (OR = 1.02, 95% CI = 1.01 to 1.04, per mg/dl) was independently associated with mortality and its lowering statistically explained the outcome benefi t of the intervention. P464 Mortality predictors in septic HIV patients in the ICU P Vidal-Cortés1, P Lameiro-Flores1, A Aller-Fernández2, M Mourelo-Fariña2, P Fernández-Ugidos1, R Gómez-López1, M Alves-Pérez1, E Rodríguez-García1 1CHU Ourense, Spain; 2CHU A Coruña, Spain Critical Care 2013, 17(Suppl 2):P468 (doi: 10.1186/cc12406) Introduction Sepsis in HIV patients carries a high mortality. Our purpose is to identify mortality predictors in this population. Introduction Sepsis in HIV patients carries a high mortality. Our purpose is to identify mortality predictors in this population. Methods A retrospective study of HIV patients admitted to our ICU between January 2005 and December 2009. We identify septic patients and analyzed demographic factors, etiology, organ failure and outcome. We used logistic regression to calculate the relative mortality risk for each qualitative variable and the beta (β) coeffi cient for quantitative variables. Conclusion Depending on the patient and the cause of admission, ICU admission may represent an excellent opportunity as a screening method to determine HIV status. Given the greater effi cacy of HAART at present, most patients with medical or surgical conditions unrelated to HIV infection will be eligible to join the ICU. People with HIV can and should benefi t from using reasonable and individualized care in an ICU. References q Results Sixty-two septic patients were admitted to our ICU during the study period (71% men, mean age: 40.59  ±  8.12). A total of 56.5% came from the ED, mean hospital stay before ICU admission: 6.14 ± 10.96 days. A total of 66.1% had a history of intravenous drug use, other comorbidities: COPD (9.7%), cirrhosis (8.1%), solid or hematologic malignancy (12.9%). A total of 40.3% were under HAART. Mean CD4 count at admission: 219.62 ± 353.93 cells/mm3. Mean viral load: 4.57  ±  3.25 log. Mean albumin levels: 2.3  ±  0.53 g/dl. APACHE II: 21.98  ±  7.97. The lung was the most frequent focus of infection (65.6%) followed by the CNS. The most common pathogen was S. pneumoniae (28.8%), followed by P. jirovecii (13.6%). In total, 62.9% needed vasopressors, 79% mechanical ventilation and 19.4% renal replacement. Mean ICU and hospital length of stay: 10.43 ± 10.52 and 34.76 ± 29.64 days. ICU mortality: 33.9%; hospital mortality: 41.9%. P464 The SMR can be used to track success when new quality measures are introduced or changes in the delivery of care are made. ICUs should report their standardized mortality ratios to evaluate performance. Figure 1 (abstract P464). Tufts surgical ICU, 15-year outcome standardized hospital mortality ratio. Conclusion The presence of decubiti on admission to the ICU is associated with longer hospitalizations even after adjusting for age, acuity, and organ supportive therapies. DU on admission to ICU provide a unique, unambiguous marker of increased resource utilization. Figure 1 (abstract P464). Tufts surgical ICU, 15-year outcome standardized hospital mortality ratio. Figure 1 (abstract P464). Tufts surgical ICU, 15-year outcome standardized hospital mortality ratio. S174 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 P466 HIV patients admitted to an ICU of a university hospital – experience of 15 years: 1995 to 2009 A Agrifoglio, M Arce, M Hernández, P Millan, C Guallar, I Pozuelo, M Jiménez Hospital Universitario La Paz, Madrid, Spain Critical Care 2013, 17(Suppl 2):P466 (doi: 10.1186/cc12404) Results We included 139 patients with HIV/AIDS admitted to the ICU. Median age was 40 (31 to 48) years and 71% were male. Severe malnutrition was common (34%). The CD4 cell count was 84 (25 to 274) cells/mm3 and viral load was 17,733 (67 to 174,214) copies/ml; 57% had at least one opportunistic infection; 55% had used antiretroviral therapy previous to ICU admission. Mechanical ventilation was used by 46% of patients and hospital mortality was 42%. Total SOFA score was 5 (2 to 9) points. Cardiovascular dysfunction was the most common on the fi rst day of stay (51%), followed by respiratory (42%), neurological (40%), renal (35%), hematological (27%) and hepatic (17%). Cardiovascular and renal dysfunctions presented with higher rate of severe dysfunction (30% and 15%, respectively). Rates of neurological (P = 0.002), renal (P  =  0.009) and hematological (P  =  0.003) dysfunctions were higher in nonsurvivors. Age, CD4 cell count, malnutrition, and opportunistic infections were included in the multivariate analysis. Neurological dysfunction was the independent risk factor for hospital mortality (odds 3.2 (1.4 to 7.2)). The presence of neurological dysfunction was dichotomized: associated or not with primary neurological diagnosis; survival was lower in the patients with neurological dysfunction and without primary neurological diagnosis (log-rank test 0.001 in the 7-day and 0.02 in the 28-day analysis). P464 Sixty-day survival was similar in primary and secondary neurological dysfunction, but it remained lower than in patients without neurological impairment. P466 Introduction HIV infection is a major public health problem in the world. The use of prophylaxis against opportunist infection and the introduction of HAART in 1996 increased life expectancies. The therapeutic use of ICU resources for HIV patients has been controversial, questioning the admission of these patients especially in advanced stages of the disease, given the poor prognosis. The aim of this study was to determine the experience of the past 15 years in relation to the income of these patients in an ICU. Methods A retrospective case series consisting of patients with diagnosis of HIV infection (known or unknown) admitted between January 1995 and December 2009. We collected demographic and epidemiological data, process of acquisition of the disease, infection status: known or unknown patient infected, whether or not receiving antiretroviral therapy and whether it was eff ective (undetectable viral load at the time of admission), APACHE II, cause of admission, need for mechanical ventilation (MV), pathology related or unrelated to HIV infection and ICU mortality. Conclusion Neurological dysfunction was independently associated with hospital survival, mainly in those AIDS critically patients without primary neuropathy. y Results During this period 12,607 patients were admitted to the ICU, 188 (1.5%) HIV-positive. Mean APACHE II score 17.6, median age 39 years, 73% men and 90% Spanish nationality. Principal risk behavior: addiction drugs injection (67%). Seventeen percent of patients did not know who was infected with HIV at the time of admission to the ICU. Fifty-three percent were not receiving HAART. Of the 88 patients treated, 93% were receiving HAART (eff ective in 88% of cases). Sixty percent of the patients came from the emergency department of the hospital. Main admission diagnoses: acute respiratory failure caused by infection (Streptococcus pneumoniae and Pneumocystis jirovecii), neurological disorders (coma for illicit drugs and psychotropic) and septic shock. Seventy percent required MV. Of patients whose HIV infection was not known, 93.5% were admitted for related pathology. In patients of known infection, the pathology associated with HIV was 30%. Average length of stay 9 was days. ICU mortality was 35%. Most frequent causes of death: septic shock and multiple organ failure. P464 ICU mortality increases for each day of hospital stay before ICU admission (β = 1.062, 95% CI = 1.009 to 1.118, P = 0.022), each point of: cardiac frequency at admission (β = 1.059, 95% CI = 1.024 to 1.096, P = 0.001), FiO2 (β = 1.034, 95% CI = 1.011 to 1.057, P = 0.003), pCO2 (per mmHg) (β = 1.052, 95% CI = 1.007 to 1.098, P = 0.023); and decreases for each point of: albumin (g/dl) (β = –0.307, 95% CI = 0.095 to 0.986, P = 0.047), mean arterial pressure (mmHg) (β = 0.951, 95% CI = 0.904 to 0.999, P = 0.048), pH (β = 0.002, 95% CI = 0.0 to 0.172, P = 0.006), fi rst ICU 24-hour diuresis (ml) (β = 0.999, 95% CI = 0.998 to 1.000, P = 0.004) and base excess (β = 0.912, 95% CI = 0.836 to 0.995, P = 0.039). Mortality increases threefold (β  =  3.134, 95% CI  =  1.051 to 9.345, P  =  0.04) if patients do not come from the ED, almost 10-fold if the patients need vasopressors for more than 24 hours (β = 9.975, 95% CI = 2.054 to 48.451, P = 0.004) and fi vefold if patients need renal replacement (β = 5.692, 95% CI = 1.466 to 22.099, P = 0.012). 1. Huang L, et al.: N Engl J Med 2006, 355:173-181. 1. Huang L, et al.: N Engl J Med 2006, 355:173-181. g g 2. Akgun K, et al.: J Intensive Care Med 2011, 26:151-164. 2. Akgun K, et al.: J Intensive Care Med 2011, 26:151-164. 2. Akgun K, et al.: J Intensive Care Med 2011, 26:151-164. 2. Akgun K, et al.: J Intensive Care Med 2011, 26:151-164. Prevalence and prognosis of organ dysfunctions of AIDS critically ill patients AJ Japiassu, RA Amâncio, D Medeiros, EM Mesquita, EM Motta, FA Bozza Oswaldo Cruz Foundation, Rio de Janeiro, Brazil Critical Care 2013, 17(Suppl 2):P467 (doi: 10.1186/cc12405) AJ Japiassu, RA Amâncio, D Medeiros, EM Mesquita, EM Motta, FA Bozza Oswaldo Cruz Foundation, Rio de Janeiro, Brazil Critical Care 2013, 17(Suppl 2):P467 (doi: 10.1186/cc12405) Introduction The aim was to analyze the prognosis of AIDS patients with organ dysfunctions at ICU admission. Introduction The aim was to analyze the prognosis of AIDS patients with organ dysfunctions at ICU admission. y Methods A prospective cohort study, including all patients with HIV/ AIDS diagnosis, who were admitted to a specialized ICU from November 2009 until May 2012. Patients with less than 24 hours of ICU stay were excluded. Demographics and nutritional status were collected. The organ dysfunctions were classifi ed according to the SOFA score, and categorized as absent (0 SOFA point), mild (1 to 2 points) and severe (3 to 4 points). We expressed numeric variables as median and interquartile interval (25% to 75%). We performed a multivariate analysis of possible variables associated with hospital mortality (P <0.2), and we explored the 7-day, 28-day and 60-day survival of patients with and without independent risk factors. S175 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Conclusion Neither immune status-related variables nor comorbidity or infection focus are mortality predictors. Poor nutritional status, delayed ICU admission, shock or renal failure increase the ICU relative mortality risk. Tachycardia, hypotension, hypercapnia, acidosis, and oliguria in the fi rst ICU 24 hours increase signifi cantly ICU mortality. Mechanical ventilation is not a mortality predictor. Methods A retrospective study was conducted in consecutive medical and surgical older patients with cancer admitted to a university hospital ICU in São Paulo, between 2007 and 2012. Univariate and multivariate analysis were performed to identify associated and independent factors related to hospital mortality. p y Results From 8,976 patients with cancer requiring ICU at the period, 600 patients were 80  years old or higher. Most patients were male (53%), had solid neoplasm (86%), were from medical admission (54%) and 37% had metastatic disease. The mean age was 84  years (± 3). The ICU and hospital mortality rates were 30% and 44%, respectively. Identifying risk factors for readmission to the ICU: a qualitative approach Results From 597 patients included in the study, 477 were medical admissions (79.8%) and 120 were surgical admissions (20.2%). Four hundred and twenty (70%) patients had metastasis, 222 patients (37%) required the ICU because of respiratory failure and 121 (20%) because of septic shock. The ICU and hospital mortality rates were 53.9% and 64%, respectively. In the univariate analysis, variables associated with hospital mortality were diagnosis of nonsmall-cell lung cancer, higher Charlson morbidity index, medical admission, active neoplasm, vasopressor need at admission to and at 24 hours of ICU, acute renal failure at admission, non-invasive ventilation or mechanical ventilation need at admission to and at 24 hours of ICU and a higher admission arterial lactate. By multivariate analysis, risk factors of hospital mortality were diagnosis of nonsmall-cell lung cancer (OR = 3.32; 95% CI, 2.08 to 5.29, P <0.001), medical admission (OR = 2.89; 95% CI, 1.75 to 4.77, P <0.001), acute renal failure at admission (OR = 4.09; 95% CI, 1.49 to 11.21, P <0.001), non-invasive ventilation at 24 hours of ICU (OR = 2.50; 95% CI, 1.48 to 4.23, P = 0.001) and mechanical ventilation at 24 hours of ICU (OR = 3.68; 95% CI, 1.87 to 7.26, P <0.001). DG Strange, K Tjelle, A Lindhardt, K Jensen Introduction Readmission to the ICU within 48 hours is an indicator of quality of intensive care and is associated with an increase in mortality. During the last  years several groups have published data based on multivariate logistic regression analysis to describe characteristics of patients who needed readmission to the ICU. Older age, comorbid conditions and severity of illness (APACHE score) have been among the strongest predictors for readmission. In our ICU most patients are in the groups formerly identifi ed as risk groups, which means that stratifi cation and prediction of readmission is diffi cult. Because of the unusual high severity of acute and pre-existing illnesses we could not fi nd a data match on comparable patient groups. To investigate whether we could reduce our rate of readmission we therefore decided to perform a qualitative investigation to identify risk factors related to readmission. After identifi cation of the risk factors we will take actions to optimize care and perform ongoing control of the implemented actions to secure that they decreases the rate of readmission. Conclusion Hospital survival in patients with lung cancer requiring ICU admission was 36%. P469 Outcome of intensive care treatment for lung cancer patients L Hajjar1, F Galas2, D Goto2, A Tavares2, M Santos2, A Freitas2, T Oliveira2, P Tonini2, L Cavichio2, M Bazan2, J Almeida2, M Cavalcante2, J Fukushima2, S Vieira2, E Osawa2 1Heart Institute, São Paulo, Brazil; 2Cancer Institute of University of São Paulo, Brazil Critical Care 2013, 17(Suppl 2):P469 (doi: 10.1186/cc12407) Introduction Patients with lung cancer commonly require the ICU for a variety of acute illnesses related to the underlying malignancy, treatment, or comorbid conditions. ICU admission of patients with nonresectable lung cancer has been criticized based on the high mortality rate in this population. However, recent advances in critical care may have changed this scenario. The aim of this study was to identify factors associated with hospital mortality in this group of patients. Conclusion Hospital survival in older patients with cancer requiring ICU admission is acceptable. Our results provide evidence that ICU management may be appropriate in older patients with cancer and appoint risk factors for mortality, helping to better triage cancer patients who will benefi t from ICU care. Methods A retrospective study was conducted in consecutive medical and surgical patients with lung cancer admitted to a university hospital ICU in São Paulo, between 2007 and 2012. A univariate analysis was performed to identify associated variables with hospital mortality. Selected variables were included in the multivariate model. Prevalence and prognosis of organ dysfunctions of AIDS critically ill patients In the univariate analysis, variables associated with hospital mortality were diagnosis of lung cancer, medical admission, active neoplasm, vasopressor need at 24 hours of ICU, acute renal failure at admission, mechanical ventilation need at admission to and at 24 hours of ICU and a higher admission arterial lactate. By multivariate analysis, risk factors of hospital mortality were diagnosis of lung cancer (OR = 2.32; 95% CI, 1.08 to 3.29, P <0.001), medical admission (OR = 6.473; 95% CI, 2.76 to 15.15, P <0.001), acute renal failure at admission (OR = 3.09; 95% CI, 1.49 to 9.21, P <0.001), mechanical ventilation at 24 hours of ICU (OR = 5.68; 95% CI, 2.87 to 7.26, P <0.001) and lactate levels at admission (OR = 1.02; 95% CI, 1.006 to 1.051, P <0.001). Identifying risk factors for readmission to the ICU: a qualitative approach Our results provide evidence that ICU management may be appropriate in patients with nonresectable lung cancer and appoint risk factors for mortality, helping to better triage cancer patients who will benefi t from ICU care. Methods Retrospective data on patients readmitted to the ICU within 48 hours during an 8-month period (November 2011 to June 2012) were drawn from the Critical Information System (CIS) at ICU ZIT, Bispebjerg Hospital, Denmark. ZIT is a multidisciplinary unit with 10 beds and 500 to 600 admissions/year and a median SAPS II score of 47. A group of consultants, junior doctors and nurses from the ICU and the ward each read the patient fi les with focus on pattern recognition and suggested trigger points to focus on. Data on trigger points were then drawn from the CIS system and re-evaluated. Finally, fi ve key points were identifi ed and serves as basis for future actions. Complementarity of prognosis models SAPS 3 and EuroSCORE in cardiac surgery Discrimination was assessed using the area under the ROC curve. With the standardized mortality ratio (SMR) we evaluated the agreement between predicted and observed mortality. We used multiple logistic regression for multivariate analysis Introduction The aim of this study is to evaluate the performance of SAPS 3 and EuroSCORE and whether there is complementarity between them in ICU patients admitted after cardiac surgery. Methods An observational, prospective and multicenter study of patients included in the ARIAM registry of adult cardiac surgery. We analyzed clinical variables, surgery data and postoperative compli- cations, outcomes and scores (SAPS 3 and EuroSCORE). Discrimination was assessed using the area under the ROC curve. With the standardized mortality ratio (SMR) we evaluated the agreement between predicted and observed mortality. We used multiple logistic regression for multivariate analysis. y Results A total of 5,642 patients were included, admitted to any fi ve hospitals, consecutively between 2008 and 2012. Mean age was 63.62 ± 12.80 years. Surgery room mortality was 1.4%, ICU mortality was 7.9% and in-hospital mortality was 11.2%. SAPS 3 was calculated in 5,383 patients. SAPS 3 was 40.91 ± 10.46, and the expected rate of mortality was 11.09% (with the southwestern Europe equation), with an observed hospital mortality of 9.6%, SMR = 0.87 (0.79 to 0.94). With the general equation, the expected mortality was 10.47%, SMR = 0.917 (0.843 to 0.996). Discrimination of SAPS 3 was evaluated using the area under the ROC curve, it was 0.77 (0.75 to 0.79). The 30-day mortality was 9.6%, with an expected 30-day mortality predicted by EuroSCORE of 7.86% (SMR = 1.22; CI = 1.12 to 1.32). The area under the ROC curve was 0.734 for EuroSCORE (0.712 to 0.755). Multivariate analysis with logistic regression showed complementarity between SAPS 3 and logistic EuroSCORE (vers. I). There is a relationship between hospital mortality and the probability of dying predicted by SAPS 3 (general equation), OR 1.05 (1.04 to 1.06), and logistic EuroSCORE (vers. I), OR 1.024 (1.015 to 1.032). The model with both variables has a similar discrimination to SAPS 3 alone. The area under the ROC curve of the combined model was 0.778 (0.758 to 0.799) and 0.77 (0.75 to 0.79) using SAPS 3 only. Reference 1. Breslow MJ, Badawi O: Severity scoring in the critically ill: part 1 – interpretation and accuracy of outcome prediction scoring systems. Chest 2012, 141:245-252. P474 g Methods A prospective registry involving patients who underwent LDLT in the period from October 2004 to December 2010. Postoperative 30- day clinical and laboratory data included: clinical BP, HR, temperature, ICU stay, liver functions AST, ALT, GGT, ALP, TotPtn, Alb, TBil, DBil, INR, PTT, CRP, kidney functions Crea, Na, K, blood picture Hb, Hct, TLC and Plt count. Complementarity of prognosis models SAPS 3 and EuroSCORE in cardiac surgery MJ Delgado-Amaya1, E Curiel-Balsera1, MD Arias-Verdu1, E Castillo-Lorente2, FJ Carrero-Gómez3, E Aguayo-DeHoyos4, A Herruzo-Avilés5 1Hospital Regional Carlos Haya, Málaga, Spain; 2Complejo Hospitalario de Jaén, Spain 3Puerta del Mar Hospital, Cádiz, Spain; 4Virgen de las Nieves Hospital, Granada, Spain; 5Virgen del Rocio Hospital, Seville, Spain Critical Care 2013, 17(Suppl 2):P474 (doi: 10.1186/cc12412) Are APACHE II scores better predictors of mortality than routine laboratory values? y Z Baykara, H Özocak, A Kuş, Z Arslan, B Yüksel, C Aksu, M Ertagın, M Solak, K Toker University of Kocaeli, Turkey Critical Care 2013, 17(Suppl 2):P473 (doi: 10.1186/cc12411) Introduction Even though more accurate and updated versions such as APACHE III and IV have been developed, APACHE II has remained the most widely used severity scoring system in the ICU [1]. In the present study, we aimed to develop a model mainly based on the laboratory data available upon admission to predict mortality and to compare its performance with that of APACHE II in a mixed ICU in Turkey. g y Conclusion There is complementarity between the SAPS 3 model and EuroSCORE. However, a joint model with both prognosis scores has no appreciable improvement in discrimination, compared with the SAPS 3 model. In our study, SAPS 3 shows very useful to rule out that the superior observed mortality (compared with the mortality predicted by EuroSCORE) is not attributable to inadequate care of patients in the ICU, the observed mortality being even slightly below the predicted mortality for patients undergoing cardiac surgery at the reference hospitals in which the SAPS 3 system was developed. Methods A total of 645 adult patients who were over 18  years old and who stayed in the ICU for more than 24 hours were included in this study. Using stepwise multivariate logistic regression analysis, a mortality prediction model was developed based on the laboratory data, diagnostic category and age. The adjusted probability of death, according to the diagnostic category of the APACHE II score (adj-APACHE II), was calculated. The ability of the laboratory model and the adj- APACHE II model to discriminate between survivors and nonsurvivors was assessed using receiver operating characteristic (ROC) curves. The calibration of the laboratory model and the adj-APACHE II model was assessed by the Hosmer and Lemeshow goodness-of-fi t test. Complementarity of prognosis models SAPS 3 and EuroSCORE in cardiac surgery p Conclusion Predictors of mortality were INR >3.06 (accuracy 87.5%) followed by PTT >53.3 (79.2%), HR >94 (76.8%), ICU stay >8  days (76.2%), Na >142.9 (72.9%), TBil >15 (71.3%), ALT >379 (67.5%), TProt <4.6 (63.5%), AST >145 (57.4%), Hct <24.4 (54.2%), CRP >12 (Acc 51.6%) and GGT >22 (51.4%). Complementarity of prognosis models SAPS 3 and EuroSCORE in cardiac surgery g y MJ Delgado-Amaya1, E Curiel-Balsera1, MD Arias-Verdu1, MJ Delgado-Amaya1, E Curiel-Balsera1, MD Arias-Verdu1, E Castillo-Lorente2, FJ Carrero-Gómez3, E Aguayo-DeHoyos4, A Herruzo-Avilés5 y Results Our study involved 142 recipients, 124 (87%) males and 18 (13%) females, who underwent living donor liver transplantation; mean age was 53 ± 14.8 years, ranging from 24 to 66 years; 29 cases (20.43%) at Kasr Al Aini University Hospital and 113 cases (79.57%) at the International Medical Center. The 30-day mortality rate was 13.38%. Clinical and laboratory parameters showed gradual resolution towards normal values in survivors or deterioration in nonsurvivors, allowing one to predict mortality at various cutoff points. Clinical parameters: HR >94 cutoff (Acc 76.8%, Sens 89.5%, Spec 62.6%, PPV 27, NPV 97) and ICU stay >8 days (Acc 76.2%, Sens 75.3%, Spec 95.7%, PPV 95.7, NPV 35.3). Laboratory parameters: INR >3.06 cutoff (Acc 87.5%, Sens 92.9%, Spec 78.6%, PPV 96.3, NPV 64.7), followed by PTT >53.3 (Acc 79.2%, Sens 78.6%, Spec 75%, PPV 93.6, NPV 42.9), Na >142.9 (Acc 72.9%, Sens 80%, Spec 72.3%, PPV 26.7, NPV 96.6), TBil >15 (Acc 71.3%, Sens 95.5%, Spec 50%, PPV 92.4, NPV 63.6), ALT >379 (Acc 67.5%, Sens 72.2%, Spec 73.8%, PPV 28.9, NPV 94.7), TProt <4.6 (Acc 63.5%, Sens 55.6%, Spec 73.3%, PPV 24.4, NPV 91.4), AST >145 (Acc 57.4%, Sens 50%, Spec 84.4%, PPV 32.1, NPV 92), Hct <24.4 (Acc 54.2%, Sens 47.4%, Spec 81.5%, PPV 34.6, NPV 88.2), CRP >12 (Acc 51.6%, Sens 43.7%, Spec 73.3%, PPV 90.5, NPV 17.7), GGT >22 (Acc 51.4%, Sens 83.3%, Spec 36.9%, PPV 16.3, NPV 93.7). 1Hospital Regional Carlos Haya, Málaga, Spain; 2Complejo Hospitalario de Jaén, Spain 3Puerta del Mar Hospital, Cádiz, Spain; 4Virgen de las Nieves Hospital, Granada, Spain; 5Virgen del Rocio Hospital, Seville, Spain Critical Care 2013, 17(Suppl 2):P474 (doi: 10.1186/cc12412) 1Hospital Regional Carlos Haya, Málaga, Spain; 2Complejo Hospitalario de Jaén, Spain 3Puerta del Mar Hospital, Cádiz, Spain; 4Virgen de las Nieves Hospital, Granada, Spain; 5Virgen del Rocio Hospital, Seville, Spain Critical Care 2013, 17(Suppl 2):P474 (doi: 10.1186/cc12412) Introduction The aim of this study is to evaluate the performance of SAPS 3 and EuroSCORE and whether there is complementarity between them in ICU patients admitted after cardiac surgery. Methods An observational, prospective and multicenter study of patients included in the ARIAM registry of adult cardiac surgery. We analyzed clinical variables, surgery data and postoperative compli- cations, outcomes and scores (SAPS 3 and EuroSCORE). P470 P470 Outcome of older patients with cancer requiring ICU admission L Hajjar1, M Franca Cavalcante2, F Galas2, J T Fukushima2, M R Sundin2, F Bergamin2, C Park2, H Palomba2, D M Goto2, A Tavares2, M Santos2, A Freitas2, P Tonini2, L Cavichio2, J P Almeida2, M Bazan2 1Heart Institute, São Paulo, Brazil; 2Cancer Institute of University of São Paulo, Brazil Critical Care 2013, 17(Suppl 2):P470 (doi: 10.1186/cc12408) Results In a qualitative analysis, readmissions to the ICU are related to the following fi ve key points – discharge outside day hours, lack of infection control, stay in ICU <2 days, lung physiotherapy ordinated but not eff ectuated, and several minor organ dysfunctions (atrial fi brillation and acute kidney injury). Age, diagnosis, SAPS II score or ventilator treatment during intensive care was not diff erent in patients with successful discharge and patients readmitted in this group of patients. Conclusion It is possible and suitable to identify key points for future eff orts in a given subgroup of patients using a systematic qualitative approach. Critical Care 2013, 17(Suppl 2):P470 (doi: 10.1186/cc12408) Introduction Because the prognosis of older patients with cancer may be poor compared with younger patients, it remains controversial whether they benefi t from ICU treatment. The objective of this study was to identify factors associated with hospital mortality in older patients with cancer requiring the ICU. S176 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 P472 P472 Clinical and laboratory postoperative impact on outcome of living donor liver transplantation recipients Y Nassar, K Omar, A Battah, A Mwafy Cairo University, Giza, Egypt Critical Care 2013, 17(Suppl 2):P472 (doi: 10.1186/cc12410) 2.01 (P = 0.98) for the laboratory model and 13.2 (P = 0.10) for the adj- APACHE II model. Conclusion If the aim is to predict mortality as accurately as APACHE II, the mortality prediction model based mainly on routine admission laboratory tests can achieve this using computer technology, without labor-related costs, as soon as the patient is admitted to the ICU. However, it is necessary to perform multicenter validation studies. Reference Introduction Adult recipients of living donor liver transplantation have a wide range of postoperative medical comorbidities altering various clinical and laboratory parameters. We aimed to assess 30- day postoperative clinical and laboratory changes and their value in predicting outcome of LDLT recipients. P477 Eldicus scores for admission to our ICU: a retrospective study G Zani, B Gemelli, S Vitali, C Villani, R Ragazzi, S Spadaro, CA Volta, R Alvisi University of Ferrara, Italy Critical Care 2013, 17(Suppl 2):P477 (doi: 10.1186/cc12415) Results A total of 273 charts were reviewed. In total, 242 were categorized into A (n = 126), B (n = 79) or C (n = 37). D (n = 31) consisted mainly of patients with hematological malignancies (n  =  12) and patients with chemotherapy or immunosuppressive treatment (n = 14). The groups diff ered in length of stay with A< B< C. During the fi rst 3 days the SOFA score was higher in A compared with C and in B compared with C. The duration of antibiotic therapy was longer in both B and C compared with A. There were no diff erences in 28-day mortality (A: 34/126 = 27%, B: 30/79 = 38%, C: 15/37 = 40%); however, the proportions of patients dying between days 8 and 28 were higher in B (14/63 = 22%) and C (7/29 = 24%) compared with A (5/95 = 5%). Introduction The indication for ICU admission is poorly defi ned and identifying patients who may benefi t from it is extremely diffi cult. As reported in literature, the Eldicus scores require further validation in centers in diff erent countries and with diff erent patient populations [1]. Previous studies have demonstrated that patients with intermediate severities of illness are those that benefi t from ICU treatment; instead, patients who are too well or too sick should not be admitted. There are many unmeasurable factors that infl uence the triage decision and can infl uence the subsequent outcome. The Eldicus scores could be the fi rst triage decision rule. The aim of this study was to confi rm the ability of the Eldicus scores to discriminate the survival status in our general ICU. Methods We retrospectively enrolled all patients >18 years admitted to the Ferrara ICU from January to June 2012. Patients’ data were recorded using Prosafe international data collection software and consulting the individual medical records. Data were chosen based on variables used in the Eldicus scores. As indicated in the Eldicus algorithm, for each patient the initial refusal score was calculated, to evaluate the too severe patients. Reference Results A total of 481 patients aged 80 or over were admitted between 1 January 2001 and 31 December 2010, representing 9.6% of the admissions during this time with minimal annual change. Admission age ranged from 80 to 96 years (median 83) with a median APACHE II score of 20 (6 to 48, IQR 16 to 25). In total, 318 (66.1%) survived to critical care discharge and 256 (53.2%) survived to hospital discharge; 228 (89.0%) went home rather than to a care facility. Those under 80 years demonstrated 80.6% critical care survival, 65.5% hospital survival and a median APACHE II score of 17 (0 to 51, IQR 13 to 23). Eighty patients died within the 22-month follow-up period with an average life expectancy of 7.4 months. None reached life expectancy (average curtailment of 5.6  years compared with age-matched and sex-matched population statistics). Neither of these fi gures changed signifi cantly through the 1. Sprung CL, et al.: Crit Care Med 2012, 40:125-131. 1. Sprung CL, et al.: Crit Care Med 2012, 40:125-131. Comparing intensive care treated primary, secondary and tertiary sepsis with respect to length of stay, organ failure and mortality: a pilot study Conclusion Older people represent a growing proportion of the population although their representation in the critical care population remained constant in this 10-year study. These patients had a slightly higher median APACHE II score and 14.5% greater critical care mortality than the younger patients. The majority of survivors were able to go home; however, 31% died within 22  months with signifi cant life expectancy curtailment, surviving on average only 7.4  months after discharge; this has not changed in the last 10 years. Those who survived this initial period (69%) had a much better outlook. This information may be vital to patients and physicians when discussing admission to critical care. p p y Methods The Swedish ICU registry was used to identify all adult patients diagnosed with severe sepsis or septic shock in a single Swedish ICU during 2006 to 2011. The charts were reviewed and patients categorized into four groups based on whether the patient had suff ered systemic infl ammatory-inducing insults previous to the septic episode. The patients were categorized as: (A) primary sepsis, no recent (<30 days) trauma/infection, ongoing systemic infl ammatory or immunosuppressive disease or malnutrition; (B) secondary sepsis, recent (<7  days) trauma/infection or systemic infl ammatory disease, but no immunosuppressive disease or malnutrition; (C) tertiary sepsis, >1 fulfi lled criteria of secondary sepsis during the last 10 days or immunosuppressive disease or malnutrition; and (D) patients not attributable to A to C. After the categorization, physiological and laboratory parameters during the ICU stay as well as therapy and outcome were extracted from the charts. Only signifi cant diff erences (P  <0.05) are presented; Mann–Whitney and chi-squared tests were used. P477 Then we counted the fi nal triage score, to discover whether some patients were too healthy to be admitted to the ICU. Correlation between the severity of the scores and patients’ outcome was estimated using the Wilcoxon’s rank-sum test. Conclusion In this retrospective material it was possible to categorize 88.6% of all patients as having primary, secondary or tertiary sepsis. The categories diff ered in clinical picture at presentation as well as in outcome. A prospective study is warranted to validate the results of this study. P476 Comparing intensive care treated primary, secondary and tertiary sepsis with respect to length of stay, organ failure and mortality: a pilot study yi Results The areas under the ROC curves of the laboratory models and the adj-APACHE II scores for the prediction of mortality were 0.80 (95% CI: 0.76 to 0.85) and 0.78 (95% CI: 0.74 to 0.83), respectively (P >0.05, z statistic). The Hosmer–Lemeshow statistic had a chi-squared value of y S Castegren1, M Jonasson1, J Sjölin2, M Castegren1 1Centre for Clinical Research Sörmland, Uppsala University, Uppsala, Sweden; 2Medical Sciences, Uppsala University, Uppsala, Sweden Critical Care 2013, 17(Suppl 2):P475 (doi: 10.1186/cc12413) S Castegren1, M Jonasson1, J Sjölin2, M Castegren1 1Centre for Clinical Research Sörmland, Uppsala University, Uppsala, Sweden; 2Medical Sciences, Uppsala University, Uppsala, Sweden Critical Care 2013, 17(Suppl 2):P475 (doi: 10.1186/cc12413) S177 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 decade. However, 10-year follow-up on 2001/2002 admissions showed that, despite this early mortality eff ect, the average post-discharge lifespan was 5.1 years with 2.2 years shortfall in life expectancy. Conclusion Older people represent a growing proportion of the population although their representation in the critical care population remained constant in this 10-year study. These patients had a slightly higher median APACHE II score and 14.5% greater critical care mortality than the younger patients. The majority of survivors were able to go home; however, 31% died within 22  months with signifi cant life expectancy curtailment, surviving on average only 7.4  months after discharge; this has not changed in the last 10 years. Those who survived this initial period (69%) had a much better outlook. This information may be vital to patients and physicians when discussing admission to critical care. decade. However, 10-year follow-up on 2001/2002 admissions showed that, despite this early mortality eff ect, the average post-discharge lifespan was 5.1 years with 2.2 years shortfall in life expectancy. Introduction It may be hypothesized that a patient’s infl ammatory status might aff ect the clinical picture and outcome of a subsequent septic insult. To test this hypothesis, patients were categorized according to previous infl ammatory activity and analyzed with respect to clinical picture and outcome in a retrospective study. Introduction It may be hypothesized that a patient’s infl ammatory status might aff ect the clinical picture and outcome of a subsequent septic insult. To test this hypothesis, patients were categorized according to previous infl ammatory activity and analyzed with respect to clinical picture and outcome in a retrospective study. Reference 1. Offi ce of National Statistics Population Data [http://www.ons.gov.uk/ons/ taxonomy/index.html?Nscl=Population] 1. Offi ce of National Statistics Population Data [http://www.ons.gov.uk/ons/ taxonomy/index.html?Nscl=Population] P476 Survival of older patients after critical care D Ashton-Cleary, R Sinclair Royal Cornwall Hospital, Truro, UK Critical Care 2013, 17(Suppl 2):P476 (doi: 10.1186/cc12414) Survival of older patients after critical care D Ashton-Cleary, R Sinclair Royal Cornwall Hospital, Truro, UK Critical Care 2013, 17(Suppl 2):P476 (doi: 10.1186/cc12414) g Results Ninety-eight patients were fi nally enrolled. Data are presented as median ± SD. The SAPS II score was 33 ± 13 and was similar to those reported in the literature [1]. The Karnofsky performance status scale was 69 ± 20. The Eldicus initial score was 56 ± 30. The fi nal score was 70 ± 48. Seventy-eight percent of the analyzed patients were alive at hospital discharge. A signifi cant correlation between the severity scores and in-hospital outcome was found (P <0.001). Limitations of our study were the low sample size and the fact that patients not admitted to the ICU were not considered. Introduction We aimed to assess the survival of older patients after admission to critical care compared with the population in general. Introduction We aimed to assess the survival of older patients after admission to critical care compared with the population in general. p admission to critical care compared with the population in general. Methods We reviewed admission data for a 12-bed critical care unit in a district general hospital in the UK between 2001 and 2010. We calculated critical care and hospital discharge survival rates and studied the trend in these through the decade. We searched hospital records to establish whether these patients were still alive at November 2012 to determine life-expectancy (minimum 22-month follow-up on all patients). This was compared with UK Offi ce of National Statistics data for the local population [1]. Discharge destination (care facility or home address) was recorded as a surrogate marker of change in quality of life post discharge. Conclusion The Eldicus scores appear to be useful in identifying patients that may benefi t more from ICU admission. Further studies should confi rm the ability of these scores in helping identify those patients who really benefi t from ICU admission. Reference Prediction of sequences of organ dysfunction in critical patients studied with Bayesian analysis M Sandri1, P Berchialla2, D Gregori3, L Gottin1, V Sweiger1, E Polati1, RA De Blasi4 1University of Verona, Italy; 2University of Torino, Italy; 3University of Padova, Italy; 4University of Rome ‘La Sapienza’, Rome, Italy Critical Care 2013, 17(Suppl 2):P479 (doi: 10.1186/cc12417) p p g p p Results Lower age, NT-Pro-BNP and TnI were signifi cantly associated with 1-year survival. We built a neural network model using the training database. We used the test database to compare our model with the IGS 2 score. The diff erence between the AUC of the biomarker score and the IGS 2 score was statistically signifi cant (Figure 1). Introduction The multiorgan dysfunction syndrome (MODS) is a dynamic process involving simultaneously or consecutively two or more organ systems [1]. The organ dysfunction’s degree can be assessed by three severity scores (SOFA [2], MODS [3], LODS [4]), but they have some limitations: they do not allow the evaluation of the clinical course of a patient, they are not reliable in populations diff erent from the reference one, and they do not support clinicians’ decisions. Because MODS implies a systemic infl ammatory reaction leading to microcirculatory dysfunction, our hypothesis was that organ failures follow a predictable sequence of appearance. Our aims were to verify the presence of more likely organ failure sequences and to assess an online method to predict the evolution of MODS in a patient. The high mortality and morbidity rate of MODS in ICUs can in fact be reduced only by a prompt and well-timed treatment [5]. y gi g Conclusion Our new neural network model built using age, NT-PRO- BNP and TnI succeeded the internal-validation processes and predicts 1-year mortality better than IGS 2. P481 Signifi cance of preadmission quality of life for mortality in the ICU: a prospective cohort study RB Bukan1, A Moeller1, M Henning1, K Mortensen2, T Klausen1, T Waldau1 1Herlev University Hospital, Herlev, Denmark; 2University of Copenhagen, Denmark Critical Care 2013, 17(Suppl 2):P481 (doi: 10.1186/cc12419) Figure 1 (abstract P480). Comparison of area under ROC curves (AUC) of the biomarker score and IGS 2. Figure 1 (abstract P480). Comparison of area under ROC curves (AUC) of the biomarker score and IGS 2. Methods We selected 73 patients consecutively admitted to the ICU of Sant’Andrea Hospital from January to June 2012. The inclusion criteria were at least two organ systems with SOFA ≥2, ICU length of stay >48 hours. Developing a screening instrument for predicting psychological morbidity after critical illness Ö A Schandl1, M Bottai2, E Hellgren1, Ö Sundin3, P Sackey1 1Karolinska University Hospital Solna, Stockholm, Sweden; 2Karolinska Institutet, Stockholm, Sweden; 3Mid Sweden University, Östersund, Sweden Critical Care 2013, 17(Suppl 2):P478 (doi: 10.1186/cc12416) Introduction The purpose was to develop a predictive screening instrument for use at ICU discharge to identify patients at risk for post- traumatic stress, anxiety or depression. Introduction The purpose was to develop a predictive screening instrument for use at ICU discharge to identify patients at risk for post- traumatic stress, anxiety or depression. S178 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Methods Potential risk factors for psychological problems were prospectively collected at ICU discharge. Two  months after ICU discharge 252 ICU survivors received the questionnaires Post- Traumatic Stress Symptom scale-10 (PTSS-10) and Hospital Anxiety and Depression Scale (HADS) to estimate the degree of post-traumatic stress, anxiety and depression. Sepsis-Related Problems of the European Society of Intensive Care Medicine. Intensive Care Med 1996, 22:707-710. Marshall JC, et al.: Multiple organ dysfunction score: a reliable descriptor of a complex clinical outcome. Crit Care Med 1995, 23:1638-1652. Le Gall JR, et al.: The logistic organ dysfunction system: a new way to assess organ dysfunction in the intensive care unit. JAMA 1996, 276:802-810. Kilica YA, et al.: Visualizing multiple organ failure: a method for analyzing temporal and dynamic relations between failing systems and interventions. Crit Care 2007, 11:417. y Results Of the 150 responders, 31% had adverse psychological outcome, defi ned as PTSS-10 >35 and/or HADS subscales ≥8. After analysis, six predictors with weighted risk scores were included in the screening instrument: major pre-existing disease, being a parent to children younger than 18 years of age, previous psychological problems, in-ICU agitation, being unemployed or sick-listed at ICU admission and appearing depressed in the ICU. Each predictor corresponded to a given risk score. The total risk score, the sum of individual risk scores, was related to the probability for adverse psychological outcome in the individual patient. The predictive accuracy of the screening instrument, as assessed with area under the receiver operating curve, was 0.77. When categorizing patients in three risk probability groups – low (0 to 29%), moderate (30 to 59%) and high (60 to 100%) risk – the actual prevalence of adverse psychological outcome in respective groups was 12%, 50% and 63%. 6. Prediction of sequences of organ dysfunction in critical patients studied with Bayesian analysis For each patient we calculated the SOFA since the beginning of the inclusion criteria and daily for 8 days. For the statistical analysis we used Dynamic Bayesian Networks (DBNs) [6]. DBNs were applied to model SOFA changes in order to identify the most probable sequences of organs failures in a patient who experienced a fi rst known failure. gi Results We created a DBN for the analysis of MODS studying the relations between organ failures at diff erent times. The DBN was made so that each organ failure is dependent on the previous one. We also considered a corrective factor to take account that not all patients completed the observation. Using software (GeNie) we obtained the probabilities of the organ failure sequences. Figure 1 (abstract P480). Comparison of area under ROC curves (AUC) of the biomarker score and IGS 2. Figure 1 (abstract P480). Comparison of area under ROC curves (AUC) of the biomarker score and IGS 2. g Conclusion The use of DBNs, although with our limited set of data, allowed us to identify the most likely organ dysfunction sequences associated with a fi rst known one. Capability to predict these sequences in a patient makes DBNs a promising prognostic tool for physicians in order to treat patients in a timely manner, or to test a treatment effi cacy. References Developing a screening instrument for predicting psychological morbidity after critical illness Ö Murphy K, et al.: Modelling Gene Expression Data using Dynamic Bayesian Networks. Technical report. University of California; 1999. Comparison of IGS 2 score and a new neural network biomarker score performance in predicting 1-year mortality Comparison of IGS 2 score and a new neural network biomarker score performance in predicting 1-year mortality C Romdhani, I Labbene, M Belhadj Amor, Z Hajjej, A Massoudi, M Ferjani Military Hospital of Tunis, Tunisia Critical Care 2013, 17(Suppl 2):P480 (doi: 10.1186/cc12418) C Romdhani, I Labbene, M Belhadj Amor, Z Hajjej, A Massoudi, M Ferjani Military Hospital of Tunis, Tunisia Critical Care 2013, 17(Suppl 2):P480 (doi: 10.1186/cc12418) Introduction The aim of this study was to compare the IGS 2 score with a predictive model built using clinical characteristics and biomarkers associated with 1-year mortality from procalcitonin, C-reactive protein, troponin I (TnI) and NT-Pro-BNP. Conclusion The preliminary screening instrument may aid ICU clinicians in identifying patients at risk for adverse psychological outcome after critical illness. Prior to wider clinical use, external validation is needed. Methods We conducted a prospective observational cohort study in a polyvalent ICU. We calculated the IGS 2 severity score in the fi rst 24  hours. We dosed NT-Pro-BNP, TnI, procalcitonin and C-reactive protein at admission to the ICU. To test the biomarker model, the patient database was divided into training (70%) and test (30%) databases. A neural network model was trained from variables associated with 1-year mortality. All statistics were done using R v.2.15.1. We used the pROC package to plot and compare ROC curves.i P481 Signifi cance of preadmission quality of life for mortality in the ICU: a prospective cohort study RB Bukan1, A Moeller1, M Henning1, K Mortensen2, T Klausen1, T Waldau1 1Herlev University Hospital, Herlev, Denmark; 2University of Copenhagen, Denmark Critical Care 2013, 17(Suppl 2):P481 (doi: 10.1186/cc12419) Predicting physical function and mental health in trauma intensive care patients 2 years after hospitalisation Predicting physical function and mental health in trauma intensive care patients 2 years after hospitalisation L Aitken1, B Macfarlane1, W Chaboyer2, M Schuetz1, C Joyce1, AG Barnett3 1Princess Alexandra Hospital, Brisbane, Australia; 2Griffi th University, GoldCoast, Australia; 3Queensland University of Technology, Brisbane, Australia Critical Care 2013, 17(Suppl 2):P482 (doi: 10.1186/cc12420) gi Results We present the results of an 18-month period, 9 months prior to and 9 months after the introduction of the SwissDRG. Data of 1,491 and 1,492 patients were analyzed, respectively. When all patients were included, we found prior to and after the introduction of the DRGs a comparable length of stay on the ICU (mean ± SD of 52.1 ± 2.2 hours vs. 50.82 ± 2.2 hours), no diff erence in the severity of illness at discharge detected by the SAPS II (mean ± SD of 27.9 ± 0.3 vs. 28.4 ± 0.3) and the number of readmissions (91 vs. 92). There was also no signifi cant diff erence when only percentiles 6 to 94 were included or when the three subgroups were analyzed. p y p L Aitken1, B Macfarlane1, W Chaboyer2, M Schuetz1, C Joyce1, AG Barnett3 1Princess Alexandra Hospital, Brisbane, Australia; 2Griffi th University, GoldCoast, Australia; 3Queensland University of Technology, Brisbane, Australia Critical Care 2013, 17(Suppl 2):P482 (doi: 10.1186/cc12420) Introduction Long-term compromise after traumatic injury is signifi cant; however, few modifi able factors that infl uence outcome have been identifi ed. The aim of this study was to identify acute and early post-acute predictors of long-term recovery amenable to change through intervention. g p y Conclusion Up to now, the introduction of the SwissDRG has no infl uence on patients’ discharge characteristics from a large ICU. Data assessment will continue and further data analysis has to be performed. Methods Adults (>17 years) admitted to the ICU, Princess Alexandra Hospital, Australia following injury were prospectively followed. Data were collected on demographics, pre-injury health, injury characteristics and acute care factors. Psychosocial measures (self- effi cacy (SE), illness perception (IP), post-traumatic stress disorder (PTSD) symptoms and psychological distress) and health status (SF-36) were collected via questionnaire 1, 6, 12, and 24 months post injury. Outcomes of interest were the Physical Function (PF) and Mental Health (MH) subscales of the SF-36. Regression models were used to estimate predictors of physical function and mental health over a 2-year period. Do ICU patients’ discharge characteristics change with Swiss Diagnosis Related Groups? Do ICU patients’ discharge characteristics change with Swiss Diagnosis Related Groups? Do ICU patients’ discharge characteristics change with Swiss Diagnosis Related Groups? G Rauchenstein-Ziltener, CA Rüst, P Fodor, S Blumenthal Tiemli City Hospital, Zurich, Switzerland Critical Care 2013, 17(Suppl 2):P483 (doi: 10.1186/cc12421) g G Rauchenstein-Ziltener, CA Rüst, P Fodor, S Blumenthal Tiemli City Hospital, Zurich, Switzerland Critical Care 2013, 17(Suppl 2):P483 (doi: 10.1186/cc12421) Results Preliminary results, based upon data from 175 included patients, reveal an ICU mortality of 12.6% and 30-day mortality of 22.9%. No patients were lost to follow-up. When the Physical Component of SF-36 (PCS) was used as an estimate of preadmission quality of life, the area under the curve for model B (AUC = 0.80) was comparable with model E (AUC = 0.81), and better than model A (AUC = 0.85). The General Health item of SF-36 (GH), used as an estimate of preadmission quality of life, gave an AUC = 0.76 (model D). All models were controlled for sex and age. Introduction Swiss Diagnosis Related Groups (SwissDRG) have been eff ective since 1 January 2012. The infl uence of this new system on patients’ discharge characteristics from a large ICU is not known. With the introduction of the DRG we expect patients to be discharged after a shorter length of stay on the ICU and with higher severity of illness. Methods The ICU of the City Hospital Triemli in Zurich has an inter- disciplinary organization with surgical and internal medical patients, with a maximum occupancy of 18 beds and a center function for the surrounding hospitals. In this ongoing prospective observational study, we collect and analyze the anonymized data of all patients discharged from our ICU prior to and after the introduction of the SwissDRG. The primary endpoint was the length of stay on the ICU in hours. The secondary endpoints were the severity of illness of the patients at the time of discharge, detected by the scoring system SAPS II as well as measured by the number of readmissions to the ICU. Initially all patients were analyzed and in a second step only patients within percentiles 6 to 94 were considered. We also analyzed the subgroups of patients referred internally, patients sent back to referring hospital and patients regionalized to a homebase hospital. The statistics have been done with SPSS and P <0.05 was considered signifi cant. P481i P481 Signifi cance of preadmission quality of life for mortality in the ICU: a prospective cohort study RB Bukan1, A Moeller1, M Henning1, K Mortensen2, T Klausen1, T Waldau1 1Herlev University Hospital, Herlev, Denmark; 2University of Copenhagen, Denmark Critical Care 2013, 17(Suppl 2):P481 (doi: 10.1186/cc12419) Signifi cance of preadmission quality of life for mortality in the ICU: a prospective cohort study p p y RB Bukan1, A Moeller1, M Henning1, K Mortensen2, T Klausen1, T Waldau1 1Herlev University Hospital, Herlev, Denmark; 2University of Copenhagen, Denmark 1. Levy MM, et al.: 2001 SCCM/ESICM/ACCP/ATS/SIS International Sepsis Defi nitions Conference. Intensive Care Med 2003, 29:530-538. Levy MM, et al.: 2001 SCCM/ESICM/ACCP/ATS/SIS International Sei 2. Vincent JL, et al.: The SOFA (Sepsis-Related Organ Failure Assessment) score to describe organ dysfunction/failure. On behalf of the Working Group on Critical Care 2013, 17(Suppl 2):P481 (doi: 10.1186/cc12419) S179 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Introduction Assessing whether a critically ill patient should be admitted to an ICU remains diffi cult and mortality amongst ICU patients is high. To render intensive care with no prospect of success is an immense emotional burden for both patient and relatives, and a great socioeconomic burden for society as well. Therefore, validated strategies that can help identify patients who will benefi t from intensive care are in demand. This study seeks to investigate whether preadmission quality of life can act as a predictor of mortality amongst patients admitted to the ICU. Predictors of PF included IP (β = –1.5, 95% CI = –3.1 to –1.1, P <0.01), SE (β = 1.8, 95% CI = 1.3 to 2.6, P <0.01), hospital length of stay (β = –1.7, 95% CI = –2.0 to –0.8, P <0.01), never having been married (β = 1.8, 95% CI = 0.3 to 5.5, P = 0.03), and having injury insurance (β = –2.7, 95% CI = –6.9 to –1.9, P <0.01). Predictors of MH included PTSD symptoms (β = –2.4, 95% CI –3.4 to –1.4, P <0.01), psychological distress (β = –6.9, 95% CI = –8.9 to –5.2, P <0.01), SE (β = 0.6, 95% CI = 0.2 to 1.1, P <0.01), and unemployment (β = –2.3, 95% CI = –5.0 to –0.2, P = 0.04). P481i Predictors of PF included IP (β = –1.5, 95% CI = –3.1 to –1.1, P <0.01), SE (β = 1.8, 95% CI = 1.3 to 2.6, P <0.01), hospital length of stay (β = –1.7, 95% CI = –2.0 to –0.8, P <0.01), never having been married (β = 1.8, 95% CI = 0.3 to 5.5, P = 0.03), and having injury insurance (β = –2.7, 95% CI = –6.9 to –1.9, P <0.01). Predictors of MH included PTSD symptoms (β = –2.4, 95% CI –3.4 to –1.4, P <0.01), psychological distress (β = –6.9, 95% CI = –8.9 to –5.2, P <0.01), SE (β = 0.6, 95% CI = 0.2 to 1.1, P <0.01), and unemployment (β = –2.3, 95% CI = –5.0 to –0.2, P = 0.04). p y β Conclusion Trauma ICU patients experience compromised physical function and mental health 24  months after injury. Psychological distress, self-effi cacy and illness perception infl uence outcomes and are potentially amenable to change in response to interventions initiated during hospital stay. p Methods All patients (>18 years) admitted to the ICU for more than 24 hours are included. In order to assess preadmission quality of life, the patient or close relatives complete the Short-Form 36 (SF-36) within 72 hours after ICU admission. Mortality is evaluated from ICU admission until 30  days hereafter. Logistic regression and receiver operating characteristic analyses are employed to assess predictive value for mortality using fi ve models: SF-36 Physical Component Summary (PCS) and APACHE II (model A), SF-36 PCS (model B), SF-36 General Health (GH) and APACHE II (model C), SF-36 GH (model D), and APACHE II (model E). Classifi cation tables are composed in order to assess sensitivity, specifi city, positive and negative predictive values and likelihood ratios. Do ICU patients’ discharge characteristics change with Swiss Diagnosis Related Groups? Conclusion Preliminary results indicate that the SF-36 GH and the SF-36 PCS are as good as APACHE II to predict mortality 30 days after ICU admission. However, in order to conclude whether preadmission quality of life can contribute to triage, by successfully identifying patients suitable for intensive care, fi nal analyses, due in 2013, are awaited. These results will clarify whether future randomized controlled trials, in which preadmission quality of life acts as a supplement to triage, are justifi able. P484 Do Diagnosis Related Groups change admission practice to a large Swiss ICU? K Homburg, CA Rüst, P Fodor, S Blumenthal Triemli City Hospital, Zurich, Switzerland Critical Care 2013, 17(Suppl 2):P484 (doi: 10.1186/cc12422) P484 Do Diagnosis Related Groups change admission practice to a large Swiss ICU? K Homburg, CA Rüst, P Fodor, S Blumenthal Triemli City Hospital, Zurich, Switzerland Critical Care 2013, 17(Suppl 2):P484 (doi: 10.1186/cc12422) Predicting physical function and mental health in trauma intensive care patients 2 years after hospitalisation A subject-specifi c intercept in a mixed model was used to account for repeated data from participants over time. P484 Do Diagnosis Related Groups change admission practice to a large Swiss ICU? Introduction The new Swiss fi nancing system by means of Diagnosis Related Groups (DRG), eff ective 1 January 2012, forms the basis of a performance-based system to reimburse general hospital services. There are only few data on the infl uence of DRG on ICU patients [1,2]. We expect that the introduction of the DRG in Switzerland will change the number of admissions from external hospitals to a large ICU with a centre function and will infl uence the severity of disease of the admitted patients. Introduction The new Swiss fi nancing system by means of Diagnosis Related Groups (DRG), eff ective 1 January 2012, forms the basis of a performance-based system to reimburse general hospital services. There are only few data on the infl uence of DRG on ICU patients [1,2]. We expect that the introduction of the DRG in Switzerland will change the number of admissions from external hospitals to a large ICU with a centre function and will infl uence the severity of disease of the admitted patients. p p p Results Participants (n  =  123) were young (median 37, IQR 22 to 55  years), predominantly male (83%) and spent on average 3  days in the ICU and 3 weeks in hospital. Response rates were over 55% at each follow-up, with responders similar to nonresponders except for being generally older. PF and MH scores improved over time, although the averages remained below the Australian norms at 24  months. S180 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Methods The ICU of the Triemli City Hospital in Zurich has an inter- disciplinary organisation with surgical and internal medical patients, with a maximum occupancy of 18 beds and a centre function for the surrounding hospitals of the region. In this prospective ongoing observational study, we collect and analyse the anonymised data of all patients admitted to our ICU from an external hospital during 12 months prior to (1 January to 31 December 2011) and after (1 January to 31 December 2012) the introduction of the DRG in Switzerland. Exclusion criteria are admissions by the emergency department, self-assignments into the hospital and internal relocations. The primary endpoint is the number of admissions from an external hospital to our ICU. References References 1. McLaughlin AM, et al.: Intensive Care Med 2009, 35:2135-2140. 2. Rossi C, et al.: Intensive Care Med 2006, 32:545-552. References 1. McLaughlin AM, et al.: Intensive Care Med 2009, 35:2135-2140. 2. Rossi C, et al.: Intensive Care Med 2006, 32:545-552. P486 Variable cost of a Greek ICU E Tsigou1, D Karabatsou1, E Boutzouka1, V Psallida1, G Baltopoulos1, M Tsironi2 1A. Anargiroi Hospital, Athens, Greece; 2University of Peloponnisos, Sparti, Greece Critical Care 2013, 17(Suppl 2):P485 (doi: 10.1186/cc12423) Critical Care 2013, 17(Suppl 2):P485 (doi: 10.1186/cc12423) Introduction The ICU consumes almost 20% of total hospital resources, despite accounting for less than 10% of hospital beds. Medical cost can be divided into a fi xed part, consisting of personnel and accommodation expenses, and a variable part, determined by the needs of each patient (medications, consumables, examinations). In order to rationalize medical cost, estimation of expenses must be real and individualized. The aim of this study was to assess the variable cost of critically ill patients in a new, seven-bed, adult, general ICU, using bottom-up costing methodology. Conclusion The latest audit follows introduction of a referral system directly to the ICU consultant and may account for the reduction in numbers of referrals attended by junior doctors. ED/medicine persist as the main source of referral to the ICU. Discussion with the referring team consultant may reduce inappropriate referrals. ICU staffi ng should not be reduced. Methods All 138 patients who were treated during 2011, and stayed for at least 24 hours, were included in the study. Data were retrospectively collected from patient records and included demographics, cause of admission, APACHE II score at admission, length of stay (LOS) and outcome. Cost was recorded for every day and for every patient, based on a hospital-specifi c cost catalogue and on national agreements. Data are presented as mean ± SEM. Analysis of data was carried out using Graph Pad Prism 5.0., applying Student’s t test. References 1. Ahmad M, et al.: Chest 1988, 93:176-179. 2. Thomas F, et al.: Chest 1987, 91:418-423. 1. Ahmad M, et al.: Chest 1988, 93:176-179. Results There were 105 occasions when a junior doctor left the unit in 2011, totalling 118 hours and 38 minutes (5 days), a continuation of the upward trend from the previous 2 years. In 2012 this reduced to 47 occasions totalling 40 hours and 55 minutes. Most attended referrals were during the daytime, 54% in 2011 and 68% in 2012. The majority of referrals originated from the ED/medicine, combined numbers 58% in 2011 and 38% in 2012. In 2011 only 30% of ED referrals had been discussed with a consultant of the referring team and only 32% of medical referrals. Just two of the 14 inappropriate ED referrals were discussed with the referring consultant, and none of the six inappropriate medical referrals were discussed with a medical consultant. In 2012 just 33% of ED/medical referrals had prior discussion with a consultant from the referring team. At the point of leaving, the ICU was left without a doctor on 22 occasions in 2011 and six occasions in the 2012 audit. Most occasions occurred at either night shift/weekend (86% in 2011/100% in 2012). Referral resulted in a patient being admitted to the ICU on 20 occasions in 2011 and 12 in 2012. 2. Thomas F, et al.: Chest 1987, 91:418-423. Audit of the medical staffi ng of and referrals to a mixed adult ICU in a teaching hospital g p A Needham, T Lyndham, D O’Neil, J Greiff University Hospitals of Leicester, UK Critical Care 2013, 17(Suppl 2):P486 (doi: 10.1186/ Introduction Leicester Royal Infi rmary is a (funded) 15-bed ICU, annual admission 1,102 patients per year (ICNARC data 2011 to 2012). It houses a broad range of specialty, has over 1,000 beds and an emergency department (ED) responsible for 297 admissions over the 2011 to 2012 period. Unit guidelines state that a doctor should be immediately available to the unit at all times, ideally physically present on the unit. This audit looked in detail at each occasion a junior doctor left the unit, the timings, reason, and outcome of each episode. Results We present the preliminary data for 10 months (in each case January to October) before and after the introduction of the DRG. We observed an increase of 9.2% (391 vs. 427 patients) of admissions to our ICU after the introduction of the DRG. The severity of disease determined by the SAPS II score is unchanged (mean 26.7 vs. 26.0 points, P = 0.466). The severity of disease determined by the APACHE II score is signifi cantly lower (15.4 vs. 14 points, P  =  0.017). We also noted that after the introduction of the DRG the patients were earlier transferred from an external hospital to our ICU (mean time until transfer 29.9 vs. 18.7 hours), but this value was not signifi cant (P = 0.55). Conclusion Up to now the introduction of the DRG in Switzerland has had a complex infl uence on the number and the kind of patients admitted from an external hospital to an ICU with centre function. Data assessment and analysis will continue. g p Methods Between 17 October 2011 and 14 November 2011, and between 2 July 2012 and 30 July 2012, junior doctors on the ICU completed a data-capture form after each occasion that they left the unit for a clinical need. Data collected were matched to the objectives. The 2011 audit results were communicated at the University Hospitals of Leicester Trust ICU Audit Meeting. A system was introduced so that daytime referrals were directed to the ICU consultant. The impact of this intervention was assessed by the 2012 re-audit. Do Diagnosis Related Groups change admission practice to a large Swiss ICU? Secondary endpoints are the severity of the disease of the admitted patients, detected by the scoring systems SAPS II and APACHE II as well as the length of stay in external hospitals before admission. The statistical analysis is descriptive. variable cost of intensive care treatment. Among drugs, antibiotics accounted for the largest part. A medical cause of admission was correlated with higher costs. variable cost of intensive care treatment. Among drugs, antibiotics accounted for the largest part. A medical cause of admission was correlated with higher costs. Impact of the time elapsed between ICU request and actual admission on mortality and length of stay Introduction The World Bank has warned that the rapid growth of the world’s urban population can only be accommodated safely if cities adequately develop key infrastructure, such as the provision of acute care resources. Yet, even basic descriptive information on urban acute care supply and demand is extremely limited. We therefore conducted a pilot assessment across seven diverse urban settings across the world. Methods We selected a convenience sample of seven large cities with varying geographical and socioeconomic characteristics: Boston, Paris, Bogota, Recife, Liaocheng, Chennai, and Kumasi. To estimate acute care supply, we developed an instrument to collect data on acute and critical care infrastructure. We collected data from municipal authorities and local research collaborators. We expressed the burden of acute disease as the number of deaths due to acute illnesses, estimated from the 2008 Global Burden of Disease Study. Results were expressed as acute care supply and acute deaths per 100,000 population and acute care supply per 100 acute deaths. Introduction The World Bank has warned that the rapid growth of the world’s urban population can only be accommodated safely if cities adequately develop key infrastructure, such as the provision of acute care resources. Yet, even basic descriptive information on urban acute care supply and demand is extremely limited. We therefore conducted a pilot assessment across seven diverse urban settings across the world. Methods We selected a convenience sample of seven large cities with varying geographical and socioeconomic characteristics: Boston, Paris, Bogota, Recife, Liaocheng, Chennai, and Kumasi. To estimate acute care supply, we developed an instrument to collect data on acute and critical care infrastructure. We collected data from municipal authorities and local research collaborators. We expressed the burden of acute disease as the number of deaths due to acute illnesses, estimated from the 2008 Global Burden of Disease Study. Results were expressed as acute care supply and acute deaths per 100,000 population and acute care supply per 100 acute deaths. Results In total, 413 patients were included, 300 of which belonged to the LWP group (65.4%). For the entire cohort, the mean APACHE II score was 19 ± 7, the mean age was 52 ± 22 years, and 211 patients were male (51.1%). The LWP group did not show diff erence in the APACHE II score (19 ± 7 vs. 18 ± 8, P = 0.13), but was older (55 ± 20 vs. Impact of the time elapsed between ICU request and actual admission on mortality and length of stay 49 ± 23, P = 0.01). LWP also had a higher incidence of primary bloodstream infection (23.8% vs. 10.4%, P = 0.01) and catheter-associated urinary tract infection (10.2% vs. 1.9%, P = 0.01). LWP patients had higher mortality (37.8% vs. 25.9%, P = 0.02) and longer ICU LOS (21 ± 47 vs. 14 ± 18 days, P = 0.01). Relative risk for death in the LWP was 1.74 (95% CI: 1.11 to 2.72). Conclusion Despite showing no signifi cant diff erences on APACHE II scores from the SWP group, patients from the LWP group presented greater incidence of primary bloodstream infection, catheter- associated urinary tract infection, higher mortality outcomes and longer ICU LOS. pp y p Results The supply of hospital beds varied from 72.4/100,000 population in Kumasi to 245.8/100,000 in Boston. ICU beds with capacity for invasive mechanical ventilation and intensive nursing services ranged from 0.4/100,000 in Kumasi to 19/100,000 population in Boston. The number of ambulances varied 70-fold between cities. The gap between cities widened when demand was estimated based on disease burden, with a 70-fold diff erence between cities in ICU beds/acute deaths. In general, most of the data were unavailable from municipal authorities. P490f P490 Diff erences in severity and resource utilization for medical and surgical ICU patients BH Nathanson1, WT McGee2, E Lederman2, TL Higgins2 1OptiStatim LLC, Longmeadow, MA, USA; 2Baystate Medical Center, Springfi eld, MA, USA Critical Care 2013, 17(Suppl 2):P490 (doi: 10.1186/cc12428) Impact of the time elapsed between ICU request and actual admission on mortality and length of stay GM Filho1, TA Silva1, AR Santana1, FB Soares1, LJ Almeida1, LG Godoy1, TA Rodrigues1, MO Maia2, JA Neto2, AP Amorim3, EB Moura2, FF Amorim1 1Escola Superior de Ciências da Saúde, Brasília, Brazil; 2Unidade de Terapia Intensiva Adulto do Hospital Santa Luzia, Brasilia, Brazil; 3Liga Academica de Medicina Intensiva do Distrito Federal (LIGAMI), Brasilia, Brazil Critical Care 2013, 17(Suppl 2):P487 (doi: 10.1186/cc12425) Results The age of participants (84 men and 54 women, 71 medical and 67 surgical) was 68.75 ± 1.18, APACHE II score was 18.64 ± 0.61, LOS was 18.46 ± 2.54, and mortality was 19%. The majority of the patients (61%) were mechanically ventilated. The total days of ICU stay were 2,548 and the total variable cost was €1,460,465 (€573.18 per patient and per day). Cost per patient was subdivided as follows: medication (including drugs, fl uids, blood products, nutrition): 56.49%, examinations (including laboratory, microbiological assays and diagnostic procedures): 22.23%, consumables: 21.26%. As for medication cost, the largest part was comprised of antibiotics (45.69%), followed by blood products (17.61%) and cardiovascular drugs (12.39%). Costs for medical patients were signifi cantly higher than those for surgical patients (P <0.0001). Conclusion The total average cost per patient and per day was found to be €573.18. Medication expenditure was responsible for the highest Introduction Measures to ensure an appropriate early treatment for critically ill patients result in signifi cant decreases in mortality [1,2]. This study aims to evaluate the impact of the time elapsed from request until admission to the ICU on mortality and ICU length of stay (LOS). Methods A retrospective cohort study performed on patients in the ICU of Hospital Regional de Samambaia over a period of 4 years, from January 2008 to December 2011. The patients were allocated into two groups: patients who waited longer than 6 hours, long waiting period S181 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 (LWP, n = 300); and patients whose waiting time was equal to or less than that period, short waiting period (SWP, n = 113). (LWP, n = 300); and patients whose waiting time was equal to or less than that period, short waiting period (SWP, n = 113). Diff erences in severity and resource utilization for medical and surgical ICU patients BH Nathanson1, WT McGee2, E Lederman2, TL Higgins2 1OptiStatim LLC, Longmeadow, MA, USA; 2Baystate Medical Center, Springfi eld, MA, USA Methods Several disasters are studied. The Fukushima Daiichi Nuclear plant explosion after the Higashinihon earthquake 2011 was medically reviewed based on the total 30-day stay on-site in addition to several days around the site. The Chernobyl incident 1986 was inspected 15 years after the incident. Other nuclear disasters are included. p gi Critical Care 2013, 17(Suppl 2):P490 (doi: 10.1186/cc12428) Introduction Medical and surgical patients use the ICU diff erently. Resources may not always be allocated by severity of illness, but by custom or habit, particularly if diff erent groups administer bed control and triage. Specialty-specifi c diff erences may exist even when a single team controls triage. Variability in resource utilization has important implications for cost-containment and triage. Results Many serious problems were revealed in the medical teams, which are as follows: inappropriate basic preparedness against large special disasters, including nuclear disaster; lack of appropriate education and training for medical teams against nuclear disaster  – that is, most members of Japan DMAT or the disaster medical assistance team are still laypersons; incorrect standard/rules of Japan DMAT, which were excessively focused upon cure of the usual type of injury and planned short period or nearly 48 hours, which should be abandoned; and insuffi cient consideration to the weak/vulnerable people or CWAP, children, (pregnant) women, aged people, and the poor people/sicker patients. Many of them died because of an insuffi cient emergency transportation system from their contaminated houses or hospital. Methods Patients admitted to a single, closed medical/surgical ICU with full-time intensivists and unifi ed triage control in a large, university-affi liated hospital were evaluated during 2011 to 2012. Patients who died in the ICU were excluded. The day of discharge (D/C) and severity using APACHE IV and its related Acute Physiology Score Figure 1 (abstract P490). y Conclusion In order to cope with the special disasters, such as NBC or nuclear, biological and chemical disaster, it is insuffi cient to take makeshift measures or use cheap tricks. Working out the systematization of disaster medicine, based upon the academic viewpoints and philosophy/reliability, is essential to protect the people and the nation. g References 1. Amorim FF, et al.: J Clin Med Res 2012, 4:410-414. 1. Amorim FF, et al.: J Clin Med Res 2012, 4:410-414. 2. Rivers E, et al.: N Engl J Med 2001, 345:1368-1377. 1. Amorim FF, et al.: J Clin Med Res 2012, 4:410-414. 2. Rivers E, et al.: N Engl J Med 2001, 345:1368-1377. 2. Rivers E, et al.: N Engl J Med 2001, 345:1368-1377. Medical problems: review from the major nuclear incidents Medical problems: review from the major nuclear incidents Y Haraguchi1, Y Tomoyasu2, H Nishi3, M Hoshino4, M Sakai5, E Hoshino6 1Disaster Medicine Compendium Team, Japan, Tokyo, Japan; 2Shirahigebashi Hospital, Tokyo, Japan; 3Tokyo Disaster Medical Center, Tokyo, Japan; 4Shisei Hospital, Saitama, Japan; 5Tokyo Women’s Medical College, Tokyo, Japan; 6Chiba Nursing Association, Chiba, Japan Critical Care 2013, 17(Suppl 2):P488 (doi: 10.1186/cc12426) Medical problems: review from the major nuclear incidents Y Haraguchi1, Y Tomoyasu2, H Nishi3, M Hoshino4, M Sakai5, E Hoshino6 1Disaster Medicine Compendium Team, Japan, Tokyo, Japan; 2Shirahigebashi Hospital, Tokyo, Japan; 3Tokyo Disaster Medical Center, Tokyo, Japan; 4Shisei Hospital, Saitama, Japan; 5Tokyo Women’s Medical College, Tokyo, Japan; 6Chiba Nursing Association, Chiba, Japan Critical Care 2013, 17(Suppl 2):P488 (doi: 10.1186/cc12426) Conclusion The provision of acute care services, a key aspect of urban infrastructure, varied substantially across the seven diverse urban settings we studied. Furthermore, the local municipal authorities generally appeared to have little knowledge of their acute care infrastructure, with implications for future planning and development. Introduction Intensivists are expected to have many roles during and after a major disaster/catastrophe; that is, triage, intensive care, education for people, and so forth. The roles of intensivists against special disaster or nuclear disaster are studied based on actual experiences. P489 Variation in acute care burden and supply across diverse urban settings S Murthy1, S Austin2, H Wunsch3, NK Adhikari4, V Karir2, K Rowan5, ST Jacob6, J Salluh7, F Bozza8, B Du9, Y An10, B Lee2, F Wu2, C Oppong11, R Venkataraman12, V Velayutham13, D Angus2 1Hospital for Sick Children, Toronto, Canada; 2University of Pittsburgh, PA, USA; 3Columbia University, New York, NY, USA; 4University of Toronto, Canada; 5Intensive Care National Audit & Research Centre, London, UK; 6University of Washington, Seattle, WA, USA; 7D’OR Institute for Research and Education, Rio de Janeiro, Brazil; 8Oswaldo Cruz Foundation, Rio de Janeiro, Brazil; 9Peking Union Medical College Hospital, Beijing, China; 10Peking University People’s Hospital, Beijing, China; 11Komfo Anokye Teaching Hospital, Kumasi, Ghana; 12Apollo Hospital, Chennai, India; 13Stanley Medical College Hospital, Chennai, India Critical Care 2013, 17(Suppl 2):P489 (doi: 10.1186/cc12427) Figure 1 (abstract P490). S182 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Introduction The management of emergency medical admissions has been a subject of recent clinical incidents. There was a high percentage of patients that were referred to the ICU by staff in training, and 21% of referrals were made by junior doctors. Consultant physicians had no knowledge of the case in 57% of referrals. Introduction The management of emergency medical admissions has been a subject of recent clinical incidents. There was a high percentage of patients that were referred to the ICU by staff in training, and 21% of referrals were made by junior doctors. Consultant physicians had no knowledge of the case in 57% of referrals. (APS) component were calculated daily for the fi rst 7 days. Trend was assessed across days by Cuzick’s test. y y Results A total of 719 surgical and 925 medical patients met inclusion criteria. In total, 20.2% of surgical and 21.3% of medical patients had an ICU LOS <1; P = 0.58. Admission severity was correlated with length of stay, P = 0.014 for both medical and surgical patients. Medical patients are sicker on admission and D/C from the ICU than surgical patients (P <0.05) (Figure 1). g Methods A prospective study of 21 cases of referrals and admissions to the ICU was conducted at the Glasgow Victoria Infi rmary Hospital from 8 to 21 September 2012. P489 A questionnaire was produced relating to the referrals, admissions, seniority involvement, cause of referral, and time of patient review by the ICU consultant after ICU admission. They were distributed to specialist registrars and the ICU consultants. All data were electronically recorded into an Excel database. Questionnaires that were not completely fi lled were further investigated using patient clinical notes and contact with medical staff . Information that may identify a patient or clinician was removed from the questionnaire for confi dentiality purposes. g Conclusion ICU utilization diff ered by patient type even with unifi ed triage control within a single unit. Surgical patients were less severely ill on admission to and D/C from the ICU. A signifi cant percentage of medical and surgical patients are D/C within 1 day and may be more effi ciently served in a less resource-intensive environment. The reasons for the diff erences in ICU utilization for surgical versus medical patients require clarifi cation and may have implications for both resource utilization and cost. Results Twenty-one complete questionnaires were collected. Fifty- seven percent (12/21) of cases involved admission to the ICU. Nine percent of the cases involved contacting either a specialist registrar or ICU consultant intensivist for assistance in practical procedures. Of the patients admitted to the ICU, 33% (4/12) were from medical wards, 33% were admitted from A&E. Consultants were the most common professionals who referred patients to critical care (48%; 10/21). Fourteen percent of cases (3/21) involved the referral of patients into ICU by a junior doctor, but only one of the referrals was accepted by the ICU intensivist. Consultants referred or were aware of the referral in 71% (15/21) of cases. Of admissions, 58% (7/12) were accepted by the ICU consultant and the remaining by the specialist registrars. All accepted were acknowledged by the ICU consultant. After admission all of the patients were reviewed by the ICU consultant and the time of review after admission was on average 1 hour 23 minutes (25 minutes to 3 hours 45 minutes). Educational eff ectiveness of introductory training for the Rapid Response System in Japan p y p T Kodama1, M Nakagawa2, E Kawamoto3, S Fujiwara4, H Imai3, S Fujitani5, K Atagi6 y T Kodama1, M Nakagawa2, E Kawamoto3, S Fujiwara4, H Imai3, S Fujitani5, K Atagi6 g 1The University of Texas Southwestern Medical Center at Dallas, TX, USA; 2Social Insurance Kinan Hospital, Tanabe-City, Japan; 3Mie University Hospital, Tsu-City, Japan; 4NHO Ureshino Medical Center, Ureshino-City, Japan; 5Tokyobay UrayasuIchikawa Medical Center, Urayasu-City, Japan; 6Osaka City General Hospital, Osaka-City, Japan Critical Care 2013, 17(Suppl 2):P491 (doi: 10.1186/cc12429) 1The University of Texas Southwestern Medical Center at Dallas, TX, USA; 2Social Insurance Kinan Hospital, Tanabe-City, Japan; 3Mie University Hospital, Tsu-City, Japan; 4NHO Ureshino Medical Center, Ureshino-City, Japan; 5Tokyobay UrayasuIchikawa Medical Center, Urayasu-City, Japan; 6Osaka City General Hospital, Osaka-City, Japan Critical Care 2013, 17(Suppl 2):P491 (doi: 10.1186/cc12429) Introduction Interest in safety and clinical outcomes of inpatients has been growing in Japan, because the 100,000 Lives Campaign was introduced under the Japanese Patient Safety Act in 2008. In this act, an introduction of the Rapid Response System (RRS) was one of the mainstreams to inpatients’ care. However, many Japanese healthcare providers cannot understand how to achieve the introduction of the RRS, because there are few who have knowledge of the system. Therefore, we developed a new introductory training course for the RRS. The educational eff ectiveness was analyzed through the surveillance questionnaires after the course. Conclusion There is still an issue with junior doctors referring patients to the ICU without the acknowledgement of consultant physicians, resulting in unnecessary admissions and decreased time that ICU trainees spend in the ICU. There are more appropriate ICU admissions when there is involvement with seniority. Contact with ICU staff to perform practical procedures outside the ICU and not about admissions should be explored further. Methods The educational program includes a lecture series con cern- ing the outline and management methods, introduction of facilities that have already deployed, small group discussions, and teaching methods-of-training for the medical emergency team using a simulator. Evaluation was made in the fi ve-point scale by 82 participants (58 physicians, 16 nurses and eight other professions) throughout seven courses. The questionnaires are: A. understanding of RRS, B. knowledge acquisition about patient safety, C. expectation for decreasing the cardiopulmonary arrest by RRS, and D. expectation for decreasing the psychological burden by RRS. P491 P491 Educational eff ectiveness of introductory training for the Rapid Response System in Japan T Kodama1, M Nakagawa2, E Kawamoto3, S Fujiwara4, H Imai3, S Fujitani5, K Atagi6 1The University of Texas Southwestern Medical Center at Dallas, TX, USA; 2Social Insurance Kinan Hospital, Tanabe-City, Japan; 3Mie University Hospital, Tsu-City, Japan; 4NHO Ureshino Medical Center, Ureshino-City, Japan; 5Tokyobay UrayasuIchikawa Medical Center, Urayasu-City, Japan; 6Osaka City General Hospital, Osaka-City, Japan Critical Care 2013, 17(Suppl 2):P491 (doi: 10.1186/cc12429) Educational eff ectiveness of introductory training for the Rapid Response System in Japan Reference 1. Gillbe C, Gunning K: Standards for Consultant Staffi ng of Intensive Care Units. ICS & IBTICM Standards. Intensive Care Society; 2006. P493 Knowledge of confi dentiality, consent and information disclosure is not aff ected by position or experience in adult critical care M Lowings1, A Molokhia2, C Anderson3, S Cowman4, T Dixson5, K El-Boghdadly6, L Williams2 1St Thomas’ Hospital, London, UK; 2Lewisham University NHS Trust, London, UK; 3King’s College Hospital, London, UK; 4Great Ormond Street Hospital, London, UK; 5Queen Elizabeth Hospital, Woolwich, UK; 6Princess Royal University Hospital, Bromley, UK Critical Care 2013, 17(Suppl 2):P493 (doi: 10.1186/cc12431) y g y Results Seventy-three participants (89.0%) answered the question- naires. The numbers of participants who scored more than four points were as follows: A. was 71 (97.2%), B. was 70 (95.9%), C. was 64 (87.7%), and D. was 68 (93.2%), respectively. The majority of participants obtained the correct knowledge, and had a solid understanding for the RRS. It was evident that providing abundant material and didactic lectures traced from the introduction to management, and collecting and resolving the questions, promoted comprehension. However, there is a limitation of whether or not the participants introduce the RRS into their own institutions. It is essential to improve the course and continue to support the activities of the participants. Introduction Teaching of medical ethical issues including confi den- tia lity and consent have long been a small part of the medical curriculum. These issues are more complex in an ICU where patients may lack capacity. Documents such as Good Medical Practice 1995, Confi dentiality 2009 and the Mental Capacity Act 2005 give guidance to medical professionals in these matters in the UK. Conclusion Our training course may promote the introduction and dissemination of the RRS in Japan. Methods A questionnaire was distributed amongst staff in four ICUs in South London. Results were analysed according to level of experience and background (medical/nursing or allied health professional (AHP)). Results Of 225 questionnaires distributed, the response rate was 66% (31% doctors, 56% nurses and 13% AHP). Staff with either less than 1 year experience or greater than 10 years experience had the greatest exposure to the Mental Capacity Act and Data Protection Act, suggesting a gap in knowledge in staff with intermediate experience. Knowledge of the Caldicott principles were unaff ected by experience, with many experienced respondents having ‘No Idea’. The majority of respondents (unaff ected by experience) felt that References 1. Leape LL, et al.: JAMA 2005, 293:2384-2390. 2. Jones D, et al.: N Engl J Med 2011, 365:139-146. References 1. Schiza et al.: J Clin Sleep Med 2012, 8:21-26. 2. Kaw et al.: Br J Anaesth 2012, 109:897-906. 1. Schiza et al.: J Clin Sleep Med 2012, 8:21-26. Survey of alcohol-related admissions to critical care and the resource and fi nancial implications Survey of alcohol-related admissions to critical care and the resource and fi nancial implications M Slattery, P Temblett, M Heatley, N Singatullina, B Jones ABMU University Trust, Swansea, UK Critical Care 2013, 17(Suppl 2):P494 (doi: 10.1186/cc12432) Introduction Alcohol-related hospital and ICU admissions are known to have a huge impact on healthcare resources in the UK. Excessive use of alcohol is independently associated with sepsis, septic shock and hospital mortality among ICU patients. This study assesses the relationship between alcohol abuse and intensive care resource utilisation in a mixed medical, surgical and neurosurgical ICU. gi p p y Conclusion Undiagnosed SDB is highly prevalent among cardiac surgical patients. Clinical trajectories of individuals with severe SDB are described by a prolonged recovery of pulmonary function, delayed weaning and a pronounced infl ammatory response after surgery. Screening for SDB might identify patients that are susceptible for a complicated postoperative course. Methods A prospective survey of emergency alcohol-related admissions over a 1-year period was undertaken at a tertiary university adult general and neurosurgical ICU. All patients were screened for acute and chronic alcohol abuse on admission. Acute alcohol abuse was defi ned as being intoxicated with alcohol at the time of admission and chronic alcohol abuse was defi ned as chronic alcohol use exceeding recommended UK national guidelines on consumption. The amount of alcohol consumption was obtained, diagnosis on admission, ICU and hospital mortality, length of stay, and total cost were recorded. All patients were screened for alcohol-related comorbidities. Comparative retrospective data were obtained for the same time period for nonalcohol-related emergency ICU admissions. Data were analyzed using SPSS. P492 Standards of referrals and admissions of critically ill patients to the ICU A Ng Glasgow Victoria Infi rmary Hospital, Glasgow, UK Critical Care 2013, 17(Suppl 2):P492 (doi: 10.1186/cc12430) A Ng Glasgow Victoria Infi rmary Hospital, Glasgow, UK Critical Care 2013, 17(Suppl 2):P492 (doi: 10.1186/cc12430) S183 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 is signifi cantly less than previously reported. Our study reveals interspecialty variations in demographic data, APACHE II scores, mortality and cost of admission. Neurosurgical alcohol-related admissions bear higher mortality and result in greater resource utilisation relative to nonalcohol-related neurosurgical admissions. Alcohol continues to burden both our patients and critical care. when giving information to relatives face to face, relatives should be kept fully informed. When giving information over the telephone, most doctors felt the response should be tailored to the knowledge of the person being spoken to whilst nurses were split between tailoring the response, giving full information, setting up a password system and not giving any information at all. Most respondents felt date of birth and hospital number constituted ‘Patient Identifi able Information’. However, experienced staff did not appreciate the importance of unusual diagnosis and clinical photographs as also being able to identify patients. Similarly, the majority knew that the patient themselves identifi ed the ‘Next of Kin’ but 7% (unaff ected by experience) felt this was decided by the family and felt the family could decide on resuscitation status. When consent is required for an elective procedure in a patient who lacks capacity, doctors tended to have a better understanding of the need to delay the procedure where possible than nurses, the majority of which felt this could be decided by the next of kin or two consultant doctors. Most doctors felt that ‘Acting in the Patient’s Best Interests’ would mean doing what would give the patient the best outcome rather than doing what the patient would have wanted (unaff ected by experience). The majority of staff , on answering this questionnaire, felt that they lacked suffi cient knowledge on the subject and most felt annual reminders would be useful. P495 P495 95 Association of sleep disordered breathing with clinical trajectories in patients undergoing cardiac surgery J Roggenbach, B Tan, E Von der Leyen, A Weymann, M Karck, E Martin, S Hofer University of Heidelberg, Germany Critical Care 2013, 17(Suppl 2):P495 (doi: 10.1186/cc12433) Introduction The prevalence of sleep disordered breathing (SDB) is presumably high among individuals with cardiac diseases [1], nonetheless SDB remains predominantly undiagnosed. However, unrecognized SDB might have relevant impact on the postoperative course of patients undergoing cardiac surgery [2]. g g g y Methods Polygraphic recordings of 181 patients, without previous diagnosis of SDB, undergoing standard cardiac surgical procedures with extracorporeal circulation were obtained during a preoperative night. The apnea–hypopnea index (AHI  – the number of apneas, hypopneas per  hour recorded) was determined and compared with clinical characteristics and postoperative course. Conclusion The ICU is an environment where issues of consent, confi dentiality and disclosure of information occur daily. Staff feel they lack knowledge in these areas that is unaff ected by their experience. We need to ensure that all staff have the necessary knowledge to deal with these situations. p p Results The prevalence of SDB was considerably high among all examined patients. Median AHI was 20.8 (interquartile range, 10.6 to 36.4). Preoperative AHI was >30 in 32% of all examined individuals. During the fi rst three postoperative days, preoperative AHI >30 was associated with a prolonged weaning time, a reduced oxygenation index (arterial pO2/FiO2), an impaired kidney function, an augmented infl ammatory response and an overall increased length of stay in the ICU. The observed association of high preoperative AHI values with postoperative clinical characteristics remained statistically signifi cant throughout the fi rst three postoperative days. Can massage therapy help cardiac patients? A literature review plus a case study A Fenlon St Georges NHS Trust, London, UK Critical Care 2013, 17(Suppl 2):P496 (doi: 10.1186/cc12434) Introduction A literature review was performed to assess whether massage benefi ts patients postoperatively following coronary bypass grafts (CABG) and or valve replacement/repair. A case study on a patient who had suff ered a hypoxic brain post cardiac arrest was conducted. Methods A review on MEDLINE and Cochrane using search terms massage, cardiac and ICU identifi ed nine research papers on the benefi ts of massage postoperatively for the aforementioned patient group. Other papers were listed but unrelated to cardiac surgery. None of the nine papers identifi ed for this review were ICU specifi c in the title but the ICU was mentioned in the main text body. For the purpose of this review the selected papers are researching the eff ects of massage on physiological parameters, anxiety, pain, calm and perceived stress indicators in the CABG and/or valve repair/replacement. Out of these nine papers, one is British (2002). Five are American (2006 to 2012), two are Brazilian (2010) and one is an Indian paper (2010). All papers are randomised control trials (RCTs). Papers written prior to 1999 were excluded from this literature review. Results In total, 7.7% of patients were admitted with a history of acute/chronic alcohol excess. Sixty-seven per cent of alcohol- related admissions were due to acute alcohol excess. Neurosurgical patients admitted due to alcohol excess had higher ITU mortality than nonalcohol-related neurosurgical patients: 32.1% versus 14.39% (P  =  0.02), respectively. Ninety-three per cent of alcohol-related neurosurgical admissions were caused by acute alcohol intoxication. The intensive care cost was signifi cantly higher for alcohol-related (£12,396 per patient) compared with nonalcohol-related neurosurgical admissions (£7,284 per patient). Of the medical patients admitted, 60% of these admissions were due to acute alcohol excess. The cost of intensive care treatment was lower for alcohol-related medical admissions. Conclusion This is one of the largest studies of alcohol-related admissions to critical care. Our survey confi rms that alcohol-related admissions to the ICU are commonplace; however, our frequency S184 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 educational program can reduce VAP incidence in the ICU [1]. In this research we aimed to observe the reduction of VAP incidence after the implementation of VAP care bundles to ICU medical personnel. Can massage therapy help cardiac patients? A literature review plus a case study A Fenlon Results Research from 1999 states there are methodological errors in prior papers and very few large-scale studies prior to this date, destabilising the validity and reliability of research from the papers written before 1999. The later research suggests that any change in the measured physiological parameters of blood pressure, heart rate and respiratory rate are insignifi cant (P >0.05). Pain, anxiety, rest and a calm score perform better with P >4.71 overall. Another American study showed that the length of stay is reduced if a patient receives healing touch postoperatively. However, in one RCT pain and anxiety increases and in some case SpO2 decreases. A case study was chosen by the author and the results support the benefi ts of massage with the cardiac patient group.i Methods Inclusion criteria: all adult surgical patients (>18 years old) who are on ventilatory support in the surgical ICU at Siriraj Hospital. There are two groups, divided into pre-educational group (group I) and post-educational group (group II) (n = 220/group). We also observed the adherence rate to VAP care bundles according to the educational program. The pretest and post-test to determine the effi cacy of the educational program were done. The VAP care bundles consisted of weaning according to weaning protocol, sedation vacation, head- of-bed elevation, measurement of cuff pressures four times/day, 2% chlorhexidine use for mouth care and emptying of ventilator circuit condensate. Conclusion There appears to be some benefi ts but larger-scale studies are required within this and other ICUs in Britain. The author will be inquiring at her workplace whether further studies can be performed and whether she can initiate the research. She will identify further case studies and trial massage on select patients. The author recommends training for nurses interested in massage therapy so a bigger caseload can be identifi ed. Results There were 45.38 and 25.25 episodes of VAP per 1,000 ventilator- days in group I and group II, respectively (P = 0.020). The incidence of VAP was 21.82% in group I and 9.09% in group II (P = 0.000). There was signifi cant reduction in the length of ventilatory support per person (group I = 2, group II = 1 (median), P = 0.013, 95% CI = 0.319 to 0.936) and mortality rate (group I = 15.5%, group II = 8.2%, P = 0.017). There was no signifi cant diff erence in LOI, LOH and ATB cost. References 1. Apisarnthanarak A, Pinitchai U, Thongphubeth K, Yeukyen C, Warren DK, Zack JE, et al.: Eff ectiveness of an educational program to reduce ventilator- associated pneumonia in a tertiary care center in Thailand: a 4-year study. Clin Infect Dis 2007, 45:704-711. Ischemic stroke in diff erent ICU type M Damasceno, F Rodriguez, R Turon Adherence rate to VAP care bundles Bundle Pre-education Post-education P value Wean 95.04 96.08 0.723 Sedate 92.26 95.28 0.467 HOB 50.10 70.36 0.017 Cuff pressure 87.29 80.78 0.278 M care 89.60 80.86 0.204 Drain 95.42 99.42 0.130 Results A total of 3,854 admissions were registered in the period in both ICUs, with 257 (6.7%) being by ischemic stroke – 49 in GICU and 208 in NICU. Mean age of patients: 73 years in GICU and 70.1 years in NICU. Admissions from emergency unit: 44 (89.8%) GICU, 181 (87%) NICU. Mean SAPS 3 score: 50.7 (29 to 71) GICU, 50.6 (29 to 98) NICU. Patients admitted with infection: 6 (12.2%) GICU, 10 (4.81%) NICU. Mean Charlson comorbidity index points: 1.61 (median 1.0) GICU, 1.1 (median 1.0) NICU. Mean length of ICU stay: 4.8 days (median 3) GICU, 4.3 days (median 3) NICU. Mean length of hospital stay: 12.2  days (median 8) GICU, 14.5  days (median 8) NICU. Predicted hospital mortality (mean  ±  SD): 20.07%  ±  13.48 GICU, 20.4%  ±  13.83 NICU. Hospital mortality rate: 12.5% GICU, 5.39% NICU. Observed-to-expected (O/E) mortality ratios: 0.62 GICU, 0.26 NICU. Conclusion The educational program and the implementation of VAP care bundles can reduce the incidence of VAP, length of ventilatory support and mortality rate in the ICU. Reference y Conclusion Despite the similar proportions numbers for patients in both ICUs, the mortality rate and the O/E mortality ratio for ischemic stroke were higher in patients of the GICU when compared with the NICU. Can massage therapy help cardiac patients? A literature review plus a case study A Fenlon The pretest scores were 15.53 and 17.53 on average from 40 medical personnel in group I and group II, respectively (P = 0.000). The head-of-bed elevation adherence rate was improved after the educational program (group I = 50.1%, group II = 70.36%, P = 0.017). But the adherence to other bundles was not improved. See Tables 1 and 2. Ischemic stroke in diff erent ICU type M Damasceno, F Rodriguez, R Turon Hospital de Clinicas de Niterói, Rio de Janeiro, Brazil Critical Care 2013, 17(Suppl 2):P497 (doi: 10.1186/cc12435) Table 1 (abstract P498). Impact of the educational program on outcomes Outcome Pre-education Post-education P value VAP 45.38 25.25 0.020 Ventday 2 1 0.013 LOI 3 3 0.054 LOH 17 17 0.960 ATB cost 2,842.50 2,098.00 0.994 % death 15.5 8.2 0.017 Table 2 (abstract P498). Adherence rate to VAP care bundles Bundle Pre-education Post-education P value Wean 95.04 96.08 0.723 Sedate 92.26 95.28 0.467 HOB 50.10 70.36 0.017 Cuff pressure 87.29 80.78 0.278 M care 89.60 80.86 0.204 Drain 95.42 99.42 0.130 Conclusion The educational program and the implementation of VAP care bundles can reduce the incidence of VAP, length of ventilatory support and mortality rate in the ICU. Reference 1. Apisarnthanarak A, Pinitchai U, Thongphubeth K, Yeukyen C, Warren DK, Zack JE, et al.: Eff ectiveness of an educational program to reduce ventilator- associated pneumonia in a tertiary care center in Thailand: a 4-year study. Clin Infect Dis 2007, 45:704-711. P499 Development of a severe infl uenza critical care curriculum and training materials for resource-limited settings JV Diaz1, P Lister2, JR Ortiz3, NK Adhikari4, N Adhikari5 1World Health Organization, San Francisco, CA, USA; 2Great Ormond Street Hospital, London, UK; 3University of Washington, Seattle, WA, USA; 4Sunnybrook Health Sciences Centre and University of Toronto, Canada; 5Health Sciences Centre and University of Toronto, Canada Critical Care 2013, 17(Suppl 2):P499 (doi: 10.1186/cc12437) Table 1 (abstract P498). Impact of the educational program on outcomes Introduction In our hospital there are a general intensive care unit (GICU) and a neurointensive care unit (NICU). Despite the preference for the NICU, in both there are admissions for ischemic stroke. There are diff erent staff for each ICU, with the same physician’s leadership. We have decided to evaluate the performance of both ICUs, analysing whether there are diff erences in results as some authors publish best results in specialized ICUs. Methods Using prospectively collected data, we undertook a retrospective evaluation of all patients admitted to the GICU and NICU of our hospital with the diagnosis of ischemic stroke, from December 2010 to November 2012. In both ICUs there are intensivists, but in the NICU the intensivists have special expertise in neuroscience. Data were collected from Epimedmonitor. Table 2 (abstract P498). P497 Ischemic stroke in diff erent ICU type M Damasceno, F Rodriguez, R Turon 1. Varelas PN, et al.: Neurocrit Care 2008, 9:293-299. 2. Berchad EM, et al.: Neurocrit Care 2008, 9:287-292. P499 Development of a severe infl uenza critical care curriculum and training materials for resource-limited settings j p CH Pisitsak, O Chaiwat Siriraj Hospital, Bangkok, Thailand JV Diaz1, P Lister2, JR Ortiz3, NK Adhikari4, N Adhikari5 1World Health Organization, San Francisco, CA, USA; 2Great Ormond Street Hospital, London, UK; 3University of Washington, Seattle, WA, USA; 4Sunnybrook Health Sciences Centre and University of Toronto, Canada; 5Health Sciences Centre and University of Toronto, Canada Critical Care 2013, 17(Suppl 2):P499 (doi: 10.1186/cc12437) Introduction VAP has continued to be a major cause of morbidity and mortality in critically ill patients in Thailand for decades. Previous research found that the implementation of VAP care bundles and the S185 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Introduction The 2009 H1N1 pandemic caused surges of severely ill patients with viral pneumonia requiring ventilation and particularly aff ected high-dependency medical services such as ICUs in settings that lack suffi cient personnel and resources to provide optimal care. Ministries of Health of several countries asked the World Health Organization (WHO) for clinical management guidance. Introduction The 2009 H1N1 pandemic caused surges of severely ill patients with viral pneumonia requiring ventilation and particularly aff ected high-dependency medical services such as ICUs in settings that lack suffi cient personnel and resources to provide optimal care. Ministries of Health of several countries asked the World Health Organization (WHO) for clinical management guidance. Figure 2 (abstract P500). Compliance with pre-ICU resuscitation bundle. g ( ) g g Methods We developed (2009 to 2011) and piloted (2011 to 2012) curriculum and training materials targeted at clinicians without formal critical care training who care for adult and paediatric patients with severe acute respiratory infections in ICUs in resource-limited settings. Results With contributions from 37 global experts, we developed a 3-day course including 14 learning units in early recognition, patho- physiology, oxygen therapy, infl uenza diagnostics, infection control, resuscitation of septic shock, antimicrobial therapy, monitoring, lung- protective ventilation for acute respiratory distress syndrome, sedation, weaning, preventive care, quality improvement, and ethics. Teaching techniques are appropriate for adult learners and include short slideshow-based lectures, interactive small-group role-play sessions and a toolkit with practical resources such as checklists. With funding from the WHO and local Ministries of Health, the training course was piloted among 67 clinician participants in the Caribbean and Indonesia (29 and 38, respectively) and taught by external critical care specialists. Participants’ daily evaluations rated all units as at least very good. P501 Safety climate perception for staff nurses in an ICU RL Rivera-Romero, M Torres-Campos, MJ Delgado-Amaya, E Curiel-Balsera, JG Quesada-García Hospital Regional Carlos Haya, Málaga, Spain Critical Care 2013, 17(Suppl 2):P501 (doi: 10.1186/cc12439) Introduction Following our study of severe sepsis care across three centres [1], we aimed to introduce a rapid feedback mechanism into our rolling audit programme. Whilst previous audits raised awareness of severe sepsis, only whole organisation performance was reported and no feedback was given to individual clinicians. It is recognised that such feedback loops can improve clinical practice [2]. Introduction When we talk about safety culture, we speak of being aware that things can go wrong. We must be able to recognize mistakes and learn from them, sharing that information fairly and impartially to try to prevent its recurrence. Organizations such as the Agency for Healthcare Research and Quality (AHRQ) have developed tools to help organizations measure their safety culture and there is little information about our country. Methods Patients admitted to critical care (58 beds, four units) with a primary admission diagnosis of infection were screened for severe sepsis. Pre-ICU care was then audited against the Surviving Sepsis Guidelines [3]. Time zero is defi ned as when criteria for severe sepsis were fi rst met. An individualised traffi c-light report was then generated and emailed to the patient’s consultant and other stakeholders involved in care (Figure 1). We aimed to report cases within 7 days of critical care admission. A cumulative report is generated monthly to track organisation-wide performance. Methods A descriptive survey study. We sent the Spanish version of the questionnaire on patient safety culture (AHRQ) to the nursing staff of a polyvalent ICU of 42 beds in a tertiary hospital. Results The questionnaire was sent to 179 nurses, receiving correctly answered 88 surveys (response rate of 49.16%). On a scale of 0 to 10, 6.97 points was obtained to estimate the safety climate for staff respondents. The item best scored was teamwork in the unit (65.9%). Detected as a fortress, ‘communication between nurses at shift changes’ (76.1% positive responses). The worst rating was obtained in the section on human resources, followed by management support in the fi eld of patient safety.f Figure 1 (abstract P500). Example report. Figure 1 (abstract P500). Example report. Conclusion The perception of safety culture in an ICU by nursing staff is far from optimal levels. Quality assurance in severe sepsis: an individualised audit/feedback system results in substantial improvements at a UK teaching hospital p M Simmonds, E Blyth, W Robson, M Chikhani Nottingham University Hospitals NHS Trust, Nottingham, UK Critical Care 2013, 17(Suppl 2):P500 (doi: 10.1186/cc12438) P499 Development of a severe infl uenza critical care curriculum and training materials for resource-limited settings Test scores to assess participant critical care knowledge improved signifi cantly from before the training to immediately after (Caribbean, 58 to 80%; Indonesia, 56 to 75%; P <0.001 for both). Results Since November 2011, 153 cases of severe sepsis have been audited and reported back to clinicians. Compliance with antibiotics in <1  hour has risen from 35 to 75% and compliance with the pre- ICU elements of the resuscitation bundle has risen from 20 to 70% (Figure  2). Feedback from clinicians has been encouraging as our reports highlight both positive and negative examples of practice. Conclusion Individualised feedback on sepsis care has led to substantial improvements in guideline compliance. This concept could be translated to other time-dependent patient pathways. References 1. Simmonds MJR, Chikhani M, Smith P, et al.: Multi-departmental system analysis is needed for evaluation of severe sepsis care: a multi-centre study [abstract]. Presented at the BMJ International Forum on Quality and Safety in Healthcare; 2011; Amsterdam. Conclusion It was feasible to develop and deliver an advanced critical care curriculum and related training materials in a short, interactive workshop for noncritical care trained clinicians in resource-limited settings. However, the small-scale programme was labour and time intensive. Widespread dissemination of these materials requires identifi cation of target countries, engagement of Ministries of Health, and secure funding. Longer-term evaluation is necessary to determine whether such programmes improve processes of care and clinical outcomes for critically ill patients. 2. Jamtvedt G, Young JM, Kristoff ersen DT, et al.: Audit and feedback: eff ects on professional practice and health care outcomes. Cochrane Database Syst Rev 2006, 2:CD000259. 3. Dellinger RP, Levy MM, Carlet JM, et al.: Surviving Sepsis Campaign: international guidelines for management of severe sepsis and septic shock: 2008. Crit Care Med 2008, 36:296-327. First-hour protocol clarifi es resource management and nursing staff education in the ICU First-hour protocol clarifi es resource management and nursing staff education in the ICU M Arbelius-Iltanen, R Siren, J Heinilä, P Korhonen, T Sutinen, V Jalkanen, T Ahonen, S Karlsson Tampere University Hospital, Tampere, Finland Critical Care 2013, 17(Suppl 2):P503 (doi: 10.1186/cc12441) Results A 10% increase in the proportion of ICU nurses with a bachelor’s degree or higher was associated with 2% lower odds of death while controlling for patient and hospital characteristics. Patients cared for in better work environments experienced 11% lower odds of risk- adjusted death than those cared for in poorer ICU work environments. Conclusion Patients cared for in hospitals with a greater proportion of bachelor’s prepared ICU nurses and in better ICU work environments experienced signifi cantly lower odds of death. As the demand for critical care services increases, attention to the education level of ICU nurses and ICU work environment may be warranted to optimize currently available resources and potentially yield better outcomes. M Arbelius-Iltanen, R Siren, J Heinilä, P Korhonen, T Sutinen, V Jalkanen, T Ahonen, S Karlsson Tampere University Hospital, Tampere, Finland Critical Care 2013, 17(Suppl 2):P503 (doi: 10.1186/cc12441) Introduction The fi rst-hour protocol determines the patient-specifi c resources for the start of an ICU stay [1]. Staff resources are decided through triage. Task charts direct the start of intensive care. Our primary goal is to improve patient care. g p p Methods A triage method (red, yellow, green) is used to manage ICU resources according to the severity of illness. For example, one doctor and one nurse would admit a stable (green) patient coming from the operating room for postoperative ICU care. A patient in septic shock with multiple organ disorder (red), on the other hand, would be admitted by a team of two doctors and three nurses. Each staff member has a task chart in a checking-list format. Also, an admission chart is used to improve data collection. The use of the protocol started as a pilot study in early 2012. Simulation education for staff members started in August 2012 and has included video recordings and debriefi ng of each simulated ICU admission. Primary goal-directed therapy goals have been mean arterial pressure (MAP  >65 mmHg), SpO2 >94%, timing of the laboratory tests, start of antibiotics, and blood glucose level 6 to 8  mmol/l. Quality indicators have been followed from the data provided by The Finnish Intensive Care Consortium. P504l Methods In a survey, we evaluated the study course. Therefore, a questionnaire was distributed among all students including the topics course contents, learning materials, time management, supervision, and overall impression. The students were asked to score their agreement to the statements ‘content is well structured’, ‘content extent is appropriate’ and ‘content is relevant for medical purposes’ on a scale ranging from 1 (fully disagree) to 5 (fully agree). P502 Physico-technical medicine: teaching technical skills to the intensivist Introduction The requirements for the intensivist in handling medical technology are constantly growing. It appears necessary to acquire technological competences particularly within the fi elds of medical technology and physics. In the master’s degree program ‘MasterOnline Physico-Technical Medicine’, such technical authority is conveyed. To cope with the intensive vocational situation of the physician, this study course follows the blended-learning concept; that is, it is conceived as an online study course with small portions of intermittent presence phases. Within the fi rst  year, technical basic skills such as ‘measurement technique’, ‘informatics’, and ‘advanced physics’ are covered. Subsequently, two of various advanced courses in diff erent fi elds of medical technology (‘technology in intensive care medicine’, ‘technology in surgery’, ‘technical cardiology’, ‘radiology’, and other) are selected. Conclusion The fi rst-hour protocol has helped us in resource management, start of the patients’ intensive care and education of nursing staff . Reference 1. Brown R, et al.: Aust Crit Care 2012, 25:178-187. 1. Brown R, et al.: Aust Crit Care 2012, 25:178-187. 1University of Pittsburgh, PA, USA; 2University of Pennsylvania, Philadelphia, PA, USA Critical Care 2013, 17(Suppl 2):P504 (doi: 10.1186/cc12442) Introduction Demand for critical care services is increasing yet a comprehensive understanding of how critical care nurses – the largest group of ICU direct care providers – impact outcomes remains unclear. The purpose of this study was to determine how critical care nurse education (hospital proportion of bachelor’s prepared ICU nurses) and ICU work environment infl uenced 30-day mortality of mechanically ventilated older adults. Results The students participated actively in this study course with highest motivation and large commitment. The students’ workload was in the targeted range of about 10 hours/week. Content structure was scored with 4.3 ± 0.1, content extent with 4.1 ± 0.2 and medical relevance with 4.3 ± 0.2. Conclusion The blended-learning concept fulfi lls the requirements for occupation-accompanying continued medical education, since it off ers the possibility to study self-employed accessing text documents, lecture recordings, and electronic lectures and to convert in concentrated presence phases this knowledge into practical exercises. Methods A multi-state cross-sectional nurse survey was linked to hospital administrative data and Medicare claims (2006 to 2008). The fi nal sample included 55,159 mechanically ventilated older adults in 303 hospitals. Logistic regression modeling was employed to jointly assess the relationship of critical care nurse education, work environment and staffi ng on 30-day mortality while adjusting for hospital and patient characteristics and accounting for clustering. First-hour protocol clarifi es resource management and nursing staff education in the ICU Questionnaires for the staff members have been used to evaluate opinions about the fi rst- hour protocol. P505 Infl uence of critical care nurse education and work environment on outcomes of mechanically ventilated older adults DM Kelly1, LH Aiken2 1University of Pittsburgh, PA, USA; 2University of Pennsylvania, Philadelphia, PA, USA P501 The team work dimension was identifi ed as the most valued by workers, with the transmission of information on shift changes the most valued item. Figure 1 (abstract P500). Example report. S186 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 P502 Physico-technical medicine: teaching technical skills to the intensivist J Seifried, S Richter, J Guttmann, S Schumann University Medical Center Freiburg, Germany Critical Care 2013, 17(Suppl 2):P502 (doi: 10.1186/cc12440) nurses considered that the protocol has no eff ect, and none thought it to be adverse for patient care. Corresponding numbers for our ICU doctors were 87% (benefi cial n = 13/15), 13% (no eff ect n = 2/15) and 0% (adverse). Furthermore, 82.5% (n = 66/80) of the nurses replied that education of new nurse staff members has improved because of the fi rst-hour protocol. A total of 17.5% (n = 14/80) thought there has been no eff ect, and none considered the protocol harmful for education. For ICU doctors the protocol did not bring either clear educational advantages or disadvantages. The variable life-adjusted display curves (The Finnish Intensive Care Consortium) have shown improvement in our patient care after the implementation of the fi rst-hour protocol. However, we cannot determine whether it is a signifi cant factor in our intensive care results.i Are general wards suffi ciently staff ed to care for level 1 patients? G Rajendran, C Tjen, S Hutchinson, S Fletcher Norfolk & Norwich University Hospital, Norwich, UK Critical Care 2013, 17(Suppl 2):P506 (doi: 10.1186/cc12444) y gi Results The survey was completed by 129 ICU workers. The global prevalence of ISQ in ICU was 67.4%. ISQ was observed in 45% of physicians and 82% of nurses and nurses assistant (P <0.001). Sleep medication was used by 13.3% of the ICU team. Univariate analysis showed that ISQ was signifi cantly associated with sex (73% vs. 43%, P = 0.03 in women and men, respectively), marital status (84% vs. 61%, P  =  0.01 in single and couple workers, respectively), more than 60 hours working in the last week (76% vs. 61%, P = 0.07) and less than 6 sleeping  hours (95% vs. 54%, P  <0.0001). Multivariable analysis demonstrated that a sleep duration less than 6  hours was independently associated with ISQ (OR = 24.5; 95% CI = 5.2 to 115.8; P <0.0001). Furthermore, pathologic sleepiness was present in 59.3% of ICU workers. Sleepiness was independently associated with use of sleep medication (OR = 5.9; 95% CI = 1.2 to 28.5; P = 0.025). Introduction There are several defi nitions of level 1 (L1) care, all refer to a group at risk of clinical deterioration on the ward [1-3]. There is evidence that ward patients who become acutely unwell often receive suboptimal care [4]. A regional study commissioned by Norfolk, Suff olk & Cambridgeshire Critical Care Network (NSCCCN) found that a majority of ward patients may be of L1 dependency and death rates appear to be correlated with L1 status. We aim to examine the relationship between the ward distribution of illness acuity, staffi ng and patient outcome. fi Methods Data were collected as part of NSCCCN’s observational prevalence study in 2010. Ward surveys included acuity of illness, staffi ng levels and skill mix. Secondary data were obtained from the Patient Administration System. Emergency, oncology, paediatric and maternity units were excluded. Conclusion The prevalence of ISQ and sleepiness is very high among ICU workers. Those disturbances are independently associated with a sleep duration less than 6 hours, and sleep medication use, respectively. These results highlights that strategies to decrease ISQ and sleepiness in ICU clinicians are urgently needed to improve work performance, improve quality of care provided and prevent adverse events. Results Complete datasets were obtained from 1,402 patients in 22 wards in our university hospital over two seasons. This constitutes 98.3% of inpatients from those wards. P505 Activity and case-mix changes in a medical ICU after the geographical transfer of a third-level university hospital JC Cebrián, FM Monsalve, JB Bonastre Hospital Universitario y Politecnico La Fe, Valencia, Spain Critical Care 2013, 17(Suppl 2):P505 (doi: 10.1186/cc12443) Introduction Information about big hospital geographical transfer is scarce in the medical literature. On 20 February 2011 our hospital (in fact, a big university complex) was transferred from their previous location in the north-center of our city towards a new southern peripheral, geographical location. This transfer has been done without any changes in assisted population or nursing/medical staff . The only change was a slight increase in bed number (21 to 24). Our aim is to analyze changes in activity indexes (length of stay, occupancy rate, and so forth) and case mix (origin, previous quality of life and NYHA score, main diagnostic groups, severity scores, in-ICU and in-hospital mortality). Results According to the questionnaire replies, 80% (n = 64/80) of our nurses estimate that the fi rst-hour protocol has improved the starting process of our patients’ intensive care. Twenty percent (n = 16/80) of the S187 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Methods To compare our number of admissions, related activity and case-mix indicators 1 year before and after the geographical change was done. We analyzed our whole number of patients admitted to the ICU. We used the chi-square test for categorical variables and one-way analysis of variance for quantitative data. Minitab and Statbas statistical programs were used. We plotted activity data using the Barber– Johnson 1 diagram. [http://www.nice.org.uk/cg50] 3. AUKUH Acuity/Dependency Tool. Association of UK University Hospitals; 2007. [http://www.aukuh.org.uk/index.php/affi liate-groups/directors-of-nursing/ patient-care-portfolio] [http://www.nice.org.uk/cg50] 3. AUKUH Acuity/Dependency Tool. Association of UK University Hospitals; 2007. [http://www.aukuh.org.uk/index.php/affi liate-groups/directors-of-nursing/ patient-care-portfolio] 4. 4. An Acute Problem? NCEPOD; 2005. [http://www.ncepod.org.uk/2005report/index.html] Results A total of 2,774 cases (63% males; mean age 61 years) were admitted to our ICU during the period (1  year before and after the transfer). No diff erences between both groups were founded in demographic data, Knaus score and NYHA status. Regarding their origin, we found more patients admitted from other hospital centers (20 vs. 29%; P <0.001). APACHE II score increased from 17.24 to 19.08% (P <0.001) and a slight increase change in SAPS 3 score was also found (52.29 to 53.75; P  <0.01). Work-related stress amongst doctors in intensive care, anaesthetics, accident and emergency and general medicine g y g  Tuthill, MS Ahmed, G Mathew, AC Bolton, AA Molokhia JI Tuthill, MS Ahmed, G Mathew, AC Bolton, AA Molokhia University Hospital Lewisham, London, UK Introduction Work-related stress is a potential problem among doctors and is associated with anxiety, depression, reduced job satisfaction, days off work, errors and near misses [1]. To compare stress levels between diff erent groups of doctors and identify causes of stress, we conducted a survey at University Hospital Lewisham using the UK Health and Safety Executive’s Management Standards (HSEMS). HSEMS is a validated tool developed to identify work conditions that warrant interventions to reduce stress levels across organisations [2]. y y Conclusion Better outcome with improved SPr may be unsurprising, although if proven conclusively would signifi cantly inform workforce planning. Lack of correlation between staffi ng levels and occupancy or acuity is also interesting given that we know L1 criteria are associated with worse outcome. P505 Our in-ICU mortality remains lower (15.5 to 15.6%) whereas observed mortality decreased (22.37 to 19.88%; P <0.001). An increase in our neurologic patients has been the most consistent change regarding diagnostic groups. The activity indexes show a slightly decrease in occupancy rate (79.2 vs. 76.8). 1. Levels of Critical Care for Adult Patients. Intensive Care Society; 2002. [http://www.ics.ac.uk/professional/standards_safety_quality/standards_and_ guidelines/levels_of_critical_care_for_adult_patients] 2. Acutely Ill Patients in Hospital. NICE Guideline 50. NICE; 2007. P508 Work-related stress amongst doctors in intensive care, anaesthetics, accident and emergency and general medicine JI Tuthill, MS Ahmed, G Mathew, AC Bolton, AA Molokhia University Hospital Lewisham, London, UK Critical Care 2013, 17(Suppl 2):P508 (doi: 10.1186/cc12446) Work-related stress amongst doctors in intensive care, anaesthetics, accident and emergency and general medicine 1. Yates J: Hospital Beds: A Problem for Diagnostic and Management. Heinemann Medical Books; 1982. Are general wards suffi ciently staff ed to care for level 1 patients? G Rajendran, C Tjen, S Hutchinson, S Fletcher Norfolk & Norwich University Hospital, Norwich, UK Critical Care 2013, 17(Suppl 2):P506 (doi: 10.1186/cc12444) The mean ward occupancy rate was 94% (10th to 90th percentile: 85% to 100%). At least one L1 acuity criterion was scored by 898 (64%) patients, with 25% from geriatrics followed by orthopaedics (17%) and general surgery (10%). Each ward had an average of eight qualifi ed nursing staff (range: 4 to 12) equating to an average staff :patient ratio (SPr) of 0.253. There was no correlation between ward occupancy and nursing staff (Pearson correlation, corr: 0.55), nor between prevalence of L1 criteria and staffi ng (corr: 0.34). The admission rate to intensive care was noted to be higher if the patients were nursed in a ward with lower than average SPr compared with higher SPr (2.7% vs. 1.2%, P = 0.058 Fisher’s exact), but this was not statistically signifi cant. Senior nursing (Band 6) staff were part of the skill mix on only nine of 44 ward surveys. P508 Reference 1. Yates J: Hospital Beds: A Problem for Diagnostic and Management. Heinemann Medical Books; 1982. Methods A survey was conducted in six Uruguayan ICUs. The sleep quality was evaluated on the basis of the Pittsburgh score (PS), and the sleepiness was identifi ed by the Epworth scale. ISQ was defi ned as PS greater than 5 points and sleepiness by an Epworth scale higher than 6 points. ICU’s, patient’s, and clinician’s characteristics were assessed for their association with the prevalence of ISQ. All variables with P <0.2 in univariate analysis were included in a model of ordinal regression. P <0.05 was considered statistically signifi cant. P507 Sleep quality among ICU workers h 1 k h 1 h 2 Sleep quality among ICU workers G Burghi1, I Serkochian1, B Frache2, P Alzugaray2, K Goinheix1, M Rodriguez Verde3, H Bagnulo1, E Azoulay4 1Hospital Maciel, Montevideo, Uruguay; 2Sanatorio Americano, Montevideo, Uruguay; 3Hospital de Paysandú, Uruguay; 4Hôpital Saint Louis, Paris, France Critical Care 2013, 17(Suppl 2):P507 (doi: 10.1186/cc12445) Introduction Prolonged shifts, workload, stress, and diff erent con- fl icts are associated with burnout, loss of psychological wellbeing, and probably with an inadequate sleep quality (ISQ). This relevant disturbance leads to deterioration of the work performance, may impair quality of care provided to patients and increases the incidence of serious adverse events. The objective was to determine the prevalence of ISQ and sleepiness among Uruguayan ICU workers, and to evaluate risk factors associated with ISQ. Conclusion According to the previous data our ICU seems to perform better in the new location with a decrease in the standardized mortality rate. On the other hand, we are admitting more patients transferred from other hospitals. A better occupancy rate was found. f P506 A P506 Are general wards suffi ciently staff ed to care for level 1 patients? G Rajendran, C Tjen, S Hutchinson, S Fletcher Norfolk & Norwich University Hospital, Norwich, UK Critical Care 2013, 17(Suppl 2):P506 (doi: 10.1186/cc12444) Infl uence of adverse drug events on the mortality and the length of hospital stay in ICUs in Japan: the JADE Study Y Ohta1, M Sakuma1, D Bates2, T Morimoto1 1Kinki University, Osaka, Japan; 2Harvard University, Boston, MA, USA Critical Care 2013, 17(Suppl 2):P510 (doi: 10.1186/cc12448) og dad y , J o , O oc e 1Princess Royal University Hospital, London, UK; 2University Hospital Lewisham, London, UK Critical Care 2013, 17(Suppl 2):P509 (doi: 10.1186/cc12447) Critical Care 2013, 17(Suppl 2):P509 (doi: 10.1186/cc12447) Introduction South-east London (SEL) presents unique challenges to healthcare providers due to its diverse demographic. The high levels of poverty, immigration and psychiatric illness impact delivery of obstetric care. These were identifi ed as risk factors for poor outcome in the latest CMACE report [1]. The Intensive Care National Audit and Research Centre (ICNARC) produced data on obstetric critical care admissions in 2007 [2]. We reviewed the obstetric critical care admissions in three SEL hospitals and compared this with the national average determined in the ICNARC and CMACE data. Introduction Adverse drug events (ADEs) are associated with a substantial increase in morbidity and mortality in any setting. Because patients in ICUs were critically ill with complex diseases and varied organ dysfunction, the incidence of ADEs on such patients is much more crucial than the counterparts. We thus assessed the nature of ADEs and their infl uence in ICUs. l Methods We conducted a prospective cohort study at ICUs at three large tertiary-care hospitals in Japan. Trained research nurses reviewed all medical charts, incident reports and reconciliations from the pharmacy to identify suspected ADEs as well as the background of patients. ADEs are any injuries that result from the use of a drug. After suspected ADEs are collected by research nurses, physician reviewers independently evaluated them and classifi ed them as ADEs or rule violations. We used the validated methodology [1]. Methods All critical care admissions in three high-risk obstetric units in SEL (1 August 2009 to 31 July 2011) were screened for patients who were currently or recently pregnant. We compared local results with national data by ICNARC and CMACE. Figure 1 (abstract P509). Causes for critical care admission in SEL, CMACE and ICNARC. Results We included 459 patients with 3,231 patient-days. The median age was 70 years and the median length of stay was 3 days. In total, 70 patients (15%) had at least one ADE during their stay in the ICU. The median ICU stay in patients who had ADEs was 14 days while 2 days in patients who had no ADEs (P <0.0001). The median length of the ADE onset days since admission was 3 days. References Methods We conducted an anonymous survey of doctors working in anaesthetics, intensive care, general medicine and accident and emergency (A&E) departments over 6 weeks using the HSEMS S188 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 question naire. We also surveyed awareness of the Trust’s stress management services and whether staff had a designated supervisor or mentor. Results were analysed using the HSEMS Analysis Tool, which rates stressors with a score from 1 to 5 (5 represents the lowest amount of stress). We compared the Trust’s results against HSEMS national standards. Results There were 68 obstetric critical care admissions in the SEL hospitals within the audited time frame. The mean age was 30.05 in ICNARC data compared with 33.93 in SEL. Average APACHE II scores were lower in SEL compared with the ICNARC data, but length of stay was greater in SEL (2.72 days) compared with ICNARC (1.5 days). Haemorrhage was the most common reason for admission in SEL, whilst sepsis was the leading cause of death according to the latest CMACE report (Figure 1). Results Seventy-two doctors completed the survey. Lowest stress levels were found in doctors working in intensive care (n = 12, mean 3.63, SD 0.39). This was followed by medicine (n  =  26, mean 3.55, SD 0.47), anaesthetics (n = 27, mean 3.40, SD 0.44), and A&E (n = 7, mean 3.11, SD 0.65), which had the highest stress levels. There was no signifi cant diff erence in stress levels between diff erent grades of doctors. When compared with HSEMS targets, staff relationships and peer support exceeded national standards. However, management of organisational change and demands at work need improvement. The majority of doctors (82%) had no idea what stress management services were provided by the Trust. Seventy-nine per cent of doctors had an allocated supervisor or mentor, 91% of those felt able to approach their supervisor. g Conclusion Data from national audits may guide protocol, but services must be tailored to local circumstances. SEL has unique population characteristics and obstetric critical care admissions diff er signifi cantly from national statistics; in particular, haemorrhage is over-represented in our region. Critical care services were generally required for a short period of time; during this period, routine postpartum care may be omitted as treatment priorities diff er. Morimoto T, et al.: Qual Saf Health Care 2004, 13:306-314. References 1. Kerr et al.: HSE management standards and stress-related work outcomes. Occup Med (Lond) 2009, 59:574-579. 1. Kerr et al.: HSE management standards and stress-related work outcomes. Occup Med (Lond) 2009, 59:574-579. 2. Health and Safety Executive Management Standards [http://www.hse.gov. uk/stress/standards/index.htm] 2. Health and Safety Executive Management Standards [http://www.hse.gov. uk/stress/standards/index.htm] References Dedicated critical care services on the labour ward may be a way to combine postnatal care with transient high-dependency requirements. This may enhance patient experience and prove cost-eff ective. References Conclusion These survey results provide reassurance that stress levels in intensive care compare well, despite critically unwell patients and higher mortality rates. We identifi ed areas that need improvement within the Trust and will present these results to all relevant departments. With the support of hospital management we will initiate HSEMS-validated measures to reduce stress. 1. Centre for Maternal and Child Enquiries: Saving mothers’ lives: reviewing maternal deaths to make motherhood safer: 2006–08. The Eighth Report on Confi dential Enquiries into Maternal Deaths in the United Kingdom. BJOG 2011, 118(Suppl 1):1-203. 2. Female Admissions (Aged 16–50 Years) to Adult, General Critical Care Units in England, Wales and Northern Ireland, Reported as ‘Currently Pregnant’ or ‘Recently Pregnant’ [http://www.oaa-anaes.ac.uk/assets/_ managed/editor/File/Reports/ICNARC_obs_report_Oct2009.pdf] References P510l Infl uence of adverse drug events on the mortality and the length of hospital stay in ICUs in Japan: the JADE Study Y Ohta1, M Sakuma1, D Bates2, T Morimoto1 1Kinki University, Osaka, Japan; 2Harvard University, Boston, MA, USA Critical Care 2013, 17(Suppl 2):P510 (doi: 10.1186/cc12448) P509 Audit of obstetric critical care admissions in a high-risk population K El-Boghdadly1, J Aron2, DN Onwochei1 1Princess Royal University Hospital, London, UK; 2University Hospital Lewisham, London, UK Critical Care 2013, 17(Suppl 2):P509 (doi: 10.1186/cc12447) P511 Attitude and knowledge of intensive care staff concerning donation in Hungary: it is the fi rst step to change g yi p A Smudla1, S Mihály2, J Fazakas1 A Smudla , S Mihály , J Fazakas 1Semmelweis University, Budapest, Hungary; 2Hungarian National Blood Transfusion Service, Budapest, Hungary Critical Care 2013, 17(Suppl 2):P511 (doi: 10.1186/cc12449) Conclusion Introduction of a SN:OD and a clinical pathway has led to the identifi cation of previously missed potential organ donors in the ED. Several patients have subsequently been admitted to critical care solely to facilitate organ donation. Introduction In Hungary, despite the high level of social support, the number of organ recoveries from deceased donors has not changed signifi cantly. The donation activity shows a positive relationship with the level of education of staff in ICUs as well as with their attitude towards transplantation. The aim of this cross-sectional study is to estimate the attitude and knowledge of intensive care specialists and nurses as regard donation and transplantation. Infl uence of adverse drug events on the mortality and the length of hospital stay in ICUs in Japan: the JADE Study Y Ohta1, M Sakuma1, D Bates2, T Morimoto1 1Kinki University, Osaka, Japan; 2Harvard University, Boston, MA, USA Critical Care 2013, 17(Suppl 2):P510 (doi: 10.1186/cc12448) Regarding the mortality, 73 patients (16%) were dead during their ICU stay: 12 deaths (17%) in patients who had ADEs and three of 12 deaths were caused by an ADE, and 61 deaths (16%) in counterparts (P = 0.8). There were no signifi cant diff erences of patients’ characteristics between patients with ADEs and without ADEs (Table 1). Conclusion ADEs were associated with longer stay and caused a part of death in ICU (4%) although they did not increase the mortality. Because the characteristics of patients were not associated with ADEs, early detection and intervention for ADEs could be important to improve the morbidity and reduce the death caused by ADEs in ICUs. Reference Figure 1 (abstract P509). Causes for critical care admission in SEL, CMACE and ICNARC. Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 S189 Table 1 (abstract P510). Patient characteristics Patients Patients with without ADEs ADEs Characteristic (n = 70) (n = 389) P value Age ≥65 years, n (%) 49 (70) 227 (58) 0.08 Male, n (%) 40 (57) 250 (64) 0.2 Department Medicine 35 (50) 228 (59) Surgery 35 (50) 161 (41) 0.2 Unconsciousness, GCS ≤8 13 (19) 91 (23) 0.4 Heart failure, NYHA = 4 37 (53) 216 (56) 0.7 Respiratory failure 44 (62) 262 (67) 0.5 Kidney failure 6 (9) 58 (15) 0.2 Clinical laboratory measurements, median (25%, 75%) Blood urea nitrogen (mg/dl) 21 (15, 39) 19 (14, 31) 0.2 Creatinine (mg/dl) 0.8 (0.5, 1.5) 0.9 (0.7, 1.7) 0.8 Aspartate aminotransferase (IU/l) 27 (20, 47) 30 (22, 48) 0.5 Alanine transaminase (IU/l) 20 (13, 42) 20 (12, 33) 1.0 Total protein (g/dl) 6.7 (5.9, 7.4) 6.6 (5.9, 7.3) 0.8 Number of medications on admission ≥4, 38 (54) 223 (57) 0.6 n (%) Number of medications on admission, 5 (3, 7) 5 (4, 8) 0.7 median (25%, 75%) GCS, Glasgow Common Scale; NYHA, New York Heart Association. Table 1 (abstract P510). Patient characteristics knowledge regarding the law and ethics of donation (P <0.01), donor management (P  <0.01), living and deceased donor transplantation (P <0.01) and joining Eurotransplant (P <0.01). Older professionals had more information about all fi elds (P <0.01). Nurses had less knowledge concerning donor management (P <0.01), law and ethics (P <0.01) and deceased donor transplantation (P <0.01) than doctors. y Reference 1. UK Organ Donation Taskforce: Organs for Transplants: A Report from the Organ Donation Taskforce. Department of Health; 2008. Specialist nurse for organ donation in an emergency department will increase organ donation Introduction In the UK, three people die each day awaiting trans plan- tation, due to the unavailability of donor organs. Traditionally, donor identifi cation has been restricted to the ICU. However, following the UK Organ Donation Taskforce report in 2008 [1], a number of emergency departments (EDs) have been working with specialist nurses for organ donation (SN:OD) to identify potential donors and approach their families for consent in the ED. We present our initial experience after the introduction of a SN:OD to an Irish teaching hospital’s ED. Methods We conducted a retrospective review of deaths in our ED during a 28-month period. For those who died in the ED, case notes were reviewed to identify those suitable for organ donation. Referral and donation rates were compared in two cohorts, pre and post introduction of a SN:OD. Fisher’s exact test was used to assess diff erences between groups. f Results Ninety-one deaths occurred in the study period. Following introduction of the SN:OD, referrals increased from zero to eight. Of the eight referred, three received consent and were transferred to the ICU, two of whom became successful donors. The number of missed potential donors fell from six to one (P = 0.009). Infl uence of adverse drug events on the mortality and the length of hospital stay in ICUs in Japan: the JADE Study Y Ohta1, M Sakuma1, D Bates2, T Morimoto1 1Kinki University, Osaka, Japan; 2Harvard University, Boston, MA, USA Critical Care 2013, 17(Suppl 2):P510 (doi: 10.1186/cc12448) knowledge regarding the law and ethics of donation (P <0.01), donor management (P  <0.01), living and deceased donor transplantation (P <0.01) and joining Eurotransplant (P <0.01). Older professionals had more information about all fi elds (P <0.01). Nurses had less knowledge concerning donor management (P <0.01), law and ethics (P <0.01) and deceased donor transplantation (P <0.01) than doctors. Conclusion Education about organ donation needs to be part of specialist training of intensive care staff , and refresher courses every fi fth year as well. The course should include knowledge regarding brain death, donor management and communication with family. This is the fi rst step to improve the number of transplantations. Reference Sepsis in HIV patients admitted to the ICU d l 1 l 1 l Sepsis in HIV patients admitted to the ICU P Vidal-Cortés1, P Lameiro-Flores1, M Mourelo-Fariña2, A Aller-Fernández2, R Gómez-López1, P Fernández-Ugidos1, M Alves-Pérez1, E Rodríguez-García1 1CHU Ourense, Spain; 2CHU A Coruña, Spain Critical Care 2013, 17(Suppl 2):P513 (doi: 10.1186/cc12451) Sepsis in HIV patients admitted to the ICU P Vidal-Cortés1, P Lameiro-Flores1, M Mourelo-Fariña2, A Aller-Fernández2, R Gómez-López1, P Fernández-Ugidos1, M Alves-Pérez1, E Rodríguez-García1 1CHU Ourense, Spain; 2CHU A Coruña, Spain Critical Care 2013, 17(Suppl 2):P513 (doi: 10.1186/cc12451) nurses as regard donation and transplantation. Methods The self-completed questionnaire that consisted of 20 items was completed at the Congress of Hungarian Society of Anesthesiology and Intensive Therapy in 2011. Besides the epidemiological data, the intensive care specialists (n = 179) and nurses (n = 103) were asked about donation activity, participation in an organ donation course, self- reported knowledge of joining Eurotransplant, donor management, legislation, and transplantation. The data were analyzed by SPSS 17.0. Results A total of 53.6% of physicians and 16.7% of nurses attended an earlier organ donation course (P <0.01). The average age of those who participated in training was signifi cantly higher among doctors (P <0.01). Fifty-nine percent of doctors and 65.1% of nurses did not even want to participate in such training. Donation activity was higher among staff who joined training (P  <0.01). Independently from accepting the presumed consent legislation (91.1%), 66% of physicians agreed with the hospital practice that requests the adult donor’s relatives to consent to organ recovery. This standpoint did not depend on donation activity, participation in an organ donation course, opinion about legislation and the nature of staff . A total 95.4% of participants consented to their organ retrieval after death. The staff who participated in an organ donation course had more g Methods The self-completed questionnaire that consisted of 20 items was completed at the Congress of Hungarian Society of Anesthesiology and Intensive Therapy in 2011. Besides the epidemiological data, the intensive care specialists (n = 179) and nurses (n = 103) were asked about donation activity, participation in an organ donation course, self- reported knowledge of joining Eurotransplant, donor management, legislation, and transplantation. The data were analyzed by SPSS 17.0. Introduction Our objective was to analyze septic HIV patients admitted to intensive care. Introduction Our objective was to analyze septic HIV patients admitted to intensive care. Table 2 (abstract P515). Unit and hospital mortality Methods A retrospective study analyzed children with hematological disorders admitted to our ICU between April 2005 and June 2012. All of the included children required emergency admission and invasive mechanical ventilation. Those who did not need intubation, or required intubation only for therapeutic intervention and died within 24 hours of ICU admission were excluded. The survival group was defi ned as patients who were discharged from the ICU, and the nonsurvival group was defi ned as those who died in the ICU or within 7 days after discharge from the ICU. The PELOD score and PIM-II were applied as morbidity scoring systems Patients ICU mortality, Hospital mortality, (n) n (%) n (%) Haematology 163 52 (31.9%) 81 (49.7%) Controls 237 73 (30.8%) 99 (41.8%) P value 0.827 0.126 Conclusion Unit mortality of critically ill patients with HM was similar to those without HM. Hospital mortality in patients with HM was higher than those without HM, although not statistically signifi cant. Severity of illness at presentation to critical care is the main determinant of outcome in patients with HM. Results Twenty-seven patients, including 18 males and nine females, with a median age of 6.1 years (range, 0.2 to 16.6 years) were analyzed. Sixteen patients had leukemia, fi ve had hemophagocytic syndrome, six had solid tumors. The average predicted mortality rate was 31.3% in PIM-II. The survival group included 15 patients (56%) and the nonsurvival group included 12 patients (44%). When the survival group was compared with the nonsurvival group, there were no signifi cant diff erences in the systolic blood pressure (101.3  ±  13.9 mmHg vs. 92.8  ±  25.4 mmHg; P  =  0.15), the proportion of patients requiring continuous renal replacement therapy (33.3% vs. 50.0%; P = 0.30), and PELOD score (15.5 ± 10.4 vs. 21.8 ± 15.4; P = 0.22). In the nonsurvival group, the PIM-II was higher than that in the survival group (27.9 ± 10.4 vs. 35.7 ± 9.0; P = 0.06); the PaO2/FiO2 (272.5 ± 136.7 vs. 153.3 ± 123.3; P = 0.03) and oxygenation index (6.7 ± 8.1 vs. 14.1 ± 9.5; P = 0.04) were signifi cantly worse in the nonsurvival group than in the survival group. Conclusion The data show that respiratory failure is more strongly associated with mortality than other organ failures in pediatric hematology patients requiring intensive care. Sepsis in HIV patients admitted to the ICU d l 1 l 1 l Mean viral load: 4.57 ± 3.25 log (vs. 2.257 ± 1.96, P = 0.001). Of sepsis patients, 62.1% were on their CD4 nadir (vs. 34.5% in nonseptic patients, P = 0.009). Mean albumin levels were 2.3 ± 0.53 g/ dl (vs. 2.92 ± 0.94, P <0.001). APACHE II at admission was 21.98 ± 7.97 (vs. 18.15 ± 8.47, P = 0.046). At admission, 52.8% were on severe sepsis and 44.3% on septic shock. The lung was the most frequent source of infection (65.6%) followed by CNS (16.4%), UTI (4.9%) and IE (4.9%). The most common pathogen isolated on these patients was S. pneumoniae (28.8%), followed by P. jirovecii (13.6%), toxoplasma (8.5%), E. coli (5.1%) and H. infl uenzae (5.1%). In total, 62.9% needed vasopressors (vs. 28.6% in non-infected patients, P = 0.001), 79% mechanical ventilation (vs. 42.85%, P  <0.001) and 19.4% renal replacement (9.5% in no septic patients, P = 0.173). Mean ICU and hospital LOS was 10.43 ± 10.52 and 34.76 ± 29.64 days in septic patients versus 6.04 ± 8.45 (P = 0.026) and 20.54 ± 27.93 days (P = 0.016). ICU mortality: 33.9% (19% in nonseptic patients, P = 0.098), hospital mortality: 41.9% (vs. 23.8%, P = 0.057). Conclusion Sepsis is a common reason for admission to the ICU in HIV patients and is accompanied by high mortality. Pneumonia is the most frequent source of infection. Septic patients are less frequently under HAART and have a worse inmune status (lower CD4 count and higher viral load). Despite a higher APACHE II, and a higher need for hemodynamic and respiratory support, there is no statistically signifi cant diff erence in ICU and hospital mortality between septic and nonseptic patients. (2.4% in non-infected patients, P = 0.223) and 40.3% were under HAART (64.3% in patients admitted without infection, P = 0.016). Mean CD4 count at admission: 219.62 ± 353.93 cells/mm3 (vs. 370.22 ± 362.56, P = 0.048). Mean viral load: 4.57 ± 3.25 log (vs. 2.257 ± 1.96, P = 0.001). Of sepsis patients, 62.1% were on their CD4 nadir (vs. 34.5% in nonseptic patients, P = 0.009). Mean albumin levels were 2.3 ± 0.53 g/ dl (vs. 2.92 ± 0.94, P <0.001). APACHE II at admission was 21.98 ± 7.97 (vs. 18.15 ± 8.47, P = 0.046). At admission, 52.8% were on severe sepsis and 44.3% on septic shock. Table 2 (abstract P515). Unit and hospital mortality These results also suggest that mechanical ventilation intervention in patients with respiratory failure must occur earlier to improve the outcomes for these patients. Sepsis in HIV patients admitted to the ICU d l 1 l 1 l The lung was the most frequent source of infection (65.6%) followed by CNS (16.4%), UTI (4.9%) and IE (4.9%). The most common pathogen isolated on these patients was S. pneumoniae (28.8%), followed by P. jirovecii (13.6%), toxoplasma (8.5%), E. coli (5.1%) and H. infl uenzae (5.1%). In total, 62.9% needed vasopressors (vs. 28.6% in non-infected patients, P = 0.001), 79% mechanical ventilation (vs. 42.85%, P  <0.001) and 19.4% renal replacement (9.5% in no septic patients, P = 0.173). Mean ICU and hospital LOS was 10.43 ± 10.52 and 34.76 ± 29.64 days in septic patients versus 6.04 ± 8.45 (P = 0.026) and 20.54 ± 27.93 days (P = 0.016). ICU mortality: 33.9% (19% in nonseptic patients, P = 0.098), hospital mortality: 41.9% (vs. 23.8%, P = 0.057). References 1. Hampshire PA, et al.: Crit Care 2009, 13:R137. 2 M i PB t l C it C M d 2002 30 2260 2 References References References 1. Tamburro RF, et al.: Pediatr Crit Care Med 2008, 9:270-277. 2. Chima RS, et al.: Pediatr Crit Care Med 2012, 13:e336-e342. P515 Survival of critically ill patients with haematological malignancies compared with patients without haematological malignancy R Pugh1, P Hampshire2, P Hajimichael3 1Glan Clwyd Hospital, Rhyl, UK; 2Royal Liverpool University Hospital, Liverpool, UK; 3Christie Hospital, Manchester, UK Critical Care 2013, 17(Suppl 2):P515 (doi: 10.1186/cc12453) P515 Survival of critically ill patients with haematological malignancies compared with patients without haematological malignancy R Pugh1, P Hampshire2, P Hajimichael3 1Glan Clwyd Hospital, Rhyl, UK; 2Royal Liverpool University Hospital, Liverpool, UK; 3Christie Hospital, Manchester, UK Critical Care 2013, 17(Suppl 2):P515 (doi: 10.1186/cc12453) Introduction Critically ill patients with haematological malignancies (HM) have high hospital mortality [1]. Severity of illness scores may underestimate mortality in such patients [2]. Introduction Critically ill patients with haematological malignancies (HM) have high hospital mortality [1]. Severity of illness scores may underestimate mortality in such patients [2]. Methods Data collection was conducted at three hospitals from 2008 to 2011. Patients with any active HM condition were matched with two control patients at two hospitals and with one control at Christie Hospital. Control patients had the same APACHE II (within 2 points) and admission diagnosis, but no HM. Readmissions and planned surgical cases were excluded. p p y Conclusion Sepsis is a common reason for admission to the ICU in HIV patients and is accompanied by high mortality. Pneumonia is the most frequent source of infection. Septic patients are less frequently under HAART and have a worse inmune status (lower CD4 count and higher viral load). Despite a higher APACHE II, and a higher need for hemodynamic and respiratory support, there is no statistically signifi cant diff erence in ICU and hospital mortality between septic and nonseptic patients. Results A total of 163 patients with HM were compared with 237 control patients. Seventy-four admissions with HM were identifi ed at two hospitals, and each was matched with two control patients. Eighty- nine admissions with HM from Christie Hospital were identifi ed. These were matched with 89 controls. Patients with HM spent signifi cantly longer in hospital before ICU admission (Table  1). Unit and hospital mortality rates were not statistically diff erent between patients with HM and without HM (Table 2). Mortality related to respiratory failure among pediatric hematology patients requiring intensive care H Okuno, K Atagi, H Shimaoka Osaka City General Hospital, Osaka, Japan Critical Care 2013, 17(Suppl 2):P514 (doi: 10.1186/cc12452) Table 1 (abstract P515). Patient characteristics Haematology Controls P value Age, mean (SD) 56.5 (15.1) 57.1 (15.7) 0.717 APACHE II, mean (SD) 22.7 (6.1) 23.1 (6.0) 0.484 ICU LOS (median) 3.2 (1.8 to 6.2) 3.1 (1.3 to 8.4) 0.948 LOS prior (median) 6.5 (2 to 17) 2.0 (0 to 7) <0.0001 bl ( b P ) i d h i l li Table 1 (abstract P515). Patient characteristics Table 1 (abstract P515). Patient characteristics H Okuno, K Atagi, H Shimaoka Introduction Although recent reports show an improvement in out- comes for pediatric hematology patients requiring intensive care [1,2], respiratory failure remains one of the major risks of pediatric mortality. This study was conducted to assess our hypothesis that mortality associated with respiratory failure is higher than that for other organ failures in pediatric hematology patients admitted to our ICU. Table 2 (abstract P515). Unit and hospital mortality Sepsis in HIV patients admitted to the ICU d l 1 l 1 l Methods A retrospective study of HIV patients admitted to our ICU between January 2005 and December 2009. We identify patients admitted to the ICU with sepsis and analyze demographic factors, etiology, organ failure and outcome, and we compare immune status, frequency of organ failure and outcome between these patients and those admitted for other reasons. We use Student’s t test to compare quantitative variables, and the chi-square test for qualitative data. Methods A retrospective study of HIV patients admitted to our ICU between January 2005 and December 2009. We identify patients admitted to the ICU with sepsis and analyze demographic factors, etiology, organ failure and outcome, and we compare immune status, frequency of organ failure and outcome between these patients and those admitted for other reasons. We use Student’s t test to compare quantitative variables, and the chi-square test for qualitative data. Results A total of 104 HIV patients were admitted to our ICU during the study period, 62 with sepsis (71% men, mean age: 40.59 ± 8.12). Of sepsis patients, 56.5% were admitted from the ER and 38.7% from a medical ward; 66.1% had history of intravenous drugs use, other comorbidities: COPD (9.7%), cirrhosis (8.1%), solid or hematologic malignancy (12.9%); 8.1% patients were unaware of their condition Results A total of 53.6% of physicians and 16.7% of nurses attended an earlier organ donation course (P <0.01). The average age of those who participated in training was signifi cantly higher among doctors (P <0.01). Fifty-nine percent of doctors and 65.1% of nurses did not even want to participate in such training. Donation activity was higher among staff who joined training (P  <0.01). Independently from accepting the presumed consent legislation (91.1%), 66% of physicians agreed with the hospital practice that requests the adult donor’s relatives to consent to organ recovery. This standpoint did not depend on donation activity, participation in an organ donation course, opinion about legislation and the nature of staff . A total 95.4% of participants consented to their organ retrieval after death. The staff who participated in an organ donation course had more S190 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 (2.4% in non-infected patients, P = 0.223) and 40.3% were under HAART (64.3% in patients admitted without infection, P = 0.016). Mean CD4 count at admission: 219.62 ± 353.93 cells/mm3 (vs. 370.22 ± 362.56, P = 0.048). Automated, electronic monitoring for early detection of sepsis S Rauch1, M Fischer1, C Martin2, A Sablotzki3 Critical Care 2013, 17(Suppl 2):P517 (doi: 10.1186/cc12455) Introduction In the year 2009, the organizational structure of the ICU in the Kanazawa University Hospital changed from an open to a semi- closed format ICU. The objective of this study was to evaluate the eff ect of this organizational change on outcome in high-risk surgical patients. Methods The medical records of all consecutive high-risk surgical patients admitted to the ICU from 2006 to 2009 (open format, n = 1,598) and from 2009 to 2012 (semi-closed format, n = 1,521) were reviewed. Parameters studied were mortality and ICU length of stay. Introduction Early detection of sepsis is important for a suffi cient treatment to reduce mortality. We hypothesized that using modifi ed systemic infl ammatory response syndrome criteria over 1 hour using an electronic software program facilitates the clinical diagnosis of sepsis. Methods After IRB approval and informed consent we enrolled in this prospective, observational, single-center study 1,119 consecutive patients (age 68.6  ±  16.4, female/male 476/649) admitted over a 6-month period to a surgical ICU. A total 149 of them met modifi ed systemic infl ammatory response criteria. Patients were monitored by an electronic software program using live data from the laboratory and bedside monitors to detect modifi ed systemic infl ammatory response syndrome criteria persisting over 1 hour. The physicians were blinded to the software program alerts that notifi ed in real time when modifi ed systemic infl ammatory response syndrome criteria were detected and persisted over 1 hour, but did not provide treatment recommendations. Results There was a total of 149 modifi ed systemic infl ammatory response syndrome criteria alerts. Seventy-four were confi rmed as true sepsis cases by physicians. The overall incidence of sepsis was 7%. Patients were categorized into length of stay <24 hours, 24 to 96 hours and >96 hours. The overall sensitivity of our system for detecting sepsis was 68% and the specifi city was 91%. The positive predictive value is 34% and the negative predictive value is 98%. y g y Results Mortality of ICU patients was 9.9% in the open format group and 6.6% in the closed format group (P <0.05). The average length of hospital stay was 4.9 days in the open format group and 4.8 days in the closed format group. Outcome of critically ill patients with haematological malignancy admitted to the ICU as an emergency T Lofaro1, S Easdale2, S Rowe2, M Ostermann2, R Carr2 1Queen Elisabeth Hospital, Woolwich, UK; 2Guys’ and St Thomas’ Hospital, London, UK Introduction ICU admission policies regarding patients with haemato- logical malignancy still vary despite data showing an acceptable prognosis. Our aim was to review ICU and 6-month outcomes in this S191 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 group when requiring emergency admission to the ICU in a tertiary cancer centre. Figure 1 (abstract P518). Starch administration between November 2008 and November 2012. Figure 1 (abstract P518). Starch administration between November 2008 and November 2012. Methods A retrospective review of medical notes between 2004 and 2012. Results A total of 249 patients were admitted, of whom 54 had more than one admission. There were 310 episodes in total. Leukaemia n = 85; lymphoma n = 90; myeloma n = 36. We compared the characteristics of those who survived ICU admission with those who failed to survive to discharge from ICU. The two populations were similar (age 51 vs. 57; males 59% vs. 57%). Those who survived had a lower APACHE II score on admission (19 vs. 23; P <0.001), lower mean organ failure scores (1 vs. 2; P <0.05), lower requirements of inotropes (26% vs. 50%; P = 0.001), ventilation (31% vs. 64%; P  =  0.001) and fi ltration (11% vs. 26%; P = 0.004). There was no diff erence in the prevalence of sepsis at the time of admission (64% vs. 70%). Both groups included patients with prior bone marrow transplant (38% vs. 40%). Of note, ICU and 6-month survival were 27% and 50%, respectively. These values are lower than those reported in the literature to date. Figure 1 (abstract P518). Starch administration between November 2008 and November 2012. Methods Our e-prescribing software enables users to prescribe intravenous fl uids from a series of menus. One of these is a template that has several fl uids available to use as a bolus when instructed by a clinician. We removed starch as an option from the template in April 2009. Starch could still be prescribed elsewhere on the prescribing system. Data on the use of starch from November 2008 to November 2012 were analysed as the mean volume of starch infused per patient per month. The mean of each set of parameters was then compared using a Student’s t test. Outcome of critically ill patients with haematological malignancy admitted to the ICU as an emergency Conclusion ICU and 6-month mortalities were 27% and 50%, respectively. Patients with haematological malignancy stand to benefi t from intensive care, and should be off ered admission based on clinical need. References 1. Cuthberson et al.: The outcome of haematological malignancy in Scottish intensive care units. J Intensive Care Soc 2008, 9:135-140. 2. Evison JM, et al.: Intensive care unit admission in patients with haematological disease: incidence, outcome and prognostic factors. Swiss Med Wkly 2001, 131:681-688. g Results The mean volume of starch per patient administered before and after electronic prescription options were altered was 480 ml and 21 ml, respectively (P = 0.004). See Figure 1. y 3. Beed et al.: Intensive care management of patients with haematological malignancy. Conti Edu An Crit Care Pain 2010, 10:167-171. 4. Kleber et al.: Comorbidity as a prognostic variable in multiple myeloma: comparative evaluation of common comorbidity scores and use of a novel MM–comorbidity score. Blood Cancer J 2011, 1:e35. Conclusion Despite clinicians intending to reduce the use of starch it was still regularly administered on our ICU. The removal of a default prescribing option dramatically reduced the volume of starch used whilst not restricting the ability to make a conscious choice to prescribe it. Adjusting default options has potential to infl uence clinical decisions and ensure more reliable, evidence-based care. Reference 5. McGrath S, et al.: ICU and 6-month outcome of oncology patients in the intensive care unit. QJM 2010, 103:397-403. P517f 1. Schultz MJ, et al.: J Crit Care 2005, 20:199-204. Eff ect of organizational structure of the ICU on the prognosis: open format versus semi-closed format T Taniguchi, M Okajima Kanazawa University Hospital, Kanazawa, Japan Critical Care 2013, 17(Suppl 2):P517 (doi: 10.1186/cc12455) T Taniguchi, M Okajima P518 Use of defaults on an electronic prescribing tool infl uences the type of fl uid received by patients L Herbert, C Bordeaux, M Thomas, L Burrows, J Bewley, T Gould Bristol Royal Infi rmary, Bristol, UK Critical Care 2013, 17(Suppl 2):P518 (doi: 10.1186/cc12456) Use of defaults on an electronic prescribing tool infl uences the type of fl uid received by patients Automated, electronic monitoring for early detection of sepsis S Rauch1, M Fischer1, C Martin2, A Sablotzki3 Conclusion Our results suggest that a semi-closed format is a more favorable setting than an open format to improve mortality in the ICU and to warrant safe outcome in this patient group. P519 Kanazawa University Hospital, Kanazawa, Japan Automated, electronic monitoring for early detection of sepsis S Rauch1, M Fischer1, C Martin2, A Sablotzki3 1Alb Fils Kliniken, Göppingen, Germany; 2Fa. Löser, Leipzig, Germany; 3Klinikum Sankt Georg, Leipzig, Germany Critical Care 2013, 17(Suppl 2):P519 (doi: 10.1186/cc12457) Use of defaults on an electronic prescribing tool infl uences the type of fl uid received by patients l y p L Herbert, C Bordeaux, M Thomas, L Burrows, J Bewley, T Gould Bristol Royal Infi rmary, Bristol, UK Critical Care 2013, 17(Suppl 2):P518 (doi: 10.1186/cc12456) l y p L Herbert, C Bordeaux, M Thomas, L Burrows, J Bewley, T Gould Bristol Royal Infi rmary, Bristol, UK yi y Critical Care 2013, 17(Suppl 2):P518 (doi: 10.1186/cc12456) Introduction Many evidence-based interventions are not delivered to patients [1]. This may not be due to a clinician’s intentional decisions. The aim of this project was to compare the use of starch before and after removing it as an option from an e-prescribing template. Conclusion Real-time alerts using an automated, electronic monitoring of modifi ed systemic infl ammatory response syndrome criteria facili- tate the clinical diagnosis of sepsis. S192 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 P520 score correlations between three raters were excellent (r  =  0.80 to 0.94). Internal consistency in 98 videotaped patient presentations was acceptable (α = 0.78). Application to 22 IDRs led by 14 diff erent intensivists in three ICUs in two hospitals demonstrated that indicators could be unambiguously rated. The staff and management of all three ICUs that were rated had considered their IDRs to be adequately performed, and they were surprised by these study results. score correlations between three raters were excellent (r  =  0.80 to 0.94). Internal consistency in 98 videotaped patient presentations was acceptable (α = 0.78). Application to 22 IDRs led by 14 diff erent intensivists in three ICUs in two hospitals demonstrated that indicators could be unambiguously rated. The staff and management of all three ICUs that were rated had considered their IDRs to be adequately performed, and they were surprised by these study results. Conclusion This study showed that the quality of IDRs can be reliably assessed for patient plan of care and process. The IDR-Assessment Scale had satisfactory inter-rater reliability, excellent overall item score correlations, and acceptable internal consistency. Our instrument may provide feedback for ICU professionals and managers to develop adjustments in quality of care. Testing the IDR-Assessment Scale in other ICUs may be required to establish general applicability. Reference P520 Admission to the ICU overnight: is it really a bad thing? Leadership training and quality improvement of interdisciplinary rounds in the ICU EC Ten Have, JE Tulleken Universitair Medical Center Groningen, the Netherlands Critical Care 2013, 17(Suppl 2):P522 (doi: 10.1186/cc12460) Introduction The development of patient-centered care by inter- disciplinary teams in the ICU has focused attention on leadership behavior. The purpose of this intervention study was to measure the eff ect of leadership training on the quality of performed inter- disciplinary rounds (IDRs) in the ICU. Methods In this nonrandomized intervention study, participants included nine intensive care medicine fellow trainees (intervention group) and 10 experienced intensivists (control group). Participants in the intervention and control groups previously were untrained in leading IDRs in the ICUs. After each participant led an IDR that was videotaped, the fellow trainees participated in a 1-day leadership training, which was consistent with principles of adult learning and behavioral modeling. After training, each fellow trainee led another IDR that was videotaped. Quality of the performed IDRs was measured by review of videotapes of the 19 IDRs lead by 19 intensivists, including 198 patient discussions subdivided into four ICUs, and assessment with the IDR-Assessment Scale. Conclusion There is no signifi cant diff erence between the mortality of patients admitted overnight and patients admitted during the day to our unit. The hospital length of stay is increased in patients who are admitted overnight to intensive care; however, ICU length of stay is not aff ected. Adjustment for other confounders such as current bed occupancy and staffi ng ratios during the entire patient stay may help to understand the diff erences seen in the hospital length of stay. Reference 1. Kuijsten HA, et al.: Intensive Care Med 2010, 36:1765-1771. f References References y Methods A literature search was performed to identify criteria for instruments about assessing team processes in the ICU. Then, 10 videotaped patient presentations led by diff erent intensivists were analyzed by Delphi rounds. Appropriate and inappropriate behaviors were highlighted. The IDR-Assessment Scale was developed and statistically tested. The inter-rater reliability was evaluated by rating nine randomly selected videotaped patient presentations by three raters. Finally, the scale was applied to 98 videotaped patient presentations during 22 IDRs in three ICUs for adults in two hospitals in Groningen. 1. Ten Have ECM, et al.: Assessing the quality of interdisciplinary rounds in the intensive care unit. J Crit Care 2013, in press. 1. Ten Have ECM, et al.: Assessing the quality of interdisciplinary rounds in the intensive care unit. J Crit Care 2013, in press. 2. Miller A, et al.: Uni- and interdisciplinary eff ects on round and handover content in intensive care units. Hum Factors 2009, 51:339-353. 3. Pronovost PJ, et al.: Perspective: physician leadership in quality. Acad Med 2009, 84:1651-1656. Reference 1. Reader TW, et al.: Developing a team performance framework for the intensive care unit. Crit Care Med 2009, 37:1787-1793. y g y Results Crude ICU and hospital mortality rates in patients admitted during the day and overnight were examined. There was no signifi cant diff erence in unit mortality (day 22.3% vs. night 22.7%, OR = 1.02, 95% CI = 0.91 to 1.16, P = 0.718) or hospital mortality (day 30.7% vs. night 29.1%, OR = 0.93, 95% CI = 0.83 to 1.04, P = 0.203). The mean unit length of stay showed no diff erence in patients admitted during  daytime compared with those admitted overnight (4.27  days vs. 4.09  days, P = 0.162). The mean hospital length of stay was decreased in patients admitted during daytime compared with patients admitted overnight (19.3 days vs. 21.7 days, P = 0.004). The average age of patients was less in those admitted out of hours (night 56.5 years vs. day 59.2 years, P = <.0001). There was no signifi cant diff erence in APACHE II scores of patients between the groups (day 19 vs. night 19, P = 0.580).if Assessing the quality of interdisciplinary rounds EC Ten Have, JE Tulleken Universitair Medical Center Groningen, the Netherlands Critical Care 2013, 17(Suppl 2):P521 (doi: 10.1186/cc12459) Results Comparison of the intervention versus control groups shows that the intervention group has more yes scores on the IDR-Assessment Scale than the control group. This diff erence was signifi cant in 12 of the, in total, 19 quality indicators. g , Critical Care 2013, 17(Suppl 2):P521 (doi: 10.1186/cc12459) Conclusion Quality of leadership will be reliably trained and measured in the context of IDRs in ICUs. Training in a simulation environment, with real-life IDR scenarios including confl icting situations, and workplace- based feedback in the preparation and feedback phases, appears to be eff ective to train leadership behaviour. Introduction Interdisciplinary rounds (IDRs) in the ICU are increasingly recommended to support quality improvement and to reduce confl icts, but uncertainty exists about assessing the quality of IDRs. We developed, tested, and applied a scoring instrument to assess the quality of IDRs in ICUs. Use of defaults on an electronic prescribing tool infl uences the type of fl uid received by patients M Adams, P Dean, K MacDowall, P Stenhouse, A Mackay Victoria Infi rmary, Glasgow, UK Critical Care 2013, 17(Suppl 2):P520 (doi: 10.1186/cc12458) Introduction Admission to hospital overnight has been shown to increase mortality and decrease hospital length of stay [1]. The objective of this study was to determine whether this relationship is valid in patients admitted to our ICU, and whether length of stay was aff ected. y y y Conclusion This study showed that the quality of IDRs can be reliably assessed for patient plan of care and process. The IDR-Assessment Scale had satisfactory inter-rater reliability, excellent overall item score correlations, and acceptable internal consistency. Our instrument may provide feedback for ICU professionals and managers to develop adjustments in quality of care. Testing the IDR-Assessment Scale in other ICUs may be required to establish general applicability. Reference f Methods A retrospective data collection identifi ed 5,827 patients admitted to a fi ve-bed ICU from April 1994 to November 2012. Data regarding patient age, sex, APACHE II score and ICU admission date and time were collected along with the length of stay in the unit and hospital. Defi nitions of day and night were set to local ICU standards of 9:00 am to 8:00 pm. Patients were then separated into two groups and analysed using Analyse-It software for Excel. Let’s go round again! Quality improvement through intentional roundingi g Results The IDR-Assessment Scale had 19 quality indicators, subdivided into two domains: Patient Plan of Care, and Process. The domain Patient Plan of Care refl ects the technical performance from the initial identifi cation of a goal to the evaluative phase. The domain Process refl ects the team processes that are important to ensure that the appropriate plan of care is agreed, understood, and executed as planned by all care providers. Indicators were essential or supportive. The inter-rater reliability of nine videotaped patient presentations among three raters was satisfactory (κ  =  0.85). The overall item P Doyle, F Cox, R Tollyfi eld, A Serajii P Doyle, F Cox, R Tollyfi eld, A Serajii Harefi eld Hospital, Harefi eld, UK ii Critical Care 2013, 17(Suppl 2):P523 (doi: 10.1186/cc12461) Introduction Harefi eld Hospital is a 150-bed cardiothoracic tertiary referral centre with transplantation, artifi cial heart, ECMO and primary angioplasty services. Our 35-bed critical care department consists of 18 intensive therapy unit, seven recovery and 10 high-dependency S193 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 (572.08  ±  214.68 vs. 168.6  ±  97.27 seconds, P  <0.001) but are also associated with higher distractions particularly during evening shifts. Conclusion A higher percentage of nurses received handover training; nursing handovers are longer and more inclusive of other components of patient management; perceived importance of components of handover varies among healthcare professionals; distractions are common during handovers and associated with longer duration, by nurses and in the evening shifts; and higher confi dence level in patient’s management following the handover is associated with better satisfaction. (572.08  ±  214.68 vs. 168.6  ±  97.27 seconds, P  <0.001) but are also associated with higher distractions particularly during evening shifts. beds. Intentional rounds or proactive patient rounds were recognised by the Royal College of Physicians and the Royal College of Nursing [1] as structured, evidence-based processes for nurses to carry out regular checks with individual patients at set intervals. The senior nursing team decided to adapt this initiative to the intensive care setting in order to address clinical challenges and provide guidance for shift leaders to focus on key elements of care. Methods Our intentional rounds, performed once per shift (twice daily), include two components. First, pressure area care – this component involves the shift leader checking whether key elements of pressure sore prevention have been performed. P524 Prospective observational study of handover in a medical ICU A Mukhopadhyay1, B Leong1, A Lua2, R Aroos2, J Wong2, N Koh2, N Goh2, K See1, J Phua1, Y Kowitlawakul3 1National University Health System, Singapore; 2National University of Singapore, Singapore; 3Alice Lee School of Nursing, National University of Singapore, Singapore Critical Care 2013, 17(Suppl 2):P524 (doi: 10.1186/cc12462) Results Over a 6-month period, 131 teleconsultations (114 patients) were done. Mean age was 50.1  years, 57.1% was male and mean APACHE II score was 24.3. A total of 64.8% originated from the ICU and 35.2% from the ED. Main consultation diagnoses were sepsis (31.3%); stroke (29.8%); survival from cardiac arrest (6.1%); trauma (6.1%); and acute myocardial infarction (5.3%). TM improved diagnosis in 14.5% and infl uenced the clinical management in 85.5% of the consultations. Invasive procedures were indicated in 61.1%. Life-saving procedures were TM related in seven patients (6.1%): stroke thrombolysis (n = 6) and limb amputation (n = 1). Seven patients (6.1%) were transferred and submitted to surgical procedures (heart surgery (n = 2), neurosurgery (n = 4) and liver transplantation (n = 1)). The majority of the patients remained at HMMD and were discharged. g p g p Critical Care 2013, 17(Suppl 2):P524 (doi: 10.1186/cc12462) Introduction Handovers are often associated with poor communi ca- tion. ICU patients with multiple complex problems are ideal to study naturally occurring handovers. However, few studies have been conducted in the ICU. Methods We conducted questionnaires of physicians and nurses involved and observed handovers in real time of medical ICU patients over 1 month. Results We interviewed 580 of 672 physicians and nurses involved (86.3%) and observed 90 real-time handovers (45 patients, 26.8%) of 168 patients. Mean duration of handover was 391.3 (± 263.6) seconds, 78.5% were face to face and 1.26 (± 1.75) distractions per handover were noted, person-to-person calling being the commonest mode of distraction (46.7%). Nurses received training during induction in signifi cantly higher numbers, covered allied specialties more and reviewed the patients early (all P  <0.05). Perception of the relative importance of diff erent components of the handover varied signifi cantly between donors, recipients, physicians and nurses. P525 Impact of knowledge transfer through the implementation of a telemedicine program in a community hospital in Brazil CA Abreu Filho1, M Steinman1, A Andrade2, R Cal1, N Akamine1, J Teixeira2, E Silva1, A Kanamura1, M Cenderoglo1, C Lottenberg1 1Hospital Israelita Albert Einstein, São Paulo, Brazil; 2Hospital Municipal Dr. Moysés Deutsch, São Paulo, Brazil Critical Care 2013, 17(Suppl 2):P525 (doi: 10.1186/cc12463) Results The incidence of pressure ulcers in the 4  months since the initiative began has averaged 2.25 per month compared with 7.8 per month prior to commencement of intentional rounding. Added to the rounding tool at the end of September 2012, RRT rates in the preceding 4 months averaged 31.5 ml/kg/hour over 24 hours, an 11.9% reduction from the previous average of 35.75 ml/kg/hour. If the pattern of RRT was to continue, this could equate to a cost saving of UK£40,000 per annum. 1Hospital Israelita Albert Einstein, São Paulo, Brazil; 2Hospital Municipal Dr. Moysés Deutsch, São Paulo, Brazil C l C (S l ) P (d / ) Introduction Emergency survival rates vary signifi cantly according to the quality of care, which depends on human and technological resources. Emergency and critical care medicine physicians must make fast decisions; the presence of experienced consultants improves survival. In developing countries, there is a shortage of skilled doctors. The aim is to describe the fi rst Brazilian initiative of real- time teleconferencing telemedicine (TM) providing 24/7 emergency department (ED) and ICU coverage. Introduction Emergency survival rates vary signifi cantly according to the quality of care, which depends on human and technological resources. Emergency and critical care medicine physicians must make fast decisions; the presence of experienced consultants improves survival. In developing countries, there is a shortage of skilled doctors. The aim is to describe the fi rst Brazilian initiative of real- time teleconferencing telemedicine (TM) providing 24/7 emergency department (ED) and ICU coverage. Conclusion The use of a modifi ed targeted intentional rounding tool by the nursing shift leader can help ensure that best practice guidelines are adhered to. This strategy can improve patient outcomes and provide potentially signifi cant fi scal benefi ts. f 1. RCP, RCN: Ward Rounds in Medicine. Principles for Best Practice. London: Royal College of Physicians, Royal College of Nursing; 2012. 1. RCP, RCN: Ward Rounds in Medicine. Principles for Best Practice. London: Royal College of Physicians, Royal College of Nursing; 2012. References 1. Ye K, et al.: Handover in the emergency department: defi ciencies and adverse eff ects. Emerg Med Australas 2007, 19:433-441 1. Ye K, et al.: Handover in the emergency department: defi ciencies and adverse eff ects. Emerg Med Australas 2007, 19:433-441 2. Apker J, et al.: Communicating in the ‘gray zone’: perceptions about emergency physician hospitalist handoff s and patient safety. Acad Emerg Med 2007, 14:884-894. Let’s go round again! Quality improvement through intentional roundingi These include completion of the Waterlow risk assessment tool [2], noting the frequency of repositioning, use of lateral positioning and pressure-relieving pads. Second, renal replacement therapy rates – this element was identifi ed as an area for focus after we established that our haemofi ltration fl uid use per hour of therapy was twice that of a near identical clinical setting. This pattern continued even after adopting similar therapy guidelines. The shift leader was guided to check whether therapy rates had been adjusted in line with latest biochemical results. P525 g Methods Since May 2012 a TM program has been implemented at two hospitals in São Paulo, Brazil  – Hospital Municipal Dr. Moysés Deutsch (HMMD), a public, secondary hospital, and Hospital Israelita Albert Einstein (HIAE), a tertiary private philanthropic entity – due to a partnership with the Brazilian Health Ministry. TM Central Command was located at HIAE with Endpoint 97 MXP Cisco® Solution and a mobile Intern MXP ISDN/IP Cisco® for the remote hospital (HMMD) via dedicated GB/sec connection. Imaging examinations were evaluated using PACS technology. Every recruited patient was assessed by the Central Command through TM with an experienced consultant. 2. Waterlow J: The importance of accurate risk assessment and appropriate intervention in tissue viability. Br J Nurs 2009, 18:1090. 2. Waterlow J: The importance of accurate risk assessment and appropriate intervention in tissue viability. Br J Nurs 2009, 18:1090. P524 Both physicians and nurses seldom (39.7%) reviewed the available electronic past medical records of the patient before handover, which in addition to training in handover and overall confi dence level in the management following handover are signifi cantly associated with better satisfaction in univariate analysis; only the confi dence level in patient management remained signifi cant after multivariate analysis. However, agreement between donor and recipient on overall satisfaction was poor (P >0.05). Nursing handovers were signifi cantly longer than physicians’ Conclusion A TM program is feasible to be implemented in a community hospital. The major benefi t is expertise medical transfer from the tertiary hospital to the community setting, improving diagnosis and management of critical care patients, and avoiding routine transfer to a major urban center. P526 P526 Utilization of telemedicine to improve burn care in a developing country J Knittel, G Fuzaylov Massachusetts General Hospital, Boston, MA, USA Critical Care 2013, 17(Suppl 2):P526 (doi: 10.1186/cc12464) Alternative to improve palliative care for all patients and families in critical care units: development and preliminary evaluation following MRC guidance of the King’s Psychosocial, Assessment and Care toolf Results Since 2011 we have provided consultation on 14 patients in Lviv, Ukraine, ranging in age from 15 months to 63 years. Each patient had an average of six consultations. We present two of these cases as examples of the capabilities of our telemedicine program. The fi rst case involved a 15-month-old female with 40% TBSA from scald injury, where telemedicine was instrumental in the primary assessment as well as to arrange a direct assessment from a nearby burn surgeon. The second case resulted from a house fi re with multiple casualties, where physicians in Boston were able to utilize telemedicine to guide the initial resuscitation and airway management of three critically burned children, as well as to arrange for transport of one of the victims, an 11-year-old male with 87% TBSA, from Ukraine to the USA for acute management. Multiple diffi culties were overcome in implementing the system between the two countries including: time zone diff erences, language barrier, and diff erent approaches to patient care. I Higginson, C Rumble, J Koff man, P Hopkins, S Heenen, W Prentice, R Burman, S Leonard, O Dampier, J Noble, M Morgan, C Shipman King’s Health Partners AHSC, London, UK Critical Care 2013, 17(Suppl 2):P528 (doi: 10.1186/cc12466) Introduction More than one in fi ve people admitted to an ICU will die there. Research has highlighted concerns about support for patients and families and decision-making in this context [1,2]. Here, we describe the development and evaluation of a tool to improve palliative care in a 32-bed general ICU in a central London teaching hospital. Methods Medical Research Council guidance for complex interventions Phase 0 to I comprised literature review, theoretical modelling, obser- vation and qualitative interviews and focus groups with staff and families exploring concerns and views of interventions identifi ed in the literature review. Phase II comprised intervention development, implementation and evaluation of tool feasibility and eff ects using staff survey, observation, audit of records and relative survey.f f Conclusion We have established a telemedicine program linking physicians in Boston, MA, USA with City Hospital #8 in Lviv, Ukraine to improve care in pediatric and adult burn patients. Our program has provided consultation on 14 patients since 2011, and it highlights the capabilities of telemedicine for acute consultation as well as triage and transport of critically ill patients to tertiary-care facilities. Diff erent aspects of therapy limitation: a comparative study of the nurse’s view É O Szűcs1, G Élő1, L Szabó1, C Varga2, J Gál1, L Zubek1 1Semmelweis University, Budapest, Hungary; 2University of Kaposvár, Hungary Critical Care 2013, 17(Suppl 2):P527 (doi: 10.1186/cc12465) O Szűcs1, G Élő1, L Szabó1, C Varga2, J Gál1, L Zubek1 1Semmelweis University, Budapest, Hungary; 2University of Kaposvár, Hungary Critical Care 2013, 17(Suppl 2):P527 (doi: 10.1186/cc12465) Introduction During the last few  years the frequency of end-of- life decisions (EOLD) signifi cantly increased in ICUs. The method of nurse involvement in making EOLD is diff erent worldwide [1,2]. The purpose of this study was to analyze opinions of nurses about therapy restriction. We have examined with a multicenter study the opinions of the medical stuff about end-of-life care in Hungarian ICUs. Methods We performed a questionnaire evaluation among physicians and nurses of ICUs about infl uencing factors of therapy restriction, the method of the decision-making process, and the frequency of diff erent EOLD. The questionnaire, containing 21 questions, was delivered electronically to Hungarian ICUs, and then we analyzed the responses anonymously. The retrieved 302 answers (191 physicians, 102 nurses) were analysed using a nonparametric Student’s test. Conclusion K-PACE is a feasible tool to improve the palliative care of patients and their families in the ICU. Further refi nement is needed and planned, with consideration of roll-out into the wider medical centre. References 1. Azoulay E, et al.: Half the families of ICU patients experience inadequate communication with physicians. Crit Care Med 2000, 28:3044-3049 1. Azoulay E, et al.: Half the families of ICU patients experience inadequate 1. Azoulay E, et al.: Half the families of ICU patients e communication with physicians. Crit Care Med 2000, 28:3044-3049.l Results A total 71% of the nurse responders work in university clinics, 2% in regional centrum, 24% in municipal hospital, 3% in other ICUs. The nurses found both human (2.72/5 vs. 1.98/5) and material (2.81/5 vs. 2.12/5) resources more restrictive factors during patient admission than physicians (P = 0.025, P = 0.0024). Nurses working in municipal hospital were more strongly infl uenced by lack of material and human resources (3.34/5, 3.3/5) than nurses working in university clinics (2.2/5, 2.43/5), P = 0.01, P = 0.025. Younger nurses (working between 6 and 10 years) were more interested in the patient’s or surrogate’s wishes than older nurses (working more than 10  years). P528 P528 Alternative to improve palliative care for all patients and families in critical care units: development and preliminary evaluation following MRC guidance of the King’s Psychosocial, Assessment and Care tool I Higginson, C Rumble, J Koff man, P Hopkins, S Heenen, W Prentice, R Burman, S Leonard, O Dampier, J Noble, M Morgan, C Shipman King’s Health Partners AHSC, London, UK Critical Care 2013, 17(Suppl 2):P528 (doi: 10.1186/cc12466) country y J Knittel, G Fuzaylov Massachusetts General Hospital, Boston, MA, USA Critical Care 2013, 17(Suppl 2):P526 (doi: 10.1186/cc12464) Introduction Our objective is to present our experience from Shriner’s Hospital and Massachusetts General Hospital in Boston, MA, USA in S194 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 physicians slightly involved them in the end-of-life decision-making process (2.1/5 vs. 2.4/5 P = 0.0001). using telemedicine to provide acute burn and critical care consultation on pediatric and adult burn patients in Lviv, Ukraine, as well as in triage and transport of critically ill patients from Lviv to a tertiary-care facility in the USA for further management. using telemedicine to provide acute burn and critical care consultation on pediatric and adult burn patients in Lviv, Ukraine, as well as in triage and transport of critically ill patients from Lviv to a tertiary-care facility in the USA for further management. Conclusion We found that the workplace, level of medical attendance, godliness, work experience, and position in medical staff strongly infl uenced making EOLD. While limitation of the therapy should be team work, nurses felt their opinions were hardly taken into consideration, although nurses seemed to be more realistic in the decision-making process. Methods Using a new telemedicine learning center established at City Hospital #8 in Lviv, Ukraine, consultations regarding acutely injured burn victims occurred between physicians in Ukraine and physicians at Shriners Hospital and Massachusetts General Hospital in Boston. After the initial presentation, each patient was reviewed on a daily basis by physicians in Boston. Skype, an Internet-based communication tool, was used in communication with the Burn Center in Lviv. Radiographic images were scanned and digitalized using an electronic scanner, and JPEG image compression was used to facilitate the transmission of radiographic images and patient charts. Informed consent and HIPPA guidelines were followed in transmitting any patient-related information. Diff erent aspects of therapy limitation: a comparative study of the nurse’s view É Religion did not infl uence patient admission and forego therapy; however, religious nurses compared with atheists and nonpracticing believers preferred to prolong therapy against the patient’s will (P = 0.04). Nurses felt that communication with physicians. Crit Care Med l 2. Asch DA, et al.: Confl icts between physicians’ practices and patients’ wishes. Am J Respir Crit Care Med 1995, 151:288-292. P527f P527 Diff erent aspects of therapy limitation: a comparative study of the nurse’s view O Szűcs1, G Élő1, L Szabó1, C Varga2, J Gál1, L Zubek1 1Semmelweis University, Budapest, Hungary; 2University of Kaposvár, Hungary Critical Care 2013, 17(Suppl 2):P527 (doi: 10.1186/cc12465) Diff erent aspects of therapy limitation: a comparative study of the nurse’s view É References References . Prendergast et al.: A national survey of end-of-life care for critical 1. Prendergast et al.: A national survey of end-of-life care for critic 1. Prendergast et al.: A national survey of end-of-life care for critically ill patients. Am J Respir Crit Care Med 1998, 158:1165-1167. 2. Banbenishty et al.: Nurse involvement in end-of-life decision making: the ETHICUS Study. Intensive Care Med 2006, 32:129-132. 2. Banbenishty et al.: Nurse involvement in end-of-life decision making: the ETHICUS Study. Intensive Care Med 2006, 32:129-132. Alternative to improve palliative care for all patients and families in critical care units: development and preliminary evaluation following MRC guidance of the King’s Psychosocial, Assessment and Care toolf y y Results Phase I: 47 staff and 24 family members were interviewed. The short time between decisions for treatment withdrawal and death, plus concerns for support management, communication and decision-making, highlighted a need to ensure excellent psychosocial assessment for all. Phase II: as part of integrated care guidelines, we developed the King’s Psychosocial Assessment and Care tool (K-PACE). K-PACE is used for all patients entering the ICU, completed within 24  hours of admission. It contains psychosocial assessment of the family and patient needs, and identifi es key individuals for contact. Educational training was supported by K-PACE and was implemented in two waves. Post-implementation survey of 95 ICU staff found that most (80%) were aware of K-PACE. Eighty-two per cent of nurses but only 17% of doctors had completed the tool. In total, 158/213 (74%) family members responded to the survey (additionally three patients responded). There were high levels of satisfaction for symptom control and psychosocial care but concerns continued regarding explanation of treatment and care. P529 d End-of life thoughts in the ICU: results of a survey A Yaguchi, M Namiki, N Saito, R Nagai, M Takeda, T Harada, R Moroi Tokyo Women’s Medical University, Tokyo, Japan Critical Care 2013, 17(Suppl 2):P529 (doi: 10.1186/cc12467) End-of life thoughts in the ICU: results of a survey A Yaguchi, M Namiki, N Saito, R Nagai, M Takeda, T Harada, R Moroi Tokyo Women’s Medical University, Tokyo, Japan Critical Care 2013, 17(Suppl 2):P529 (doi: 10.1186/cc12467) y y y p Critical Care 2013, 17(Suppl 2):P529 (doi: 10.1186/cc12467) Introduction The decision of terminal care in the ICU is a very tough issue because the law, ethics, traditions and futility should S195 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 that withholding and withdrawing of treatment were ethically equal. A DNR would be used most often. Withdrawal of inotropes or antibiotics would be used more often than withdrawal of mechanical ventilation or extubation. Termination of artifi cial hydration would be rarely used in practice. be concerned involving the family’s will. Especially, stopping or withdrawing therapy is a quite diffi cult operation in Japan because of legal issues. Our hypothesis is that some diff erence exists in thoughts between physicians and nurses for terminal patients in the ICU. The aim of this study is to know their real thoughts. y g Methods A questionnaire survey was performed on physicians and nurses in our medico-surgical ICU. The questionnaire consists of 11 questions with fi ve optional answers related to the thoughts of participants about treatment of hopeless or brain death patients. Concretely, the questions were; whether to withhold therapy or not, whether to accept to withdraw therapy or not and with family’s will, whether to accept to immediately stop therapy and with family’s will, whether to positively or not donate organs from a brain death patient, necessity of ICU care for brain death patients, and feeling guilty and stress for stopping or withdrawing therapy. The optional answer has fi ve gradations from ‘Yes’ to ‘No’ for all questions. The participants were asked to answer the questionnaire by expressing themselves without regarding legal issues or the consensus. It was guaranteed to be anonymous for them in the data analysis. The answers were compared between physicians and nurses. The Mann–Whitney U test was used for statistical analysis. P <0.05 was considered statistically signifi cant. P529 d P531 Attitudes of intensivists in the UK to withdrawal of futile therapy M Mariyaselvam, M Irvine, J Carter, M Blunt, P Young Queen Elizabeth Hospital, Kings Lynn, UK Critical Care 2013, 17(Suppl 2):P531 (doi: 10.1186/cc12469) Introduction We aimed to determine the current practice and attitudes of consultants in intensive care medicine when withdrawing futile life-sustaining therapy. Published guidelines suggest variation in withdrawal of futile life-sustaining therapy and are therefore not prescriptive [1]. Although there is an awareness of diff ering practices, the extent of these variations is not established. Methods We surveyed a convenience sample of delegates at the Intensive Care Society (UK) State of the Art Meeting (2012) on attitudes and practice regarding withdrawal practice. Anonymised data were collected using surveymonkey.com. i Results There were in total 52 participants (response rate 98.1%) with 20 physicians and 32 nurses. Withdrawing therapy was signifi cantly accepted in nurses than in physicians (83% vs. 55%, P = 0.039), when the family well understood. Withholding therapy should not be operated for brain death patients for physicians (65%), while it seemed a diffi cult judgement for nurses (23%, P = 0.021). ICU care for brain death patients is less necessary for physicians than nurses (80% vs. 53%, P = 0.016). There were no signifi cant diff erences in other questions between physician and nurses such as feeling guilty or stress for stopping or withdrawing therapy.f Results Of 457 consultant attendees from the UK, 149 completed the survey (33%). For 58% of consultants there was no formal institutional protocol for withdrawal of futile therapy. When deciding to withdraw therapy, 57% of consultants routinely seek and document a second opinion. Regarding donation after cardiac death (DCD), 93% of consultants were happy to delay withdrawal to facilitate successful donation, 85% have already done so in their practice and 14% routinely withdraw therapy in theatres rather than on the ICU. Even if it would impact on the care of other patients, 48% would delay withdrawal of therapy to facilitate DCD. For patients accepted for DCD, 36% think that some intensivists withdraw more aggressively (in essence, hasten death) in the hope of improving the likelihood of a successful organ donation and 29% have felt pressurised to withdraw therapy more quickly than their usual practice. Furthermore, 45% experienced pressure to refer a patient for DCD when it they felt it was not appropriate. End-of-life decisions in Slovenian ICUs: a cross-sectional survey S Grosek1, M Orazem2, M Kanic2, G Vidmar3, U Groselj1 Conclusion This survey confi rms variation in the practice and attitudes to withdrawal of futile therapy amongst UK consultant intensivists. Formal protocols were frequently unavailable to guide withdrawal and second opinions were often not sought. Nearly one-half of the intensivists delay withdrawal to facilitate donation, even if this may impact on the care of other patients. Many intensivists have felt pressure to refer for donation when they feel this is inappropriate and there is a perception that some intensivists may withdraw care more aggressively in those who are accepted for DCD to improve the likelihood of a successful donation. This survey may help inform debate in this ethically challenging area. Critical Care 2013, 17(Suppl 2):P530 (doi: 10.1186/cc12468) Introduction The purpose of our study was to assess the attitudes of Slovenian intensivists towards end-of-life (EOL) decision-making and to analyze the decision-making process in their clinical practice. Methods A cross-sectional survey among Slovenian intensivists and intensive care medicine residents from 35 diff erent ICUs was performed using a questionnaire containing 43 questions about views on EOL decision-making. Fisher’s exact test and the Fisher–Freeman–Halton test were applied to cross-tabulated data; signifi cance level was set at P ≤0.001 due to the large number of tested hypotheses. Reference Reference g yp Results The response rate was 72.1% (267 questionnaires were returned out of 370 distributed), which represented roughly the same percentage of all Slovenian intensivists. Termination of futile treatment was assessed as ethically acceptable (P  <0.001). The statement that there is no ethical distinction between withholding and withdrawing of treatment could not be confi rmed (the answers ‘there is a diff erence’ and ‘undecided’ were less frequent, but not statistically signifi cant; P = 0.216). A do-not-resuscitate order (DNR) was used more often than other withholding treatment limitations (P <0.001). A DNR was used most frequently in internal medicine ICUs (P <0.001; compared with paediatric and surgical ICUs). Withdrawal of inotropes or antibiotics was used more often than withdrawal of mechanical ventilation or extubation (66.7% vs. 12.0%; P  <0.001). Withdrawal of mechanical ventilation or extubation was more often used in the paediatric ICUs (21.7%) as compared with the internal medicine ICUs (19.6%) and the surgical ICUs (3%) (P <0.001). Over two-thirds (70.6%) of intensivists were against termination of hydration, which would be more often used in the internal medicine ICUs (P <0.001). Thirty-one percent of intensivists used written DNR orders. 1. Organ Donation after Circulatory Death 1. Organ Donation after Circulatory Death [http://www.ics.ac.uk/professional/standards_and_guidelines/dcd] 1. Organ Donation after Circulatory Death [http://www.ics.ac.uk/professional/standards_and_guidelines/dcd] P529 d Conclusion Some of end-of-life thoughts in the ICU showed diff erences between physicians and nurses. P530 P530 End-of-life decisions in Slovenian ICUs: a cross-sectional survey S Grosek1, M Orazem2, M Kanic2, G Vidmar3, U Groselj1 1University Medical Centre Ljubljana, Slovenia; 2 University of Ljubljana, Slovenia; 3University Rehabilitation Institute, Republic of Slovenia, Ljubljana, Slovenia Critical Care 2013, 17(Suppl 2):P530 (doi: 10.1186/cc12468) Feasibility of neuromuscular electrical stimulation in critically ill patients p J Segers, G Hermans, F Bruyninckx, G Meyfroidt, D Langer, R Gosselink UZ/KU Leuven, Belgium Critical Care 2013, 17(Suppl 2):P535 (doi: 10.1186/cc12473) J Segers, G Hermans, F Bruyninckx, G Meyfroidt, D Langer, R Gosselink UZ/KU Leuven, Belgium Critical Care 2013 17(Suppl 2):P535 (doi: 10 1186/cc12473) Results Seven terminally ill patients wishing to go home for dying were transferred home. Diagnoses varied: end-stage lung disease, cancer, surgical complications. Ages ranged from 68 to 84 years. All patients survived transport home, and time at home varied from a few hours to 4 days. Later contacts with patients’ families indicated that both patient and family were grateful, and that they did not experience the patient having pain or dyspnea at home. Introduction Survivors of critical illness often have a prolonged stay on the ICU. These patients may suff er from ICU-acquired weakness. It has been shown that reduction in muscle mass and muscle strength occurs early after admission to the ICU. However, in the very early stage on the ICU, patients are often sedated and not able to participate in any active mobilizations. Therefore the use of neuromuscular electrical stimulation (NMES) is becoming a treatment of interest in the ICU. The aim was to study the feasibility and safety of NMES in a surgical and medical ICU of a large, tertiary referral university hospital. Conclusion Sending critically ill patients home to die is not common. Anyhow, our experiences doing that are only positive. Terminally ill patients, awake to make a decision of their own, and in a condition making it possible, should have the choice to go home to die, with our help in logistics, planning and transportation. Methods Fifty patients with an expected prolonged stay on the ICU of 5 more  days (judged on  day 3) with no trauma or neurological disease were included. They then received daily a NMES session (DUO 500; Gymna, Belgium) for 25 minutes on the quadriceps bilaterally during their entire stay on the ICU. The main outcome was the ability to produce a contraction of the quadriceps through NMES. The muscle contraction was quantifi ed on a 5-point scale: 1 (no contraction palpable and visible) up to 5 (contraction very well palpable and visible). Patients were classifi ed as responders when an adequate muscle bulk was obtained in ≥75% of the sessions. Getting critical care patients home for end-of-life care LS Nielsen Kolding Sygehus, Kolding, Denmark Critical Care 2013, 17(Suppl 2):P533 (doi: 10.1186/cc12471) Introduction Despite our eff orts in making patients healthy and going home, critical illness has a mortality, in Danish ICUs, of 10 to 12%. Approximately 90% of deaths in ICUs happen after life-sustaining therapy has been withheld or withdrawn. Although trying to provide patients and family with what we suppose is a good death, most patients would prefer dying at home, and sometimes patients and family ask for the possibility of doing that. The last 2 years we transferred seven patients from our unit to end-of-life care in their own home. Conclusion The 8-week exercise intervention resulted in statistically signifi cant improvements in fi tness at 9 weeks while focus group participants highlighted the positive eff ects of the exercise intervention leading to enhanced energy levels, motivation and achievement. Psychological benefi ts of the exercise programme are apparent from the focus group, emphasising the important link between physical and mental health. Methods After making the decision of withholding or withdrawing intensive care therapy, the care of the patient changes from an active, medical, technological treatment to relief and care. In that period we determine whether the patient’s condition is stable enough to go home. We try to fi nd out if it is a wish for the patient to go home, and if the family has resources to take care of the patient at home. If that is the case, we start planning care at home, arrange for transportation, and contact the primary care physician and nurse. Due to the patient’s condition on the day of transferring, we plan following the patient by either a nurse or a doctor. P535 Parent’s perception on end-of-life care in Brazil P Lago, J Piva, G Halal, M Halal Hospital de Clinicas de Porto Alegre, Brazil Critical Care 2013, 17(Suppl 2):P532 (doi: 10.1186/cc12470) Parent’s perception on end-of-life care in Brazil P Lago, J Piva, G Halal, M Halal Hospital de Clinicas de Porto Alegre, Brazil Critical Care 2013, 17(Suppl 2):P532 (doi: 10.1186/cc12470) Introduction The aim of the study is evaluate the perceptions of parents of children who died in two Brazilian pediatric ICUs. Introduction The aim of the study is evaluate the perceptions of parents of children who died in two Brazilian pediatric ICUs. Methods An exploratory-descriptive study with a qualitative approach in the PICU of Hospital São Lucas and Hospital de Clinicas de Porto Alegre involving 15 parents of children who died. Data collection was performed through three steps: (a) the researchers contacted the parents through a telephone call to invite them to attend the hospitals; (b) at the hospital, the doctors who assisted the children clarifi ed doubts about the therapy off ered; and (c) an interview was carried out by two researchers not involved in the care. Data analysis was performed using the technique of thematic content analysis. Conclusion Termination of futile treatment was found to be ethically acceptable for Slovenian intensivists, although they were not convinced S196 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 arm of the study, we examined the patient experience after critical illness, their views of the exercise programme and the eff ects on their HRQoL. Results and conclusion The research shows that the diffi culty of com- muni cation is a factor that impacts negatively on the grieving process. Moreover, it stresses the importance for parents to rediscuss the moment of their child’s death with health professionals. Methods A randomised controlled trial was undertaken in adult survivors of ICU admission. They were allocated to receive an 8-week in-hospital supervised aerobic programme consisting of two cycle ergometry and one unsupervised session per week (exercise group) or no exercise (control group). Primary outcomes were the anaerobic threshold (in ml O2/kg mass/minute), physical function and mental health scores (SF-36 questionnaire), measured at weeks 9 and 26. Participants were then allocated to focus groups where the interpretation of experiences was compared with outcomes from the PIX study. 1. Meyer E, Ritholz M, Burns J, Truog R: Improving the quality of end-of-life care in the pediatric intensive care unit: parents priorities and recommendations. Pediatrics 2006, 3:469-457. 2. Meyer E: On speaking less and listening more during end-of-life conferences. Feasibility of neuromuscular electrical stimulation in critically ill patients The potential factors associated with the feasibility were: gender, age, body mass index (BMI), diagnosis of sepsis, Barthel index prior to admission to the hospital, APACHE II score, Glasgow Coma Scale (GCS), fi ve questions for adequacy, stimulus intensity and leg edema. A multiple regression analysis was performed to identify the factors determining whether or not a contraction could be expected in a patient. Safety of NMES was assessed through heart rate, blood pressure, oxygen saturation and respiratory rate. 1. Lusardi P, et al.: The going home initiative: getting critical care patients home with hospice. Crit Care Nurse 2011, 31:46-57. P534 Project PIX (Post Intensive care eXercise): impact on physical fi tness and focus group analysis of quality of life following exercise rehabilitation Parent’s perception on end-of-life care in Brazil P Lago, J Piva, G Halal, M Halal Hospital de Clinicas de Porto Alegre, Brazil Critical Care 2013, 17(Suppl 2):P532 (doi: 10.1186/cc12470) Crit Care 2004, 32:1609-1611. 3. Piva, JP, Lago P, Othero J, Garcia PC: Evaluating end of life in ten Brazilian pediatric and adults intensive care units. J Med Ethics 2010, 36:344-348. 3. Piva, JP, Lago P, Othero J, Garcia PC: Evaluating end of life in ten Brazilian pediatric and adults intensive care units. J Med Ethics 2010, 36:344-348. 4. Meert, K et al.: Parents’ perspectives on physician-parent communication near the time of a child’s death in the pediatric intensive care unit. Pediatr Crit Care 2008, 9:2-7. 4. Meert, K et al.: Parents’ perspectives on physician-parent communication near the time of a child’s death in the pediatric intensive care unit. Pediatr Crit Care 2008, 9:2-7. y Results Fifty-nine patients were recruited to the study. The anaerobic threshold increased at week 9 in the exercise group by a clinically and statistically signifi cant amount of 2 ml O2/kg mass/minute (90% CI, 1 to 3 ml/kg/minute). There was further improvement in fi tness levels in both groups by week 26 (although no signifi cant diff erence between groups). No signifi cant diff erence in HRQOL measures between groups was demonstrated; however, the exercise group did show an improvement in their mental health scores. The focus groups centred on feelings of isolation, abandonment, vulnerability, dependency and reduced physical activity post hospital discharge. Many reported a lack of social inclusion as they did not have the energy or confi dence to venture outside. However, those in the exercise group felt that the rehabilitation programme was motivating, built up confi dence, improved fi tness, helped social interaction and gave them a sense of achievement. P533 G i Getting critical care patients home for end-of-life care LS Nielsen Kolding Sygehus, Kolding, Denmark Critical Care 2013, 17(Suppl 2):P533 (doi: 10.1186/cc12471) Implementation of an ICU follow-up clinic: outcomes and patient satisfaction after 1 year Implementation of an ICU follow-up clinic: outcomes and patient satisfaction after 1 year Implementation of an ICU follow-up clinic: outcomes and patient satisfaction after 1 year y Results Sixty-two patients matched inclusion criteria and were contacted. Thirty-three patients (90.9% males; age 38.7 ± 14.3 years; mean SAPS II 33.75  ±  11.79) were interviewed. Mean ICU stay was 25.7 ± 15.3 days. Fifteen patients (45.5%) had undergone temporary tracheostomy. Twenty-six patients were employed at the moment of trauma: of these, 11 (42.3%) stated to be jobless and one was pensioned at follow-up. In the whole cohort, the unemployment rate increased from 12.1 to 45.6%. Mean PCS was 43.59 ± 6.43, mean MCS was 41.04 ± 7.69. At follow-up, the 14 employed patients had a lower MCS versus the 19 jobless patients (37.94 ± 7.14 vs. 43.33 ± 7.44; P = 0.04), while no diff erences concerning PCS were found. The three patients that were living on their own when the trauma occurred showed, at follow-up, a lower mean MCS score towards the rest of the cohort (32.2 ± 0.47 vs. 41.93 ± 7.51; P <0.001). Introduction The aim was to analyse the outcomes and patient satisfaction of a recently implemented ICU follow-up clinic. These clinics are National Institute for Clinical Excellence recommended [1]. Introduction The aim was to analyse the outcomes and patient satisfaction of a recently implemented ICU follow-up clinic. These clinics are National Institute for Clinical Excellence recommended [1]. Methods A retrospective analysis of prospective collected data from January to December 2012. The clinic is run monthly by an ICU consultant and a critical care outreach sister. Criteria to be invited to the clinic are mechanical ventilation ≥3 days. Patients fi lled an anonymous satisfaction survey after the clinic. Methods A retrospective analysis of prospective collected data from January to December 2012. The clinic is run monthly by an ICU consultant and a critical care outreach sister. Criteria to be invited to the clinic are mechanical ventilation ≥3 days. Patients fi lled an anonymous satisfaction survey after the clinic. y Results Our attendance rate is 50% (26 patients), which is similar to other series reported in the literature. Those patients who attended the clinic required a longer length of mechanical ventilation (5.3 days vs. 7.1) and a longer length of stay in the ICU (7.6 vs. 13) and in hospital (14 vs. 28). Follow-up and counseling service in trauma patients: needs and goals – a preliminary study DL Grieco, M Biancone, SL Cutuli, G Pintaudi, MT Santantonio, ES Tanzarella, F Toni, AG De Belvis, MG Bocci, C Sandroni, M Antonelli Catholic University School of Medicine, Rome, Italy Critical Care 2013, 17(Suppl 2):P536 (doi: 10.1186/cc12474) g p y y DL Grieco, M Biancone, SL Cutuli, G Pintaudi, MT Santantonio, ES Tanzarella, F Toni, AG De Belvis, MG Bocci, C Sandroni, M Antonelli Catholic University School of Medicine, Rome, Italy Critical Care 2013, 17(Suppl 2):P536 (doi: 10.1186/cc12474) Conclusion This survey demonstrates that the provision of ICU clinics in the UK is not well established, with only 48% currently providing a regular service. Currently 67% of clinics are not funded and further resources should be employed so this service becomes an integral part of the ICU pathway. Despite recent evidence demonstrating that diaries reduce new-onset PTSD, only 44% of ICUs in our study provide this service. Ninety-one per cent of ICUs do not provide family support groups; similarly, it appears that fi nancial constraints are the limiting factor. Introduction Trauma is the most common cause of morbidity in young people. It has a high social impact both because of the high cost of the acute treatments and because of the physical and psychological consequences that it may cause. A prospective, observational, single- center study on quality of life 12 to 24 months after trauma was carried out. The aim of the study is to evaluate life quality after trauma and to identify the most important needs of the patients, in order to improve the level of care after an ICU stay and to implement a faster and more eff ective reintegration into the active and productive society. References 1. DH and Modernisation Agency: The National Outreach Report. London: NHS Modernisation Agency; 2003. 2. Jones C, et al.: Crit Care Med 2010, 14:168. 3. Orcutt TA:. Nurs Crit Care 2010, 5:33-37. 1. DH and Modernisation Agency: The National Outreach Report. London: NHS Modernisation Agency; 2003. f Methods All patients admitted to the 18-bed ICU of our university hospital between 5 May 2010 and 28 February 2011 because of a trauma who stayed in the ICU for at least 48 hours were eligible for the study. Patients were contacted and invited to attend a multidisciplinary interview. Data concerning family, working and economic status, main disabilities and medical needs were collected. The SF12 questionnaire was administered to defi ne physical component summary (PCS) and mental component summary (MCS) scores. SPSS 20.0 was used to perform statistical analysis. Project PIX (Post Intensive care eXercise): impact on physical fi tness and focus group analysis of quality of life following exercise rehabilitation Critical Care 2013, 17(Suppl 2):P534 (doi: 10.1186/cc12472) Introduction This study assessed the eff ect of a hospital-based aerobic exercise programme on physical fi tness and health-related quality of life (HRQoL) for survivors of ICU admission. Including the qualitative Results In 48% of the patients we were able to achieve adequate muscle contractions in more than 75% of the sessions. GCS (P = 0.047), edema S197 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 (P = 0.001) and sepsis (P = 0.010) were signifi cantly diff erent between responders and nonresponders. Responders had a lower mean GCS (7 ± 3 vs. 9 ± 3), lower amount of edema and were less likely to have had sepsis. In a multiple regression analysis, sepsis, edema, BMI and age explained 51% of the variance. As for safety, none of the parameters changed signifi cantly. incidence of new-onset PTSD [2]. Family support groups may also play a pivotal role [3]. We performed a national survey via the Intensive Care Society (ICS) to determine the provision of follow-up clinics, patient diary services and family support groups. Methods An online structured questionnaire was sent via the ICS to all ICU linkmen at 124 hospitals in the UK. Responses were received from 77 ICUs, a 62% response rate. Conclusion In patients with a better neurological condition, sepsis and/ or leg edema it was more diffi cult to obtain an adequate quadriceps contraction with NMES. NMES is safe to apply on the ICU. Results Out of 77 ICUs, 37 (48%) run a follow-up clinic. The majority of clinics (51%) only invite patients who have been admitted for more than 3 to 4 days. Only 10 clinics (30%) receive funding from the ICU budget or PCT, with the majority (67%) receiving no funding at all. Only 44% (34) of ICUs use patient diaries, mostly as a nonfunded service (68%). Additionally, 91% of ICUs do not run family support groups; from the minority that do, these are mostly held quarterly and are largely not funded (55%). Implementation of an ICU follow-up clinic: outcomes and patient satisfaction after 1 year We identifi ed a wide range of physical and nonphysical morbidities on these patients (Figure  1). We referred them to the appropriate specialities. Patients were very satisfi ed with this new service (Figure 2). Conclusion Trauma is associated with an increase of the unemployment rate in young people. Patients that are employed at follow-up seemed to have a worse mental health status towards the unemployed ones. Conversely, the employed patients did not show any trend to a lower physical health status. Thus a counseling service that provides psychological care would be useful to implement more eff ective work reintegration for trauma patients. Figure 1 (abstract P538). Physical and nonphysical morbidity. Follow-up and counseling service in trauma patients: needs and goals – a preliminary study Follow-up and counseling service in trauma patients: needs and goals – a preliminary study DL Grieco, M Biancone, SL Cutuli, G Pintaudi, MT Santantonio, ES Tanzarella, F Toni, AG De Belvis, MG Bocci, C Sandroni, M Antonelli Catholic University School of Medicine, Rome, Italy Critical Care 2013, 17(Suppl 2):P536 (doi: 10.1186/cc12474) Introduction Optimal patient evaluations of ICU rehabilitation therapy remain unclear. Introduction Optimal patient evaluations of ICU rehabilitation therapy remain unclear. Methods One hundred ICU patients with acute respiratory failure were randomized to receive early rehabilitation (ER) or usual-care (UC). Cohort 1 (n = 50) received ER as one physical therapy (PT) session/day versus UC; Cohort 2 (n = 50) received ER as 2 PT/day with the second session resistance training, versus UC. UC was without ER. Blood was drawn for cytokines through day 7. Cohort 2 underwent strength and physical functional assessments using the Short Physical Performance Battery (SPPB), a valid and reliable measure of physical function consisting of walking speed, balance, and repeated chair stands. It is a well-studied composite measure in older persons, but has not been used in ICU survivors. Small changes of 0.5 to 0.6 points in the SPPB have been shown to be clinically meaningful. Methods This study is a prospective randomised controlled trial with a repeated-measures crossover design. Eligible participants, requiring mechanical ventilation for 4 or more  days, completed two exercise activities routinely used in early critical care rehabilitation, a passive chair transfer (PCT) and active sitting on the edge of the bed (SOEOB). The oxygen consumption and cardiovascular parameters were measured to quantify and compare the exercise response between the two activities. Results Data are presented as the median (interquartile range). Data for fi ve patients have been collected, aged 68 years (23), with an ITU stay of y g Results Baseline parameters were similar between groups. Median days from enrollment to fi rst PT were 4 (IQR 1 to 7.25). Deaths occurred in eight UC subjects and four in ER (P = 0.22). For both arms, ventilator days, ICU days and hospital days were not statistically diff erent. ER had ventilator-free days of 22, 95% CI = 19.9 to 24.6, where UC had 22.3 days, 19.9 to 24.6, P = 0.99. ICU-free days for ER was 21, 95% CI = 19.1 to 23.6, and that for UC was 21.0, 18.8 to 23.2, P = 0.84. Similarly, hospital days for ER was 16.7, 95% CI = 11.8 to 21.4, and for UC was 18.2, 13.8 to 22.7, P = 0.45. TNF, IL-6 and IL-8 through day 7 were not diff erent between groups. Despite similar baseline acuity and infl ammatory profi les, Cohort 2 ER group strength scores were numerically but not statistically higher. P537 Post-traumatic stress disorder – are we doing all we can? A UK national-based survey X Watson Ashford and St Peter’s Hospital, Surrey, UK Critical Care 2013, 17(Suppl 2):P537 (doi: 10.1186/cc12475) Introduction Post-traumatic stress disorder (PTSD) is a well-recognised complication in patients discharged from the ICU. ICU clinics have been recommended to treat physical and psychosocial problems post discharge, with guidelines issued by the Department of Health UK [1]. Recent evidence has advocated the use of patient diaries to reduce the Figure 1 (abstract P538). Physical and nonphysical morbidity. Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 S198 Critical Care 2013, Volume 17 Suppl 2 http://ccforum com/supplements/17/S2 pp http://ccforum.com/supplements/17/S2 Figure 2 (abstract P538). Patient satisfaction survey. Figure 2 (abstract P538). Patient satisfaction survey. Conclusion Our follow-up clinic has enabled us to identify a wide range of complications related to ICU admission and coordinate their future management. This clinic improved patient satisfaction. Reference 15 days (10.5) and duration of mechanical ventilation 8 days (12), at the point of intervention. Exercise response results are reported (Table 1). Conclusion Intensive care patients with prolonged mechanical ventilation demonstrate a higher rate of oxygen consumption when actively sitting on the edge of the bed, compared with a passive chair transfer. This may have important consequences for early mobilisation of the critically ill. 1. Rehabilitation after Critical Illness [www.nice.org.uk/CG83] References References 1. Unroe et al.: Ann Intern Med 2010, 153:167-175. 2. Thomas: Phys Ther Rev 2011, 16:46-57. Acute exercise response in the critically ill N Collings1, R Young2 g g 1University Hospital Southampton, UK; 2Sheffi eld Hallam University, Sheffi eld, UK Randomized, controlled pilot study of early rehabilitation strategies in acute respiratory failure Critical Care 2013, 17(Suppl 2):P539 (doi: 10.1186/cc12477) Introduction This study aims to quantify the acute exercise response to early passive and active activities in order to inform exercise prescription when designing rehabilitation programmes for the critically ill. Critical care survival is often associated with a poor functional outcome [1], with recent investigations presenting the case for early rehabilitation in order to optimise functional recovery [2]. There, remains, however, a scarcity of research investigating the immediate response to exercise and subsequent exercise prescription, in the acute phase following critical illness. p y D Files, P Morris, S Shrestha, S Dhar, M Young, J Hauser, E Chmelo, C Thompson, L Dixon, K Murphy, B Nicklas, M Berry Wake Forest University School of Medicine, Winston Salem, NC, USA Critical Care 2013, 17(Suppl 2):P540 (doi: 10.1186/cc12478) D Files, P Morris, S Shrestha, S Dhar, M Young, J Hauser, E Chmelo, C Thompson, L Dixon, K Murphy, B Nicklas, M Berry Wake Forest University School of Medicine, Winston Salem, NC, USA Critical Care 2013, 17(Suppl 2):P540 (doi: 10.1186/cc12478) Introduction Optimal patient evaluations of ICU rehabilitation therapy remain unclear. Grip strength, as a percentage of predicted for ER was 66 versus Table 1 (abstract P539) Parameter PCT (n = 5) SOEOB (n = 5) Oxygen consumption (ml/minute) 270 (46.5) 334 (121) Carbon dioxide production (ml/minute) 142 (45.5) 185 (121) Heart rate (bpm) 92 (24) 77.5 (29) Mean arterial pressure (mmHg) 100.3 (20) 97.3 (13) Minute ventilation (l/minute) 11.0 (3.5) 12.1 (3.4) S199 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 39 for UC, P = 0.06. Dynamometry for ER was 211 versus 181 lbs for UC, P = 0.124. Although the diff erence in SPPB values for ER versus UC (5 vs. 3, P = 0.172) was not statistically diff erent, it was greater than the minimal clinically signifi cant diff erence. There were no diff erences in adverse events. or alleviate this stress [4]. To action this it is important to identify the main stressors from the patient’s perspective. A systematic review was performed to provide a list of what patients consider stressors in intensive care. These were then ranked in order to provide an identifi cation tool that can be used to shape appropriate care. Conclusion In this pilot study, early ICU rehabilitation was safe, and was associated with numerically although not statistically shorter hospital stay, greater strength and improved functional scores. Particularly, the SPPB demonstrated discriminatory ability in groups of ICU survivors with low physical function. Future early ICU rehabilitation studies should consider ICU survivor assessments using the SPPB due to its ease, reproducibility and discriminatory ability following ICU and hospital discharge. i Methods A systematic review was performed using MEDLINE, CINAHL, Psych INFO and Academic Search Complete. Grey literature was included and searches were not restricted to type of intensive care or country. Criteria were used to fi lter those articles that identifi ed the patients’ views of their stressor, not the patient experience. Eligible articles were critiqued using the Critical Appraisal Skills Programme for qualitative studies [5] and brought together using a narrative synthesis. All of the reviewed studies used a questionnaire as a means to identify what elements on the intensive care patients found stressful. A list of the top-10 stressors could then be expressed for each study and compared. From this information, a set of guidelines for best practice were devised. y References y p References 1. Baker C, et al.: Physical rehabilitation following critical illness. J Intensive Care Soc 2008, 9:166-169. 2. Jones C, et al.: Long term outcome from critical illness. Anaesth Intensive Care Med 2006, 7:155-156. Results In six hospitals the CQI ‘R-ICU’ is sent to relatives after receiving informed consent (n = 441), 55.1% of the respondents are the patient’s partner. Respondents seem to be most satisfi ed with the presence of a professional at fi rst entrance to the ICU. The highest need for improvement scores relate to information about meals, parking and other disciplines (for example, social worker, spiritual worker or psychologist). Factor analysis shows that quality of care is determined by four clusters of items: Support, Communication, General Information and Organisation. The reliability of the CQI ‘R-ICU’ is suffi ciently high, only Communication and Support are signifi cant predictors of total quality judgement of relatives (adj. R2 = 0.74). In addition, there is a signifi cant diff erence in mean total quality judgement between the six hospitals as well as between the four wards within Erasmus MC. None 3. CG83 Critical Illness Rehabilitation: Guideline 2009 [www.nice.org.uk/CG83] P543 Quality of care in the ICU from the perspective of relatives M Van Mol1, E Bakker2, A Rensen3, I Menheere3, L Verharen3 1Erasmus MC, Rotterdam, the Netherlands; 2OU, Heerlen, the Netherlands; 3HAN, Nijmegen, the Netherlands Critical Care 2013, 17(Suppl 2):P543 (doi: 10.1186/cc12481) P543 Quality of care in the ICU from the perspective of relatives M Van Mol1, E Bakker2, A Rensen3, I Menheere3, L Verharen3 1Erasmus MC, Rotterdam, the Netherlands; 2OU, Heerlen, the Netherlands; 3HAN, Nijmegen, the Netherlands Critical Care 2013, 17(Suppl 2):P543 (doi: 10.1186/cc12481) Introduction This study describes the development and validation of the Consumer Quality Index Relatives in ICUs (CQI ‘R-ICU’), which aims to measure the satisfaction of relatives and to identify aspect of care that need improvement in the ICU in a reliable and valid way. According to the quality standards of the Dutch Society of Intensive Care, every ICU needs to record the satisfaction of relatives [1]. At this moment there is insuffi cient insight into the quality of care off ered to relatives on the ICU because an evidence-based Dutch measurement instrument is missing. Results At baseline measurements, there were no statistical diff erences in age, gender, length of stays or APACHE II scores between the two groups. Results indicate that the exercise group (n = 10) had signifi cantly greater improvements in cardiopulmonary fi tness (P  <0.001) and balance (P <0.05) compared with the control group (n = 10). Greater improvements were also evident in anxiety, depression and grip strength in the exercise group, although not statistically signifi cant. i Conclusion This pilot study highlights that a 6-week supervised exercise programme can signifi cantly improve cardiopulmonary fi tness and balance in post-ICU patients. Further recruitment to the study and 6-month/1-year follow-up is needed. Methods The CQI ‘R-ICU’ has been developed based on a scientifi c and standardised method [2]. A mixed design method is used, consisting of qualitative and quantitative survey studies. Factor analyses are carried out to determine the underlying structure of the newly developed questionnaire. Multiple regression analysis is used to explore the relationship between demographic variables and the perceived quality of care. P541 P541 Early results of a 6-week exercise programme in post-ICU patients C Battle1, K James1, P Temblett1, H Hutchings2 1Morriston Hospital, ABMU Health Board, Swansea, UK; 2Swansea University, Swansea, UK Critical Care 2013, 17(Suppl 2):P541 (doi: 10.1186/cc12479) Early results of a 6-week exercise programme in post-ICU patients C Battle1, K James1, P Temblett1, H Hutchings2 1Morriston Hospital, ABMU Health Board, Swansea, UK; 2Swansea University, Swansea, UK Critical Care 2013, 17(Suppl 2):P541 (doi: 10.1186/cc12479) Results A total 1,424 articles were systematically assessed for suitability and applicability. Of these, 14 articles remained eligible for review. The stressors were ranked by their frequency in the individual studies’ top- 10 lists. In rank order: 1, Tubes. 2, Pain. 3, Sleep Diffi culties. 4, Thirst. 5, Lack of Patient Understanding. Overall, stressors were found to be similar throughout all of the studies.i Early results of a 6-week exercise programme in post-ICU patients C Battle1, K James1, P Temblett1, H Hutchings2 g 1Morriston Hospital, ABMU Health Board, Swansea, UK; 2Swansea University, Swansea, UK 1Morriston Hospital, ABMU Health Board, Swansea, UK; 2Swansea University, Swansea, UK Introduction The aim of this study was to investigate the eff ect of a 6-week exercise programme on outcomes in post-ICU patients. With improvements in intensive care medicine, increasing numbers of patients are surviving catastrophic illness [1]. Severe weakness is common in patients with prolonged critical illness and results in considerable morbidity, mortality and healthcare costs [2]. The NICE83 guidelines Rehabilitation in Critical Care recommend follow-up for post-ICU patients and that further research is needed in this fi eld [3]. Conclusion The review identifi ed a list of the most pertinent common stressors. Awareness of these and ranking in priority may enable plans of care to be instigated to eff ectively alleviate patient stress. References References 1. Jones C, et al.: Clin Intensive Care 1998, 9:199-205. 2. Scragg P, et al.: Anaesthesia 2001, 56:9-14. 3. Perrins J, et al.: Intensive Crit Care Nurs 1998, 14:108-116. 4. Ballard KS: Issues Ment Health Nurs 1981, 3:89-108. 5. Public Health Resource Unit: Critical Appraisal Skills Programme. 10 Questions to Help You Make Sense of Qualitative Research. [http://www.medev.ac.uk/ static/uploads/workshop_resources/166/166_Qualitative_Appraisal_Tool.pdf] References 1. Jones C, et al.: Clin Intensive Care 1998, 9:199-205. 2. Scragg P, et al.: Anaesthesia 2001, 56:9-14. 3. Perrins J, et al.: Intensive Crit Care Nurs 1998, 14:108-116. 4. Ballard KS: Issues Ment Health Nurs 1981, 3:89-108. 5. Public Health Resource Unit: Critical Appraisal Skills Programme. 10 Questions to Help You Make Sense of Qualitative Research. [http://www.medev.ac.uk/ static/uploads/workshop_resources/166/166_Qualitative_Appraisal_Tool.pdf] 1. Jones C, et al.: Clin Intensive Care 1998, 9:199-205. i Methods Patients who have been discharged home from hospital following an ICU stay of 48 hours or more were recruited to the study. Patients were only excluded if they were not considered safe for exercise. Baseline measurements were completed prior to stratifi ed (age, gender, APACHE II score) random allocation to either the exercise or control group. Outcome measures included cardiopulmonary fi tness (6-minute walk test), balance (Berg Balance Scale), grip strength (JAMAR grip dynamometer) and hospital anxiety and depression (HAD score). The exercise group completed a 6-week supervised exercise programme, twice a week for up to 1 hour. In the seventh week, all patients repeated the baseline measurements. An unpaired Student’s t test was used to compare any diff erences between the control and exercise groups.f Introduction Optimal patient evaluations of ICU rehabilitation therapy remain unclear. Acknowledgement Supported by NIH grants 1R011186-01 and P30 AG21332. Family satisfaction in the ICU: enhancing patient experience E Kursumovic, J Bilinska, A Molokhia University Hospital Lewisham, London, UK Critical Care 2013, 17(Suppl 2):P544 (doi: 10.1186/cc12482) p p J Othero, AB Cavalcanti, JC Mouro, K Normilio-da-Silva, R Pusch, F Moreira, AA Kodama, O Berwanger Hospital do Coração – HCor, São Paulo, Brazil Critical Care 2013, 17(Suppl 2):P545 (doi: 10.1186/cc12483) Introduction Following recommendations made by the Scottish Intensive Care Society [1], we have introduced an annual Family Satisfaction Survey in the intensive care unit (FS-ICU), in anticipation that this will become an integral part of how we measure, improve and strive to advance patient care in the future. We aim to recognise factors impacting patient care and highlight areas for improvement. Improving understanding of patients’ needs is currently a UK national goal for CQUIN 2011/2012. An estimated £6.9 billion per year can be saved by reaching these goals [2] and a positive patient experience results in improved long-term outcomes and shorter hospital stays [2]. Methods We performed a study to assess Family Satisfaction over a 10- week period in a 16-bed critical care unit (CCU). A modifi ed version of an FS-ICU published in US and Canadian studies [3] was distributed to up to two family members per patient. Five-point Likert scale responses were linearly transformed to give percentage scores. Higher values represented a greater degree of satisfaction. Introduction We are conducting a cluster randomized trial with two parallel arms to evaluate strategies to improve family satisfaction with the care that themselves and their critically ill relatives receive in the ICUs of nonacademic Brazilian public hospitals. Here we report the results of the baseline phase of this trial. Introduction We are conducting a cluster randomized trial with two parallel arms to evaluate strategies to improve family satisfaction with the care that themselves and their critically ill relatives receive in the ICUs of nonacademic Brazilian public hospitals. Here we report the results of the baseline phase of this trial. Methods In this baseline phase, we interviewed the family member most closely involved with the care of critically ill patients who stayed in the ICU for at least 72 hours. We applied a form with 24 questions divided into four domains: overall ICU experience, communication, decision-making, and questions related to end-of-life care for patients who died in the ICU. Each question scored from 0 (very poor) to 100 (excellent). The form was adapted from the Family Satisfaction with Care in the ICU (FS-ICU 24). Recognition of the primary stressors aff ecting intensive care patients: a systematic review p y S Birch, S Elliot S Birch, S Elliot Leeds Teaching Hospitals, Leeds University Teaching Hospitals Trust, Leeds, UK Critical Care 2013, 17(Suppl 2):P542 (doi: 10.1186/cc12480) , Leeds Teaching Hospitals, Leeds University Teaching Hospitals Trust, Leeds, UK Critical Care 2013, 17(Suppl 2):P542 (doi: 10.1186/cc12480) Introduction Intensive care patients suff er psychological and physiological distress that may have debilitating and long-lasting eff ects [1-3]. Healthcare professionals are in a position to help avoid S200 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 Critical Care 2013, Volume 17 Suppl 2 http://ccforum.com/supplements/17/S2 References 3. Wall et al.: Refi nement, scoring, and validation of the Family Satisfaction in the Intensive Care Unit (FS-ICU) survey. Crit Care Med 2007, 35:271-279. 1. Vos et al.: Quality measurement at intensive care units: which indicators should we use? J Crit Care 2007, 22:267-274. 2. Sixma et al.: Handboek CQI Ontwikkeling: richtlijnen en voorschriften voor de ontwikkeling van een CQI meetinstrument. Utrecht: Nivel; 2008. 2. Sixma et al.: Handboek CQI Ontwikkeling: richtlijnen en voorschriften voor de ontwikkeling van een CQI meetinstrument. Utrecht: Nivel; 2008. References of the demographic variables such as sex, age, education, race and length of stay had an eff ect on perceived quality of care. 1. Quality Indicators for Critical Care in Scotland [http://www.sicsag.scot.nhs. uk/SICSQIG-report-2012-120209.pdf] 1. Quality Indicators for Critical Care in Scotland [http://www.sicsag.scot.nhs. uk/SICSQIG-report-2012-120209.pdf] 1. Quality Indicators for Critical Care in Scotland [http://www.sicsag.scot.nhs. uk/SICSQIG-report-2012-120209.pdf] f Conclusion The CQI ‘R-ICU’ turned out to be a valid, reliable, sensitive and feasible instrument. Large-scale implementation is recommended. Acknowledgements Thanks to researchers of Ziekenhuis Gelderse Vallei, Kennemer Gasthuis, Catharinaziekenhuis, Ziekenhuis Rivieren- land and Scheperziekenhuis. References 2. Using the Commissioning for Quality and Innovation (CQUIN) Payment Framework – Guidance on National Goals for 2011/12 [http://www.dh.gov. uk/prod_consum_dh/groups/dh_digitalassets/@dh/@en/@ps/documents/ digitalasset/dh_123009.pdf] 2. Using the Commissioning for Quality and Innovation (CQUIN) Payment Framework – Guidance on National Goals for 2011/12 [http://www.dh.gov. uk/prod_consum_dh/groups/dh_digitalassets/@dh/@en/@ps/documents/ digitalasset/dh_123009.pdf]i Family satisfaction with patient care in ICUs of nonacademic Brazilian public hospitals Family satisfaction with patient care in ICUs of nonacademic Brazilian public hospitals J Othero, AB Cavalcanti, JC Mouro, K Normilio-da-Silva, R Pusch, F Moreira, AA Kodama, O Berwanger Hospital do Coração – HCor, São Paulo, Brazil Critical Care 2013, 17(Suppl 2):P545 (doi: 10.1186/cc12483) Family satisfaction in the ICU: enhancing patient experience E Kursumovic, J Bilinska, A Molokhia University Hospital Lewisham, London, UK Critical Care 2013, 17(Suppl 2):P544 (doi: 10.1186/cc12482) As many questions assessed the quality of intensivist care or communication, the interview was applied by a psychologist or a nurse. y g Results Families of 564 patients were interviewed. A total 45/564 (8.3%) died in the ICU. Most respondents were satisfi ed with overall ICU experience (mean ± SD score 85.5 ± 11.9). However, family satisfaction with communication (67.8 ± 18.0) and decision-making (69.5 ± 21.1) resulted in somewhat lower scores. Most families of patients who died in the ICU (38/45 (82.6%)) considered that their relative’s life was neither extended nor shortened unnecessarily. Also, most of the families believed that their relative did not suff er or suff ered little in the ICU (37/46 (80.4%)) and felt supported by the healthcare team (40/46 (87.0%)).i Results We received and analysed 32 completed surveys. Seventy- eight per cent of relatives reported that the treatment of the patient was excellent, including symptom control such as pain, breathlessness and agitation (72%, 69%, 100%, respectively). Ninety-one per cent felt the care and frequency of communication provided by nurses was excellent or very good, compared with 81% by doctors. Only 9% of family members were off ered spiritual support during their CCU experience. Forty-seven per cent felt they would have liked more involvement in the decision-making process. Conclusion Most families were satisfi ed with the care themselves and their critically ill relatives received in the ICU. Also, most relatives of patients who died in the ICU felt that end-of-life care was adequate. Although we believe there is much room for improvement in communication, decision-making and support critically ill patients and their families, as their baseline satisfaction with patient care is quite high, it may be hard to demonstrate substantial improvement after interventions. Conclusion The vast majority of relatives rated their overall experience on CCU as excellent or very good. The study highlighted two main areas for improvement: provision of spiritual support as well as family and patient involvement in the decision-making process. We will arrange multi-disciplinary teaching sessions focusing on the positive impact, and therefore importance of patient satisfaction. A poster providing information about available spiritual support will be displayed around our CCU. Further studies are required to evaluate these measures.
https://openalex.org/W2414700531
https://hal.archives-ouvertes.fr/hal-01330139/file/article_mpsv.pdf
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Morphological processing of stereoscopic image superimpositions for disparity map estimation
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To cite this version: Jean-Charles Bricola, Michel Bilodeau, Serge Beucher. Morphological processing of stereoscopic image superimpositions for disparity map estimation. 2016. ￿hal-01330139￿ Morphological processing of stereoscopic image superimpositions for disparity map estimation Jean-Charles Bricola, Michel Bilodeau and Serge Beucher PSL Research University – MINES ParisTech CMM – Centre de Morphologie Math´ematique 35 rue St-Honor´e 77300 Fontainebleau, France PSL Research University – MINES ParisTech CMM – Centre de Morphologie Math´ematique 35 rue St-Honor´e 77300 Fontainebleau, France Abstract. This paper deals with the problem of depth map computa- tion from a pair of rectified stereo images and presents a novel solution based on the morphological processing of disparity space volumes. The reader is guided through the four steps composing the proposed method: the segmentation of stereo images, the diffusion of superimposition costs controlled by the segmentation, the resulting generation of a sparse dis- parity map which finally drives the estimation of the dense disparity map. An objective evaluation of the algorithm’s features and qualities is provided and is accompanied by the results obtained on Middlebury’s 2014 stereo database. Keywords: stereo vision, geodesic distances, 3D watershed, image seg- mentation, sparse disparity measurements, dense estimation HAL Id: hal-01330139 https://hal.science/hal-01330139v1 Preprint submitted on 27 Jun 2016 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Public Domain 1 Introduction The problem of computing a depth map from a pair of rectified stereo images is undoubtedly a classic one in computer vision. When a point of the scene projects onto the two image planes, it does so with the same ordinates but with different abscissa. The difference of abscissa corresponds to what is commonly referred to as the disparity and is inversely proportional to the point’s depth being sought for. Finding point correspondences between the left and right views of the stereo pair is relatively easy across non-uniformly textured areas. However, homogeneous regions are the source of matching ambiguities whilst the occlusion phenomenon makes it impossible for some pixels to have a correspondence and thus require their disparity to be estimated according to a suitable model. In order to overcome these two difficulties, it is usual to devise algorithms which ensure, on the one hand, that disparities evolve smoothly across the low- textured areas of the image for which the depth map is estimated and, on the other hand, that disparities remain consistent with the resulting warping of stereo images. Depth discontinuities must be tolerated though in order to han- dle the presence of different objects in the scene. Image gradients and prior segmentations are, to this end, often used as pertinent cues on natural scenes. Furthermore, occluded areas will never, despite correct disparities, yield a mean- ingful superimposition with the other image of the stereo pair. Bad superimposi- Bricola J.-C., Bilodeau M., Beucher S. 2 tions for such areas should therefore not prevent the algorithm from giving them the right disparities. tions for such areas should therefore not prevent the algorithm from giving them the right disparities. Methods able to satisfy the aforementioned specifications typically belong to the category of global approaches. The general idea is to formulate an energy function which, for a given disparity map, equals the sum of superimposition or warping costs plus a regularisation term, penalising non-smooth disparity transi- tions. Finding the disparity map minimising this energy may be achieved using gradient descent algorithms [1] or, in the context of maximum-a-posteriori or MAP inference, using 3D graph-cuts [2], alpha-expansion [3] and belief propa- gation methods [4–6]. The work of [5] is a good example of how the occlusion phenomenon is taken into account within the MAP inference. 2 Segmentation This section explains how both images of the stereo pair are segmented. Our seg- mentation scheme produces partitions which are slightly over-segmented across textured regions, which preserve all contrasted objects even when they are thin and which capture most of the remaining objects’ contours. This choice has been made in the view of the constrained cost diffusion process presented in section 3. Section 2.1 recalls the fundamentals of the watershed transformation, as it is used here in our segmentation procedure but also in sections 4 and 5. Section 2.2 goes into more details about the segmentation algorithm employed in this approach. 2.1 The watershed transformation controlled by markers Let S : N2 →N be a discrete elevation surface, mapping every point p of the image domain to its altitude S[p]. We define the accompanying image of lakes as L : N2 →N, mapping every point p to a label L[p]. We impose that a point p belongs to a lake if and only if L[p] > 0. The watershed transformation of S, controlled by an initial image of lakes L0, is the result of an iterative process which assigns all pixels {p|L0[p] = 0} to a lake existing in L0. The recurrence relationship between Lt and Lt−1 is defined as follows: 1. Let St be the set of points reached at altitude t, i.e. St = {p | S[p] ≤ et St be the set of points reached at altitude t, i.e. St = {p | S[p] ≤t} t p , t {p | [p] ≤} 2. The lakes of Lt−1 are propagated in an isotropic fashion to all points p ∈St having Lt−1[p] = 0 and belonging to a path leading to any lake of Lt−1. 3. The meeting points of lakes of different labels belong to the watershed. 2. The lakes of Lt−1 are propagated in an isotropic fashion to all points p ∈S having Lt−1[p] = 0 and belonging to a path leading to any lake of Lt−1. 3. The meeting points of lakes of different labels belong to the watershed. 3. The meeting points of lakes of different labels belong to the watersh When t reaches the highest altitude of S, say t∗, the iterative process stops and the image of lakes Lt∗is completely filled. Although the algorithm is presented for two-dimensional elevation surfaces, it applies equally well to any higher di- mension. The reader may find more details on the watershed transformation as well as computationally efficient algorithms based on hierarchical queues in [10]. Morphological processing of stereoscopic image superimpositions Morphological processing of stereoscopic image superimpositions 3 3 1 Introduction Former methods perform a similar kind of optimisation on each horizontal scanlines independently, so as to warp the corresponding image rows together [7]. Given the disparity space image, the warping is obtained by finding a shortest path going through the corresponding array of accumulated costs, which turns out to be nothing else than a particular type of geodesic distance function. Due to the fact that backtracing, the component of dynamic programming responsible for recovering the shortest path, is employed, the concept cannot easily extend to 3D grids. The combination of scanline optimisations along different axes is nevertheless the main constituent of the semi-global matching [8], which still serves as an essential component in state-of-the-art methods such as [9]. In this article, we propose a novel approach to depth map computation, based on the morphological processing of a disparity space volume, characteris- ing the superimpositions of the considered stereo images for different disparities. A disparity space volume, abbreviated as DSV, is in fact a stack of disparity space images, piled according to an increasing order of disparities. We show how geodesic distance functions computed across a DSV may be employed with the watershed transformation controlled by markers [10], so as to obtain a separat- ing hyperplane between the foreground and background voxels belonging to the DSV. Furthermore, our method draws on the idea of [7] which is to integrate ground control points in the process. As a matter of fact, an effort has been made to systematically provide a sparse disparity map carrying a reasonable amount of information, while minimising the number of invalid matches. To fulfil that objective, we got inspired by the research of [11] and [12] in order to diffuse costs inside the disparity space volumes while resorting to the image segmentation so as to prevent incoherent cost aggregations. The paper starts with four sections which sequentially go through each step of the algorithm. Section 2 presents the morphological segmentation in general and shows how images of the stereo pair are initially partitioned. These partitions add a constraint on our diffusion mechanism presented in section 3, resulting in the generation of sparse disparity maps described in section 4. Then, section 5 provides all the details on the dense disparity map estimation based on the 3D watershed transformation. Finally, section 6 is devoted to the evaluation of the proposed methods on the Middlebury 2014 database. 2.2 Image partitioning with little over-segmentation In order for Lt∗to constitute a relevant image partition, both the topographical surface S and the markers L0 must be chosen appropriately. It is common to define S as a colour gradient, for instance the supremum of the red, green and blue channels’ gradients, when dealing with images of unknown nature. The most trivial choice for the lake’s initialisation then consists of taking each of the gradient’s minima as a marker with a distinctive label, but this typically leads to a severe image over-segmentation, as shown in Figure 2. This is accounted for by the fact that the gradient is, on natural images, composed of a multitude of minima. Filtering the image or the gradient beforehand helps reducing the amount of minima, but care must be taken not to deform the relevant contours. Bricola J.-C., Bilodeau M., Beucher S. Bricola J.-C., Bilodeau M., Beucher S. 4 We propose to alter each level set of the gradient function by applying a series of closings, which decrease exponentially in strength as the altitude of the elevation surface increases. This is exactly the method proposed by [13] for regularising watersheds by oil flooding. The resulting gradients have less min- ima and their watershed transformation produces therefore less over-segmented partitions as testifies Figure 2. Besides, since the closing has little strength at high altitudes, it has little effect on the corresponding level sets and thus sharp contours are not deformed. Threshold(0,tc) Threshold(0,tf) Label Label AreaOpen(¾c) AreaOpen(¾f) Threshold(1,+1) AreaOpen(¾c) Label Merge + W.T. W.T. g – S L L S Mc Mf Mc Mf PartitionBuild Marker function Mask function + – W.T. L S g Lfinal Merge Fig. 1. Flowchart of the image segmentation procedure. Fig. 1. Flowchart of the image segmentation procedure. The partition frontiers obtained at this stage of the proceedings contains the frontiers of the final partition Lfinal. Figure 1 schematises the full segmentation algorithm. Let us briefly describe the operators involved in this system: – AreaOpen(σ) is an area opening operator which removes any cell of the par- tition having an area inferior or equal to σ pixels. – AreaOpen(σ) is an area opening operator which removes any cell of the par- tition having an area inferior or equal to σ pixels. – Label is a labelling operator which, given a binary image as input, assigns a unique label to every connected component. 2.2 Image partitioning with little over-segmentation Pixels set to 0 in the binary image are set to 0 in the labelled image. – Label is a labelling operator which, given a binary image as input, assigns a unique label to every connected component. Pixels set to 0 in the binary image are set to 0 in the labelled image. – Merge is responsible for merging two images of markers while ensuring that each of them receives a unique label in the output image. – Merge is responsible for merging two images of markers while ensuring that each of them receives a unique label in the output image. – PartitionBuild takes two images of lakes Lmask and Lmarker as input. The out- put Lout is an image of lakes of identical dimensions, defined by the following relation: – PartitionBuild takes two images of lakes Lmask and Lmarker as input. The out- put Lout is an image of lakes of identical dimensions, defined by the following relation: Lout[p] = ( Lmask[p] if ∃p′ | Lmask[p′] = Lmask[p] ∧Lmarker[p′] > 0 0 otherwise Morphological processing of stereoscopic image superimpositions 5 – Threshold(t0, t1) denotes the standard threshold operator mapping all pixels of the input image in the range [t0, t1] to 1, the others to 0. – Threshold(t0, t1) denotes the standard threshold operator mapping all pixels of the input image in the range [t0, t1] to 1, the others to 0. – W.T. is the watershed transformation described in section 2.1. The algorithm’s input g is nothing else than the altered gradient we have pre- viously described. The system represented in the top flowchart yields two inter- mediate images of lakes, called M c and M f. The first is meant to hold lakes splitting on very sharp gradient areas while tolerating thin structures. The sec- ond, on the contrary, is more sensitive to smaller contrast but imposes lakes of a certain area. The contrast and area parameters must therefore be chosen, such that tc > tf and σc < σf. The system in the bottom flowchart simply shows how the thin structures in M c are appended to the image of lakes of M f and how this results in the final segmentation. Fig. 2. Sample segmentations for Adirondack, Jadeplant and Motorcycle images from Middlebury 2014 database. 3 Costs diffusion The left and right images of the stereo pair, denoted by I0 and I1 respectively, are superimposed for different horizontal shifts corresponding to the disparities being tested. The superimpositions are stored in a disparity space volume V : N3 →R. The entry V[x, y, d] reflects how well the pixels of coordinates (x, y) in I0 and (x −d, y) in I1 match. In this approach, V[x, y, d] corresponds to the Hamming distance between the two pixel values in their corresponding Census transformed images [14], encoding the rising edges of the standard gradient according to different directions. This choice makes the superimposition costs insensitive to illumination changes and has become a standard in many stereo applications. The purpose of costs diffusion is to filter the disparity space volume V so as to highlight the locations where the stereo images effectively superimpose. A trivial choice would be to use a moving average filter which operates on each disparity plane independently, which of course wouldn’t be a good solution. The primary reason is that the moving average filter may obviously aggregate the costs of pixels representing different objects in the scene. This is an issue if the moving average window spans two or more objects with different depths because their costs will be mixed together for the same disparity. In [11], a segmentation of the left image was used to restrict the domain of the aggregation window to the pixels which supposedly belong to the same object as the one of the window centre. In our method, we resort to both the left and right segmentations of the stereo pair, obtained by the algorithm presented in section 2. The domain inside which the diffusion may freely operate, is constrained by the intersection of the left segmentation with the right segmentation shifted according to the considered disparity. This way of proceeding ensures that the costs obtained across the portions of the left image being occluded in the right image do not again mix with the costs obtained across non-occluded areas. g While the chosen segmentations facilitate the construction of quite large ag- gregation supports across homogeneous areas and still consistent ones across thin image structures, the fact that filtering is, at this stage, only applied to each disparity plane independently constitutes a limitation with respect to the processing of tilted and non-planar surfaces. 3 Costs diffusion This is why, we shall integrate a basic warping technique to the diffusion scheme. 2.2 Image partitioning with little over-segmentation The top row shows the result of the watershed transforma- tion of the colour gradient, despite the image pre-filtering, using the gradient’s minima as markers. The middle row is the watershed transformation on the altered gradient g using the gradient’s minima as markers. The bottom row is the output of the algorithm presented in section 2.2. Fig. 2. Sample segmentations for Adirondack, Jadeplant and Motorcycle images from Middlebury 2014 database. The top row shows the result of the watershed transforma- tion of the colour gradient, despite the image pre-filtering, using the gradient’s minima as markers. The middle row is the watershed transformation on the altered gradient g using the gradient’s minima as markers. The bottom row is the output of the algorithm presented in section 2.2. Regarding the choice of the four parameters, we opted for a solution that is dependant on the image characteristics. tc corresponds to the 45th percentile of the intensity values observed in the initial colour gradient excluding 0, and tf Bricola J.-C., Bilodeau M., Beucher S. 6 corresponds to the 10th percentile. Furthermore, it is the ratio of the opening parameter with respect to the full image area which is fixed. In our experiments, this ratio has been set to 5 · 10−5 for σc and to ten times more for σf. 3.1 Erosions and distance functions The proposed diffusion algorithm resorts to two morphological operators which, for the sake of completeness, are briefly recalled in this section. Morphological operators are typically controlled by structuring elements which, in the discreet case, are described by sets of points. We will refer to B as an arbitrary structuring element. Morphological processing of stereoscopic image superimpositions 7 Erosions The erosion operator ε under structuring element B transforms a vol- ume V into another volume εB(V) according to equation 1. εB(V)[p] = inf p′∈B V[p + p′] (1) (1) If structuring element B is just composed of one point, such that B = {p′}, the erosion turns into a simple translation in the direction of −p′. In the rest of this article, such a translation will be represented by εp′. Distance functions The distance function DB(M) associated to a binary volume M : N3 →{0, 1}, and controlled by structuring element B, is expressed by the following recurrence relationship: Dt = min {Dt−1, εB(Dt−1) + 1} (2) setting D0[p] = ( 0 if M[p] = 0 +∞ otherwise (2) DB(M) = Dt∗, for t∗satisfying Dt∗= Dt∗+1. In fact, DB(M)[p] corresponds to the minimum number of erosions being necessary for voxel p to get reached by any deactivated voxel of mask M, using structuring element B. Bricola J.-C., Bilodeau M., Beucher S. Bricola J.-C., Bilodeau M., Beucher S. 8 Bricola J.-C., Bilodeau M., Beucher S. The way algorithm 1 is presented suggests that the diffusion is still con- tained inside each disparity plane. The extension to multiple disparity planes is however quite straightforward and does not change the proposed template. For a given direction t = (tx, ty, 0)⊺, we define a new structuring element Bt = {(tx, ty, +1)⊺, (tx, ty, −1)⊺}. The cost update rule in line 5 transforms into: VUPD ←V + min {εt(VOUT), εBt(VOUT) + ξ} where the parameter ξ controls the regularity of the diffusion. To put it in a nutshell, it is a partial scanline optimisation which is performed along the chosen direction using the left and right image segmentations as a boundary constraint. Algorithm 1 Diffusion of superimposition costs Algorithm 1 Diffusion of superimposition costs 1: function DirectionalDiffusion(V, t, n) 2: t ←0 3: VOUT ←V 4: while t < n do 5: VUPD ←V + εt(VOUT) 6: VSEL ←Binary volume highlighting Dt(Mt) > t 7: VOUT ←VUPD · VSEL + VOUT · (1 −VSEL) 8: t ←t + 1 9: end while 10: return VOUT 11: end function 12: function DiffuseCosts(V, Vs, n) 13: VXL ←DirectionalDiffusion(V, tl, n) 14: VXR ←DirectionalDiffusion(V, tr, n) 15: VX ←(VXL + VXR) ÷ (min(n, Dtl(Mtl)) + min(n, Dtr(Mtr)) + 2) 16: VYU ←DirectionalDiffusion(VX, tu, n) 17: VYD ←DirectionalDiffusion(VX, td, n) 18: VY ←(VYU + VYD) ÷ (min(n, Dtu(Mtu)) + min(n, Dtd(Mtd)) + 2) 19: return VY 20: end function Algorithm 1 Diffusion of superimposition costs 1: function DirectionalDiffusion(V, t, n) 2: t ←0 3: VOUT ←V 4: while t < n do 5: VUPD ←V + εt(VOUT) 6: VSEL ←Binary volume highlighting Dt(Mt) > t 7: VOUT ←VUPD · VSEL + VOUT · (1 −VSEL) 8: t ←t + 1 9: end while 10: return VOUT 11: end function 12: function DiffuseCosts(V, Vs, n) 13: VXL ←DirectionalDiffusion(V, tl, n) 14: VXR ←DirectionalDiffusion(V, tr, n) 15: VX ←(VXL + VXR) ÷ (min(n, Dtl(Mtl)) + min(n, Dtr(Mtr)) + 2) 16: VYU ←DirectionalDiffusion(VX, tu, n) 17: VYD ←DirectionalDiffusion(VX, td, n) 18: VY ←(VYU + VYD) ÷ (min(n, Dtu(Mtu)) + min(n, Dtd(Mtd)) + 2) 19: return VY 20: end function 3.2 Diffusion algorithm Let Vs : N3 →N be the volume encoding the intersections of L(0) and L(1), i.e. the partitions of I0 and I1 respectively, for different disparities. Each entry of Vs is computed as: Vs[x, y, d] = L(0)[x, y] + L(1)[x −d, y] · max x,y L(0)[x, y] (3) (3) Suppose that the costs were propagated along a particular direction −t, such that the disparity space volume V would transform into 1 2(V + εt(V)). The set {p | Vs[p] ̸= εt(Vs)[p]} determines all the voxels for which costs from different regions would have been mixed. Let Mt stand for the binary mask attributing the value 0 to such voxels and the value 1 to the others. Furthermore, consider the following directions: tl = (−1, 0, 0), tr = (+1, 0, 0), tu = (0, −1, 0) and td = (0, +1, 0). Suppose that the costs were propagated along a particular direction −t, such that the disparity space volume V would transform into 1 2(V + εt(V)). The set {p | Vs[p] ̸= εt(Vs)[p]} determines all the voxels for which costs from different regions would have been mixed. Let Mt stand for the binary mask attributing the value 0 to such voxels and the value 1 to the others. Furthermore, consider the following directions: tl = (−1, 0, 0), tr = (+1, 0, 0), tu = (0, −1, 0) and td = (0, +1, 0). Algorithm 1 describes the proposed diffusion mechanism. Similarly to [12], the costs are first diffused along the horizontal axis, providing a new filtered disparity space volume, of which the costs in turn are diffused along the vertical axis. However, for each voxel, the aggregation of costs stops as soon as a frontier between two different regions in Vs is crossed or when the maximum diffusion’s scope n has been attained along a particular direction. This is what is enforced by lines 6 and 7 in algorithm 1. As a result, the aggregations along two opposite directions do not necessarily have the same weight and thus more importance is given to the direction where a region border is the farthest away from the considered voxel. 8 4 Sparse disparity map generation The initial disparity measures are recovered from the filtered disparity space volume computed in section 3, by calculating the disparity that minimises the superimposition cost at each pixel. In addition, cross-checking [15] is performed to ensure that the disparity measures remain consistent between the left and the right images of the stereo pair. The disparity measures which do not satisfy this criterion are discarded. Figure 3 shows some of the resulting disparity maps on the Middlebury 2014 dataset. Morphological processing of stereoscopic image superimpositions Morphological processing of stereoscopic image superimpositions 9 Fig. 3. Sparse disparity maps obtained by the diffusion algorithm presented in section 3.2. n is set to 25 pixels. ξ is set to 20% of the maximum possible cost in the disparity space volume V. Pixels appearing in black are those which did not satisfy the cross- checking criterion [15]. Top row shows the disparity maps obtained for Adirondack, Jadeplant, and Motorcycle stereo images. Bottom row shows the disparity maps ob- tained for Pipes, PlaytableP, and Vintage stereo images. Fig. 3. Sparse disparity maps obtained by the diffusion algorithm presented in section 3.2. n is set to 25 pixels. ξ is set to 20% of the maximum possible cost in the disparity space volume V. Pixels appearing in black are those which did not satisfy the cross- checking criterion [15]. Top row shows the disparity maps obtained for Adirondack, Jadeplant, and Motorcycle stereo images. Bottom row shows the disparity maps ob- tained for Pipes, PlaytableP, and Vintage stereo images. 4.1 Detection and pruning of bad measures Despite their perceptual appeal, these initial disparity maps are subject to some artefacts. It is preferable to detect and remove them if sparse disparity maps were to be used as initialisers of the dense estimation algorithm. Firstly, we observe that artefacts are not predominant in the initial disparity maps. This means that clusters of smoothly evolving disparities should be preserved if they span a significant area of the image. At the opposite extreme, peaky measure- ments should be eliminated without further consideration. The cases in-between demand some more investigation. Clusters computation In order to find the clusters of smoothly evolving dis- parities, the holes of the initial disparity map are filled in using the watershed transformation presented in section 2.1. The available disparities play the role of initial markers, and the image gradient plays the one of the topographical sur- face controlling the flooding. The gradient of the filled disparity map indicates where disparities do not smoothly evolve. We threshold this gradient between the values 0 and 1 so as to highlight the connected components which uniquely iden- tify the desired clusters. After the labelling, each pixel attributed to a disparity measure receives the identifier of the cluster it belongs to. Clusters selection The selection of clusters is primarily achieved by the area opening operator introduced in section 2.2. According to the aforementioned specifications, two area openings are performed. The strongest one keeps the Bricola J.-C., Bilodeau M., Beucher S. 10 largest clusters while the weakest one removes small impurities. Structures con- served by the weak area opening but removed by the stronger area opening typically correspond to thin image structures or erroneous measures. To tell them apart, it suffices the look at the image gradient and observe that erroneous measures mainly occur across homogeneous areas. Occlusion artefact filtering Another source of error originates from the diffusion of contour disparities to regions being both homogeneous and textureless. This may be observed, for instance, on Jadeplant (Figure 3) between the top of the elongated box and the background. Since the disparity measures are virtually the same from either side of the frontier, the sole selection of clusters does not suffice to prune the wrong measures, in that scenario. These wrong measures constitute fattening artefacts. 4.1 Detection and pruning of bad measures In order to detect and remove them, it is important to notice that fattened disparity measures lie near region borders, and are usually not consistent with the disparities found within the interior of the regions. Therefore, we propose to perform the filtering of the fattening artefacts at a regional scale, as follows: each cell of the partition corresponding to the image for which we aim at computing its disparity map, is eroded using an isotropic structuring element of size equal to the diffusion’s scope. The processing then concerns the cells of the partition, which have not been completely destroyed by the erosion. For each of these cells, only the pixels belonging to the cluster(s) covering some area of the corresponding eroded cell, may be restored with their disparity measures. Since the fattened disparities and the interior disparities should belong to different clusters, and that the pixels attributed to the fattened disparities should not belong to the eroded cells, then fattened disparities should not be reconstructed, as desired. 5 Dense disparity map estimation We aim at estimating the final disparity map by means of a 3D watershed trans- formation. Similarly to any watershed transformation (cf. section 2.1), the suc- cess of the procedure depends on the definition of the initial markers L0 : N3 →N and the topographical surface being flooded, S : N3 →N. Since the disparity map is in fact a representation of the hyperplane separating the disparity space volume into the foreground and background voxels, only two markers designat- ing the foreground and background regions are required. Therefore, the markers are initialised so that L0[x, y, d] is equal to the background label ℓg > 0 if d = 0, to a distinct foreground label ℓf > 0 if d equals the maximum disparity consid- ered with respect to V, and to 0 elsewhere. The most demanding part of this 3D segmentation will be the definition of the topographical surface S. 5.1 Interpolation and distance functions The topographical volume S should be chosen so that the watershed passes by the points {(xi, yi, di)} for any valid pixel (xi, yi) attributed to disparity di in Morphological processing of stereoscopic image superimpositions 11 (a) (b) (c) (d) (e) (f) Fig. 4. Illustration of the filtering stage. (a) Left view of AustraliaP, (b) An initial disparity map containing many artefacts, (c) The large clusters of continuous dispari- ties, (d) The union of both large clusters and smaller clusters spanning areas holding sufficient gradient information, (e) Close-up showing the occasional fattening effect on homogeneous areas, (f) Result of the occlusion artefact filtering deduced from the segmentation of the left view. (c) (a) (b) (a) (c) (b) (a) (d) ( ) (f) (e) (e) (f) (d) Fig. 4. Illustration of the filtering stage. (a) Left view of AustraliaP, (b) An initial disparity map containing many artefacts, (c) The large clusters of continuous dispari- ties, (d) The union of both large clusters and smaller clusters spanning areas holding sufficient gradient information, (e) Close-up showing the occasional fattening effect on homogeneous areas, (f) Result of the occlusion artefact filtering deduced from the segmentation of the left view. the filtered sparse disparity map described in section 4. One way of proceeding is to resort to the binary distance function defined by equation 2. In that case, the accompanying mask of control points has to be exclusively set to 0 for all the chosen {(xi, yi, di)} and B must correspond to an isotropic structuring element. In order to drive the watershed transformation, S must therefore equal the in- verted distance function. However, the binary distance function has one major drawback: the fact that the disparity space volume computed in section 3 would be totally ignored from the interpolation process. To overcome this limitation, the geodesic distance controlled by the DSV V may be used instead. The latter is described by equation 4. Dt = min {Dt−1, εB(Dt−1) + V} (4) (4) In fact, this equation is a simple alteration of the binary distance update rule (cf. equation 2), where V acts as a viscosity function, delaying the time it takes for a voxel to get reached by one of its neighbours. Figure 5 illustrates the comparison between the segmentations controlled by the additive inverses of the binary and the geodesic distance functions. 5.2 Generation of the topographical volume S Upon convergence at t = t∗, the topographical volume is finally given by equation 5. S = −Dt∗+ max (x,y,d) Dt∗[x, y, d] (5) (5) 5.2 Generation of the topographical volume S Now, let us explain what the generation of the topographical volume S consists of. At our disposal, we have the segmentation of the left view L(0), the disparity Bricola J.-C., Bilodeau M., Beucher S. 12 (a) (b) (c) (d) Fig. 5. Distance functions and segmentation. (a) Viscosity function, (b) The experi- ment’s settings: in black are shown the pixels from which the distance functions are computed. The coloured rows represent the markers controlling the watershed segmen- tation in conjunction with the inverted distance functions. (c) Binary distance function and resulting watershed shown in yellow. (d) Geodesic distance function controlled by the viscosity functions and resulting watershed segmentation. (d) (c) (a) (b) (b) (a) (d) (c) Fig. 5. Distance functions and segmentation. (a) Viscosity function, (b) The experi- ment’s settings: in black are shown the pixels from which the distance functions are computed. The coloured rows represent the markers controlling the watershed segmen- tation in conjunction with the inverted distance functions. (c) Binary distance function and resulting watershed shown in yellow. (d) Geodesic distance function controlled by the viscosity functions and resulting watershed segmentation. space volume V with the aggregated superimposition costs, and a filtered sparse disparity map D. Let D be a distance function, having the same dimensions as V. D is initialised as follows: D0[x, y, d] = ( 0 if D[x, y] = d ∨∥∇L(0)∥[x, y] > 0 +∞ otherwise The points set to zero in this distance function are composed of the control points originating from the sparse disparity map plus the segmentation boundaries of L(0) added to every disparity plane. These boundaries not only guarantee that the interpolation is applied independently on each cell of the partition but also that the hyperplane will fold appropriately at such locations, so as to allow discontinuities within the resulting disparity map. Once initialised, the computation of the distance function is accomplished using the update rule provided by equation 4. There is one alteration though: similarly to the cost diffusion of section 3, we enforce some regularity by adding a supplementary contribution ζ when accumulating distances across different dis- parity planes. In order to do that, B has to be decomposed into two structuring elements B1 and B2, the first holding the directions fronto-parallel to the image plane, the second holding the tilted directions. The term εB(Dt−1) in equation 4 is then replaced by min{εB1(Dt−1), εB2(Dt−1) + ζ}. 6 Experiments and Results Our method has been tested on the Middlebury 2014 dataset [16], using the quarter resolution images. Both our sparse and dense results are compared to Morphological processing of stereoscopic image superimpositions 13 the ground truth disparity maps acquired using the method described in [17]. This section provides the reader with the parameters chosen to produce all the disparity maps. Then the quality of the sparse disparity measures is evaluated, while discussing the impact of the filtering stage described in section 4. Finally, we analyse the results obtained on the dense disparity maps. 6.1 Parameters The choices of the segmentation’s parameters are given and explained in section 2. In section 3, two parameters were introduced. ξ, the extra contribution added to the costs aggregated from different disparity planes, is set to 20% of the worst superimposition cost while n, the maximal scope of the diffusion along a particular direction, is fixed to 25 pixels. It should be noted that reducing n is likely to cause the disparity map to become sparser and to be subject to more measurement errors. In section 4, large disparity clusters are supposed to cover at least 0.5% of the image plane against 0.005% for the small clusters. Finally, the regularisation term ζ presented in section 5 is fixed to 50% of the worst superimposition cost. 6.2 Sparse disparity measures 6.2 Sparse disparity measures Adirondack ArtL Jadeplant Motorcycle MotorcycleE Piano PianoL Pipes Playroom Playtable PlaytableP Recycle Shelves Teddy Vintage 0.000 0.100 0.200 0.300 0.400 0.500 0.600 0.700 Additional bad pixels (before filtering) Bad Pixels Additional valid pixels (before filtering) Valid Pixels Image occupancy ratio Fig. 6. Occupancy of valid and “bad” disparity measures for the training set of Mid- dlebury 2014 database, before and after the filtering stage. Fig. 6. Occupancy of valid and “bad” disparity measures for the training set of Mid- dlebury 2014 database, before and after the filtering stage. Some of the initial sparse disparity maps have been presented in Figure 3. Based on the ground truth provided for the training set of the database, the statistics regarding the density and the accuracy of these disparity maps as well as the impact of the post filtering have been gathered in Figure 6. On average, 54% of the pixels covering the non-occluded areas of the image plane have been attributed to a disparity measure. If we consider that erroneous measures are those having a disparity error higher than 2 pixels, then the initial measures have an average error percentage of 12.4%. The filtering stage reduces the latter to 2.80%, while it preserves about 88% of the initial disparity measures which Bricola J.-C., Bilodeau M., Beucher S. 14 were correct. Note that the Playtable instance has been discarded from these averages, since the matching errors are the result of severe vertical disparities between the left and right images. The visual appeal of the disparity maps is probably best explained by the fact that they remain consistent with respect to the segmentations computed in section 2, that the absence of measures is recurrent across occluded areas, and that the disparities of pixels being classified as erroneous are still not too different from the ground truth. The root-mean- square error reflects this perfectly well on the benchmark: considering the full image plane, including occluded areas, this error evaluates to 2.2 pixels (quarter resolution), which, at the time of writing, ranks our sparse disparity maps third among those obtained using the other methods evaluated in the benchmark. Adirondack Jadeplant Motorcycle Pipes PlaytableP Vintage AustraliaP Bicycle2 Classroom Djembe Newkuba Livingroom Fig. 7. Sparse disparity maps resulting from the diffusion algorithm, combined with the filtering step presented in section 4. Newkuba Livingroom Djembe Fig. 7. 6.2 Sparse disparity measures Sparse disparity maps resulting from the diffusion algorithm, combined with the filtering step presented in section 4. Morphological processing of stereoscopic image superimpositions 15 6.3 Dense disparity map estimation Disparity maps resulting from the interpolation process (cf. section 5) are shown in Figure 8. As desired, the disparity maps are consistent with the provided segmentations, which is again reflected by the RMS error. However, disparities are still subject to inaccuracies across homogeneous areas and the interpolated disparity functions lack of smoothness. As part of a future work, it would in- teresting to study the effect of the viscous watershed transformation [13] on the topographical surface controlling the 3D watershed, since it exhibits interest- ing regularisation properties. Furthermore, the costs obtained across occluded areas should deserve a complementary treatment so that they do not perturb the interpolation when decreasing the regularisation parameter ζ. Finally, the interpolation process could benefit from disparity plane hypotheses, similarly to [18], which could be made on a regional basis, thanks to the sparse disparity measures. Adirondack Motorcycle PlaytableP Fig. 8. Disparity maps obtained using the interpolation algorithm presented in section 5, controlled by the corresponding sparse disparity maps. Fig. 8. Disparity maps obtained using the interpolation algorithm presented in section 5, controlled by the corresponding sparse disparity maps. References 1. Aydin, T., Akgul, Y.S.: Stereo depth estimation using synchronous optimization with segment based regularization. Pattern Recognition Letters 31(15) (2010) 2389–2396 2. Prince, S.: Models for grids. In: Computer vision: models, learning, and inference. Cambridge University Press (2012) 3. Boykov, Y., Veksler, O., Zabih, R.: Fast approximate energy minimization via graph cuts. IEEE Transactions on Pattern Analysis and Machine Intelligence 23(11) (2001) 1222–1239 4. Sun, J., Li, Y., Kang, S.B., Shum, H.Y.: Symmetric stereo matching for occlusion handling. In: IEEE Computer Society Conference on Computer Vision and Pattern Recognition, 2005. CVPR 2005. Volume 2., IEEE (2005) 399–406 ( ) 5. Yang, Q., Wang, L., Yang, R., Stew´enius, H., Nist´er, D.: Stereo matching with color-weighted correlation, hierarchical belief propagation, and occlusion handling. IEEE Transactions on Pattern Analysis and Machine Intelligence 31(3) (2009) 6. Facciolo, G., de Franchis, C., Meinhardt, E.: Mgm: A significantly more global matching for stereovision. In: Proceedings of the British Machine Vision Conference (BMVC), BMVA Press (2015) 90.1–90.12 ( ), ( ) 7. Bobick, A.F., Intille, S.S.: Large occlusion stereo. International Journal of Com- puter Vision 33(3) (1999) 181–200 8. Hirschm¨uller, H.: Stereo processing by semiglobal matching and mutual informa- tion. IEEE Transactions on Pattern Analysis and Machine Intelligence (2008) 9. Zbontar, J., LeCun, Y.: Stereo matching by training a convolutional neural network to compare image patches. arXiv preprint 1510.05970 (2015) 10. Beucher, S., Meyer, F.: The morphological approach to segmentation: the water- shed transformation. Optical Engineering 34 (1992) 433–481 11. Hosni, A., Bleyer, M., Gelautz, M.: Near real-time stereo with adaptive support weight approaches. In: International Symposium on 3D Data Processing, Visual- ization and Transmission (3DPVT). (2010) 1–8 12. Cigla, C., Alatan, A.A.: Information permeability for stereo matching. Signal Processing: Image Communication 28(9) (2013) 1072–1088 13. Vachier, C., Meyer, F.: The viscous watershed transform. Journal of Mathematical Imaging and Vision 22(2-3) (2005) 251–267 14. Zabih, R., Woodfill, J.: Non-parametric local transforms for computing visual correspondence. In: Third European Conference on Computer Vision 1994 Pro- ceedings, Volume II. Springer Berlin Heidelberg (1994) 151–158 g , p g g ( ) 15. Fua, P.: A parallel stereo algorithm that produces dense depth maps and preserves image features. Machine vision and applications 6(1) (1993) 35–49 ( ) ( ) 16. : Middlebury stereo database. http://vision.middlebury.edu/stereo/ 17. Scharstein, D., Hirschm¨uller, H., Kitajima, Y., Krathwohl, G., Neˇsi´c, N., Wang, X., Westling, P.: High-resolution stereo datasets with subpixel-accurate ground truth. In: Pattern Recognition. 7 Conclusion In this study, we have investigated the use of morphological operators within the analysis of stereo image superimpositions and deduced the corresponding disparity maps. The watershed transformation plays a pivotal role in that re- spect, since it is employed for the segmentation of the two images of the stereo pair, the clustering of disparity measures required by the filtering stage, and the interpolation mechanism leading to the dense disparity maps. An important aspect of this work has been the use of the left and right segmentations, in order to avoid irrelevant cost aggregations, perform the pruning of fattening artefacts appearing in the initial sparse disparity maps, and constrain the computation of distance functions used within the interpolation of the final disparity maps. The proposed method yields very good results for the sparse disparity map genera- tion. The dense estimation is the first of its kind to resort to a 3D watershed. While the results are quite encouraging, future work should concentrate on the regularisation of the interpolation process. Bricola J.-C., Bilodeau M., Beucher S. Bricola J.-C., Bilodeau M., Beucher S. 16 References Springer (2014) 31–42 18. Sinha, S.N., Scharstein, D., Szeliski, R.: Efficient high-resolution stereo matching using local plane sweeps. In: IEEE Conference on Computer Vision and Pattern Recognition (CVPR), IEEE (2014) 1582–1589
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Política de Educação Especial: sobre ambivalência, tensão e indeterminação
Educação & Realidade
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Educação & Realidade, Porto Alegre, v. 44, n. 1, e84860, 2019. http://dx.doi.org/10.1590/2175-623684860 Introdução Questões sobre a melhor forma de harmonizar educação e defici- ência são uma constante desde o Iluminismo. Quem deve receber uma Educação Especial e quem deve ensiná-la? Como identificar esses alu- nos e suas necessidades educacionais? Como e onde ensinar? Se certa dificuldade de consenso em relação a essas questões e sua base concei- tual marca a história da Educação Especial, paradoxos contemporâne- os enfatizam sua posição desafiadora (Richardson; Powell, 2011). Isso sustenta a tese, defendida por alguns teóricos, de que as questões mais importantes da área são igualmente contemporâneas e recorrentes: “Nunca completamente resolvidas, elas devem ser revisitadas a cada geração” (Kauffman et al., 2011, p. 15). Contemporaneidade e recorrência; insolubilidade de uma ques- tão em coexistência com seu permanente dever de reflexão. O que uma proposição dessa natureza, com sua carga de ambivalência e tensão, pode ter a pretensão de expressar? Como aceitar o caráter incessante dessas questões enquanto se busca contribuir de modo propositivo para o debate nesse contexto? De que modo pensar sobre essa modalidade de educação escolar em nosso país a partir de tal afirmação, decorrida uma década da publicação da Política Nacional de Educação Especial na Perspectiva da Educação Inclusiva – PNEEPEI (Bras il, 2008)? Partindo dessa problemática e com o objetivo de contribuir com a reflexão sobre os efeitos da PNEEPEI depois de 10 anos, consideramos produtivo interpretar os efeitos dessa política mediante a avaliação da pertinência, na atualidade, de questões que gestaram a sua produção; questões que, em princípio, apontavam para a existência de um equí- voco, para a possibilidade de um entrave ou uma dificuldade. Enfim, de algo a ser então esclarecido, confrontado ou por ela superado. Isso posto, propomos neste artigo retomar a discussão teórica que influenciou de forma decisiva a política de Educação Especial corrente, para, a partir de sua situação atual, refletir sobre seus efeitos diante da expectativa por ela gerada. Trata-se, portanto, de recuperar questões que aludiam à emergência de um pensamento e um posicionamento crítico sobre esse tema e justificariam o chamamento à perspectiva in- clusiva como possibilidade de ressignificar práticas – pedagógicas, po- líticas e sociais – capazes de segregar e estigmatizar os alunos da Edu- cação Especial. SEÇÃO TEMÁTICA: EDUCAÇÃO ESPECIAL, PSICANÁLISE E EXPERIÊNCIA DEMOCRÁTICA Política de Educação Especial: sobre ambivalência, tensão e indeterminação SEÇÃO TEMÁTICA: EDUCAÇÃO ESPECIAL, PSICANÁLISE E EXPERIÊNCIA DEMOCRÁTICA Wladimir Brasil UllrichI IUniversidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre/RS – Brasil RESUMO – Política de Educação Especial: sobre ambivalência, tensão e in- determinação. O artigo propõe retomar a discussão teórica que influenciou a Política Nacional de Educação Especial na Perspectiva da Educação Inclu- siva, com o objetivo de refletir sobre seus efeitos após uma década da publi- cação. Revisitamos questionamentos que constituem o debate nessa área, tomando por eixo central a análise de sua base conceitual – a adjetivação especial e a ideia de inclusão – considerando-a sob o registro da modernida- de. Além disso, destacamos uma estrutura dilemática a sustentar questões fundamentais em Educação Especial, bem como advertir sobre a força de certa ambivalência e tensão que as políticas e os discursos que engendram a Educação Especial pretendem superar. ç p p p Palavras-chave: Educação Especial. Inclusão. Política Educacional. Mo- dernidade. Conflito de Valores. ABSTRACT – Special Education Policy: on ambivalence, tension and inde- terminacy. The article proposes to resume the theoretical discussion that influenced the Brazilian National Policy on Special Education in the Pers- pective of Inclusive Education, with the aim of reflecting on its effects after a decade of its implementation. We revisit issues that constitute the debate in the area, taking as its central axis the analysis of its conceptual basis – the adjective special and the idea of inclusion – considering it under the aegis of Modernity. In addition, we highlight a dilemmatic structure that sup- ports fundamental issues in Special Education, as well as warn about the strength of a certain ambivalence and tension that policies and speeches engendering Special Education intend to overcome. g g p Keywords: Special Education. Inclusion. Educational Policy. Modernity. Value Tensions. 1 Educação & Realidade, Porto Alegre, v. 44, n. 1, e84860, 2019. http://dx.doi.org/10.1590/2175-623684860 Política de Educação Especial Educação & Realidade, Porto Alegre, v. 44, n. 1, e84860, 2019. A Modernidade não Poderia ser sobre Exclusão Pensar os efeitos da PNEEPEI uma década após sua publicação re- quer que se considere o seguinte lembrete: “[…] os problemas endereça- dos pela Educação Especial são problemas modernos; as necessidades que ela identifica são necessidades modernas” (Armstrong, 2003, p. 08). Ou seja, a junção das palavras educação e especial sob um único termo é algo que remonta fundamentalmente ao pensamento iluminista, ten- do ganho, porém, mais corpo e justificativa a partir do Estado de bem- -estar social forjado após a Segunda Guerra Mundial, quando “[…] os discursos dos direitos humanos foram colocados na cena primordial do espaço social” (Birman, 2009, p. 71). q g p Ou seja, a junção das palavras educação e especial sob um único termo é algo que remonta fundamentalmente ao pensamento iluminista, ten- do ganho, porém, mais corpo e justificativa a partir do Estado de bem- -estar social forjado após a Segunda Guerra Mundial, quando “[…] os discursos dos direitos humanos foram colocados na cena primordial do espaço social” (Birman, 2009, p. 71). Em outras palavras, o que antes da Idade Moderna não era um problema principal – a questão da inclusão e o papel da educação no processo de construção e manutenção de certa ideia de homem – passa a exigir resposta e dar a tônica de um conflito que ainda nos acompanha: “A Modernidade não era, e logicamente não poderia ser, sobre exclusão” (Armstrong, 2003, p. 10). Portanto, é nesse tempo que será colocado em destaque aquilo que todo Iluminismo tem em comum: a crença de que o homem é por natureza moralmente neutro e maleável pela educação; “[…] ou, na pior das hipóteses, com profundas deficiências, mas capaz de um melhoramento radical e indefinido pela educação racional e por circunstâncias favoráveis [...]” (Berlin, 2002, p. 293). Disso decorre um movimento que deixou marcas indeléveis na- quilo que nas últimas décadas se convencionou nomear como Educa- ção Especial. Trata-se, como destaca Joel Birman, de um bem conhecido signo constitutivo da modernidade no Ocidente dos séculos XVIII e XIX: “[…] a passagem crucial da problemática da salvação para a da cura” e o processo de “[…] medicalização do campo social como um todo”, com a enunciação, no centro estratégico desse processo, das categorias de normal, anormal e patológico. Introdução Busca-se, assim, revisitar questionamentos que constituem o de- bate nessa área, tomando por eixo central a análise de sua base con- ceitual – como, por exemplo, a adjetivação especial, principalmente por meio de seu correlato necessidades educacionais especiais, e a ideia de inclusão – considerando-a sob o registro da modernidade. A referência à modernidade tem dupla função, atuando como pano de fundo para iluminar o que consideramos uma estrutura dilemática a sustentar questões fundamentais em Educação Especial e como advertência so- bre a força de certa ambivalência e tensão que algumas políticas e os 2 Educação & Realidade, Porto Alegre, v. 44, n. 1, e84860, 2019. Ullrich Ullrich discursos que engendram a Educação Especial pretendem, talvez de modo incauto, superar. discursos que engendram a Educação Especial pretendem, talvez de modo incauto, superar. Educação & Realidade, Porto Alegre, v. 44, n. 1, e84860, 2019. A Modernidade não Poderia ser sobre Exclusão Tais categorias passaram a orientar os discursos e as práticas sociais e educacionais, levando esse modelo a se inscrever em um registro teórico e moral, “[…] de modo que o campo dos valores se imbricou intimamente com o dos conceitos” (Birman, 2009, p. 24-26). Por conseguinte, definições clínicas de vários tipos e graus de deficiência e subnormalidade se tornaram a prerrogativa dos médicos, contribuindo para que o diagnóstico médico-psicológico assumisse po- sição central na determinação de práticas pedagógicas, limitando, so- bretudo, as formas de conhecer em Educação Especial (Vasques, 2015). A centralidade e a utilidade dessas definições clínicas, por sua vez, não são de difícil compreensão. O diagnóstico e o uso rotineiro de rótu- los, tais como necessidades educacionais especiais, dificuldades de apren- dizagem e os correlatos que os antecederam, não são apenas exemplos de respostas sociais à diferença percebida em relação à norma, desempe- nhando a partir disso um papel importante nos processos de manutenção 3 Política de Educação Especial dos limites e da ordenação social. Eles revelam, também, a supremacia e a força dos valores médicos (Edwards, 2009), os quais objetivam o alívio do sofrimento ao estabelecer, mediante um consenso hegemônico e tranqui- lizador para a maioria, aquilo que significa ser um ser humano normal. Em outras palavras: dos limites e da ordenação social. Eles revelam, também, a supremacia e a força dos valores médicos (Edwards, 2009), os quais objetivam o alívio do sofrimento ao estabelecer, mediante um consenso hegemônico e tranqui- lizador para a maioria, aquilo que significa ser um ser humano normal. Em outras palavras: A deficiência é também um conceito normativo que refle- te ideias sobre o tipo de ser que os humanos devem ser, tanto mental como fisicamente, e como a sociedade deve ser construída para tratar seus membros igualmente. O termo deficiência implica que indivíduos considerados como deficientes não possuem habilidades humanas es- senciais ou possibilidades de se qualificarem como pes- soas (em um sentido moralmente significativo) ou para viver uma vida boa (Vehmas, 2004, p. 213). Fazer uso de diagnósticos e rótulos – de um modo ainda mais ra- dical no âmbito educacional nomeado especial – é, portanto, mais do que decidir sobre normalidade e patologia; é, provavelmente, marcar a deficiência e decidir sobre o outro de maneira até mesmo incontorná- vel, trazendo à tona o problema da validade da ação educativa sob um viés fundamentalmente ético. Educação & Realidade, Porto Alegre, v. 44, n. 1, e84860, 2019. A Modernidade não Poderia ser sobre Exclusão Essa situação provoca uma série de questionamentos no meio acadêmico e conduz a uma revisão conceitual e teórica nesse contexto, favorecendo as condições para a criação de uma nova política de Edu- cação Especial brasileira. Voltando ao quadro que inspirou a política, pode se dizer, grosso modo, que os questionamentos estavam relacio- nados, em um primeiro plano e momento, à crítica a uma perspectiva tradicional em Educação Especial, individualmente orientada, positi- vista, com raízes na psicologia, tendo como objetivo desenvolver me- lhores métodos educacionais para grupos de diagnóstico. Assim, seja ao perguntar em que sentido se justificava uma forma especial de en- tender e produzir uma educação para certos sujeitos (Skliar, 1997); seja no questionamento da primazia do modelo clínico-terapêutico e dos enfoques tecnicistas biomédicos ou psicologizantes comportamenta- listas (Ferre, 2001); seja na crítica ao conceito ambíguo de necessidades especiais, acusado de legitimar a exclusão de pessoas do sistema regular de educação e criar um subsistema especial (Tomlinson, 1982); o que se tinha, minimamente, era um apelo ao esmaecimento da fronteira dis- criminatória estabelecida entre educação e Educação Especial, tomada então como mais do que simplesmente um direito à educação. Essas questões desdobravam-se, por sua vez, em um segundo pla- no e momento, ainda na crítica à persistente retórica médico-psicoló- gica e seu destaque aos déficits individuais. Porém, não mais apenas por advogar a adoção de uma perspectiva social e contextual em edu- cação, mas também pela presumida qualificação dessa perspectiva por meio da adesão a um discurso de inclusão que ganhou maior promoção e força no contexto mundial a partir da primeira metade da década de 1990, colocando a Educação Especial no debate geral sobre igualdade e democracia e, majoritariamente, sob as regras desse debate. Com isso, Educação & Realidade, Porto Alegre, v. 44, n. 1, e84860, 2019. 4 Ullrich tratava-se, naquele momento e “[…] em definitivo, de avançar a uma educação para todos, com todos e para cada um” (Guijarro, 2005, p. 10). No entanto, já soava um alerta sobre distintos pressupostos e prá- ticas encontrados sob a bandeira da inclusão, que garantiria um con- senso aparente, pois acomodava posições divergentes. A Modernidade não Poderia ser sobre Exclusão Desse modo, seja ao questionar certa confusão feita pela maioria dos discursos oficias no uso de palavras tais quais diferença, diversidade e igualdade, além de alertar para o receio de que o diferente fosse apenas equalizado com os demais, como parte de um processo de inclusão iluminista (Veiga-Neto, 2005); seja ao criticar a transformação do debate em Educação Especial em um embate radical, em decorrência da imposição de uma concep- ção única e não consensual de educação, fundamentada no princípio da inclusão total (Mendes, 2006); seja ao identificar aí um campo que poderia ser definido pela superficialidade e interrogar em que medida um debate sobre inclusão tinha até então contribuído para que a edu- cação dos sujeitos considerados diferentes pudesse ser pensada como parte da educação (Baptista, 2004); o que estava em jogo pode ser inter- pretado, principalmente, como reiterada convocação a um necessário pensamento crítico sobre o tema. As questões estavam postas, forçando um, nem sempre desejado, olhar para trás. Todavia, um olhar necessário para que fosse possível, em algum sentido, atentar de modo crítico à finalidade da educação e ao sig- nificado de ser educado sob essa modalidade nomeada especial. Educação & Realidade, Porto Alegre, v. 44, n. 1, e84860, 2019. Uma Escola, Duas Educações Esta adota um “[…] paradigma educacional fundamentado na concep- ção de direitos humanos, que conjuga igualdade e diferença como va- lores indissociáveis” (Brasil, 2008). Assim, espera superar uma lógica de integração, bem como o modelo clínico-terapêutico na abordagem edu- cativa especial e a própria noção de políticas especiais. A escola parece retomar assim seu protagonismo na superação da lógica de exclusão. De acordo com essa política, na perspectiva de uma inclusão, “[…] a Educação Especial passa a constituir a proposta pedagógica da esco- la, definindo como público-alvo os alunos com deficiência, transtornos globais de desenvolvimento e altas habilidades/superdotação” (Brasil, 2008). Por fim, há uma proposta de articulação entre essas duas formas de educação, com objetivo de que sejam atendidas as necessidades es- peciais dos alunos. No turno inverso ao da classe comum ou das turmas comuns de ensino regular mantém-se o Atendimento Educacional Espe- cializado (Brasil, 2008). Pode se dizer, então, que há duas educações – a dos alunos comuns e a dos alunos especiais, bem como seus diferentes tipos de professores, os comuns e os especialistas – mesmo que haja ape- nas uma escola. Entretanto, ao nos voltarmos para a força dos conceitos que dão forma à política, e procurar entender de modo mínimo o que eles envol- vem, primeiramente cabe observar – mesmo que esse seja um truísmo – que só há um atendimento educacional especializado porque há alunos compreendidos como aqueles com necessidades educacionais especiais. Isso, por si só, já precisaria ser encarado com muita seriedade, já que: É impossível definir algo como uma ‘necessidade es- pecial’ na prática educativa sem alguns critérios de que tipos de resultados de aprendizagem (conhecimentos e habilidades) são desejáveis, importantes ou, talvez, até mesmo necessários. Em última análise, fazer a distinção entre necessidades comuns e especiais não é uma ques- tão de fato empírico. Em vez disso, é uma questão de fazer julgamentos de valores normativos do que é bom e valioso para os alunos e as pessoas em geral (Vehmas, 2010, p. 91). Uma Escola, Duas Educações A modernidade construiu uma forma específica de se referir ao fato educacional ao ancorar-se na escola; a escola se ocupou, por seu turno, da missão da modernização do homem. Uma razão para isso é que “[…] a promessa da modernidade na construção de um homem capaz de, constituindo-se a si e ao mundo, chegar à autonomia, à li- berdade e à justiça, gerou a articulação de ações políticas, com vistas à efetivação desses valores” (Hermann, 1996, p. 46). A escola moderna – partícipe de um sistema universal de ensino – surge então como me- diação necessária ao cumprimento de certas promessas. Cabe destacar, nesse sentido, que a questão da igualdade, como a denominamos, ou seja, sob um aspecto universal, não cabia na lógica da escola da antiguidade clássica, onde a diferença entre os homens era uma diferença de natureza, sendo desnecessária “[…] a sutil tecnologia social que faz da escola um mecanismo de incorporação do outro” por meio da promessa da igualdade (Maximiliano; Masschelein; Simons, 2017, p. 179). Em outras palavras, se na escola da antiguidade a igualda- de era um requisito para dela participar, na escola da modernidade ela se torna a finalidade da participação. É nessa linha de raciocínio que pode ser, mesmo que não exclu- sivamente, pensada a invenção moderna da Educação Especial e, pos- teriormente, a crítica a sua ênfase integracionista e a adesão a uma 5 Educação & Realidade, Porto Alegre, v. 44, n. 1, e84860, 2019. Política de Educação Especial abordagem educacional em consonância com uma ideologia de inclu- são. Com essa abordagem, a ênfase foi deslocada da discussão sobre as necessidades especiais para a agenda dos direitos, o que exigiu uma rein- terpretação da própria Educação Especial, como proposto na PNEEPEI. Esta adota um “[…] paradigma educacional fundamentado na concep- ção de direitos humanos, que conjuga igualdade e diferença como va- lores indissociáveis” (Brasil, 2008). Assim, espera superar uma lógica de integração, bem como o modelo clínico-terapêutico na abordagem edu- cativa especial e a própria noção de políticas especiais. A escola parece retomar assim seu protagonismo na superação da lógica de exclusão. abordagem educacional em consonância com uma ideologia de inclu- são. Com essa abordagem, a ênfase foi deslocada da discussão sobre as necessidades especiais para a agenda dos direitos, o que exigiu uma rein- terpretação da própria Educação Especial, como proposto na PNEEPEI. Uma Escola, Duas Educações Não obstante essa consideração, embora se argumente que o ter- mo necessidades educacionais especiais tenha sido introduzido buscan- do um afastamento de categorias de déficit, pode-se entender que uma das razões de seu malogro guarda relação justamente com a aceitação não problematizada do conceito de necessidade especial, o que pare- ce pressupor a existência de processos de avaliação infalíveis que irão diagnosticar e definir corretamente as necessidades dos alunos. Ou seja, trata-se de um conceito que não se afasta do horizonte interpreta- tivo ainda dominado pelo discurso médico-científico. É nessa esteira que se pode confirmar a força dos valores médicos e compreender possíveis fracassos na tentativa de rompimento com a 6 Educação & Realidade, Porto Alegre, v. 44, n. 1, e84860, 2019. Ullrich lógica da deficiência em educação – conforme empreendido, por exem- plo, por meio do Relatório Warnock, em 1978 –, bem como sustentar o fato de que o conceito de necessidade especial seja usado na prática como espécie de termo semitécnico ou especializado que cria a impres- são de que todo mundo sabe do que se está falando (Vehmas, 2010, p. 88-89). Pois, embora as necessidades educacionais especiais – conceito estabelecido por aquele relatório – aludissem aos recursos e ao supor- te para atender às diferenças relacionadas à aprendizagem, permanece forte a ideia de que ter uma necessidade especial é ter uma condição indesejável, restando o foco na deficiência. E como resultado teve-se o surgimento de uma supercategoria com uma série de rótulos que, em seu uso oficial, veio a ser sinônimo das dificuldades dos alunos em so- breposição ao possível suporte a ser a eles oferecido. Isso levou a uma contestável expansão da Educação Especial, motivando fortemente a crítica sociológica que contra ela se insurgiu. Porém, apesar de correta sob certo aspecto, a crítica pode ser vista tam- bém como excessiva, reforçando a estrutura dilemática por meio da qual se apresenta a Educação Especial. Pois, ao nos voltarmos para o Relatório Warnock, fica claro seu objetivo central de apontar o que era necessário, como suporte, para que cada criança pudesse fazer progres- sos em sua aprendizagem, em vez de ressaltar a sua deficiência. Inclu- sive, o dilema entre identificar as necessidades educacionais de alguns alunos e os efeitos negativos da possível rotulagem e estigmatização já era da ciência do próprio Comitê: Argumentar-se-á que o efeito prático de nossa proposta será apenas substituir um rótulo por outro. Educação & Realidade, Porto Alegre, v. 44, n. 1, e84860, 2019. Uma Escola, Duas Educações Acreditamos, no entanto, que o termo que propusemos, o qual será usa- do para fins descritivos e não para qualquer propósito de categorização, é preferível ao rótulo existente, pois dá mais indícios da natureza das dificuldades e é menos provável que estigmatize a criança (Warnock Report, Seção 3.26). Outro ponto a ser destacado diante da crítica sociológica, e seus desdobramentos em nossa política, é o fato de que o entendimento de que seria melhor que crianças com necessidades educacionais espe- ciais fossem educadas na escola regular não é uma novidade trazida pela Educação Inclusiva. Esse entendimento estava presente desde o movimento pela integração escolar em nosso país, mesmo que conco- mitantemente ainda estivesse implícita uma visão acrítica da escola, e a integração pudesse ser pensada como uma forma pragmática e politica- mente neutra de prestação de serviços (Mendes, 2006, p. 395). Assim, mesmo que a Educação Inclusiva carregue uma forte carga ideológica que parece poder traçar novos rumos para a Educação Es- pecial e fomentar um debate crítico sobre o tema, pode-se argumentar que a distinção entre inclusão e integração não justifica a separação dos conceitos e o tratamento da integração de forma negativa (Nor- wich, 2008). Aliás, pode-se destacar a própria intercambialidade entre os conceitos de integração e inclusão, encontrada em textos-chave para 7 7 Política de Educação Especial se pensar a Educação Especial, como a Declaração de Salamanca, de 1994, bem como a sua tradução em conformidade com a terminologia educacional de cada país. O que pode servir para confundir, mas tam- bém para elucidar, porque a própria história da Educação Especial pode ser compreendida como uma reação de inclusão, ao possibilitar que, “[…] no início do século XXI, a inclusão aparecesse como preocupação central e como um dos fins da educação nacional para as pessoas com deficiência” (Lopes; Fabris, 2013, p. 85-87). Uma Escola, Duas Educações Em suma, se não levarmos em conta a ideia de que as necessidades podem ser necessárias e contingentes, ao mesmo tempo que é extrema- mente difícil avaliar o peso moral dos vários objetivos que definem essas necessidades; se pretendermos ler a política de Educação Especial abrin- do mão de certa noção de continuidade e conexão entre as ideias de in- tegração e inclusão; se não compreendermos que o conceito de inclusão está fortemente vinculado a uma ideologia de justiça social, com toda a carga abstrata e multidimensional que isso comporta, levando a Educa- ção Especial a ser explicada majoritariamente como política; se descon- siderarmos a interpretação da inclusão como imperativo e do jogo com- plexo que jogamos sob o paradoxo inclusão/exclusão (Veiga-Neto, 2001); apenas estaremos regando as sementes de confusão por vezes plantadas por políticas de Educação Especial (Warnock, 2010). Assim, a pergunta sobre os objetivos da Educação Especial ou, sob outro aspecto, a pergunta sobre o seu sentido, são ainda de difícil resposta. Além disso, essas perguntas se desdobram em outras, como: “O que representa a manutenção da noção de especial nas práticas que constituem uma Educação Inclusiva?” (Lopes; Fabris, 2013, p. 113). E de que modo isso contribui para o estabelecimento de “[…] grandes ambi- guidades na política brasileira de escolarização das crianças com ne- cessidades especiais” (Plaisance, 2015, p. 236), incorrendo-se no risco de que ela não se desenvolva em desejável oposição à estigmatização e às delimitações impostas pelos tão criticados diagnósticos médicos? Um exemplo ilustrativo dessa ambiguidade e do risco se dá, por exemplo, quando se toma ciência do crescimento do número de Sa- las de Recursos Multifuncionais disponibilizadas; ou seja, do saliente apoio técnico e financeiro aos sistemas públicos de ensino para a ga- rantia do atendimento educacional especializado nos primeiros anos após a publicação da política de Educação Especial vigente. Algo que poderia, de modo rápido, ser visto como um avanço, mas que, ao não estar acompanhado em contrapartida pelo apoio técnico e financeiro naquilo que concerne às salas de ensino regular e à formação de seus professores, parece apenas contribuir para o protagonismo do atendi- mento especializado em um contexto que se quer inclusivo, causando possivelmente a impressão de que apenas novos nomes são dados às indesejadas classes especiais. Uma Escola, Duas Educações Portanto, é nesse contexto que cabe perguntar: “Quais são os pres- supostos implicados na valorização da sala de recursos como o espa- ço prioritário para o apoio especializado aos alunos com deficiência?” (Baptista, 2011, p. 61). Ou, ainda: “Em que medida as políticas inclusivas 8 Educação & Realidade, Porto Alegre, v. 44, n. 1, e84860, 2019. Ullrich de Educação Especial desconstroem sentidos que relacionam a dife- rença à desigualdade, à anormalidade e à ineducabilidade?” (Vasques; Moschen; Gurski, 2013, p. 81). Seria possível argumentar, o que guarda certa ironia, que a Educação Especial brasileira se dá na atualidade sob a perspectiva da Educação Inclusiva, mas certos diagnósticos (Asperger, Williams, Rett, entre outros, uma vez nomes próprios), bem como a força do manual que os compila e os significa, não tiveram atenuada sua fun- ção normalizadora e espacializante. Em palavras esclarecedoras: de Educação Especial desconstroem sentidos que relacionam a dife- rença à desigualdade, à anormalidade e à ineducabilidade?” (Vasques; Moschen; Gurski, 2013, p. 81). Seria possível argumentar, o que guarda certa ironia, que a Educação Especial brasileira se dá na atualidade sob a perspectiva da Educação Inclusiva, mas certos diagnósticos (Asperger, Williams, Rett, entre outros, uma vez nomes próprios), bem como a força do manual que os compila e os significa, não tiveram atenuada sua fun- ção normalizadora e espacializante. Em palavras esclarecedoras: Não precisamos questionar a motivação psicológica bá- sica da inclusão, nem os conceitos básicos de que ela faz uso: ninguém é contra a compaixão, a justiça ou a fra- ternidade; a dificuldade é antes identificar uma teoria coerente que coloca esses sentimentos e conceitos em al- guma forma racional e defensável. Resumidamente, nós precisamos saber o que queremos dizer com ‘inclusão’ [...]. Sem isso, temos apenas um conjunto do que se pode chamar de intuições apaixonadas, que então traduzimos de forma acrítica na prática: algo que facilmente acontece mesmo com os movimentos revolucionários ou evolutivos mais bem motivados, às vezes com resultados desastrosos (Wilson, 2000, p. 297). Uma Escola, Duas Educações Desse modo, é diante da falta de consenso e definição quanto a um conceito que orienta a política educacional e supõe fazer a diferen- ça para o ensino e a escolaridade, que algumas questões necessitam ser abordadas: Qual a validade, em termos de Educação Inclusiva, da constatação de que avançamos na oferta de apoio e aumentamos a dis- ponibilidade das salas de recursos como dispositivo pedagógico para a ação da Educação Especial, todavia sem a contrapartida no que diz res- peito ao investimento no espaço comum? Se os alunos com necessida- des educacionais especiais passam parte do seu tempo fora das salas de aula regulares, sob um atendimento especializado e espacializante, de que modo isso é consistente com a inclusão e o que estabelece o limite para essa consistência? Qual o status epistemológico da Educação Es- pecial na perspectiva da Educação Inclusiva e como tal status repercute nas discussões sobre currículo e avaliação? Já não são essas questões urgentes, do mesmo modo que pareciam ser aquelas que precederam a PNEEPEI? E, se ainda não são, seria isso um efeito da própria política? Desvio: impureza ideológica e indeterminação Em um texto onde procura recuperar a passagem da Educação Especial à Educação Inclusiva, aludindo à realidade de nosso país, Eric Plaisance estabelece o ponto central de sua crítica a nossa política: De fato, esta se define como Política Nacional de Educa- ção Especial na Perspectiva da Educação Inclusiva. Conse- quentemente, pode-se questionar a pertinência de uma coexistência do especial e do inclusivo, que tangem, no entanto, a paradigmas opostos e orientam para políticas radicalmente diferentes. Além disso, esse texto oficial de- 9 Educação & Realidade, Porto Alegre, v. 44, n. 1, e84860, 2019. Política de Educação Especial fine crianças como públicos-alvo. Ora, a própria noção de necessidades especiais sugere uma atenção às particulari- dades educativas das crianças, às suas singularidades e, por conseguinte, opõe-se à delimitação de diagnósticos médicos ou psiquiátricos. É grande o risco de novamente categorizar as crianças e se orientar para uma psicopato- logia da inclusão (Plaisance, 2015, p. 236). Como sustentar uma posição teórica que defenda a proposta de colocar a Educação Especial e a Educação Inclusiva em situação de co- existência, mesmo diante do risco que isso implica e do fato de essas concepções de educação serem, em muitos momentos, antípodas no debate educacional? Em uma resposta provável, seria possível defender justamente a ideia de suspender a oposição, recorrendo-se a uma espé- cie de perspectivismo, onde a interpretação da política e de seus efeitos dependeria, ao fim, do balanço dessas perspectivas. Porém, nesse caso, o peso a ser porventura colocado sobre uma ou outra perspectiva es- taria necessariamente relacionado ao entendimento da força de certas palavras, bem como do uso que fazemos delas, da compreensão do seu papel em um processo mais amplo que é o da produção de sentido em determinados contextos históricos. Enfim, no caso da Educação Espe- cial, não apenas do entendimento do que pode estar em jogo em relação aos conceitos especial e inclusão, mas da compreensão da modernidade como experiência histórica e subjetiva, um modo de estar no mundo que dá forma a esses conceitos. Educação & Realidade, Porto Alegre, v. 44, n. 1, e84860, 2019. Desvio: impureza ideológica e indeterminação E aqui, essa espécie de perspectivismo parece logo se deparar com um complicado nó, pois, quando a Educa- ção Especial opera sua crítica à modernidade – por exemplo, ao oferecer a denúncia da racionalidade científica e seu discurso médico com as conhecidas oposições normal/anormal – ela está, em um sentido forte, opondo-se à sua criação, uma vez que surge a partir do reconhecimento de deficiências oriundas da prevalência dessa racionalidade. Por outro lado, ao adotar a perspectiva da inclusão como contraponto, ela pode não encontrar melhor argumento para se contrapor a essa criação: ela acabará por tornar consciente para si mesma a amplitude de significa- do da inclusão que, também parte do projeto moderno, esborra um con- ceito de especial. De fato, o que está em jogo ainda é a tentativa de equilibrar valo- res modernos conflitantes. Estamos comprometidos com valores como identidade, diferença, liberdade e igualdade, entre outros. Nossa políti- ca de Educação Especial está fundamentada sob uma “[…] concepção de direitos humanos, que conjuga igualdade e diferença como valores indissociáveis” (Brasil, 2018). A inclusão, por sua vez, é geralmente en- tendida como implicando que as diferenças entre as crianças devem ser valorizadas e que a diferença é uma condição natural para a escolarida- de. No entanto, a ideia de uma condição natural está associada a uma concepção de natureza humana, à posse de certos atributos e ao desen- volvimento de certas capacidades. E é justamente sob uma concepção de natureza humana, modernamente aprimorada, que certas diferen- ças ou deficiências parecem difíceis de serem admitidas. Educação & Realidade, Porto Alegre, v. 44, n. 1, e84860, 2019. 10 Ullrich O que a nossa política de Educação Especial faz, então? Ela não adota novos valores em resposta aos valores modernos, tampouco os questiona de modo assertivo. Ela simplesmente os junta, paradoxal- mente. Não que algumas de suas proposições sejam nossa exclusivi- dade, que apenas nessa política certa ambiguidade e ambivalência estejam estabelecidas. Contudo, ao colocar as coisas de certo modo, a PNEEPEI pode ser vista não apenas como a expressão radical do telos moderno, em sua incessante aproximação e luta contra certos ideais, mas também como responsável, ao se debater contra os limites impos- tos pela contradição, por operar, ao fim, um talvez salutar curto-circui- to na lógica moderna. Educação & Realidade, Porto Alegre, v. 44, n. 1, e84860, 2019. Desvio: impureza ideológica e indeterminação Conforme expõe Philippe Bénéton, o espírito moderno é compó- sito, ele paira entre duas ideias ou interpretações que estão em ação no seio da história moderna: A primeira fundamenta-se sobre o que a pró- pria humanidade exige do homem, a segunda, sobre o que resulta da autonomia da vontade. Em decorrência dessas ideias, duas concepções de igualdade então se cruzam. Por um lado, temos uma igualdade subs- tancial, onde a qualidade de homem pertence igualmente a todos, bem como a sua dignidade a ser respeitada. Por outro lado, “[…] os homens são iguais não porque partilham qualquer coisa de substancial que lhes faz homens, mas, ao contrário, por carência de substância. O próprio do homem, sob esse aspecto, é sua indeterminação e, portanto, sua liber- dade absoluta” (Bénéton, 2002, p. 159-160). Como consequência dessas concepções, temos que ideias mo- dernas caras ao discurso educacional, como identidade, autenticidade, emancipação e liberdade, acabam por tensionar o princípio de igual- dade reivindicado pela própria modernidade e parte do seu discurso. Diante desse quadro, a PNEEPEI pode ser vista tanto como crítica à Educação Especial e ao seu histórico déficit social quanto como crítica, mesmo que enviesada, ao próprio projeto moderno no qual essa mo- dalidade educacional tem suas raízes, bem como um indicativo dos li- mites de certa perspectiva de inclusão, de abordagem radical, a qual se coloca como norte de muitos movimentos sociais, mas parece descon- siderar a própria tensão inerente ao conceito de inclusão e sua relação bipartite com a ideia de exclusão: aqui, a Educação Especial desafia a própria ideia de inclusão. Assim, explicitar um dilema da diferença no próprio título de uma política educacional, ainda mais em um contexto em que esses dilemas são centrais (Nilholm, 2006, p. 434), pode ser pensado como uma rea- ção de resistência do homem ao projeto iluminista; como uma resposta à aventada crise da modernidade e ao fracasso do processo civilizatório que conduz à crise da educação e da escola, onde somos então convoca- dos a inverter o peso da balança das interpretações que estão no seio da história moderna. Sob outra ótica, preservamos a ideia da escola como lugar de igualdade, mas não mais de uma igualdade substancial como promessa civilizatória até aqui não realizada. Desvio: impureza ideológica e indeterminação Deslocamos a igualdade para sua outra possibilidade de compreensão: como indeterminação e potência que constitui cada homem; desvinculada, portanto, da pro- 11 Política de Educação Especial messa desenvolvimentista e “[…] da temporalidade progressiva à qual a submeteu a época moderna” (Maximiliano; Maschelein; Simons, 2017, p. 186). Desse modo, não viramos as costas para metadiagnósticas que compreendem a modernidade como um “[…] movimento alternado de expectativas de determinação, racionalização e impessoalização segui- das por um contramovimento de anomia, de indeterminação e de am- biguidade” (Dunker, 2015, p. 188). No entanto, onde a indeterminação não deve ser pensada em uma lógica dicotômica, como a falta simétrica da determinação, como o problema, mas como possuindo estatuto on- tológico próprio que, mesmo negativo, ressalta o valor do encontro e da contingência (Dunker, 2015, p. 290). Em vista disso, ao se admitir a inconsistência ontológica da subje- tividade e a dificuldade do cumprimento de algumas promessas feitas pelo homem, bem como ao se considerar a importância de reconhecer, inclusive, “[…] a dimensão produtiva e constitutiva de certas experiên- cias de indeterminação” (Dunker, 2015, p. 229), pode-se mirar além das expectativas modernas e pensar então a escola como um todo e para todos, bem como a Educação Especial – mas não, de modo cínico, so- mente esta e seus alunos – sob uma outra perspectiva: uma perspectiva onde a força e o peso do político, do cultural e da racionalidade científi- ca podem ser suspensos temporariamente, fortalecendo “[…] a ideia de que na aula algo ‘pode acontecer’, a força de todos os tipos de definições e determinações podem ser anuladas” (Kohan; Masschelein; Simons, 2017, p. 170). Enfim, pode-se até sustentar a tese de que a PNEEPEI se expressa em clara ambiguidade, caindo em uma espécie de cilada provocada pe- las palavras da Educação Especial (Plaisance, 2015). Mas podemos lê-la, e essa é a aposta deste texto, como intrincada provocação, como ma- nifestação de certa impureza ideológica (Norwich, 1996) inevitável em uma estrutura dilemática constituída por valores conflitantes. Logo, não como uma política assertiva que oferece diretrizes com as mesmas características, mas que, ao não negar uma tensão que é constitutiva da Educação Especial – o que explica, ao menos em parte, a contempo- raneidade e recorrência de suas questões –, provoca-nos a pensar qual seria a solução para escapar da cilada, caso a queda tivesse realmente ocorrido. Educação & Realidade, Porto Alegre, v. 44, n. 1, e84860, 2019. 12 Considerações Finais Recebido em 16 de julho de 2018 Aprovado em 20 de outubro de 2018 Recebido em 16 de julho de 2018 Aprovado em 20 de outubro de 2018 Considerações Finais A tese que funciona como leitmotiv para a reflexão neste texto expressa algo constitutivo da Educação Especial. Aponta para a con- temporaneidade de suas questões, ao mesmo tempo em que indica a necessidade de sua revisão permanente. No entanto, ao apontar para o caráter recorrente dessas questões, não apenas indica que temos um persistente caminho pela frente, mas também nos força a tentar com- preender possíveis razões para isso. Nesse movimento, cruza passado e presente, fazendo-nos revisitar, mesmo que de modo breve, os ques- 12 Educação & Realidade, Porto Alegre, v. 44, n. 1, e84860, 2019. Ullrich tionamentos que precederam a atual política de Educação Especial em nosso país, além de parte substancial daquilo que podemos chamar de herança moderna e se apresenta como a expressão de um dilema maior: o do homem que se debate contra o próprio homem. Assim, a discussão teórica aqui proposta teve como intuito mostrar, como possível modo de compreensão dessas razões, a força dos problemas colocados pela modernidade, aos quais a Educação Especial dá chamativos contornos por meio de movimentos de ambivalência e tensão. Esses podem ser vistos, de modo equivocado, como obstáculos não apenas conceituais, mas como paredes ideológicas para a pesquisa e a reflexão nesse cam- po. Porém, se aceitamos a força histórica desses movimentos – prin- cipalmente se não negamos essa herança sob a qual nos forjamos – pas- samos então a olhá-los não como um obstáculo, mas como uma parede simplesmente a indicar que não podemos continuar por onde seguía- mos. Nessa perspectiva, sob a qual aqui interpretamos o texto de nossa política e certa crítica a ele, a indicação de que não podemos continuar por certo caminho implica não pensar a ambivalência e a tensão como objeções à reflexão, mas sim, pensar como objeção e persistente obstá- culo o modo de reflexão que procura eliminar esses aspectos do debate em Educação Especial, desviando-se de um pensamento crítico para o qual este texto pode ser pensado como modo de exercício. A questão, então, é lavar ou não lavar as mãos; não aceitar ou aceitar jogar o jogo da ambivalência, da tensão e, possivelmente, da indeterminação. Isso sem esquecer que uma estrutura dilemática aponta para a inescapibilidade do conflito, requerendo assim que Educação Especial e ética necessa- riamente andem juntas. Recebido em 16 de julho de 2018 Aprovado em 20 de outubro de 2018 Referências ARMSTRONG, Derrick. Experiences of Special Education: re-evaluating policy and practice through life stories. 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Rio de Janeiro: Civilização Brasileira, 2009. 13 Educação & Realidade, Porto Alegre, v. 44, n. 1, e84860, 2019. Política de Educação Especial BRASIL. Política Nacional de Educação Especial na Perspectiva da Educação Inclusiva. Brasilia: Ministério da Educação; Secretaria de Educação Especial, 2008. DUNKER, Christian Ingo Lenz. Mal Estar, Sofrimento e Sintoma: uma psicopa- DUNKER, Christian Ingo Lenz. Mal Estar, Sofrimento e Sintoma: uma psicopa- tologia do Brasil entre muros. São Paulo: Boitempo Editorial, 2015. , g , p p tologia do Brasil entre muros. São Paulo: Boitempo Editorial, 2015. EDWARDS, Steven. Definitions of Disability: ethical and other values. In: KRIS- TIANSEN, Kristjana; VEHMAS, Simo; SHAKESPEARE, Tom (Org.). Arguing About Disability: Philosophical perspectives. Nova York: Routledge, 2009. P. 30-41. FERRE, Nuria Pérez de Lara. Identidade, Diferença e Diversidade: manter viva a pergunta. In: SKLIAR, Carlos; LARROSA, Jorge (Org.). Habitantes de Babel: polí- ticas e poéticas da diferença. Belo Horizonte: Autêntica, 2001. P. 195-214. GUIJARRO, María Rosa Blanco. Inclusão: um desafio para os sistemas educa- cionais. In: MEC. Ministério da Educação. Ensaios Pedagógicos: Construindo Escolas Inclusivas. Brasília: MEC, SEESP, 2005. P. 7-14. HERMANN, Nadja. Educação e racionalidade: conexões e possibilidades de uma razão comunicativa na escola. Porto Alegre: EDIPUCRS, 1996. KAUFFMAN, James et al. Contemporary Issues. In: KAUFFMAN, James, HALLAHAN, Daniel (Org.). Handbook of Special Education. Nova York: Rou- tledge, 2011. P. 15-26. KOHAN, Walter Omar; MASSCHELEIN, Jan; SIMONS, Maarten. Sobre a Escola que Defendemos. In: LARROSA, Jorge (Org.). Elogio da Escola. Belo Horizonte: Autêntica, 2017. P. 161-176. LOPES, Maura Corcini; FABRIS, Eli Henn. Inclusão & Educação. Referências Belo Horizonte: Autêntica, 2013. MAXIMILIANO, Valério López; MASSCHELEIN, Jan; SIMONS, Maarten. Skholé e Igualdade. In: LARROSA, Jorge (Org.). Elogio da Escola. Belo Horizonte: Autên- tica, 2017. P. 177-193. MENDES, Enicéia Gonçalves. A Radicalização do Debate sobre Inclusão Escolar no Brasil. Revista Brasileira de Educação, Rio de Janeiro, v. 11, n. 33, p. 387-405, dez. 2006. NILHOLM, Claes. Special Education, Inclusion and Democracy. European Journal of Special Needs Education, v. 21, n. 4, p. 431-445, nov. 2006. NORWICH, Brahm. Special Needs Education or Education for All: connective specialisation and ideological impurity. British Journal of Special Education, Londres, v. 23, n. 3, p. 100-104, set. 1996. NORWICH, Brahm. Dilemmas of Difference, Inclusion and Disability: interna- tional perspectives and future directions. Nova York: Routledge, 2008. PLAISANCE, Eric. Da Educação Especial à Educação Inclusiva: esclarecendo as palavras para definir as práticas. Educação, São Paulo, v. 38, n. 2, p. 230-238, maio/ago. 2015. RICHARDSON, John; POWELL, Justin. Comparing Special Education: origins to contemporary paradoxes. California: Stanford University Press, 2011. SKLIAR, Carlos. Educação & Exclusão: abordagens sócio-antropológicas em Educação Especial. Porto Alegre: Mediação, 1997. TOMLINSON, Sally. A Sociology of Special Education. Nova York: Routledge, 1982. 14 Educação & Realidade, Porto Alegre, v. 44, n. 1, e84860, 2019. Ullrich VASQUES, Carla Karnoppi. Formas de Conhecer em Educação Especial: o diag- nóstico como escudo e lista. Revista de Educação, Campinas, v. 20, n. 1 , p. 51- 59, jan./abr. 2015. VASQUES, Carla Karnoppi; MOSCHEN, Simone; GURSKI, Roselene. Entre o Tex- to e a Vida: uma leitura sobre as políticas de Educação Especial. Educação e Pesquisa, São Paulo, v. 39, n. 1, p. 81-94, jan./mar. 2013. VEHMAS, Simo. Ethical Analysis of the Concept of Disability. U.S. National Li- brary of Medicine, Montgomery/EUA, v. 42, n. 3, p. 209-222, jul. 2004. VEHMAS, Simo. Special Needs: a philosophical analysis. International Journal of Inclusive Education, Londres, v. 14, n. 1, p. 87-96, fev. 2010. VEIGA-NETO, Alfredo. Incluir para Excluir. In: SKLIAR, Carlos; LARROSA, Jorge (Org.). Habitantes de Babel: políticas e poéticas da diferença. Belo Horizonte: Autêntica, 2001. P. 105-118. VEIGA-NETO, Alfredo. Quando a Inclusão Pode Ser uma Forma de Exclusão. In: MACHADO, Adriana Marcondes et al. (Org.). Psicologia e Direitos Humanos: educação inclusiva-direitos humanos na escola. São Paulo: Casa do Psicólogo, 2005. P. 55-70. WARNOCK COMMITTEE et al. Special Educational Needs: The Warnock Re- port. London, 1978. WARNOCK, Mary. Special Educational Needs: a new look. In: TERZI, Lorella (Org.). Educação & Realidade, Porto Alegre, v. 44, n. 1, e84860, 2019. VASQUES, Carla Karnoppi. Formas de Conhecer em Educação Especial: o diag- nóstico como escudo e lista. Revista de Educação, Campinas, v. 20, n. 1 , p. 51- 59, jan./abr. 2015. Referências Special Educational Needs: a new look. Londres: Continuum, 2010. P. 11- 46. WILSON, John. Doing justice to inclusion. European Journal of Special Needs Education, Londres, v. 15, n. 3, p. 297-304, 2000. WILSON, John. Doing justice to inclusion. European Journal of Special Needs Education, Londres, v. 15, n. 3, p. 297-304, 2000. Wladimir Brasil Ullrich é doutorando e mestre em Educação pela Univer- sidade Federal do Rio Grande do Sul (UFRGS), licenciado e bacharel em Fi- losofia também pela UFRGS. É integrante do Núcleo de Pesquisa em Psica- nálise, Educação e Cultura (NUPPEC/UFRGS). Desenvolve pesquisas nas áreas de filosofia moral e educação. ORCID: http://orcid.org/0000-0002-0138-9535 Este é um artigo de acesso aberto distribuído sob os termos de uma Licen- ça Creative Commons Atribuição 4.0 Internacional. Disponível em: <http:// creativecommons.org/licenses/by/4.0>. 15 15
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Teacher Development to Augment Pedagogical Competence in Society 5.0
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Abstract This research aims to analyze and describe the process of enhancing teachers' pedagogical competence in Society 5.0. A qualitative research methodology was employed, utilizing a case study approach. Data were collected through interviews with one principal, one vice principal, six teachers, and one administrative staff member. Data processing techniques included reduction, data presentation, and conclusion drawing. The validity of the data was ensured through credibility and confirmability. The findings indicate three stages of teacher development: (a) Awareness and behavior formation stage, which involves training needs analysis, learning design, access to self- contained material centers, teacher motivation, moral and material support, performance assessment, Yaumiyah, Tastqif, and Tahfidzul Qur'an programs. (b) The ability transformation stage focused on education quality, the application of independent learning, and learning design. (c) The ability enhancement stage involved IT-based learning and teacher collaboration. The outcomes of teacher development encompassed competence mapping, student mentoring, identification of developmental needs, fostering Islamic personality, nurturing character-driven teachers, employing Student- Centered and Project Learning models, and an increase in teachers' knowledge. Corresponding Author: Sabariah: Muhammad Arsyad Al Banjari Islamic University of Banjarmasin, Indonesia; Sabariahsabariah997@gmail.com Teacher Development to Augment Pedagogical Competence in Society 5.0 Sabariah1, Luqmanul Hakim2, Kadori3, Annisa Zahra4, Abdul Muin5 1 Muhammad Arsyad Al Banjari Islamic University of Banjarmasin, Indonesia; Sabariahsabariah997@gmail.com 2 PGRI Adi Buana University Surabaya, Indonesia; luqmanulhakim@unipasby.ac.id 3 Mulawarman University, Indonesia; haidarkadori1@gmail.com 4 Muhammad Arsyad Al Banjari Islamic University of Banjarmasin, Indonesia; nisazahra136@gmail.com 5 Muhammad Arsyad Al Banjari Islamic University of Banjarmasin, Indonesia; abdulmuin0813@gmail.com INTRODUCTION The inception of Society 5.0 heralds an era of technological amalgamation with education to respond to the evolving facilities and human resource conditions in Indonesia. This investigation analyzes the interlacing of education with advances in technology, taking into account human resource competencies, the Internet of things, augmented reality, and artificial intelligence (Sukatin et al., 2022). Education management in the Society 5.0 era integrates information technology with educational necessities in schools (N. N. L. Handayani & Muliastrini, 2020). Hence, educational processes within an institution must align with the demands of Society 5.0, embodying an innovative, creative, and superior educational system (Harun, 2021). The integration of technology in education in Society 5. In the era of Society 5.0, teachers play a pivotal role as agents of knowledge and technology transformation, leveraging advancements in information technology as the primary medium supporting educational processes (Padmawati & Pihung, 2022). Innovative educational and learning systems are key to ensuring students acquire the requisite competencies and creativity (Pinatih, 2020). Furthermore, to actualize the educational system in Society 5.0, teachers must enhance their pedagogical competence through technology-based learning centered on students (Mega, 2022). The application of pedagogical competence through learning also necessitates a futuristic learning system and apt learning models to holistically develop student potential to meet the challenges of Society 5.0 (Ma’rufah & Arsanti, 2022). Thus, teachers in the Society 5. Teacher development in Society 5.0 is one of the measures schools adopt to prepare teachers to face challenges in an era intertwined with information technology and the internet (Ayub et al., 2022). Teacher development in schools can be realized through several means such as in-service training, scholarly work development, professionalism enhancement, research development, and others that can augment teachers' capabilities in Society 5.0 (Husaini, 2018). Through development, teachers can create more innovative learning experiences that ensure maximal delivery of teaching materials to students in Society 5.0 (Anggreini & Priyojadmiko, 2022). Teacher development aims to enhance classroom teaching systems, rectify discrepancies or errors during the learning process until a conducive classroom climate is established, The research results indicate that teacher development, employing the Hakadohop approach conducted in SDN Balukon 1 and SDN Bukit Bamba 1, successfully enhanced teachers' professionalism through supervision, in-house training, evaluation meetings, and teacher file evaluations (Berliani et al., 2022). Abstrak Tujuan penelitian untuk menganalisis dan mendeskripsikan proses pembinaan kompetensi pedagogic guru di era 5.0. Metode penelitian kualitatif dengan pendekatan studi kasus. Teknik pengumpulan data: wawancara kepada 1 kepala sekolah, 1 wakil kepala sekolah, 6 guru dan 1 tenaga administrasi. Teknik pengolahan data: reduksi, penyajian data dan penarikan kesimpulan. Teknik keabsahan data: kredibelitas (triangulasi teknik dan sumber) serta konfirmabilitas. Hasil penelitian menunjukkan: (1) pembinaan guru: (a) tahap penyadaran dan pembentukan perilaku: analisis kebutuhan pelatihan, desain pembelajaran, akses pusat materi mandiri, memotivasi guru, dukungan moril dan materiil, penialaian kinerja, program Yaumiyah, program pembinaan Asosiasi Pendidikan Nasional Indonesia, program Tastqif, dan program Tahfidzul Qur`an. (b) tahap tranformasi kemampuan: mutu pendidikan, aplikasi merdeka belajar dan desain pembelajaran. (c) tahap peningkatan kemampuan: pembelajaran berbasis IT dan kolaborasi sesame guru. (2) hasil pembinaan guru: pemetaan kompetensi, pembimbing siswa, indentifikasi kebutuhan pembinaan, berkepribadian Islami, guru berkarakter, menggunakan model pembelajaran Student Centered dan Project Learning serta pengetahuan guru semakin meningkat. j g p g g g Corresponding Author: Sabariah: Muhammad Arsyad Al Banjari Islamic University of Banjarmasin, Indonesia; Sabariahsabariah997@gmail.com http://pasca.jurnalikhac.ac.id/index.php/tijie/index Sabariah et al 666| Vol. 4, No. (4) 2023: Tafkir: Interdisciplinary Journal of Islamic Education Teacher Development to Augment Pedagogical Competence in Society 5.0 This research specifically aims to elucidate and qualitatively reveal how school principals facilitate teacher development to augment pedagogic competence in Society 5.0. The development process unfolds in three stages: firstly, explaining and uncovering the awareness and behavior formation stage that consists of development preparation processes, stimulating, and forming teacher behaviors for development. Second, delineating and revealing the ability transformation stage, which encompasses the transformation of knowledge and skills regarding teachers' pedagogical competence. Lastly, elucidating and introducing the ability enhancement stage that involves augmenting the knowledge and skills of teachers in schools. INTRODUCTION Another study showcases that school principal-led development pays attention to and evaluates teachers' learning administration, such as RPP, learning implementation, and learning evaluation activities, also undertaking follow-up measures post-development (Fatimah, 2022). The divergence in these two research outcomes lies in their focus; this study emphasizes schools, especially principal-led teacher development, to prepare teachers for Society 5.0 through enhancing pedagogic competence in teachers. 666| Vol. 4, No. (4) 2023: Tafkir: Interdisciplinary Journal of Islamic Education 667| Vol. 4, No. (4) 2023: Tafkir: Interdisciplinary Journal of Islamic Education RESEARCH METHOD Research concerning the development in the Society 5.0 era to enhance teachers' pedagogical competence was conducted qualitatively through a case study approach. The research was carried out to explain and describe in depth and naturally the process of awareness and behavior formation, capability transformation, and capability enhancement as well as the results of the development process in improving teachers' pedagogical competence in the Society 5.0 era at the Integrated Islamic Elementary School Robbani Banjarbaru. Data collection techniques in this research were conducted through three techniques. First, the interview technique, which involved interviewing 1 principal, 1 vice curriculum, 6 teachers, and 1 school administrative staff about the process of awareness and behavior formation, capability transformation, and teacher capability enhancement, as well as the results of pedagogic competence development process in the Society 5.0 era. Second, the observation technique, observing the process of awareness and behavior formation, capability transformation, and capability enhancement, as well as the results of the development process for 2 months, from May to June 2023. Third, the documentation technique, collecting documents in the form of development files from the principal, development documentation files, guidebooks, Data processing techniques in this research were carried out using the approach from Miles and Huberman which consists of four main steps. First, data collection, data collection in this research was carried out through interviews, observation, and documentation techniques. Second, data condensation, where the researcher collected data according to two focuses in this research, firstly grouping data based on stages in development, namely the stages of awareness and behavior formation, capability transformation, and capability enhancement. Second, grouping data based on the results of the Society 5.0 era development process in improving teachers' pedagogic competence at Integrated Islamic Elementary School Robbani Banjarbaru. Third, data presentation, where the researcher displays research results in the form of sentences or brief descriptions and tables that can explain research results based on the results of the condensation that has been carried out. Fourth, conclusion drawing, where the researcher makes conclusions on the data that has been condensed and presented in sentence form with the aim of obtaining valid research results. 668| Vol. 4, No. (4) 2023: Tafkir: Interdisciplinary Journal of Islamic Education Sabariah et al Data validity techniques in this research were carried out with two techniques, namely credibility and confirmability techniques. First, credibility techniques were carried out through technique triangulation and source triangulation. Triangulation technique was done by comparing research results from interviews that were compared with observation results and documentation results. Meanwhile, source triangulation was carried out by comparing research results from the principal with the vice curriculum, teachers, and also educational/administrative staff. Second, confirmability, which was carried out after the research results were compiled and tested for credibility. Confirmability in this research was carried out by reconfirming research results to research sources, namely the principal, vice curriculum, teachers, and administrative staff. Teacher Development to Augment Pedagogical Competence in Society 5.0 This statement was also confirmed by teachers Zulkarnaen, S. Pd and Harist Setiyawan, S. Pd who were interviewed on May 16 2023 in the teachers' room and explained as follows. This stimulation can be done by providing full support to teachers both in terms of material and non-material. By ensuring all teachers access PMM and report the achievements of their independent training by taking real action. Implementing learning communities according to level From this statement it can be concluded that stimulating awareness is carried out by providing motivation, support, material and non-material, being able to access the Independent Material Center, and forming a community according to the level of teachers at school. Meanwhile, for the behavior formation process, the results of interviews with the school principal, namely Oelistianti, ST., M. Pd. who was interviewed on May 15 2023 in the school principal's room explaining. “By assessing teacher performance through learning supervision, by completing teacher yaumiyah practices, carrying out apni coaching activities, taking part in monthly Tastqif coaching and implementing the tahfidzul qur`an program for teachers.” The principal's explanation was also clarified and strengthened by teacher M. Alfian, S. Pd who was interviewed on May 16 2023 in the teacher's room and explained. “To shape behavior, it is necessary to observe (observe) the training needs of teachers, then share ideas or experiences with fellow colleagues and reflect on the actions that have been carried out in teaching.” Fitrah Annisa as an administrative staff at the Robbani Integrated Islamic Basic School, whom the researcher interviewed on May 18 2023 in the Administration room, explained. I'm just an admin worker, I prepare administrative guidance for what the school principal I'm just an admin worker, I prepare administrative guidance for what the school principal needs. If it's related to coaching, yes, I know, teachers take online training, so they're free to learn here. There are also other school programs with an Islamic theme, such as reciting tahfidz and tasqif. I'm just an admin worker, I prepare administrative guidance for what the school principal needs. If it's related to coaching, yes, I know, teachers take online training, so they're free to learn here. There are also other school programs with an Islamic theme, such as reciting tahfidz and tasqif. Results of the Research The research on teacher training for enhancing pedagogic competence at the Integrated Islamic Elementary School Robbani Banjarbaru during the Society 5.0 era, developed from Sumodiningrat's theory regarding training stages, is explained as follows. The principal of Integrated Islamic Elementary School Robbani Banjarbaru, Oelistianti, ST., M. Pd., who was interviewed on May 15, 2023, in the principal's office, explained the preparatory phase before the implementation of teacher training by stating. Yes, before doing the coaching, I m Ask teachers to design learning that suits students' needs and characteristics according to the results of initial diagnostic assessments (cognitive and non-cognitive). This statement was further clarified by the deputy principal for curriculum, Mr. M. Usman Fauzan, S. Pd., who was interviewed on May 15 2023 in the teachers' room. “Pre-condition preparation for improving teacher competency in the era of Society 5.0 is to ensure that all teachers have accessed PMM and studied at least the 7 modules contained in it.” These two explanations were also confirmed and added again by the teacher, Mrs. Feny Jatiayu, S. Pd. And Meta Julia, S. Pd. who was interviewed on May 16 2023 in the teacher's room by explaining: “Because coaching is a continuous process, before the coaching process, training planning and needs analysis should be carried out, both strengths and weaknesses. Plan what training is needed by teachers.” So it can be concluded that in the process of preparing teacher coaching in the era of society 5.0 to improve teacher pedagogic competence, it is to analyze coaching needs, teacher weaknesses and strengths, design learning according to needs and prepare the Independent Learning Center on the independent learning platform. Then the process of stimulating teacher awareness for the coaching process was explained by the principal Oelistianti, ST., M. Pd. who was interviewed on May 15 2023 in the school principal's room, namely. “Motivating teachers about the importance of implementing an independent curriculum according to KHD teachings so that learning at Robbani is not inferior to other schools.” 668| Vol. 4, No. (4) 2023: Tafkir: Interdisciplinary Journal of Islamic Education Teacher Development to Augment Pedagogical Competence in Society 5.0 Teacher Development to Augment Pedagogical Competence in Society 5.0 It can be concluded that the formation of teacher pedagogic behavior is carried out through school programs such as Yaumiyah, coaching Indonesian National Education Association,Tastqif,and Tahfidzul Qur'an. Apart from that, this is also done by analyzing training concepts and conducting performance assessments on teachers. Furthermore, the transformation of teacher knowledge and skills in the era of society 5.0 as explained by the school principal, namely Oelistianti, ST., M. Pd. who was interviewed on May 15 2023 in the school principal's room. “By ensuring that each teacher carries out independent training on the Merdeka Mengajar Platform and forms a learning community according to their respective class levels. Meanwhile, skills include doing collaborative work and honing digital skills according to current developments, such as using Google Workspace for education.” 669| Vol. 4, No. (4) 2023: Tafkir: Interdisciplinary Journal of Islamic Education Sabariah et al This was explained further by the deputy principal for curriculum M. Usman Fauzan, S. Pd., who was interviewed on May 15, 2023, and by teacher Multi Muljiah, S. Pd who was interviewed on May 16 2023 in the teacher's room sequentially explaining about transformation of abilities and knowledge viz. For knowledge transformation, teachers are invited to learn about the quality of education, while for skills transformation, teachers are invited to become more skilled in using computers so that they can use computers and design learning in the RPS. Then for our teachers to transform their knowledge, transfer it into the RPS as best as possible before teaching, and then transform their skills because it was the RPS, so our learning process is always good, innovative and so on. For knowledge transformation, teachers are invited to learn about the quality of education, while for skills transformation, teachers are invited to become more skilled in using computers so that they can use computers and design learning in the RPS. Then for our teachers to transform their knowledge, transfer it into the RPS as best as possible before teaching, and then transform their skills because it was the RPS, so our learning process is always good, innovative and so on. If a conclusion is drawn, it can be explained that the transformation of teacher knowledge is carried out by providing an understanding of the quality of education and training in independent learning applications. Meanwhile, the transformation of teacher skills is carried out by designing learning in RPS innovatively. Furthermore, increasing the knowledge and skills of teachers at the Robbani Integrated Islamic Elementary School was obtained from the results of interviews with the school principal Oelistianti, ST., M. Pd. who was interviewed on May 15 2023 in the school principal's room. If knowledge increases, that can be done by increasing the learning process, especially in today's IT-based era. Meanwhile, to improve teachers' skills, they are more skilled in teaching using computers or technology. If knowledge increases, that can be done by increasing the learning process, especially in today's IT-based era. Meanwhile, to improve teachers' skills, they are more skilled in teaching using computers or technology. Then it was explained again by the teacher, namely Feny Jatiayu, S. Pd., who was interviewed on May 16 2023 in the teacher's room and said. Sabariah et al Yes, to increase knowledge, yes, by good and innovative teaching and learning in the classroom, utilizing IT means having to be innovative based on IT. Meanwhile, to improve teachers' skills, they can be skilled and innovative in the classroom, as well as being innovative and able to collaborate with fellow teachers to share knowledge and experiences about their respective versions of innovative learning. Yes, to increase knowledge, yes, by good and innovative teaching and learning in the classroom, utilizing IT means having to be innovative based on IT. Meanwhile, to improve teachers' skills, they can be skilled and innovative in the classroom, as well as being innovative and able to collaborate with fellow teachers to share knowledge and experiences about their respective versions of innovative learning. So it can be concluded that teachers' knowledge about IT-based learning has increased. Meanwhile, increasing skills can teach innovatively by utilizing IT and collaborating with colleagues. Next, the results of research on the stages of awareness and shaping teacher behavior in the coaching preparation process, stimulating awareness and shaping behavior were explained by the school principal Oelistianti, ST., M. Pd. who was interviewed on May 15 2023 in the school principal's room as follows. Creation of mutual understanding and agreement regarding the goals and objectives of coaching, mapping of current teacher pedagogical competence, identification of training needs, and development of teacher pedagogical competence in the hope of implementing student formation as stated in the vision and mission of the school at Sdit Robbani. Every teacher has the motivation to transform into a teacher who not only transfers knowledge 670| Vol. 4, No. (4) 2023: Tafkir: Interdisciplinary Journal of Islamic Education Teacher Development to Augment Pedagogical Competence in Society 5.0 but is also able to guide students to become individuals with noble character according to Islamic teachings. Teachers have good pedagogical competence and have an Islamic personality. Then the results of research on the ability transformation stage, namely the transformation of teacher knowledge and skills, were also explained by the school principal Oelistianti, ST., M. Pd. who was interviewed on May 15 2023 in the school principal's room as follows. Formation of teacher character who can be an example for his students. Teachers carry out a transformation in pedagogical competence from those who usually transfer knowledge to teachers who can guide students according to the nature of nature and the nature of the times. Sabariah et al There is a change in learning patterns that is more in favor of students and the use of digital technology in their learning. Finally, the results of research regarding the capacity building stage, namely increasing teacher knowledge and skills, were also explained by the school principal Oelistianti, ST., M. Pd. who was interviewed on May 15 2023 in the school principal's room as follows. “Teachers have better pedagogical competence. Teachers have better pedagogical competence with Student-Centered learning and project-based learning. Teachers have better pedagogical competence in classroom learning.” Overall, the results of research on teacher development to improve pedagogic competence in the era of Society 5.0 at the Robbani Integrated Islamic Elementary School are explained in Table 1 below: Table 1. Development of Pedagogical Competency for Teachers in Society Era 5.0 Table 1. Development of Pedagogical Competency for Teachers in Society Era 5.0 Table 1. Development of Pedagogical Competency for Teachers in Society Era 5.0 Coaching Sub Coaching Coaching Results Era 5.0 Teacher Development Stage of awareness and formation of behavior - Preparation process: analysis of teacher strengths and weaknesses, analysis of coaching needs, designing learning according to student needs and accessing the Independent Material Center - The process of stimulating teacher awareness: motivating teachers to implement independent learning and training, material and non-material support, and accessCenter for Independent Materials and forming communities according to level. - Process of forming teacher behavior: Analysis of training concepts, performance assessment, Yaumiyah program, coaching programIndonesian National Education Association, programTastqif,and the Tahfidzul Qur'an program Ability Transformation Stage - Knowledge transformation: quality of education and application of independent learning training. - Transforming skills: designing learning Capacity Enhancement Stage - Increasing knowledge: IT-based learning system Table 1. Development of Pedagogical Competency for Teachers in Society Era 5.0 Coaching Sub Coaching Coaching Results Era 5.0 Teacher Development Stage of awareness and formation of behavior - Preparation process: analysis of tea and weaknesses, analysis of coa designing learning according to s d i h I d d M - Preparation process: analysis of teacher strengths and weaknesses, analysis of coaching needs, designing learning according to student needs and accessing the Independent Material Center g - The process of stimulating teacher awareness: motivating teachers to implement independent learning and training, material and non-material support, and accessCenter for Independent Materials and forming communities according to level. - Knowledge transformation: quality of education and application of independent learning training. Sabariah et al - Transforming skills: designing learning - Increasing knowledge: IT-based learning system 671| Vol. 4, No. (4) 2023: Tafkir: Interdisciplinary Journal of Islamic Education 671| Vol. 4, No. (4) 2023: Tafkir: Interdisciplinary Journal of Islamic Education Sabariah et al - Skills enhancement: learning innovation and collaboration with peers Results of Teacher Development Era 5.0 Stage of awareness and formation of behavior - Teachers understand the goals of coaching - Teachers are able to map pedagogic competencies - Teachers are able to identify coaching needs - Teachers are able to guide students according to Islamic teachings - Teachers' pedagogical competence increases and they have an Islamic personality Ability Transformation Stage - Character teachers are formed - Teachers are able to guide students' abilities - Teachers are able to teach using IT Capacity Enhancement Stage - Teachers' pedagogical competence increases - Teachers develop Student Centered and Project Learning learning models - Teacher knowledge in the classroom increases Research Discussion Based on Table 1, the results about-coaching to improve the pedagogical competence of teachers at the Robbani Banjarbaru Integrated Islamic Elementary School of Society 5.0 research can be discussed as follows: Development of Pedagogical Competency for Teachers in Society Era 5.0 Development in the era of Society 5.0 to improve the pedagogical competence of teachers at the Robbani Banjarbaru Integrated Islamic Elementary School is carried out in three stages which are explained as follows: 672| Vol. 4, No. (4) 2023: Tafkir: Interdisciplinary Journal of Islamic Education Awareness and Behavior Formation Stage The stage of awareness and behavior formation in improving the pedagogic competence of teachers at the Robbani Banjarbaru Integrated Islamic Elementary School in the era of Society 5.0 is carried out in several processes which are explained as follows: First pre-condition preparation process to facilitate the teacher development process in the Society 5.0 era. The process of preparing pre-conditions to facilitate the process of developing teachers in the Society 5.0 era at the Robbani Banjarbaru Integrated Islamic Elementary School is carried out by the principal, deputy principals in both curriculum and other fields as well as quality assurance which is carried out at the school at every school meeting. The principal, deputy principal, and quality guarantor conducted an analysis of teacher needs to face the Society 5.0 era, namely identifying new skills and knowledge needed by teachers to create a learning environment that is relevant to developments in technology and society. There is no significant influence between teacher skills and abilities and teacher performance, so teachers need to improve their skills and knowledge to improve teacher performance in the future (Azimmi & Zaitul, 2020). The aim of carrying out the pre-condition preparation process to facilitate the process of developing teachers in the Society 5.0 era at the Robbani Banjarbaru Integrated Islamic Primary School is firstly to ensure that teachers at SDIT Robbani Teacher Development to Augment Pedagogical Competence in Society 5.0 Banjarbaru have an important and strong basis for developing teacher skills to develop Society 5.0 era skills through the use of technology, integration of digital literacy, and the ability to teach students with diverse learning styles. Because teacher skills can influence student learning outcomes by as much as 37% (Moonti et al., 2021), this can have a positive impact on student achievement at school (Wicaksono et al., 2021). Second, ensure that all teachers have the same understanding of the goals and objectives to be achieved and to find out what pedagogical competencies teachers already have and which still need to be improved to create uniformity in the vision and goals of coaching, so that all teachers move in the same direction in developing skills. Teacher. By understanding competency, teachers can understand their abilities, understanding, skills and arrive at aspects of assessment that must be carried out while at school (Ramaliya, 2018). 673| Vol. 4, No. (4) 2023: Tafkir: Interdisciplinary Journal of Islamic Education Awareness and Behavior Formation Stage The process of preparing pre-conditions to facilitate the teacher development process in the Society 5.0 era at the Robbani Banjarbaru Integrated Islamic Elementary School is carried out in several ways, first analyzing the needs of both teacher strengths and weaknesses which includes assessing their strengths and weaknesses in various aspects of learning, class management and communication with students and parents. By analyzing work abilities, teachers can increase their work motivation by 54.35%, this shows that teachers can increase their pedagogical competence by 63.04% (Siswanto, 2021). Secondly, analyzing what teachers need to improve their pedagogical competence includes training in the application of educational technology, the use of digital learning resources, and teaching strategies that are in accordance with the Society 5.0 concept, which emphasizes the integration of technology in the learning process. Teachers as educational facilitators in schools are required to know and master the use of information technology to support students' level of learning achievement and understanding during learning (Lesmana et al., 2023). Third, ask teachers to design learning that suits students' needs and characteristics. Teachers need to understand their students well, recognize students' learning styles, and design learning strategies that are relevant to Society 5.0 educational goals that involve the use of technology such as e-learning, augmented reality, or the use of sophisticated educational applications. By designing learning well, this can significantly influence the quality of teacher teaching (Nurhasanah et al., 2021). Fourth, ensure that all teachers have accessed the Independent Material Center (PMM) and studied at least the 7 modules contained in it.contains training modules, online educational resources, and tools that support teacher professional development. By accessing PPM, teachers can get inspiration from teaching videos that have been validated by the ministry, learning is more innovative, learning documentation can be stored neatly and shared with other fellow teachers and teachers get lots of references regarding teaching materials that will be used in learning (Arnes et al., 2023). Both processes mstimulate teacher awareness to improve teacher pedagogic competence in the era of society 5.0. Awareness and Behavior Formation Stage Stimulating the awareness of Society 5.0 era teachers Sabariah et al in increasing their pedagogical competence at the Robbani Banjarbaru Integrated Islamic Elementary School by the principal, deputy principal, or colleagues has been carried out since SDIT Robbani became the implementer of the first generation driving school program and the implementation of the independent curriculum and the launch of the independent teaching platform for teachers . The process of stimulating the awareness of Society 5.0 era teachers in improving their pedagogical competence at the Robbani Banjarbaru Integrated Islamic Primary School was carried out in several ways, firstly providing motivation to teachers about the importance of implementing the independent curriculum according to the teachings of the Living Curriculum (KHD) so that learning at Robbani runs well.Teacher performance motivation can be done through seminars, training, or lectures that illustrate how education that focuses on independence, problem solving, and innovation is the key to success in the current era. Because teacher work motivation is in the medium category, it can influence teacher performance at school (Agustina et al., 2020). Second, stimulate teacher awareness to be able to take part in various teacher trainings to improve teacher pedagogic competence by inviting educational experts, successful practitioners, or educational technology experts to provide examples of how strong pedagogical competence can improve the quality of teaching and student learning outcomes. Apart from that, schools also provide incentives or rewards for teachers who actively participate in training. Because training and education, if done well in schools, have a significant influence on teacher performance (Audah, 2020). Third, provide full support to teachers both in terms of material and non- material.Materials may include educational facilities, devices, or resources necessary to implement current technologies and methodologies. Non-material support includes guidance, mentoring and ongoing coaching to help teachers overcome obstacles in improving teacher pedagogical competence in the era of society 5.0. Fourth, ensure all teachers accessCenter for Independent Materials(PMM) and report on the achievements of their independent training by taking concrete actions and implementing learning communities according to their level.Teachers have access to PMM and understand how to use it, so teachers must report learning achievements in independent training by integrating what teachers learn in daily learning according to educational levels to be able to collaborate and support each other in improving teachers' pedagogical competence. 674| Vol. 4, No. (4) 2023: Tafkir: Interdisciplinary Journal of Islamic Education Awareness and Behavior Formation Stage The aim of the process of stimulating the awareness of teachers in the Society 5.0 era in increasing their pedagogical competence at the Robbani Banjarbaru Integrated Islamic Elementary School is first to increase teachers' pedagogical competence, namely regarding teaching techniques, evaluation and the latest learning approaches that are in accordance with Society 5.0. Second, improve teaching strategies so that they will provide an interesting learning experience for students. By improving teachers' skills in utilizing technology and interesting learning methods, learning will be more relevant and make students more involved in the learning process. The teacher's teaching skills have a positive effect on students' learning motivation, meaning that the more skilled Teacher Development to Augment Pedagogical Competence in Society 5.0 the teacher is in preparing the lesson, the more motivated the students will be to take part in the lesson (Handayani et al., 2021). Third, to ensure that all teachers have the same understanding of the goals and objectives to be achieved,including understanding the importance of the Merdeka Curriculum, the use of the Merdeka Mengajar platform so that all teachers will work towards a uniform educational vision and mission. Fourth, to ensure learning that supports students according to the teachings of the Living Curriculum (KHD), namely teachers must be able to create a learning environment that supports students' exploration, creativity and personal growth. The independent curriculum requires teachers to conduct innovative learning that focuses on student exploration where teachers must be facilitators, innovators and inspirers for students (Pertiwi et al., 2022). The three processes of forming teacher behavior to improve teacher pedagogic competence in the era of society 5.0 at the Robbani Banjarbaru Integrated Islamic Elementary School which is carried out by the principal, deputy principal, local education office, or foundation which is carried out with the aim of influencing teachers to improve the teaching system. The next goal is to ensure the progress of education, especially at SDIT Robbani, as well as the formation of student character as stated in the school's vision and mission.Teachers can act as good role models, teaching values such as integrity, honesty, discipline and responsibility to students. 675| Vol. 4, No. (4) 2023: Tafkir: Interdisciplinary Journal of Islamic Education Sabariah et al Fourth, by completing the teacher's Yaumiyah practice,includes daily good practices that reflect Islamic moral and ethical values which are able to shape the character of teachers in education in the Society 5.0 era.The fifth took part in monthly Tastqif coaching as a vehicle for teachersto increase teachers' understanding of Islamic teachings and their relevance in education. This can help teachers understand Islamic values that need to be integrated into teacher learning. Sixth, implement the Tahfidzul Qur'an program for teachers,helping teachers improve their understanding of the Islamic religion and develop skills in reading and understanding the Koran which is the school's commitment to religious education in a modern educational environment. Religious work programs implemented and applied in schools are capable and proven to be able to comprehensively increase the level of faith or spirituality of school residents (Alfiah, 2018). Awareness and Behavior Formation Stage Things that are done to shape behavior in improving the pedagogical competence of teachers in the Society 5.0 era at the Robbani Banjarbaru Integrated Islamic Elementary School include: firstly analyzing the concepts, ideas and actions of training in teacher teaching.This involves a deep understanding of the concepts of Society 5.0 to be integrated in the learning process, technological developments that influence teaching methods. Analysis of training needs needs to be carried out well, because it will influence the process and results of the training itself. Second, by assessing teacher performance through learning supervisionclassroom observations, analysis of lesson plans, and evaluation of student learning outcomes. The results of supervision can provide the necessary feedback to help teachers improve teacher pedagogical competence. Good supervision techniques will determine the results of those supervised, therefore the principal must have and master several supervision techniques such as effective meetings to supervise the school climate, school culture and so on, then supervise with effective visits to supervise learning in the classroom and and so on (Baidowi & Syamsudin, 2022). Third, carry out APNI coaching activities,APNI (Indonesian National Education Association) coaching activities can provide a platform for sharing knowledge and experience between teachers through the exchange of ideas and effective teaching strategies in the context of Society 5.0. Educational development for all school members requires a consistent and sustainable process, the aim of which is to shape the character of school members in accordance with the school's vision and to improve the abilities of school members to be better than before. 675| Vol. 4, No. (4) 2023: Tafkir: Interdisciplinary Journal of Islamic Education Ability Transformation Stage The ability transformation stage in improving the pedagogic competence of teachers at the Robbani Banjarbaru Integrated Islamic Elementary School in the era of Society 5.0 is carried out with two activities as follows: First, knowledge transformation, knowledge transformation to increase the pedagogic competence of teachers at the Robbani Banjarbaru Integrated Islamic Elementary School in the era of society 5.0 is carried out by the head schools and all teachers in the school environment after the Merdeka Belajar platform training process. This knowledge transformation is carried out by implementing the results of the training through real action in the Independent Learning Process (PPM). With effective implementation, teachers have the knowledge and skills needed to create a learning environment that is under the school's vision and the demands of the Society 5 era. Both skill transformations, The transformation of skills to improve the pedagogical competence of teachers at the Robbani Banjarbaru Integrated Islamic Primary School in the Society 5.0 era, which is carried out by all teachers and school principals, is carried out when applying the independent curriculum in schools by creating learning experiences that are relevant, interactive, and in accordance with the demands of the Society 5.0 era. The skills transformation process is carried out by designing learning that suits students' needs and characteristics through collaborative work and honing digital skills according to current developments, such as using Google Workspace for Education. Teaching skills with a creative approach must focus on actively involving students to obtain maximum learning results (Nasihudin & Hariyadin, 2021). 676| Vol. 4, No. (4) 2023: Tafkir: Interdisciplinary Journal of Islamic Education Capacity Enhancement Stage The stage of capacity building in improving teacher pedagogic competence at the Robbani Banjarbaru Integrated Islamic Elementary School in the era of society 5.0 is carried out in the following way: Firstly increasing knowledge, increasing teacher knowledge to improve pedagogic competence at the Robbani Banjarbaru Integrated 676| Vol. 4, No. (4) 2023: Tafkir: Interdisciplinary Journal of Islamic Education Teacher Development to Augment Pedagogical Competence in Society 5.0 Islamic Elementary School in the society 5.0 era is carried out by the head schools, deputy principals and all teachers with the aim of improving the quality of schools based on the existing vision. Teachers have succeeded in creating learning experiences that are more relevant, interactive, and in line with the demands of the Society 5.0 era by combining science and technological advances. Increasing the knowledge of teachers at the Robbani Banjarbaru Integrated Islamic Elementary School in the era of society 5.0 was carried out when carrying out training on the independent learning platform. The improved knowledge is related to the independent learning teaching system in schools by combining classical learning with IT developments to create an active and innovative learning process. With comprehensive training on the Merdeka Belajar platform, teachers at SDIT Robbani Banjarbaru gain the knowledge and skills needed to create learning experiences that meet the demands of the times. With IT-based and digital learning, teachers can make students more independent in carrying out their learning tasks (Lillihata et al., 2022). Second, skill improvement, improving teacher skills at the Robbani Banjarbaru Integrated Islamic Elementary School in the Society 5.0 era involving the school principal and all teachers, and the aim is to create innovation in the learning process in the Merdeka Belajar Class so that it is in line with student character and current developments. Teachers at SDIT Robbani Banjarbaru are able to create learning experiences that are more innovative, according to student character, and in accordance with the demands of the Society 5.0 era. With innovative learning, teachers can shape good student character, especially in terms of religious beliefs and creative ways of thinking (Susanto, 2021). Improving teacher skills at the Robbani Banjarbaru Integrated Islamic Elementary School in the Society 5.0 era is an initiative that combines collaboration between teachers and the use of online platforms such as Belajar.id. Capacity Enhancement Stage The main aim is to improve teachers' pedagogical competence so that they can provide education that meets the demands of the times. Through the use of peers and online platforms such as Belajar.id, teachers at SDIT Robbani Banjarbaru can improve their skills in designing learning that is appropriate for the Society 5.0 era. The application of technology-based learning media and the use of applications or software for use in learning can influence students' comprehension of the material being studied (Wahyuni et al., 2022). 677| Vol. 4, No. (4) 2023: Tafkir: Interdisciplinary Journal of Islamic Education Results of Teacher Pedagogical Competency Development in Era Society 5.0 Results of the Awareness and Behavior Formation Stage The undertaking aimed at augmenting pedagogical competence among teachers at Robbani Banjarbaru Integrated Islamic Elementary School during the Society 5.0 era yielded significant outcomes. Primarily, a mutual understanding and consensus concerning the objectives of the coaching endeavor were cultivated, encompassing a thorough comprehension of the school's vision, mission, and the pivotal role of teacher pedagogical competence in achieving the school's objectives. Secondly, teachers engaged in a comprehensive mapping of current pedagogical competencies via self-evaluation, 677| Vol. 4, No. (4) 2023: Tafkir: Interdisciplinary Journal of Islamic Education Sabariah et al Sabariah et al peer assessment, or even external evaluation. Through this, an understanding of the strengths and weaknesses of their pedagogical competencies was attained. Thirdly, teachers were adept in identifying training needs and fostering pedagogical competencies, aiding in the formulation of training programs aligned with the areas requiring enhancement in pedagogical competencies. Fourthly, every teacher demonstrated a motivation to evolve into an educator who not merely disseminates knowledge but is adept at guiding students to embody noble characters in adherence to Islamic teachings. This transition underscores the imperative of a teacher's role in molding students' character and moral ethos in tandem with Islamic teachings. Lastly, teachers showcased exemplary pedagogical competence and Islamic personas, exhibiting proficiency in classroom management, curriculum design, employing effective teaching methodologies, and embodying Islamic attitudes and behavior, which notably influenced the caliber of teaching and learning processes in the classroom (Rosyada et al., 2021). Results of the Capability Transformation Phase The capability transformation process at the Robbani Banjarbaru Integrated Islamic Elementary School during the Society 5.0 era yielded teachers with enriched pedagogical competence. Firstly, the process molded teachers into exemplary figures for their students, transcending academic instruction to instill moral values, ethics, and virtuous behavior. The presence of virtuous teachers positively impacts student character development, laying the groundwork for a morally upright generation. Teachers characterized by commendable attitudes and behaviors serve as quintessential examples for students, fostering a conducive communication and understanding among teachers and students (Cahyati, 2020). Secondly, teachers exhibited a capacity to mentor students in alignment with the evolving societal and technological landscapes. The shift from mere knowledge transfer to a more sensitive understanding of students' needs, varying learning styles, and the application of diverse teaching methods resonates with technological advancements and contemporary societal needs. Thirdly, an alteration in teaching patterns favored students through the integration of digital technology in learning processes. This transition significantly impacted students' learning outcomes as teaching strategies employed by teachers accounted for a 64% influence on students' learning achievements (Lisaholit et al., 2021). 678| Vol. 4, No. (4) 2023: Tafkir: Interdisciplinary Journal of Islamic Education Results of the Capacity Building Phase The capacity building endeavor at the Robbani Banjarbaru Integrated Islamic Primary School in the Society 5.0 epoch enhanced the pedagogical competence of teachers. Initially, teachers acquired a profound understanding of effective and efficient teaching methodologies, child development theories, teaching strategies, and current educational trends. The indicators of pedagogical competence included an acute understanding of students, adeptness in learning design, capability in executing and 678| Vol. 4, No. (4) 2023: Tafkir: Interdisciplinary Journal of Islamic Education Teacher Development to Augment Pedagogical Competence in Society 5.0 designing learning evaluations, and the potential for nurturing student and teacher abilities within the school environment (Kinanty & Ramadan, 2021). Subsequently, teachers embraced the Student-Centered model and project-based learning paradigm. This model accentuates the individual needs of students, transforming teachers into facilitators aiding students to actively engage in their learning journey. Thirdly, teachers demonstrated enhanced mastery over classroom teaching by infusing project-based learning in their teaching approach. Project-based learning offers a real-world context for education, fostering the development of analytical, problem- solving, collaboration, and communication skills among students. This method significantly elevates the quality of education and assists students in achieving desirable learning outcomes (Nirmayani & Dewi, 2021). 679| Vol. 4, No. (4) 2023: Tafkir: Interdisciplinary Journal of Islamic Education CONCLUSION Teacher development in improving pedagogic competence in the 5.0 era at Robbani Banjarbaru Integrated Islamic Elementary School, overseen by the principal, deputy principal, and teachers, has been successful in implementing the independent curriculum. The process of enhancing teachers' pedagogical competence in the 5.0 era consists of three stages. The first stage involves raising awareness and shaping teacher behavior by addressing coaching needs, fostering awareness, and influencing behavior. The second stage focuses on transforming teacher's knowledge and skills, while the third stage concentrates on further enhancing the teacher's knowledge and skills. Results of teacher coaching to improve pedagogic competence in the 5.0 era at Robbani Banjarbaru Integrated Islamic Elementary School indicate that teachers are knowledgeable about the coaching content. They can map their competencies in the classroom, becoming effective guides for students, resulting in enhanced competencies after coaching. During the capability transformation stage, schools can shape teachers with Islamic character aligned with the school's vision. Teachers excel in guiding students and adeptly use IT for teaching. Meanwhile, in the capacity-building stage, teachers experience increased pedagogical competence, adopt Student-Centered and Project Learning models, and acquire more knowledge during classroom learning processes. The implication of this research serves as valuable knowledge for schools, principals, and other teachers in Indonesia seeking to enhance teachers' pedagogical abilities in the Society 5.0 era. A research suggestion is for schools to reduce reliance on independent learning platform training for improving teacher pedagogic competence. Instead, pedagogical competency development should occur periodically, at least twice a year, through teacher performance evaluation meetings in schools. Another suggestion for future researchers is to explore teacher development in various competencies. 679| Vol. 4, No. (4) 2023: Tafkir: Interdisciplinary Journal of Islamic Education Sabariah et al REFERENCES Agustina, A., Ibrahim, M. M., & Maulana, A. (2020). Pengaruh Motivasi Kerja Guru Terhadap Kinerja Guru Pada Mtsn Di Kecamatan Bontotiro Kabupaten Bulukumba. Jurnal Idaarah, IV(1), 111–118. ( ) Alfiah. (2018). 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Pembinaan Supervisi Akademik Melalui Observasi Class Dan Individual Conference Untuk Meningkatkan Kualitas Pembelajaran Di SDN Bojongherang Cianjur. Jurnal Al-Azhary, 8(1), 1–11. Handayani, N. D., Arief, M., Kurjono, & Mulyadi, H. (2021). Pengaruh Keterampilan Mengajar Guru dan Fasilitas Belajar Terhadap Motivasi Belajar Siswa Kelas XI OTKP SMK Nasional Bandung. Jurnal Pendidikan Ekonomi Undiksha, 13(2), 394–401. http://dx.doi.org/1 Handayani, N. N. L., & Muliastrini, N. K. E. (2020). Pembelajaran Era Disruptif Menuju Era Society 5.0 (Telaah Perspektif Pendidikan Dasar). Prosiding Seminar Nasional: Endidikan Guru Sekolah Dasar Menyongsong Society 5.0, 1–14. https://prosiding.iahntp.ac.id Harun, S. (2021). Pembelajaran Di Era 5.0. Prosiding Seminar Nasional Pendidikan Dasar “Merdeka Belajar Dalam Menyambut Era Masyarakat 5.0”, 265–276. Husaini, R. (2018). Pembinaan Profesionalisme Guru. Jurnal Ilmaih Pendidikan Agama Islam, 8(2), 1–15. Kinanty, & Ramadan, Z. A. (2021). REFERENCES Profil Kompetensi Pedagogik Guru Sekolah Dasar. Jurnal Mimbar Ilmu, 26(03), 425–430. 680| Vol. 4, No. (4) 2023: Tafkir: Interdisciplinary Journal of Islamic Education Teacher Development to Augment Pedagogical Competence in Society 5.0 Lesmana, I., Amanda Sari, L., Fairuz Husna, A., Fitrotul Unsa, F., & Furqon Akhbar, A. (2023). Strategi Pemanfataan Teknologi dan Informasi dalam Pembelajaran Guna Mempersiapkan Pelajar Berkarakter di Era Society 5.0. Proceedings Series of Educational Studies Prosiding Seminar Nasional “Peran Manajemen Pendidikan Untuk Menyiapkan Sekolah Unggul Era Learning Society 5.0” Departemen Administrasi Pendidikan Fakultas Ilmu Pendidikan, 99–108. Lillihata, S., Karesina, D. M., Alfons, A., & Pulung, R. (2022). Pemanfataan Media Pembelajaran Berbasis IT dalam meningkatkan Kemandirian Belajar siswa di era digital. Jurnal Pendidikan DIDAXEI, 3(2), 377–393. Lisaholit, S., Hentihu, V., & Umanailo, M. C. B. (2021). Pengaruh Gaya Mengajar Guru Terhadap Hasil Belajar Siswa Di SMA Negeri 1 Namlea. Media Bina Ilmiah, 16(05), 6973–6976. Ma’rufah, L. A., & Arsanti, M. (2022). Inovasi dan Strategi Pembelajaran untuk Menghadapi Era Society 5.0. Prosiding Senada (Seminar Nasional Daring): Tema “Membangun Insan Cendekia Di Era Society 5.0 Melalui Inovasi Pembelajaran,” 527–532. Mega, K. I. (2022). Mempersiapkan Pendidikan di Era Tren Digital (Society 5.0). Jurnal Belaindika :Pembelajaran Dan Inovasi Pendidikan, 4(3), 114–121. https://belaindika.nusaputra.ac.id/indexbelaindika@nusaputra.ac.id Moonti, U., Bahsoan, A., & Gumohung, A. M. (2021). Pengaruh Keterampilan Menjelaskan Guru Terhadap Hasil Belajar Siswa. Jambura Economic Education Journal, 3(1), 1–7. ( ) Nasihudin, & Hariyadin. (2021). Pengembangan Keterampilan Dalam Pembelajaran. Jurnal Pendidikan Indonesia, 2(4), 733–743. Nirmayani, L. H., & Dewi, N. P. C. P. (2021). Model Pembelajaran Berbasis Proyek (Project Based Learning) Sesuai Pembelajaran Abad 21 Bermuatan Tri Kaya Parisudha. Jurnal Pedagogi Dan Pembelajaran, 4(3), 378–385. https://ejournal.undiksha.ac.id/index.php/JP2/index Nurhasanah, A., Pribadi, R. A., & Apriliana, R. (2021). Pengaruh Perencanaan Pembelajaran Terhadap Peningkatan Kualitas Dan Mutu Pendidik Dalam Kegiatan Mengajar Di SDN Banjar 1. Didaktik : Jurnal Ilmiah FKIP Universitas Mandiri, 7(2), 619–627. Padmawati, N. N., & Pihung, E. S. (2022). Mengembangkan Pembelajaran Digitalisasi Di Era Society 5.0. Widyadari, 23(2), 378–288. Pertiwi, A. D., Nurfatimah, S. A., & Hasna, S. (2022). Menerapkan Metode Pembelajaran Berorientasi Student Centered Menuju Masa Transisi Kurikulum Merdeka. Jurnal Pendidikan Tambusai, 6(2), 8839–8848. Pinatih, N. P. S. (2020). Pembelajaran Menyenangkan dalam Menyongsong Era Society 5.0. Prosiding Seminar Nasional: Pendidikan Guru Sekolah Dasar Menyongsong Era Society 5.0, 64–76. https://prosiding.iahntp.ac.id Ramaliya. (2018). Pengembangan Kompetensi Guru Dalam Pembelajaran. Bidayah: Studi Ilimu-Ilmu Keislaman, 9(1), 77–88. Rosyada, A., Harapan, E., & Rohana. (2021). 681| Vol. 4, No. (4) 2023: Tafkir: Interdisciplinary Journal of Islamic Education 682| Vol. 4, No. (4) 2023: Tafkir: Interdisciplinary Journal of Islamic Education REFERENCES Pengaruh Kompetensi Pedagogik Guru terhadap Kualitas Pembelajaran Sekolah Menengah Atas di Kota Sekayu, Sumatera Selatan. Jurnal Manajemen Pendidikan: Jurnal Ilmiah Administrasi, Manajemen Dan Kepemimpinan Pendidikan, 3(1), 31–42. Siswanto. (2021). Analisis Kinerja Guru Akuntansi Bersetifikasi Sekolah Menengah Kejuruan (SMK) Bisnis Dan Manajemen. Jurnal Ekonomi & Pendidikan, 18(1), 17–24. 681| Vol. 4, No. (4) 2023: Tafkir: Interdisciplinary Journal of Islamic Education Sabariah et al Sukatin, Hasanah, S. A. N., Ningsi, O., Pratiwi, R. I., & Subagia, W. (2022). Perkembangan Pendidikan di Era 5.0. PIJAR: Jurnal Pendidikan Dan Pengajaran, 1(1), 78–86. Susanto, S. (2021). Dampak Inovasi Pembelajaran Berbasis Karakter Terhadap Pengembangan Karakter Positif di SD Karakter Genius Islamic School Kota Depok. QALAMUNA: Jurnal Pendidikan, Sosial, Dan Agama, 13(2), 687–708. https://doi.org/10.37680/qalamuna.v13i2.1066 Wahyuni, D. A. A., Misdalina, & Noviati. (2022). Dampak Media Pembelajaran Berbasis Teknologi Pada Aplikasi Tiktok Terhadap Daya Tangkap Dikalangan Siswa Kelas V di SDN 077 Palembang. Jurnal Pendidikan Dan Konseling, 4(4), 1853–1863. Wicaksono, B. I., Partono, & Sholah, A. (2021). Pengaruh Keterampilan Mengajar Terhadap Prestasi Belajar Siswa SMK Di Kabupaten Malang. Jurnal Teknik Otomotif Kajian Keilmuan Dan Pengajaran, 5(1), 1–6.
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Use of geoinformation systems for forecasting and recognizing crisis situations in agriculture
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© 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/). Use of geoinformation systems for forecasting and recognizing crisis situations in agriculture Ekaterina Podryadchikova1,*, Larisa Gileva2, Aleksey Dubrovsky3, and Elena Lobanova4 1Industrial University of Tyumen, 38 Volodarsky Str., 625000 Tyumen, Russia 2Omsk State Agrarian University named after P. А. Stolypin, 1 Institutskaya Square, 644008 Omsk, Russia 3Siberian State University of Geosystems and Technologies, 10 Plakhotny St., 630108 Novosibirsk Russia 4Novosibirsk State University of Economics and Management, 56 Kamenskaya St., 630099 Novosibirsk, Russia Abstract. This article covers the necessity of use of geoinformation systems for forecasting and recognizing crisis situations in agriculture. The method of creation of thematic maps was offered as a tool by the authors of the article, three thematic maps were obtained for functional zoning by the indicators of: financial state of agricultural production, ecological state expressed in monetary equivalent and forecast of crisis states in agriculture. The given method was proved within the boundaries of the 121 village councils of the central part of Novosibirsk region for 64 agricultural companies of different forms of organization. * Corresponding author: podryadchikova_ed@mail.ru E3S Web of Conferences 296, 03004 (2021) ESMGT 2021 E3S Web of Conferences 296, 03004 (2021) ESMGT 2021 https://doi.org/10.1051/e3sconf/202129603004 1 Introduction The agricultural lands represent the strategic resource of any state and predetermine the food security of the population. The efficient management of agricultural production depends in many aspects on the level of awareness of the soil condition and properties, field configuration, location of infrastructure facilities and transport flows, as well as forecasting and recognizing crisis situations [1]. The development of scientific knowledge and practical experience in forecasting and recognizing crisis situations in the agricultural production is particularly relevant in the conditions of the COVID-19 pandemic due to the common restrictions imposed and, as a result, the disruption of supply chains and economic ties in the most countries of the world. Most of the problems entailing the development of crisis situations in the agricultural production can be solved by the development and implementation of the modern production systems using geoinformation technologies. Currently, the Russian Federation is actively introducing information and communication technologies in the agricultural production, including the use of geoinformation systems. The “Smart Land Use”, “Smart Field”, “Smart Garden” projects developed on the basis of geoinformation systems as part of the action E3S Web of Conferences 296, 03004 (2021) ESMGT 2021 https://doi.org/10.1051/e3sconf/202129603004 plan for the implementation of the national platform “Digital Agriculture” can be given as an example [2]. plan for the implementation of the national platform “Digital Agriculture” can be given as an example [2]. The main and distinctive feature of the use of geoinformation systems is the ability to use a variety of algorithms for spatial analysis and operational processing of large data arrays. One of the most common and well-known areas of using geoinformation technologies in the agricultural production is the processing of the Earth remote sensing data [3]. 2 Materials and Methods The main cartographic area of geoinformation support for forecasting and recognizing crisis situations in the agricultural production is the thematic digital mapping that results in creating accurate digital maps of terrain, soils, crop conditions, climate, etc. [4]. Another important area, that develops through geoinformation support, is logistics. At present, the priority factor in the agricultural production zoning has become an orientation towards favourable climatic conditions with the best convenient further transportation of products [5]. The most well-known area of geoinformation support is the cadastre and land management system, the purpose of which is to ensure rational land use, planning, control and protection of land, including land plots as real estate located on the territory of the Russian Federation [6]. To forecast and recognize crisis situations in the agricultural production, it is necessary to provide for crisis management measures, namely, to analyse the agricultural productivity at a certain interval, clarify and update crop insurance premiums, annually compile multifactor models of crop yields [7]. The authors of the article offered the geoinformation system structure and obtained thematic maps of the functional zoning of crisis situations in agriculture as a tool for forecasting and recognizing crisis situations in the agricultural production. The main elements of the geoinformation support system for forecasting crisis situations in the agricultural production are given in Fig. 1. 2 2 E3S Web of Conferences 296, 03004 (2021) ESMGT 2021 https://doi.org/10.1051/e3sconf/202129603004 Fig. 1. Interaction of elements by using the geoinformation system for forecasting and recognizing crisis situations in agriculture Monitoring Thematic digital mapping - Digital terrain models (slope maps, slope exposures, etc.) - Soil maps - Crop condition maps - Climatic maps - Hydrologic maps - Agri-ecological zoning - Agricultural land maps - Geodetic works - Remote sensing (aerophotographs, satellite images, including multizonal and hyperspectral) - Soil surveys - GNSS Logistics - Transportation and sales optimization - Selective farming - Optimization of movement of agricultural machinery Crisis management - Specification of crop insurance premiums - Analysis of the agricultural productivity - Multifactor models of crop yields Cadastre and land management - Cadastral appraisal - Cadastral zoning - Control of compliance with the land legislation requirements - Rational nature management - Land-use planning Fig. 1. 2 Materials and Methods Interaction of elements by using the geoinformation system for forecasting and recognizing crisis situations in agriculture The method of creation of thematic maps for forecasting and recognizing crisis situations in the agricultural production can be represented as the work stages consequently performed: 1) to perform zoning of the territory according to the financial state indicator of the agricultural production; 2) to perform zoning of the territory according to the ecological state indicator expressed in the monetary equivalent; 3) to compare maps of financial and ecological states and consolidate them into an integral digital map of crisis states in agriculture. g At stage I, to perform zoning of the territory according to the financial state indicator of the agricultural production, for each minimum object of division of the territory, it is necessary to calculate the financial stability coefficient and the solvency coefficient according to the conventional formulas (1) and (2) of financial analysis. 𝑓𝑓𝐶𝑒𝑒𝐿𝑙𝑙𝑃𝐶 C𝑓𝑓𝑓𝑓= (𝐶𝐶𝑒𝑒𝑒𝑒+𝐿𝐿𝑙𝑙𝑙𝑙) 𝑃𝑃𝑡𝑡𝑡𝑡𝑡𝑡 (1) (1) where C𝑓𝑓𝑓𝑓 is the financial stability coefficient; 𝐶𝐶𝑒𝑒𝑒𝑒 is the equity capital including all available reserves of the agricultural organization; Llt is long-term loans and credits (liabilities) with the period of involvement of more than 1 year; 𝑃𝑃𝑡𝑡𝑡𝑡𝑡𝑡is the total amount of liabilities. 𝐶𝑠𝐶𝑒𝑒𝑃𝑃𝑃 𝐶𝐶𝑠𝑠= 𝐶𝐶𝑒𝑒𝑒𝑒 (𝑃𝑃1+𝑃𝑃2+𝑃𝑃3) (2) (2) 3 https://doi.org/10.1051/e3sconf/202129603004 E3S Web of Conferences 296, 03004 (2021) ESMGT 2021 where Cs is the solvency coefficient; 𝐶𝐶𝑒𝑒𝑒𝑒 is the equity capital including all available reserves of the agricultural organization; P1is the maturing liabilities, 𝑃𝑃2is the short-term liabilities, 𝑃𝑃3is the long-term liabilities. where Cs is the solvency coefficient; 𝐶𝐶𝑒𝑒𝑒𝑒 is the equity capital including all available reserves of the agricultural organization; P1is the maturing liabilities, 𝑃𝑃2is the short-term liabilities, 𝑃𝑃3is the long-term liabilities. g The range scales of the financial stability and solvency coefficients by the groups of regions of the Russian Federation are given in Table 1. Table 1. Values of financial stability and solvency coefficients Group No. Description Cfs Cs I The region where the organizations with absolute financial stability and solvency are located. 0.8-0.9 0.5-0.7 II The region where most of the organizations show financial stability but insignificant lag in one of the criteria is allowed. 0.7-0.9 0.4-0.7 III The agricultural organizations of the region are estimated as a medium, since there are organizations with normal financial stability but low solvency, and vice versa. 2 Materials and Methods 0.6-0.7 0.3-0.4 IV The region with the organizations with financial non- stability, the profit of such organizations is often lacking or very insignificant but sufficient only for making mandatory payments. 0.4-0.6 0.2-0.3 V The region where most of the organizations are chronically un-profitable, insolvent, with crisis financial state. less than 0.4 less than 0.2 Table 1. Values of financial stability and solvency coefficients At stage II, to perform zoning of the territory according to the ecological state indicator expressed in the monetary equivalent, it is necessary to calculate the costs for bringing the land plot to the maximum efficient state for use as an ecological component for each object of division of the territory according to the formula (3) presented in the article [8 ]. 𝑅𝑎𝑃𝑠𝑁𝑓𝑃𝑓𝑠𝑠𝑆𝑌𝑃𝑃𝐶𝑅𝑍𝑡𝑡𝑡𝑅 y g ( ) p [ ] ES = (𝑅𝑅𝑎𝑎× 𝑃𝑃𝑠𝑠+ 𝑁𝑁𝑓𝑓 × 𝑃𝑃𝑓𝑓+ С𝑠𝑠𝑠𝑠) × 𝑆𝑆+ 𝑌𝑌×𝑃𝑃×𝑃𝑃×𝐶𝐶×S ( 1+ 𝑅𝑅 100) + 𝑍𝑍𝑡𝑡𝑡𝑡𝑡𝑡 (3) 𝑅𝑎𝑃𝑁 (3) ES is the ecological state indicator, 𝑅𝑅𝑎𝑎 is the recommended seed application rate, t/ha; 𝑃𝑃𝑠𝑠 is the seed price, RUB/t; 𝑃𝑃𝑓𝑓 is the fertilisers price, RUB; 𝑁𝑁𝑓𝑓is the recommended fertiliser application rate, kg/ha; С𝑠𝑠𝑠𝑠 is the soil treatment cost to be determined on the basis of crop cultivation technology, RUB/ha; 𝑆𝑆 is the land plot area, ha; Y is the average crop yield for the production facility for the last 5 years, c/ha; P is the price of a centner of products, RUB; C is the correction factor considering the duration of restoration of fertility; R is the refinancing rate of the Central Bank of Russia. The values range scale is based on the percentage of costs for bringing the land plot to the maximum efficient state for use and current cadastral cost of the land plot by consolidated groups (Table 2). Table 2. Value of the ecological state indicator Group No. I II III IV V Description minimum degree average significant high very high percentage 0-20 20-40 40-60 60-80 80-100 At stage III with the use of the methods of spatial multifactor analysis, the obtained maps of the financial and ecological state are compared and consolidated into the integral digital map that can be used for forecasting and recognizing crisis situations in the agricultural production. To solve this task, the methods of spatial multifactor analysis, namely, geoprocessing and raster algebra, were used together with ArcGIS software product. Table 2. 3 Results and Discussion The object of research is the agricultural land plots in the territory of Novisibirsk region that make 62.6% of total area of the region and occupy 11,128.5 ths. ha. According to official statistics, the agricultural land plots in the territory of Novosibirsk region are mainly used as arable land for growing crops or as pastures for grazing, the rest of the agricultural land is the personal subsidiary farming and individual housing construction with household plots, vegetable gardens for growing vegetables and other crops. The above method for creation of thematic maps for forecasting and recognizing crisis situations in the agricultural production was proved within the boundaries of the 121 village council territory of the central part of Novosibirsk region, including 64 agricultural companies of different forms of organization. The results are given in Table 3. companies of different forms of organization. The results are given in Table 3. Table 3. Thematic maps for forecasting and recognizing crisis situations in the agricultural production By analysing the thematic maps, it can be stated that the most part of the territory under review, namely that of 89 village councils, belongs to ecological state groups I and II, minimum and average need in restoration. The thematic map of the financial status shows that groups I, II and III include 96 village councils that are characterized by 28%, 29% and 30%, respectively. It can be concluded from the consolidated thematic map of crisis situations there are no situations observed, ranges IV and V do not include the territory of any village council, and the main amount is within the ranges of groups II and III, 64 and 37, respectively, that evidences rather stable state of the considered territory of Novosibirsk region. NA Settlements Ranges of financial state Ranges of ecological state Ranges of crisis situations in the agricultural production Group I 32 pcs. (28%) Group II 34 pcs. (29%) Group III 30 pcs. (30%) Group IV 14 pcs. (12%) Group V 6 pcs. (5%) Group I 48 pcs. (44%) Group II 41 pcs. (37%) Group III 19 pcs. (17%) Group IV 2 pcs. (2%) Group V Group I 9 pcs. (8%) Group II 64 pcs. (58%) Group III 37 pcs. (34%) Group IV Group V Table 3. Thematic maps for forecasting and recognizing crisis situations in the agricultural production Table 3. 2 Materials and Methods Value of the ecological state indicator Group No. I II III IV V Description minimum degree average significant high very high percentage 0-20 20-40 40-60 60-80 80-100 Table 2. Value of the ecological state indicator At stage III with the use of the methods of spatial multifactor analysis, the obtained maps of the financial and ecological state are compared and consolidated into the integral digital map that can be used for forecasting and recognizing crisis situations in the agricultural production. To solve this task, the methods of spatial multifactor analysis, namely, geoprocessing and raster algebra, were used together with ArcGIS software product. At stage III with the use of the methods of spatial multifactor analysis, the obtained maps of the financial and ecological state are compared and consolidated into the integral digital map that can be used for forecasting and recognizing crisis situations in the agricultural production. To solve this task, the methods of spatial multifactor analysis, namely, geoprocessing and raster algebra, were used together with ArcGIS software product. 4 4 https://doi.org/10.1051/e3sconf/202129603004 E3S Web of Conferences 296, 03004 (2021) ESMGT 2021 3 Results and Discussion Thematic maps for forecasting and recognizing crisis situations in the agricultural production NA Settlements Ranges of financial state Ranges of ecological state Ranges of crisis situations in the agricultural production Group I 32 pcs. (28%) Group II 34 pcs. (29%) Group III 30 pcs. (30%) Group IV 14 pcs. (12%) Group V 6 pcs. (5%) Group I 48 pcs. (44%) Group II 41 pcs. (37%) Group III 19 pcs. (17%) Group IV 2 pcs. (2%) Group V Group I 9 pcs. (8%) Group II 64 pcs. (58%) Group III 37 pcs. (34%) Group IV Group V By analysing the thematic maps, it can be stated that the most part of the territory under review, namely that of 89 village councils, belongs to ecological state groups I and II, minimum and average need in restoration. The thematic map of the financial status shows that groups I, II and III include 96 village councils that are characterized by 28%, 29% and 30%, respectively. It can be concluded from the consolidated thematic map of crisis situations there are no situations observed, ranges IV and V do not include the territory of any village council, and the main amount is within the ranges of groups II and III, 64 and 37, respectively, that evidences rather stable state of the considered territory of Novosibirsk region. By analysing the thematic maps, it can be stated that the most part of the territory under review, namely that of 89 village councils, belongs to ecological state groups I and II, minimum and average need in restoration. The thematic map of the financial status shows that groups I, II and III include 96 village councils that are characterized by 28%, 29% and 30%, respectively. It can be concluded from the consolidated thematic map of crisis situations there are no situations observed, ranges IV and V do not include the territory of any village council, and the main amount is within the ranges of groups II and III, 64 and 37, respectively, that evidences rather stable state of the considered territory of Novosibirsk region. 5 5 5 https://doi.org/10.1051/e3sconf/202129603004 E3S Web of Conferences 296, 03004 (2021) ESMGT 2021 4 Conclusions The article provides the method and results of thematic mapping for forecasting and recognizing crisis situations in agriculture. Such geoinformation support is an important component for agricultural management, control of rational land use, compliance with the land legislation requirements, and formation of logistics systems. To speed up the implementation of such digital innovative technologies, regular works on the detailed mapping of agricultural lands, soil content of agricultural lands, agri-ecological indicators should be performed with the use of the Earth remote sensing data. References 1. J. A. Ingram, Food Sec., 3 (2011) 2. Departmental project «Digital Agriculture» - Federal state scientific institution «Rosinformagrotech», 48 (2019) 3. E. Tsoraeva, S. Mezhyan, M. Kataeva, L.Hugaeva, T.Rogova, GIS technologies used in zoning agricultural land for optimizing regional land use, E3S Web Conf. 224, 03001 (2020) 4. L.Klerkx, E. Jakku, P. Labarthe, Wageningen Journal of Life Sciences, 90 (2019) 5. O.A. Kosareva, M. N. Eliseev, V. P. Cheglov, A.N. Stolyarova, S. B. Aleksina, Eurasia J Biosci, 13 (2019) 6. V. V. Vershinin, T. N. Kovaleva, M. M. Demidova, P. P. Lebedev, Moscow Economic Journal, 5(1) (2018). 7. F. I. Ereshko, V. I. Medennikov, V. V. Kulba, Information society, 3 (2020) 8. E.D. Podryadchikova, L.N. Gilyova, Improvement of the methods of state cadastral assessment of agricultural lands while accounting the expenses for bringing a land plot to the state of maximum use efficiency, E3S Web Conf. 244, 03003 (2021) 6 6
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Effects of Straw Return in Deep Soils with Urea Addition on the Soil Organic Carbon Fractions in a Semi-Arid Temperate Cornfield
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6,027
OPEN ACCESS Citation: Zou H, Ye X, Li J, Lu J, Fan Q, Yu N, et al. (2016) Effects of Straw Return in Deep Soils with Urea Addition on the Soil Organic Carbon Fractions in a Semi-Arid Temperate Cornfield. PLoS ONE 11 (4): e0153214. doi:10.1371/journal.pone.0153214 Editor: Wenju Liang, Chinese Academy of Sciences, CHINA Editor: Wenju Liang, Chinese Academy of Sciences, CHINA Received: December 8, 2015 Accepted: March 26, 2016 Published: April 28, 2016 Copyright: © 2016 Zou 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. CHINA Received: December 8, 2015 Accepted: March 26, 2016 Published: April 28, 2016 Copyright: © 2016 Zou et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Copyright: © 2016 Zou et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper. Effects of Straw Return in Deep Soils with Urea Addition on the Soil Organic Carbon Fractions in a Semi-Arid Temperate Cornfield Hongtao Zou☯, Xuhong Ye☯, Jiaqi Li, Jia Lu, Qingfeng Fan, Na Yu, Yuling Zhang, Xiuli Dang, Yulong Zhang* Hongtao Zou☯, Xuhong Ye☯, Jiaqi Li, Jia Lu, Qingfeng Fan, Na Yu, Yuling Zhang, Xiuli Dang, Yulong Zhang* College of Land and Environment, Shenyang Agricultural University & Key Laboratory of Cultivated Land Conservation, Ministry of Agriculture, Shenyang, PR China a1111 ☯These authors contributed equally to this work. * yexuhong1980@163.com Abstract Returning straw to deep soil layers by using a deep-ditching-ridge-ploughing method is an innovative management practice that improves soil quality by increasing the soil organic carbon (SOC) content. However, the optimum quantity of straw return has not been deter- mined. To solve this practical production problem, the following treatments with different amounts of corn straw were investigated: no straw return, CK; 400 kg ha-1 straw, S400; 800 kg ha-1 straw, S800; 1200 kg ha-1 straw, S1200; and 1600 kg ha-1 straw, S1600. After straw was returned to the soil for two years, the microbial biomass C (MBC), easily oxidized organic C (EOC), dissolved organic C (DOC) and light fraction organic C (LFOC) content were measured at three soil depths (0–10, 10–20, and 20–40 cm). The results showed that the combined application of 800 kg ha-1 straw significantly increased the EOC, MBC, and LFOC contents and was a suitable agricultural practice for this region. Moreover, our results demonstrated that returning straw to deep soil layers was effective for increasing the SOC content. RESEARCH ARTICLE SOC of Straw Deep Return beneficial for the next crop and can decrease the rate of crop emergence[12]. In addition, because straw is in direct contact with the soil, the organic acids in straw increase crop pests and diseases that occur during straw decomposition[13]. beneficial for the next crop and can decrease the rate of crop emergence[12]. In addition, because straw is in direct contact with the soil, the organic acids in straw increase crop pests and diseases that occur during straw decomposition[13]. Competing Interests: The authors have declared that no competing interests exist. In view of the traditional problems with returning straw to the soil, our laboratory devel- oped a method for returning straw to deep soil layers that uses ridge plowing at a depth of 40cm after harvest[14]. When using this new method of straw return to the field, the bottom of the soil plow layer is broken, the soil bulk density is reduced, and the soil structure is improved [15]. Meanwhile, as straw is buried in the furrow, the next crop is planted on the ridge. Thus, the crop roots do not directly contact the straw, which could reduce the incidence of disease caused by the harmful substances produced during straw degradation. However, the optimal amount of straw return using this method has not been determined yet. The amount of straw return is related to multiple factors. Excessive amounts of straw return prevent the complete degradation of straw over a short period, and the addition of insufficient straw does not meet the needs of the carbon pool[16]. Therefore, straw return in actual production systems is an important problem that needs to be addressed. Soil organic carbon (SOC) content is the most important parameter for sustainable soil use, and the labile fractions of SOC affect soil physical, chemical and biological properties[17–18]. Labile organic carbon (C) significantly impacts soil nutrient transformations, soil formation, microbial metabolism, organic matter decomposition and the migration of soil contaminants [19]. Soil labile organic C fractions, such as microbial biomass C (MBC), easily oxidized organic C (EOC), dissolved organic C (DOC) and light fraction organic C (LFOC), are more sensitive indicators of changes in soil C than the soil total organic carbon (TOC)[20]. Active SOC changes correspond to small changes and occur before total soil carbon changes[21–22]. In addition, SOC turnover is a more sensitive indicator of soil quality and soil fertility and is affected by various tillage systemsand comprehensive soil activity. Furthermore, SOC is neces- sary for maintaining balance between the soil carbon pool and soil fertility. Thus, we can use the SOC content to determine soil productivity and predict soil environmental changes caused by changes in oil management practices[23]. Straw return is a widely recognized strategy for increasing SOC contents and improving soil quality and crop productivity[24] and increases soil microbial activity and biomass[25]. Dynamic changes in SOC in agricultural soils are mainly determined from the balance between organic material inputs and the degradation rates of existing SOC[26]. Thus, in this paper, we aim to quantify the effects of different amounts of straw return on soil MBC, EOC, DOC, and Straw return is a widely recognized strategy for increasing SOC contents and improving soil quality and crop productivity[24] and increases soil microbial activity and biomass[25]. Dynamic changes in SOC in agricultural soils are mainly determined from the balance between i i l i d h d d i f i i SOC[26] Th i hi Straw return is a widely recognized strategy for increasing SOC contents and improving soil quality and crop productivity[24] and increases soil microbial activity and biomass[25]. quality and crop productivity[24] and increases soil microbial activity and biomass[25]. Dynamic changes in SOC in agricultural soils are mainly determined from the balance between organic material inputs and the degradation rates of existing SOC[26]. Thus, in this paper, we aim to quantify the effects of different amounts of straw return on soil MBC, EOC, DOC, and LFOC contents and determine the optimal amount of straw return at deep soil depths in a semi-arid temperate cornfield. Introduction Straw is a relatively abundant resource in China, with an annual straw production of 0.79 bil- lion tons[1]. However, nearly 50% of the straw produced in China is wasted by burning, which results in serious environmental pollution[2]. As one of the most widely protective measures [3–5], returning straw to the soil is beneficial for optimizing the soil environment, preventing and controlling soil degradation, and reducing air pollution that results from burning straw[6– 9]. Currently, straw is returned to the soil in the following three ways: mechanical crushing with backward pressure return, direct mulching and leaving tall stubble[10–11]. In China, land must be planted each year, and yearly or quarterly crops are grown overshort intervals to meet food production needs because the amount of available arable land per capita is low. Straw decays slowly because it has a relatively high carbon-to-nitrogen (C/N) ratio, which is not Funding: The work was supported by the National Science and Technology Support Program (grant no: 2012BAD05B01, 2015BAD23B0203), Research Projects of Agricultural Public Welfare Industry of China (grant no: 201303125), the National Natural Science Foundation of China (grant no: 31572206, 41301255) and Liaoning Agricultural Science and Technology Innovation Talents Training Program (grant no: 2015051). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. 1 / 10 PLOS ONE | DOI:10.1371/journal.pone.0153214 April 28, 2016 Experimental design After corn harvest in October 2010, the soil was furrowed to a depth of 40cm using deep ditch- ing before covering the soil and ridge plowing to bury the maize straw. The average maize straw production was 9900-1150kg ha-1. We designed the straw return experiment to include the following treatments: one-half hectare of straw was returned to one hectare of arable land; one hectare of straw was returned to one hectare of arable land; one and one-half hectare of straw was returned to one hectare of arable land; and two hectares of straw were returned to one hectare of arable land. Straw was buried at a depth of 40 cm at the following concentra- tions: 400 kg ha-1 straw (S400), 800 kg ha-1 straw (S800), 1200 kg ha-1 straw (S1200), and 1600 kg ha-1 straw (S1600). The C/N ratio of the straw was 77:1. To achieve a C/N ratio of 25:1[27], addi- tional urea was applied at rates of 1.03 kg ha-1, 2.16 kg ha-1, 3.09 kg ha-1and 4.12 kg ha-1, respectively, with a base application of 235 kgha-1 urea, 300 kgha-1diammonium phosphate and225 kgha-1 potassium sulfate before ridge plowing. Thus, the depth of the upper soil con- taining straw was approximately 20cm. The soil was left undisturbed from the autumn of 2010 to the autumn of 2012. A control treatment (CK) without straw and with the same amount of fertilizer was also studied. The experimental plots were arranged using a random design with three replications, and each plot covered an area of 60 m2. The following spring, corn (Zheng- dan 958) was cultivated by creating a large double ridge in the ridge-sown maize treatment. Two years later, the straw added at rates of 400 kg ha-1 (S400) and 800 kg ha-1(S800) was decom- posed completely, and the straw in the other two treatments was partially decomposed. The depth of the upper soil layer above the straw and the depth of the straw were both approxi- mately 10cm. Soil samples were collected and analyzed in October 2012. Materials and Methods Study site The field experiment was performed at Fumeng County, Fuxin City, Liaoning Province, China (42°10'N, 122°00'E). The owner of the land, Mr. Wang Yi, gave us permission to conduct this study on his land. The site has a semi-arid and semi-humid continental monsoon climate with an average annual rainfall of 450 mm, an average annual evaporation of 1660 mm, a mean annual temperature of 7.6°C,and mean annual sunshine of 2760 h. The frost-free period is 154 days. The soil at the experimental site is silty sand and is classified as cinnamon soil according to the Chinese soil classification system (1992). The main properties of the soil sampled in October 2012 were as follows: bulk density, 1.45 g cm-3; organic C, 23.3 g kg-1; total nitrogen (N), 1.2 g kg-1; nitrate N, 7.4 mg kg-1;ammonium N, 10.2 mg kg-1; and pH, 7.7. 2 / 10 PLOS ONE | DOI:10.1371/journal.pone.0153214 April 28, 2016 SOC of Straw Deep Return PLOS ONE | DOI:10.1371/journal.pone.0153214 April 28, 2016 Soil sampling and analysis Soil samples were collected using “S”-type methods at five points and at three soil depths.(0– 10, 10–20 and 20–40 cm). The fresh soil samples were isolated from the straw residues and root debris by passing them through 10-mesh (2 mm) sieves before measuring the organic car- bon fractions. Soil samples were air-dried and passed through a 2 mm sieve for laboratory analysis of soil texture and SOC was measured by Walkley–Black wet oxidation method [28]. The MBC was determined using the chloroform fumigation extraction method[29]. Briefly, a field-moist soil (equivalent to 20 g dry weight) sample with a field moisture capacity of 40%was fumigated with chloroform overnight. After removing the chloroform from the soil sample, C was promptly extracted from the samples using 0.5 mol L−1 K2SO4 at a soil-to-extract ratio of 1:5. The C contents in the K2SO4-extracted solutions from the chloroform-treated and untreated soils were measured using an automated TOC Analyzer (Shimadzu, TOC-Vcph, Japan). Soil MBC was calculated by subtracting the K2SO4-extracted C of the untreated soils from theK2SO4-extracted C of the chloroform-treated soil and calibrated using an extraction effi- ciency factor (Kc) of 0.38. The EOC was analyzed via oxidation using 333-mol L-1 KMnO4[30]. Finely ground air- dried soil samples were oxidized using 25 ml of 333 mM KMnO4. The suspensions were hori- zontally shaken at 60 r min−1 for 1 h and centrifuged at 2000 r min−1 for 5 min. The superna- tants were diluted and measured using a spectrophotometer (UV2300) at 565 nm. DOC was extracted in water at 25°C[20] by shaking the sample and water mixture for 0.5 h at 250 rpm and centrifuging for 10 min at 15,000 rpm. The supernatant liquid was passed through 0.45 μm filter membranes, and the resulting filtrate was stored at −18°C until analysis. The organic C content in the extracts was measured using an automated TOC Analyzer. 3 / 10 PLOS ONE | DOI:10.1371/journal.pone.0153214 April 28, 2016 SOC of Straw Deep Return LFOC was determined using heavy liquid flotation separation[31]. Three 10 g subsamples of each soil (<2 mm) were weighed in a 100 mL plastic centrifuge tube, and 50 mL of a NaI liq- uid solution was added to the tube. The tubes were shaken on a shaker for 30 min before centrifuging at 1,000 rpm for 10 min. The floating, light fraction was immediately removed and poured into a plastic bottle. Effects of returning different amounts of straw to deep soil on SOC The changes of the SOC contents in the different soil layers are presented in Fig 1. The same treatment had a small change in three soil layers, with ranges of 19.33–22.07g kg-1, 17.43– 22.05g kg-1, 16.57–20.28g kg-1, and 16.15–18.45 g kg-1 for S400, S800, S1200, and S1600, respec- tively. S800 had the highest TOC content at a depth of10-20 cm, and the other samples had the highest TOC contents at a depth of 0–10 cm. According to the significant difference test, the TOC contents in S400 and S800 were significantly higher (P < 0.05) at depths of 10–20 cm and0-10 cm than the CK. No significant difference was observed at a depth of 20–40 cm. Soil sampling and analysis This process was repeated three times, and the supernatant materials from the replicates were poured into the same plastic bottle. The light fraction was collected by passing the solutions through a membrane filter (0.45 μm) in a Büchner funnel and washing three times with0.01 mol L−1 CaCl2 to remove excess NaI. The material was washed three more times with deionized water before drying at 60°C for 24 h, weighing, and finely grinding it for organic carbon analysis. Data analysis The data were analyzed usingSPSS17.0 (IBM, New York, USA). A single-factor analysis of vari- ance (ANOVA) was used to conduct different comparisons among the five treatments at P<0.05, and the means were separated using least significant difference (LSD). Effects of returning different amounts of straw to deep soil on MBC MBC content of 0-10cm and 10-20cm soil with S800 treatment is higher than those of CK and S1600, No significant difference between S400, S800 and S1200. (Fig 2). The MBC contents were 168.75–204.17 mg kg-1 in the S400treatment, 160.00–229.50 mg kg-1 in the S800 treatment, 181.25–191.67 mg kg-1 in the S1200 treatment, and 135.42–157.08 mg kg-1 in the S1600 treat- ment. In the 10-20cm soil layer, the S800 treatment had the highest MBC content, which was significantly higher than that in the CK. The soil MBC content decreased in the following Fig 1. Effects of straw return to the deep soil on soil microbial biomass carbon (SOC) at three soil depths. SOC values are expressed as the means±SE of three replicates. Different letters indicate significant differences among the treatments (*P<0.05). doi:10 1371/journal pone 0153214 g001 Fig 1. Effects of straw return to the deep soil on soil microbial biomass carbon (SOC) at three soil depths. SOC values are expressed as the means±SE of three replicates. Different letters indicate significant differences among the treatments (*P<0.05). Fig 1. Effects of straw return to the deep soil on soil microbial biomass carbon (SOC) at three soil depths. SOC values are expressed as the means±SE of three replicates. Different letters indicate significant differences among the treatments (*P<0.05). doi:10.1371/journal.pone.0153214.g001 doi:10.1371/journal.pone.0153214.g001 PLOS ONE | DOI:10.1371/journal.pone.0153214 April 28, 2016 4 / 10 SOC of Straw Deep Return Fig 2. Effects of straw return to the deep soil on soil microbial biomass carbon (MBC) at three soil depths. Values are expressed as the means±SE from three replicates. Different letters indicate significant differences among the treatments(*P<0.05). doi:10.1371/journal.pone.0153214.g002 Fig 2. Effects of straw return to the deep soil on soil microbial biomass carbon (MBC) at three soil depths. Values are expressed as the means±SE from three replicates. Different letters indicate significant differences among the treatments(*P<0.05). order: S800 > S1200, S400 > CK, S1600 at this depth. MBC in 20–40 cm is S1200, S400 greater than that of S1600 and CK. The straw return to deep soil treatments significantly increased the MBC distribution at 0–20 cm compared with the CK. Similar phenomena were observed in the treatments with low and medium contents of MBC at 0–20 cm and 20–40 cm; however, this difference was not sig- nificant. The MBC contents at 0–10 cm and 10–20 cm were nearly identical (Fig 2). Effects of returning different amounts of straw to deep soil on DOC The soil DOC contents at the three investigated soil depths for different treatments are shown in Fig 4. The soil DOC ranged from 297.83–351.97 g kg-1 for S400, 223.08–243.88 g kg-1 for S800, 220–254.34 g kg-1 for S1200 and 311.83–321.43 g kg-1 for S1600. Straw return to deep soil significantly decreased the soil DOC. The straw return treatments had significantly lower soil DOC than the CK at each soil depth, except for the S400 treatment at depths of 0–10 cm and 20–40 cm. The soil DOC contents were not significantly different between the soil layers in each treatment. Regarding the vertical distribution of soil DOC, the soil DOC decreased with increasing soil depth in all treatments (Fig 4). Effects of returning different amounts of straw to deep soil on easily oxidized organic carbon (EOC) The straw return treatments increased the EOC at the three soil depths (Fig 3). The soil EOC content ranged from 7.98–11.23 g kg-1 for S400, 8.80–13.47 g kg-1 for S800, 7.92–10.02 g kg-1 for S1200 and 7.58–9.42 g kg-1 for S1600. The EOC contents in the S800 treatment were the highest, and the EOC contents in the CK were the lowest at each soil depth. Some appreciable differ- ences were observed between the straw return in the different treatments, which decreased in the following order: S800 > S400 > S1200 > S1600 > CK (Fig 3). The deep soil (depth of 20–40 cm) had the lowest EOC content compared with the other two depths. In the control treatment, the soil EOC decreased as the soil depth increased from 0–10 cm to 10–20 cm. In the straw return treatments, the soil EOC was higher at 10–20 cm than at 0–10 cm. The effects of different amounts of straw return on the soil light fraction organic carbon (LFOC) The soil LFOC contents at all soil depths are shown in Fig 5. The soil LFOC in the different straw return treatments ranged from 223.12–280.37 mg kg-1 for S400, 235.67–300.32 mg kg-1 5 / 10 PLOS ONE | DOI:10.1371/journal.pone.0153214 April 28, 2016 SOC of Straw Deep Return Fig 3. Effects of straw return to deep soil on the easily oxidized organic carbon (EOC) contents at three soil depths. Fig 3. Effects of straw return to deep soil on the easily oxidized organic carbon (EOC) contents at three soil depths. Fig 3. Effects of straw return to deep soil on the easily oxidized organic carbon (EOC) contents at three soil depths. doi:10.1371/journal.pone.0153214.g003 fects of straw return to deep soil on the easily oxidized organic carbon (EOC) contents at three soil depths. doi:10.1371/journal.pone.0153214.g003 for S800, 233.78–301.32 mg kg-1 for S1200, and 218.44–268.77 mg kg-1 for S1600. The ranges of soil LFOC in all treatments decreased as follows: S1200 > S800 > S400, CK >S1600 at 0–10 cm, S800, S1200 > CK, S400 > S1600 at 10–20 cm, and S800, S1200 > S400 > S1600 > CK at 20–40 cm (Fig 5). for S800, 233.78–301.32 mg kg-1 for S1200, and 218.44–268.77 mg kg-1 for S1600. The ranges of soil LFOC in all treatments decreased as follows: S1200 > S800 > S400, CK >S1600 at 0–10 cm, S800, S1200 > CK, S400 > S1600 at 10–20 cm, and S800, S1200 > S400 > S1600 > CK at 20–40 cm (Fig 5). Discussion After two years of straw return, the straw applied at 400 kg ha-1 (S400) and 800 kg ha-1 (S800) in the two districts was completely decomposed, and the straw in the other two treatments was partially decomposed. The quantity of straw return should be determined from the treatment Fig 4. Effects of returning straw to the deep soil on soil dissolved organic carbon (DOC) at the three soil depths. Values are expressed as means ±SE from three replicates. Different letters indicate significant differences (*P<0.05). doi:10.1371/journal.pone.0153214.g004 Fig 4. Effects of returning straw to the deep soil on soil dissolved organic carbon (DOC) at the three soil depths. Values are expressed as means ±SE from three replicates. Different letters indicate significant differences (*P<0.05). d i 10 1371/j l 0153214 004 doi:10.1371/journal.pone.0153214.g004 PLOS ONE | DOI:10.1371/journal.pone.0153214 April 28, 2016 6 / 10 SOC of Straw Deep Return Fig 5. Effects of returning straw to deep soil on the soil light fraction organic carbon (LFOC) at three soil depths. Fig 5. Effects of returning straw to deep soil on the soil light fraction organic carbon (LFOC) at three soil depths. Fig 5. Effects of returning straw to deep soil on the soil light fraction organic carbon (LFOC) at three soil depths. doi:10.1371/journal.pone.0153214.g005 doi:10.1371/journal.pone.0153214.g005 with complete decomposition. In addition, many physical and chemical soil properties should be considered, especially the SOC content. The C/N ratio is a key factor that influences the decomposition of crop straw[32]. High doses of crop straw should be applied to guarantee the absorption of available N by crops[26]. A C/N ratio of 25:1 facilitates the straw decomposition and the release of N[33]. Qiu et al. [25] and Ge et al. [34] found that using straw return and adjusting the C/N ratio could increase crop production. Therefore, in this paper, appropriate amounts of nitrogen fertilizer (C/N ratio of 25:1) were added after straw was returned to the fields. Soil MBC is an active part of the SOC pool[29] and is influenced by easily degradable organic matter via processes such as the decomposition of microbial organisms, variations in soil moisture and soil management measures[31]. MBC participates in the decomposition and formation of soil organic matter, plays an important role in nutrient cycling and transforma- tions, and serves as a repository for effective nutrients in the soil[35–37]. Straw return increased the MBC contents in the upper and deep soil layers. PLOS ONE | DOI:10.1371/journal.pone.0153214 April 28, 2016 References 1. Liu F, Zhang C, Chen A, Zhou G, Wu J. Technology research and application prospect of straw return- ing. Crops. 2012; 2: 18–23. 1. Liu F, Zhang C, Chen A, Zhou G, Wu J. Technology research and application prospect of straw return- ing. Crops. 2012; 2: 18–23. 2. Zhou L. Thoughts on the domestic present condition of straw utilization. Journal of Anhui Agricultural Sciences. 2012; 40: 15853–15855. 2. Zhou L. Thoughts on the domestic present condition of straw utilization. Journal of Anhui Agricultural Sciences. 2012; 40: 15853–15855. 3. Cao Z, Hao JM, Liang L. 2009. Supporting capacity of main crop straw for Chinese residents’ food secu- rity. Transactions of the Chinese Society of Agricultural Engineering. 2009; 25: 179–184. 3. Cao Z, Hao JM, Liang L. 2009. Supporting capacity of main crop straw for Chinese residents’ food secu- rity. Transactions of the Chinese Society of Agricultural Engineering. 2009; 25: 179–184. 4. Bi YY, Wang LY. China's main number and regional distribution of straw resources. Agricultural Mecha- nization Research. 2010; 3: 1–7. 4. Bi YY, Wang LY. China's main number and regional distribution of straw resources. Agricultural Mecha- nization Research. 2010; 3: 1–7. 5. Wang HB, Qin YP, Yu K. Distribution, use and development strategies of crop straw resources. Land and Natural Resources Research. 2008; 2: 92–93. 5. Wang HB, Qin YP, Yu K. Distribution, use and development strategies of crop straw resources. Land and Natural Resources Research. 2008; 2: 92–93. 6. Tiao SZ, Ning TY, Wang Y. Effect of different tillage and straw returning on soil organic carbon concen- tration catcher. J Appl Ecol. 2010; 21: 373–378. 6. Tiao SZ, Ning TY, Wang Y. Effect of different tillage and straw returning on soil organic carbon concen- tration catcher. J Appl Ecol. 2010; 21: 373–378. 7. Chen H, Zhou J, Xiao B. Characterization of dissolved organic matter derived from rice straw at different stages of decay. J Soils Sediments. 2010; 10: 915–922. doi: 10.1007/s11368-010-0210-x 8. Lü M, Li ZJ, Zhang T, Ning T, Zhao J, Li H. Efforts of minimum or no-tillage system and straw returning on extreme soil moisture and yield of winter wheat. Transactions of the Chinese Society of Agricultural Engineering. 2010; 26: 41–44. 9. Zhao P, Chen F. Effects of straw mulching plus nitrogen fertilizer on nitrogen efficiency and grain yield in winter wheat. ActaAgron Sin. 2008; 34: 1014–1018. 10. Thorburn PJ, Meier EA, Collins K, Robertson FA. Acknowledgments The work was supported by the National Science and Technology Support Program (Grant No: 2012BAD05B01, 2015BAD23B0203), Research Projects of Agricultural Public Welfare Industry of China (Grant No: 201303125), the National Natural Science Foundation of China (Grant No: 31572206, 41301255) and Liaoning Agricultural Science and Technology Innova- tion Talents Training Program (Grant No: 2015051). Discussion This result suggests that LFOC is an active form of organic carbon that reflects the varia- tions in soil total organic C. Additionally, S800should facilitate the accumulation of LFOC. In conclusion, returning 800kg ha-1of straw to the soil with urea application generally enhanced the EOC, MBC, and LFOC contents and was a suitable agricultural practice in this region for the new straw return method. Author Contributions Conceived and designed the experiments: HTZ Yulong Zhang. Performed the experiments: XHY JQL JL. Analyzed the data: QFF NY Yuling Zhang. Contributed reagents/materials/analy- sis tools: XLD. Wrote the paper: XHY. Discussion This increase in MBC could be explained by the increased availability of C and N. However, the MBC decreased in the S1200 and S1600 treatments relative to the S800 treatment. This decrease in microbial activity could be attributed to the soil pH changes caused by the large amounts of added urea[38]. Indeed, decreases in the soil pH following urea addition are suggested to be a main factor controlling decreases in MBC[39,40]. The results also indicated that the amount of urea added and the amount of straw return could be an important combined factor that affects the soil MBC con- tent. These results were consistent with the results presented by Zhang et al. [41], who found that the microbial biomass and functional diversity decreased due to decreasing soil pH beyond a critical N loading level in a semi-arid temperate steppe. The threshold of the N loading level was 16 gNm−2year−1[42], which corresponded to the N-addition rates in the S1200 and S1600 treatments in this study. Soil EOC may reflect the effectiveness of organic C for indicating soil quality, which was more sensitive than other indicators in farmland soil[30]. The EOC content was higher at 0–20 cm than at 20–40 cm because of increased microbial activity, and the soil permeability was improved after straw was returned to the 0-40-cm soil layer. The EOC content was the highest in S800, which could suggest that the application of 800 kg ha-1 straw was the most effective for improving soil microbial activity and the soil EOC content. The DOC content influenced the PLOS ONE | DOI:10.1371/journal.pone.0153214 April 28, 2016 7 / 10 SOC of Straw Deep Return migration of soil organic and inorganic components and their transformations and degrada- tion. In addition, the soil DOC content had a relatively strong impact on the migration of heavy metals and the adsorption of inorganic ions[42]. The soil DOC content was lower in the straw return treatments than in the CK. This result potentially occurred because the crops absorbed DOC during their growth and development or because the DOC was temporarily accumulated in or transformed to other substances. Otherwise, the decrease in DOC could be associated with microbial use[42]. In addition, the environmental changes in soil chemistry should also be considered[43–45]. 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PLOS ONE | DOI:10.1371/journal.pone.0153214 April 28, 2016 References Soil SciSoc Am J. 1994; 58: 1130–1139. 23. Li S, Li YB, Li XS, Tian X, Zhao A, Wang S, et al. Effect of straw management on carbon sequestration and grain production in a maize–wheat cropping system in Anthrosol of the Guanzhong Plain. Soil Till- age Res. 2016; 157: 43–51. 24. Qiu XL, Zheng B, Lu Y, Duan Z, Yin S, Lei B. Effects of C/N regulated corn straw application on nitrogen phosphorus and potassium uptake in tobacco. ActaAgriculturaeBoreali-OccidentalisSinica. 2012; 21: 125–129. 25. Laird DA, Chang C. Long-term impacts of residue harvesting on soil quality. Soil Tillage Res. 2013; 134: 33–40. doi: 10.1016/j.still.2013.07.001 26. Wang XB, Cai DX. Effect of corn stover incorporated in dry farm on soil fertility. Agric Science China. 2000; 33: 54–61. 27. Bao SD. Soil and agricultural chemistry analysis. Beijing: China Agriculture Press; 200 28. Vance ED, Brookes PC, Jenkinson DS. Microbial biomass measurements in forest soils: the use of the chloroform fumigation-incubation method in strongly acid soils. Soil BiolBiochem. 1987; 19: 697–702. doi: 10.1016/0038-0717(87)90051-4 29. Blair G, Lefroy R, Lisle L. Soil carbon fractions based on their degree of oxidation, and the development of a carbon management index for agricultural systems. Aust J Agric Res. 1995; 46: 1459–1466. doi: 10.1071/AR9951459 30. Jiang PK, Xu QF, Xu ZH, Cao ZH. Seasonal changes in soil labile organic carbon pools within a Phyl- lostachys praecox stand under high rate fertilization and winter mulch in subtropical China. For Ecol Manage. 2006; 236: 30–36. doi: 10.1016/j.foreco.2006.06.010 31. Spedding TA, Hamel C, Mehuys GR, Madramootoo CA. Soil microbial dynamics in maize-growing soil under different tillage and residue management systems. Soil Biol Biochem. 2004; 36: 499–512. doi: 10.1016/j.soilbio.2003.10.026 32. Trinsoutrot I, Recous S, Bentz B, Linères M, Chèneby D, Nicolardot B. Biochemical quality of crop resi- dues and carbon and nitrogen mineralization kinetics under nonlimiting nitrogen conditions. Soil SciSoc Am J. 2000; 64: 918–926. doi: 10.2136/sssaj2000.643918x 33. Moreno-Cornejo J, Zornoza R, Faz A. Carbon and nitrogen mineralization during decomposition of crop residues in a calcareous soil. Geoderma. 2014; 230–231: 58–63. doi: 10.1016/j.geoderma.2014.03. 024 34. Zhang YL, Shen QR, Cao CY. Effect of organic fertilizers on soil organic phosphorus fractions and bio- availability. Journal of Nanjing Agricultural University. 1998; 21: 59–63 PLOS ONE | DOI:10.1371/journal.pone.0153214 April 28, 2016 9 / 10 SOC of Straw Deep Return 35. Ge LL, Ma LH, Bian JL, Wang ZQ, Yang JC, Liu LJ. PLOS ONE | DOI:10.1371/journal.pone.0153214 April 28, 2016 References Effects of returning maize straw to field and site- specific nitrogen management on grain yield and quality in rice. China. Rice Sci. 2013; 27: 153–160 36. Spedding TA, Hamel C, Mehuys GR, Madramootoo CA. Soil microbial dynamics in maize-growing soil under different tillage and residue management systems. Soil BiolBiochem. 2004; 36: 499–512. doi: 10.1016/j.soilbio.2003.10.026 37. Kandeler E, Tscherko D, Spiegel H. Long-term monitoring of microbial biomass, N mineralisation and enzyme activities of a chernozem under different tillage management. BiolFertil Soils. 1999; 28: 343– 351. doi: 10.1007/s003740050502 38. Gaind S, Pandey AK, Lata. Biodegradation study of crop residues as affected by exogenous inorganic nitrogen and fungal inoculants. J Basic Microbiol. 2005; 45: 301–311. doi: 10.1002/jobm.200410483 PMID: 16028202 39. Wang RZ, Filley TR, Xu ZW, Wang X, Li MH, Zhang YG, et al. Coupled response of soil carbon and nitrogen pools and enzyme activities to nitrogen and water addition in a semiarid grassland of Inner Mongolia. Plant Soil. 2014; 381: 323–336. doi: 10.1007/s11104-014-2129-2 40. Wang R, Dorodnikov M, Yang S, Zhang Y, Filley TR, Turco RF, et al. Responses of enzymatic activities within soil aggregates to 9-year nitrogen and water addition in a semi-arid grassland. Soil BiolBiochem. 2015; 81: 159–167. 41. Zhang GS, Chan KY, Li GD, Huang GB. Effect of straw and plastic film management under contrasting tillage practices on the physical properties of an erodible loess soil. Soil Tillage Res. 2008; 98: 113– 119. doi: 10.1016/j.still.2007.09.001 42. Moore TR, de Souza W, Koprivnjak J. Controls on the sorption of dissolved organic carbon by soils. Soil Sci. 1992; 154: 120–129. doi: 10.1097/00010694-199208000-00005 43. Ji B, Hu H, Zhao Y, Mu X, Liu K, Li C. Effects of deep tillage and straw returning on soil microorganism and enzyme activities. ScientificWorldJournal. 2014; 2014: 451493. doi: 10.1155/2014/451493 PMID: 24982955 44. Zou HT, Ma YB, Xu M, Yan HL, Fan QF, Huang Y, et al. Semiarid region of western Liaoning deep straw to field on soil water concentration, bulk density and corn yield. Shenyang Agricultural Univer- sity.2012; 43: 494–497. 45. Neff JC, Asner GP. Dissolved organic carbon in terrestrial ecosystems: synthesis and a model. Ecosys- tems. 2001; 4: 29–48. doi: 10.1007/s100210000058 10 / 10
https://openalex.org/W2725731183
https://revistas.pucp.edu.pe/index.php/quimica/article/download/18728/18965/
Spanish; Castilian
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Química y Farmacología de Smallanthus sonchifolius (Poepp.) H. Rob. ("Yacón")
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Resumen misma suerte que el «cardo» con su pimienta y naranja, y de esta manera se parece en el sabor al «Cardo». La presente publicación pretende resumir el conocimiento científico químico-biológico sobre el Smallanthus sonchifolius (Poepp.) H. Rob. Se incluye una descripción de los componentes químicos reportados a la fecha en las revistas científicas; entre ellos, los ol igofructanos, así como los compuestos de naturaleza fenólica y terpénica. Además, se presenta una discusión de las actividades biológicas encontradas para extractos de hojas y raíces del yacón. El yacón es el nombre vernacular más usual con el que se conoce al Smallanthus sonchifolius perteneciente a la familia Asteraceae. Para el género Smallanthus se reporta 21 especies de distribución cosmopolita, de las cuales 11 son endémicas de nuestro paísY En los últimos años el yacón ha tenido una gran aceptación por los consumidores por su agradable sabor dulce y por las propiedades atribuidas desde antigüo que empiezan a ser revaloradas. Es un cultivo alto-andino, pero ha sido llevado a Italia y a Nueva Zelandia, y de ésta última a japón en 1985. Química y Farmacología de Smallanthus sonchifolius (Poepp.) H. Rob. ("Yacón") Oiga Locka y Rosario Rojasb dDepartamento de Ciencias, Pontificia Universidad Católica del Perú, Lima 32, Perú hDepartamento de Ciencias Farmacéuticas, Facultad de Ciencias y Filosofía, Universidad Peruana Cayetano Heredia, Lima, Perú Abstract This review article covers the present knowledge on the chemistry and biological activities of Smallanthus sonchifolius (Poepp.) H. Rob. A description of the chemical compounds reported in the scientific literature (i.e. oligofructans, phenolic compounds and terpenes) is included. The biological activities of the crude extracts of leaves and roots from yacón are discussed. La infusión de las hojas secas es usada para la diabetes y para los desórdenes digestivos y renales. El emplasto de las hojas calentadas es usado para el reumatismo y dolores musculares.'·' El yacón tiene un gran futuro industrial para la obtención de inulina y fructosa, muy apropiados para enfermos diabéticos Se hace una bebida refrescante de la raíz rayada y pasada por una tela; también se prepara una «chancaca>> del jugo que se obtiene por expresión de la raíz, luego de ser concentrada. Palabras claves: Smallanthus sonchifolius, yacón, hipoglicemiante, antimicrobiano, antioxidante Los botánicos refieren que existen dos sinonimias para la especie Smallanthus sonchifolius las cuales son Polymnia sonchifolia Poeppig y Polymnia edufis Weddell. Por otro lado, se conocen varios nombres comunes para el yacón, como por ejemplo: Llacón, llakuma, llaqon, yacumpi, (v. Quechua); aricoma y aricona (v. Aymara); llacjon, taraca, jacón, jikima, jiquimilla, llacoma, polaco, colla, llamon, arboloco, jicama.3 Metabolitos primarios Dentro de los oligosacáridos en la raíz de yacón se ha encontrado desde trisacáridos a decasacáridos, confirmándose que corresponden a ~(2 ~ 1) fructool igosacáridos con sacarosa como azúcar terminal (oligofructanos tipo inulina)6, el contenido total de estos oligosacáridos es de 201 mg/g (base seca). Estos fructooligosacáridos están siendo considerados como una fuente potencial de prebióticos para la industria alimentaria y de suplementos dietéticos. 7 Se ha determinado, asimismo, azúcares libres en concentración de 350 mg/g de fructosa, 158 mg/g de glucosa y 74 mg/g de sacarosa; estos análisis se han efectuado en muestras de raíces almacenadas en condiciones suaves y 96 días después de ser cosechadas. 8 R,OtCO~H ~OH H OR, - llA OH = cafeoil H OR3 H OR4 COOH 1 : R1=R3=cafeoil. R2=R4=H 2 : R1=R4=cafeoil, R2=R3=H 3: R1=R2=R4=cafecil, R3=H ó R2=R4=cafeoil. R1=R3=H ó R1 =R3=R4=cafeoil, R2=H HO~H COOH R10 H H OH 4 : R1 = H, R2 = cafeoil 5 : R1 = R2 = cafeoil 6: R=H 7: R =OCH3 HO~H COOH R10 H H OH 4 : R1 = H, R2 = cafeoil 5 : R1 = R2 = cafeoil Se ha determinado que el yacón contiene abundante oligofructano (60-70% sobre base seca), pero que carece de almidón Y Además de los carbohidratos, la raíz de yacón contiene pequeñas cantidades de proteínas (1 ,3 a 7,3 %); grasa (0,4 a 1,0%), fibra (1,0- 5,7%) sobre base seca; y sobre base fresca, con un porcentaje de agua de 93 - 70%, se ha determinado minerales como calcio 23 mg/g, fósforo 21 mg/g y hierro 0,3 mg/g; así como niacina 0,33 mg/g, riboflavina O, 1%, ácido ascórbico 13 mg/g y retino! 1 O mg/g. 10 Para la raíz también se reporta la presencia del compuesto fenólico ácido clorogénico (8), y del aminoácido L-triptófano (9), en concentraciones de 48,5 y 14,6 mg/g (sobre muestra fresca), respectivamente. A ambos se les atribuye la actividad antioxidante del yacón. 9 Los tallos y hojas secas contienen proteínas (11-17%) y grasa (2-3%). 3 o HO~;( HO ~COH HO OH o HO~;( HO ~COH HO OH ~OH ~NJ ~H, H 8 9 o HO~;( HO ~COH HO OH ~OH ~NJ ~H, H 9 Constituyentes químicos ga/acto-oct-2-ulopiranosonico ácido) (4), y el ácido (1 R,25, 3R,4R,55, 7 R)-2,4-dihidroxi-7-hidroximeti l-2,3-bis[3-(3,4- dihidroxifeni 1)-1-oxo-2-propeni loxi]-6,8-dioxabiciclo[3 .2.1 ]- octan-5-carboxíl ico (4 ,5-d i-0-cafeoi 1-2,7 -anh idro-D-g/ icero-b-D- ga/acto-oct-2-ulopiranosico ácido) (5). 12 También se han aislado los ácidos cafeico (6) y ferúlico (7). 13 Introducción El poblador andino ha usado desde la antigüedad el yacón en su alimentación. A pesar que la parte comestible es la raíz tuberosa, el yacón ha sido considerado como un fruto por su sabor dulce. La presente revisión tiene como objetivo resumir los estudios científicos químico-biológicos reportados a la fecha en la literatura científica sobre este importante recurso natural para así propiciar su aplicación y explotación adecuada. En el libro de Soukup' se puede leer una referencia que el Padre Cobo (1653) hace sobre el yacón: «cómense crudas por frutas y tienen un buen sabor, y mucho mejor si se posan un poco al sol; suélese cortar en ruedas y preparar de la junio 2005 Revisto de QUÍMICA 31 Metabolitos secundarios Se reporta que la raíz de yacón contiene 3,8%, en base seca, de compuestos fenól icos. 9 8 Se ha aislado de las hojas de yacón el éster metílico del ácido 8-angeloil-1 (1 0), 4,11 (13)-germacratrien-12,6-ólido-14- oico, al que se le ha llamado sonchifolina (10), un nuevo compuesto sesquiterpeno tipo melampól ido. Se aislaron también otros tres melampólidos de estructuras conocidas, que son la polimatina B (11), uvedalina (12) y la enhidrina (13). La polimatina es un derivado acetoxi de sonchifolina y ha sido encontrada antes en Sma/lanthus maculata, mientras que la uvedalina y enhidrina han sido reportadas previamente en Sma/lanthus uveda/ia y Enhydra fluctuans, respectiva- Dentro de los compuestos fenólicos mayoritarios se han encontrado tres nuevos ésteres cafeoil de ácido altrárico, ácido 2,4 ó 3,5-dicafeoilaltrárico (1), ácido 2,5-dicafeoil altrárico (2) y ácido 2,3,5 ó 2,4,5-tricafeoilaltrárico (3) 11 , así como dos ésteres cafeoil derivados de ácido octulosónico que tienen un esqueleto 6,8-dioxabiciclo [3.2.1] octano, siendo uno de ellos un monocafeoil y el otro un dicafeoil éster. Ellos son: el ácido ( 1 R, 25,35 ,4R, 55,7 R)-4-h id roxi-7 -h id roxi me ti 1-3-[3-(3 ,4- dih idroxifeni 1)-1-oxo-2-propeni loxi]-6,8-dioxabiciclo[3.2.1] octan-5-carboxíl ico (4-0-cafeoi 1-2,7 -anh idro-D-g/ icero-b-D- 32 Revista de QUÍMICA Junio 2005 mente.' 4 Ultimamente también se reportó el aislamiento de otros dos nuevos melampólidos denominados como el éster metílico del ácido 8 b-tigloiloximelampolid-14-oico (14) y el éster metílico del ácido 8 b-metacriloiloximelampodic-14-oico (15), y el conocido fluctuanina (16). 15 ratas normales, en ratas con hiperglicemia transitoria y en ratas con diabetes inducida por steptozotocina (STZ). Una decocción de yacón al 10%, administrada por vía oral (8 m U Kg) o intraperitoneal (4 mL!Kg), produjo una disminución significativa de los niveles de glucosa en sangre de ratas normales. MeO OC MeO OC ~ ~ R . 1 . o o o o 10 R1 = + R2=H o ~ 11 R1 = + 13 R = R2=0Ac o o 12 R1 = 0~ R2=0Ac 16 R = + o o 14 R1 = y( R2=H o 15 R1 = ~ R2=H o En un test de tolerancia a la glucosa en ratas normales, una dosis única (8 mL/Kg i.p.) de la decocción de yacón al 10% disminuyó la concentración de la glucosa plasmática. Metabolitos secundarios En contraste, una sola dosis de la decocción de yacón por vía oral o intraperitoneal no produjo efecto alguno sobre los niveles de glucosa plasmática en ratas con diabetes inducida por STZ.' MeO OC Sin embargo, la administración de un té de yacón al 2% ad libitum en vez de agua por 30 días consecutivos produjo un efecto hipoglicémico significativo en ratas con diabetes inducida por STZ. Luego de 30 días de administración del té, las ratas diabéticas mostraron mejores parámetros corporales (glucosa plasmática, insulina plasmática, peso corporal) y renales (peso renal, relación peso renal/peso corporal, aclaramiento de creatinina, albuminuria) en comparación con las ratas diabéticas del grupo control. La administración del té de yacón produjo un aumento de los niveles circulantes de insulina. No se sabe si este incremento es por la estimulación de la síntesis o secreción de la insulina, o por la inhición de la degradación de la misma, o una conjunción de ambos mecanismos. 5 Se ha aislado y determinado que las hojas de yacón y el exudado del tricoma glandular son ricos en el diterpeno ácido ent-kaurenoico (ac. ent-kaur-16-en-19-oico) (17); se han aislado además otros derivados del ácido angeloil kaurénico, como el éster del ácido 15 a-angeloi loxi-ent-kaur-16-en-19- oico (18), el ácido 18-angeloiloxi-ent-kaur-16-en-19-oico (19) y el ácido 15 a-angeloiloxi-16-epoxi-ent-kauren-19-oico (20). 16 Los extractos acuosos y las fracciones acetato de etilo obtenidos de hojas de yacón disminuyen la producción de glucosa (tanto por gluconeogénesis como glucogenolisis) en hepatocitos normales. En células de hepatoma, las fracciones de acetato de etilo tienen un efecto similar al de la insulina, que es el de reducir la expresión de CYP2B y CYP2E mRNA. Estos resultados parecen sugerir que el efecto hipoglicémico de los extractos acuosos de S. sonchifolius (hojas) se debe probablemente a un incremento en la insulina plasmática, pero también a la inhibición de la gluconeogénesis y glicogenólisis hepática. 17•18 Actividad hipoglicemiante Aybar et al. 5 evaluaron el efecto hipoglicemiante del extracto acuoso de las hojas de Smallanthus sonchifolius (yacón) en Actividad antioxidante ©~ R, R, R, ~ 17 H CH3 H 18 'o~ CH3 H o ' CH,~O~ COOH L R 2 COOR3 19 H H 20 Van et al.9 evaluaron la actividad antioxidante in vitro de las raíces de yacón por medio del test de DPPH (1, 1-difenil- 2-picrilhidrazil); el test de desoxiribosa y el test de decoloración del ~-caroteno. Se encontró que el extracto metanój,jco de las raíces de yacón tenía mayor actividad antioxidante en el test 20 junio 2005 Revista de QUÍMICA 33 de DPPH en comparación con los extractos clorofórmicos, de acetato de etilo o acetona. El extracto metanólico fue purificado por medio de cromatografía de permeación en gel y por HPLC preparativa en fase reversa. Se lograron identificar dos compuestos antioxidantes: el ácido clorogénico y el L- triptofano. El ácido clorogénico mostró mayor actividad antioxidante que el L-triptofano en el test de DPPH. Ambos compuestos previenen la decoloración del ~-caroteno. En el test de la desoxiribosa, el L-triptofano es capaz de captar radicales hidroxilo, mientras que el ácido clorogénico en cambio, estimula en cierta medida la producción de radicales hidroxilo mediada por fierro. Por lo tanto, el mecanismo de actividad antioxidante de ambos compuestos parece ser diferente, dependiendo .de la especie radicalaria en estudio. acetilo, mientras que en la pos1C1on 8 es mejor colocar un grupo angeloi loxi en 1 ugar de un epoxiangeloi loxi para aumentar la actividad antifúngica. 14 En un estudio posterior realizado por Lin et al." se obtuvo un compuesto melampólido cuya actividad in vitro contra el hongo P. oryzae es mayor que la hallada para la sonchifolina. Al analizar la estructura de este compuesto, se puede observar que en la posición 8 es mejor la presencia de un grupo metacrilo que un grupo angeloiloxi para mejorar la actividad antifúngica in vitro. Por otro lado, al medir la actividad contra la bacteria Bacillus subtilis se obtuvo que el melampólido más activo fue la fluctuanina (16), seguido de la uvedalina (12) y la enhidrina (13). Todos estos compuestos poseen un grupo acetoxi en la posición 9, el cual parece ser necesario para la presencia de actividad antibacteriana. La actividad de la uvedalina fue algo mayor que el de la enhidrina. La única diferencia entre estos dos compuestos es la presencia de un grupo epóxido en C4/C5, el cual parece ser necesario para la actividad antibacteriana. Actividad antioxidante 15 Valentova et al.' 9 usaron el test de DPPH y el de inbición de la enzima xantina oxidasa para demostrar que las fracciones acetato de etilo, obtenidas a partir de extractos metanólicos de hojas y tubérculos de yacón, poseen actividad antioxidante in vitro. Además, estas fracciones inhiben la lipoperoxidación, inducida por tert-butilhidroperóxido, de membranas microsomales y mitocondriales de células hepáticas de rata. Sin embargo, dichas fracciones pueden ser citotóxicas para hepatocitos de rata a concentraciones mayores de 100 ¡.tg/mL. Al parecer, los compuestos responsables de dichas actividades biológicas son el ácido cafeico y el ácido clorogénico. En el test de DPPH, los valores de IC50 para estos compuestos fueron de 0,86 ± 0,005 y 2,46 ± 0,17 ¡.tg/mL, respectivamente. La actividad inhibitoria de la xantina oxidasa y de inhibición de la lipoperoxidación de membranas mitocondriales se debe principalmente a la presencia del ácido cafeico. Yacón como prebiótico Los fructooligosacáridos (FOS) contenidos en la raíz del yacón pueden comportarse como prebióticos al mejorar el balance de la microflora intestinal y al promover el crecimiento de organismos probióticos. Los prebióticos se definen como ingredientes alimenticios no digeribles que benefician al hospedero estimulando el crecimiento y la actividad de un número limitado de bacterias del intestino y por mejorar la salud del hospedero. Los probióticos se definen como microorganismos vivos añadidos en la dieta que benefician la microflora del colon. Existe un interés en buscar maneras de incrementar el número de estos organismos en el intestino. Una de estas maneras es el uso de FOS. Pedreschi et al-' demostraron que los FOS del yacón son metabolizados por 3 cepas de probióticos conocidos: Lactobacillus acidophilus NRRL-191 O, Lactobacillus plantarum NRRL B-4496 y el Bifidobacterium bifidum A TCC 15696. Actividad antimicrobiana Dado que generalmente no es necesario usar pesticidas durante el cultivo del yacón, es de suponer que las partes aéreas podrían contener compuestos antifúngicos o pesticidas. A partir de un extracto metanólico de las hojas del yacón, lnoue et al. 14 pudieron aislar un melampólido nuevo, al que se llamó sonchifolina (10), y tres ya conocidos: polimatina B (11), uvedalina (12) y enhidrina (13). De los cuatro melampólidos aislados, la sonchifolina (10) posee la mayor actividad antifúngica contra el Pyricularia oryzae, el hongo causante de la enfermedad del arroz. 34 Revisto de QUÍMICA junio 2005 Referencias yacón (Po/ymnia sonchifolia). Resúmenes Curso IX Congreso Internacional de Cultivo Andino. En Andean Roots and Tubers: ahipa, arracacha, maca, yacon. Hermann M. and Heller J. (eds,) lnstitute of Plant Genetic and Crop Plant Research, 1997. p. 222. 1. Soukup, J. Vocabulario de los Nombres Vulgares de la Flora Peruana. Ed. Salesianos, Lima, 1987. p. 379. 2. Brako, L., Zarucchi, J. Catalogue of the Flowering Plants and Cymnosperms of Peru. Missouri Botanical Carden, Missouri 1983. pp. 177-1 78 11. Takenaka, M., Yan, X., Ono, H., Yoshida, M., Nagata, T., Nakanishi, T. }. Agric. Food. Chem. 2003, 51, 793-796. 3. Brack, A. Diccionario Enciclopédico de Plantas Utiles del Perú. PNUD - Centro de Estudios Bartolomé de las Casas, Cusca. 1999. p. 460. 12. Takenaka, M., Ono, H. Tetrahedron Lett. 2003, 44, 999- 1002. 4. Crau, A., Rea, J. Yacón. Sma/lanthus sonchifolius. En Andean Roots and Tubers: Ahipa, arracacha, maca, yacón. Hermann, M. and Heller, J. (Eds.) lnstitute of Plant Genetic and Crop Plant Research. 1997. pp. 199-242. 13. Simonovska, B., Vovk, 1., Andrensek, 5., Valentová, K., Ulrichová, J. }. Chromatog A 2003, 1076, 89-98. 14. lnoue, A., Tamogami, 5., Kato, H., Nakazato, Y., Akiyama, M., Kodama, O., Akatsuka, T., Hashidoko, Y. Phytochemistry 1995, 39, 845-848. S. Aybar, M., Sánchez, A., Grau, A., Sánchez, S. }. Ethnopharmacol. 2001, 74, 125-132. 6. Gota, K., Fukay, K., Hikida, J., Nanjo, F., Hara, Y. Biosci. Biotechnol. Biochem. 1995, 59, 2246-2347. 1 S. Lin, F., Hasegawa, M., Kodama, O. 2003. Biosci. Biotechnol. Biochem., 2003, 67, 2154-2159. 7. Pedreschi, R., Campos, D., Noratto, G., Chirinos, R., Cisneros- Zevallos, L. }. Agric. Food Chem. 2003, 47, 4711-4713. 16. Kakuta, H., Takuhiko, 5., Hashidoko, Y., Mizutani, J. Biosc. Biotechnol. Bioch., 1992, 56, 1562-1564. 1 7. Valentová, K., Moncion, A., de Waziers, 1., Ulrichová, J. Ce// Bio/. Toxico/., 2004, 20, 109-120. 8. Ohyama, T., lto, 0., Yasuyoshi, 5., lkarashi, T., Minamizawa, K., Kubota, M., Tsukihashi, T., Asami, T. Soil Sci. P/ant Nutr. 1990, 36, 167-171. 18. Valentová, K., Ulrichová, J. Biomed. Papers 2003, 147, 1 19- 130. 9. Yan, X., Suzuki, M., Ohnishi, M., Sada, Y., Nakanishi, T., Nagata, T. }. Agric. Food Chem. 1999, 47, 4711-4713. 9. Yan, X., Suzuki, M., Ohnishi, M., Sada, Y., Nakanishi, T., Nagata, T. }. Agric. Food Chem. 1999, 47, 4711-4713. 10. Lizarraga, L., Ortega, R., Vargas, M.,Vidal, A. Cultivo del 19. Valentová, K., Cvak, L., Muck, A., Ulrichová, J., Simanek, V. Eur. }. Nutr. Conclusiones La presencia de un alto porcentaje de oligofructanos (60- 70% sobre base seca) en las raíces del yacón hacen de él un producto de alto interés para la industria alimentaria y de suplementos dietéticos. Del análisis de las estructuras químicas de estos 4 melampólidos versus la actividad biológica se puede deducir que en la posición 9 no es necesaria la presencia de un grupo 34 Revisto de QUÍMICA junio 2005 Por otro lado, los compuestos fenólicos, principalmente los ácidos clorogénico, ferúlico y cafeico y sus derivados explican la actividad antioxidante encontrada en extractos de yacón. agente prebióticos que promueven el crecimiento de organismos probióticos. Actualmente existen en el mercado diversos productos derivados de las raíces del yacón, a los cuales se les atribuye actividad hipoglicemiante o que pueden ser usados para el tratamiento de la diabetes. Sin embargo, es de notar que aún no existen estudios científicos que respalden dichas aseveraciones. Por otro lado, los estudios de actividad hipoglicemiante realizados a las hojas del yacón parecen indicar que el mecanismo de acción respectivo es mediante la inhibición de la gluconeogénesis y glicogenólisis hepá- tica. A los compuestos terpénicos hallados en las hojas, especialmente a los sesquiterpénicos tipo melampólido, se les atribuye la actividad antimicrobiana, mientras que a los diterpenos tipo kaurenos se les reconoce como sustancias que las plantas biosintetizan como mecanismos de defensa. Se han hecho estudios de actividad biológica de extractos de hojas y raíces del yacón. Las hojas poseen actividad hipoglicemiante y antimicrobiana, mientras que las raíces tienen actividad antioxidante in vitro y pueden ser usados como Referencias 2003, 42, 61-66. 10. Lizarraga, L., Ortega, R., Vargas, M.,Vidal, A. Cultivo del Junio 2005 Revisto de QUÍMICA 35
W4312769298.txt
https://juaps.uoanbar.edu.iq/article_175963_3439810e5db156d7406ddaf04ae5a00a.pdf
en
Effect of Microwaves on Some Homo, Co-Polymers and Blended Polymers
Maǧallaẗ ǧāmiʻaẗ al-anbār li-l-ʻulūm al-ṣirfaẗ/Maǧallaẗ ǧāmiʻaẗ al-Anbār li-l-ʻulūm al-ṣirfaẗ
2,017
cc-by
2,443
‫يجهة جايعة االنبار نهعهىو‬ ‫‪Open Access‬‬ ‫انصرفة )‪(JUAPS‬‬ ‫‪P- ISSN 1991-8941 , E-ISSN 2706-6703‬‬ ‫‪2015 ,( 9), ( 2 ) :181-185‬‬ ‫تأثير الموجات المايكروية عمى بعض البوليمرات المشتركة والمختمطة‬ ‫ضاري محمد عبد اهلل‬ ‫احمد كريم محمد‬ ‫جامعة االنبار – كمية العموم‬ ‫الخالصة‪:‬‬ ‫معمومات البحث‪:‬‬ ‫فييه ى ي ا العمييلت ييم امي ثةم ولويية بييوليمراي رليمييية فييه العيينالةت فيييه مم عييةة المي ايرينت م عييةة‬ ‫تاريخ التسليم‪2013/00/00 :‬‬ ‫تاريخ القبول‪2014/5/6 :‬‬ ‫تاريخ النشر‪2012 /05/03 :‬‬ ‫مكموريية اليينيييلوت وم عيةة ميويييل ميويا كيريليو و يم عريشيييا الير فييعة الميكرووييول و ييم امي عمال بييوليمر‬ ‫‪DOI:10.37652/juaps.2017.175963‬‬ ‫م جييانو والبييوليمراي المف ي ركة و البييوليمراي المثموطيية بام ي ثةام العييي اليييةويل و جريييي ة ارميياي ال جةليية‬ ‫بام ثةام فرن الميكروويو ال جاري فه الميكروويو مث مية قيو م‪021‬ت‪281‬ت‪221‬ت‪081‬ت‪W021‬و فيه وقيي‬ ‫انكهًات انًفتاحية‪:‬‬ ‫محةة م‪ 5‬وانيةو لجميع العينايل و م عريض العيناي لإلفيعاع الميكرووييو بطاقية واب ية ميع االوقياي ال اليية‬ ‫‪Microwave,‬‬ ‫م‪1‬ل‪1‬ت ‪5‬ت ‪01‬ت ‪05‬ت ‪21‬و وانيةت و بع أوير االفعة مع انثياض فيه م وميط ازوةان الجةيليية م‪MW‬ت‪Mn‬و‬ ‫‪Degradation,‬‬ ‫‪Polyvinyl Chloride,‬‬ ‫والةياة والنقعان فه قياماي اليحوعاي الميكانيكيةل‬ ‫‪.Blended Polymers‬‬ ‫المقــدمــة‬ ‫لنة مميط فعة الموجاي الةقيقة ‪ Microwave‬لمر المواة البوليمرية‬ ‫م عةة المويل ميواكريليي م‪PMMA‬و الفكل م‪1‬و ال ي ىو لبار لن‬ ‫فإن فعة م‪MW‬و ةثل إلر ةاثل الجةيلةت ف عمل لمر ةياة طاقة‬ ‫ماة لةالنية فيافة لةيمة المون وةرجة ليون يا )‪(Softening Point‬‬ ‫جميماي الماة لمر لكو الطاقاي الح اررية وازفعة ازثرىل حيث‬ ‫كون لمرت وقو عاةميا ومقاوم يا لألحوال البيلية فشل من م عةة‬ ‫عمل لمر ال ةاثل مع جميماي الماة فه المطح الثارجه وم ن قل‬ ‫الم ايرينم‪3‬ول‬ ‫إلر ةاثل ال ركييم‪28‬وت ل لك فإن ال جةلة بالموجاي الةقيقة م‪MW‬و‬ ‫موو كون مريعة جةاً ويحةث ال يكك بفكل مرع من ام ثةام نواع‬ ‫الطاقاي ازثرى فه ى ه الةرامة ام ثةم جياة فرن لمموجاي الةقيقة‬ ‫م‪Oven Microwave‬و و لوحة حكم رقمية ويعمل بطاقاي م عةة ت‬ ‫حيث يعمل مولة الموجاي الةقيقة )‪Magnetron‬و إلن اج فعة‬ ‫الموجاي الةقيقة القابمة لمر النيا إلر ةاثل جمم جةيلة البوليمر‬ ‫و حطيم ازواعر وان اج‬ ‫جةيلاي‬ ‫جةية‬ ‫نا جة لن‬ ‫فكل م‪1‬و معاةلة بممر اليم‪PMMA‬و‬ ‫جةلة‬ ‫البوليمرايم‪1‬ت‪2‬ول‬ ‫وين ج بفكل قشبان وقوالي وعيالح و نابييت ولقة وعل إن اجو‬ ‫و م ام ثةام ولث بوليمراي ىه ‪-:‬‬ ‫‪ -1‬متعدد‬ ‫مثيل‬ ‫ميثا‬ ‫اكرياليت‪2-‬‬ ‫فه لام )‪ (1969‬إلر حواله )‪ (350‬مميون باونةل إن الثاعية الممية‬ ‫‪poly(methyl‬‬ ‫لي ا البوليمر ىه الفيافية البعرية وفقةان المونل ل لك يم ثةم حالياً‬ ‫)‪methacrylate‬‬ ‫فه عنيع لةماي العين لنة ام بةال العةماي ازعمية؛ بمبي غير‬ ‫يعة مونومير المويل ميوااكريليي ماللً لةيم المون لو ةرجة‬ ‫لةمة العين )‪(Cataracts‬ل وبمبي مألم و مع نمجة البفر يم ثةم‬ ‫انعيار)‪ (M.P)(-48°C‬ويغمه )‪ (P.P‬فه )‪ (100°C‬وي حول إلر‬ ‫فه ال جبيرت إ يم عمل ماة العقة مرابطةو ل وبيي العظم المةروع‬ ‫مكان العظم ازعمه الميقوةت وكما يم ثةم فه عنالة طقم ازمنان‬ ‫‪* Corresponding author at: Department of Chemistry ,College‬‬ ‫‪of Science, University of Anbar,Anbar.Iraq.‬‬ ‫‪E-mail address: dean_coll.science@yahoo.com‬‬ ‫)‪ (Dentures‬وفه عنالة المعي )‪(Toys‬ل كما يم ثةم حالياً فه‬ ‫‪181‬‬ ‫‪Open Access‬‬ ‫يجهة جايعة االنبار نهعهىو‬ ‫انصرفة )‪(JUAPS‬‬ ‫‪P- ISSN 1991-8941 , E-ISSN 2706-6703‬‬ ‫‪2015 ,( 9), ( 2 ) :181-185‬‬ ‫عنالة ازقراص الميةرية )‪ (Laser disc‬المم عممة فه عنالة‬ ‫ومنوت فشلً لن م عو باليفافة وةرجة لوينة قل من ‪100oC‬ت إ إنو‬ ‫)‪ (CDs‬و)‪ (DVDs‬وفه عنالة االةواي الموميقية )‪ (guitars‬م‪4‬ول‬ ‫يبة بيقةان وباي فكمو فه حةوة ‪ 82 oC‬مما يحجم من ام ثةامو فه‬ ‫كما ن م عةة مويل ميوااكريليي ‪ PMMA‬ال ي وةنو الجةيله‬ ‫بعض المجاالي ال ه طمي مقاومة العةماي وةرجاي الحرار العاليةل‬ ‫بحةوة م‪150000 - 80000‬و يم عمل فه طمه المواة المعةنيةل‬ ‫إن ثواص م عةة الم ايرين أور بوةنو الجةيلهت فكمما ةاة ى ا الوةن‬ ‫و م يمك عنالة المياراي ممن لةماي و غطية جية الشوء‬ ‫حمني ثواعو الميكانيكيةت ويم ثةم فه مجاالي م عةة ىميا فه‬ ‫وازقراص المةرجةو كور من نعو ‪ PMMA‬المن جت وان عيالحو‬ ‫ال عبلةت وان اج العبواي مث مية االم عمال والعبواي اي االم ثةام‬ ‫)‪ (Sheets‬م ثةم بوعييا ةجاجاً مقاوماً لمكمر فه الفبابيك وازبواي‬ ‫الواحةت وازواثت والبراةاي وبعض ازجية المنةلية والمث برية م‪8‬ول‬ ‫بةالً من الةجاج االل ياةي ولمةيكور فه ازبنية والمنفآيل ما بوليم ار و‬ ‫‪ -3‬متعدد (كموريد الفينايل) ‪Poly vinyl chloride P.V.C‬‬ ‫المف ركة )‪ (Co-Polymers‬ف م عمل بعور وامعة ار نجاي عمة‬ ‫ماة بلم يكية كوير االم عمال وىو من كور المن جاي‬ ‫ح اررياًت إ م عمل فه عنالة المينة )‪ (enamel‬و ال غميو الةجاجهل‬ ‫الومينة لمعنالة الكيمياليةتوم عةة مكمورية اليينايلو من البوليمراي‬ ‫كما و م ثةم بوليمراي م عةة اكريليي وم عةة ميوااكريليي اي الوةن‬ ‫ال ه عنع منيا كمياي كبير جةاً لبر البممر بطريقة الج ور‬ ‫الجةيله المنثيض بفكل وامع كمونوميراي كبير الحجم فه حشير‬ ‫الحرم‪9‬ول لالمياً كور من ‪ %51‬من م عةة مكمورية اليينايلو‬ ‫البوليمراي المف ركة المقولبة مالمجمعةو والمطعمة م‪5‬ول‬ ‫المعنع يم ثةم فه البناء كماة إنفالية ؛ زنو رثيص وميل‬ ‫‪ -2‬متعدد الستايرين (‪Polystyrene ( PS‬‬ ‫ال ركييم‪10‬ول وفه المنواي ازثير ام بةل م عةة مكمورية اليينايلو‬ ‫يعة م عةة الم ايرين من البوليمراي الةجاجية ‪Glassy Polymers‬‬ ‫مكان مواة إنفالية كوير فه العةية من المفاريع لمر الرغم من‬ ‫الميمة عنالياًت إ بة إن اجو عنالياً فه واثر الولوينياي وىو من‬ ‫وجوة مثاوو حول أوير م عةة مكمورية اليينايلو لمر البيلة‬ ‫البوليمراي الرثيعة نمبياًت ويعة من البلم يكياي المطاولة لمحرار ولو‬ ‫والعحة البفريةل إ‬ ‫إن ى ا البوليمير بمبي كونو غير مم قر‬ ‫الكوير من االم ثةاماي البلم يكية كعنالة الحاجياي المنةلية وفه‬ ‫جاه الشوء والحرار ت وى ا يؤةي إلر غيراي وامعة فه البنية؛‬ ‫عنالة البلم ك الممامه ‪ Rigid foam‬العمةل وي م الحعول لمر‬ ‫و لك بمبي إةالة ‪ HCl‬النا ج من أوير الظروو الجويةت وى ا‬ ‫م عةة الم ايرين ببممر الم ايرين بحمي المعاةلة اآل ية‬ ‫م‪6‬و‬ ‫يؤور يشاً لمر ثواعو الييةيالية والكيميالية والميكانيكيةل يحشر‬ ‫فكل م‪2‬ول‬ ‫ميم‬ ‫وم عةة مكمورية اليينايلو من المونيمركمورية اليينيل وىو مركي ٌ‬ ‫جةاً فه العنالة كما فه المعاةلة فكل م‪3‬وم‪11‬ول‬ ‫فكل م‪2‬و معاةلة بممر م عةة الم ايرين‬ ‫فكل م‪3‬و معاةلة بممر م عةة مكمورية اليينايلو‬ ‫و لك ب قنية البممر المعمقة و االم حلبية و البممر بالك مة بوجوة‬ ‫الجـــــــــزء العممي‬ ‫وميط من نوع ةكمير و فوق حة ازكامية لنة ةرجة حرار ‪200-‬‬ ‫م ام ثةام طريقة القولبة اليةوية ‪Hand lay-up molding‬‬ ‫‪ 100oC‬والحعول لمر الحبيباي الم بممر غير المفكمة بكوافة ‪102‬‬ ‫حشير العينايت و مثص بالثطواي اآل ية‪-:‬‬ ‫‪ g/cm3‬يمكن فكيل الحبيباي لنة ةرجة حرار ‪ 180-160oC‬و لك‬ ‫م‪7‬و‬ ‫بام ثةام القولبة بالبوق والحقن وال أليو الحراري والرغوي‬ ‫فه‬ ‫وال‪ -‬قبل البةء بعممية حشير العيناي م نظيو ازلواح المعةنية‬ ‫لإن برة‬ ‫والةجاجية المم ثةمة‬ ‫مواعياي م عةة الم ايرين ىه الفيافيةتوميولة ال موينت والثمول‬ ‫بإةالة المواة العالقة وم غمميا جيةاً بالماء‬ ‫والعابونت وبعةىا وشع فه فرن حراري لمة م‪20-15‬و ةقيقة‬ ‫النمبه جاه الكيمياويايت والعةل الحراري والكيرباله الجيةت ورثص‬ ‫‪182‬‬ ‫‪Open Access‬‬ ‫يجهة جايعة االنبار نهعهىو‬ ‫انصرفة )‪(JUAPS‬‬ ‫بةرجة ‪ °C‬م‪60-50‬و لم جييوت و م لممية ييلة القالي لعي‬ ‫لقة م ام ثةام جياة م ‪ Durometer Hardness‬و نوع‬ ‫العيناي كما فه الفكلم‪1-2‬و وكما فه الثطواي اآل ية‪- :‬‬ ‫‪ -1‬ييلة قالة من الةجاج بأبعاة‪mm3‬م‪10×300×350‬و‬ ‫م ‪Shore D‬و والمعنع من قبل فركة م‪TIME GROUP INC.‬و فه‬ ‫إجراء اث بار العلة بام ثةام ةا غرة نقطيةت حيث ي م غمغل ةا‬ ‫و لك‬ ‫الغرة النقطية ةاثل مطح الماة ت و لك ن يجة الشغط لمر الجياة‬ ‫الم عماليا قالة ماميةل‬ ‫‪-2‬‬ ‫ييلة‬ ‫‪P- ISSN 1991-8941 , E-ISSN 2706-6703‬‬ ‫‪2015 ,( 9), ( 2 ) :181-185‬‬ ‫ليلمو مطح ةا ال غمغلم ‪ Pressure foot‬و ماماً مع مطح العينة‬ ‫فرطة ةجاجية بأبعاة ‪mm3‬م‪10×10×270‬و و‬ ‫‪mm3‬م‪10×10×320‬و الم عماليا جةر ٍ‬ ‫ان لمقالي ماإلطارول‬ ‫تو كرار العممية ولث مراي فه ماكن مث مية من مطح العينة لمرالا‬ ‫العوامل المؤور لمر ةقة القراء مول جانو مطح العينة ونعوم يا‬ ‫‪ -3‬غمو القالة الةجاجية وازفرطة بماة اليابمون الملعق لمنع‬ ‫وثموىا من الفوالي واليجواي في م قراء قيمة العلة ل‬ ‫ال عاق الماة ازماو مع قالة القالي واإلطار ل‬ ‫اختبار متانة االنحناء ‪: Bending Test‬‬ ‫‪ -4‬ام ثةام الميمكون الحراري لمة اليراغاي بين ازفرطة الةجاجية من‬ ‫ىو جياة اث بار ولوه النقاط نوع (‪Ceramic Instruments-‬‬ ‫الثارج والةاثل وك لك ركان القاليل‬ ‫‪ -5‬ييلة لوح ةجاجه ي بعاة ‪mm3‬م‪10×300×350‬و يم ثةم‬ ‫‪ ,) Sassvolo-Itaiy‬حيث وشع العينة ال ه كون بفكل م واةي‬ ‫ل غطية مطح الماة الم راكبة وشغطيا قبل ال عمي لشمان الحعول‬ ‫المم طيلي لمر ممنةين الممافة بينيما واب ة ت وم يممط حمل لمر‬ ‫لمر ال جانو فه ممك الم راكي المن ج لممياًل‬ ‫فكل فه نقطة المن عو لمعينة بفكل م ٍ‬ ‫ماو فيحةث ليا انحناء فه‬ ‫تشعيع العينات بالموجات الدقيقة ( المايكروويف )‬ ‫فكميا حي أوير إجياة فةت واجياة انشغاطت واجياة قص ل ومن‬ ‫م فعيع العيناي بوماطة فرن جاري من نوع‬ ‫ثلل القراء لممقياو الثاص بالقو ومعرفة طول العينة ولرشيا‬ ‫‪ GL‬رقمه و ي‬ ‫وممكيا ي م حماي م انة االنحناءل‬ ‫طاقاي مث مية يمكن ال حكم بيا من ثلل لوحة حكمت و م عريض‬ ‫م إجراء اث بار حماي مقاومة االنشغاط‬ ‫العيناي إلر معةر مبافر لمموجاي الةقيقة وبحمي وقيي ي م شبطو‬ ‫اختبار االنضغاطية‬ ‫بوماطة لوحة ال حكم الثاعة باليرن من حيث الطاقة المم ثةمة‬ ‫بام ثةام المكبو الييةروليكه من نوع م ‪Leybold Horris‬‬ ‫والةمنت و كون الةقة لالية جةاً؛ زن المؤقي رقمه ل‬ ‫‪No.36110‬و لحيث ي م وبيي العينة لمر القالة الم حركة ورفعيا‬ ‫فحوصات الخواص الميكانيكية‪-:‬‬ ‫إلر لمر بوماطة راع الجياة ح ر يعبح مطح العينة فه حالة‬ ‫م إجراء اث بار الفة لمر العيناي المحشر فام عمل‬ ‫ماو مع الطرو العموي لمجياة وم مميط الحمل بعور ةريجية لمر‬ ‫جياة م‪ )JIANQ Test Equipment‬المعنع من فركة م‪Instron‬و‬ ‫العينة اي ازبعاة القيامية ح ر ي م حعول اليفلت إ يمول قعر‬ ‫اإلنجميةيةت ب مميط قو فة ةةاة ةريجياً لحين ففل و كمر العينةت ي م‬ ‫حمل مممط قيمة مقاومة االنشغاط القعوى ليام‪12‬ول‬ ‫اختبار الشد‬ ‫النتائج والمناقشة‪:‬‬ ‫لنةىا إيجاة مقاومة الفة ب قميم قعر حمل حممو الماة لمر مماحة‬ ‫المقطع العرشه وبحمي العلقة ( ‪ F / A‬‬ ‫‪ ) ‬وحةا يا م‪Mpa‬و‬ ‫إن فعيع البوليمراي الم جانمة والمف ركة‬ ‫والمثاليط البوليمرية بأفعة الموجاي الةقيقة م ‪MW‬و وبطاقاي‬ ‫مث ميةم‪120,180,220,280,320 W‬و لنة ةمن وابيم‪5 second‬و‬ ‫إختبار الصدمة ‪: Impact Test‬‬ ‫ولنة طاقة واب ة م‪320 W‬و بةمن مث مو م‪5,10,15,20,25 S‬و‬ ‫م ام ثةام جياة فحص العةمة نوع م ‪( ISOD Impact‬‬ ‫‪ Test‬رقمه م‪Digital‬والمعنع فه فركة )‪ (GAMA‬الينةيةل إ ؤث‬ ‫وب رةة وابي فه كل الحال ين مقةاره م ‪2450Hz‬وت ةي إلر حعول‬ ‫لينة االث بار و وشع فه المكان المثعص ليات ومن وم يرفع البنةول‬ ‫ياللي ةاثل نواع البوليمراي المم ثةمةت و ظيري أوي اًر واشحاً‬ ‫إلر ازلمر ويوبي فه لمر الجياةت بعةىا يعير الجياة ل ولنة‬ ‫لمر ثواص البوليمراي الميكانيكية والييةيالية والطييية ل‬ ‫حرير البنةول يقوم باالعطةام بالعينة وكمرىا وبحركة أرجحية بحيث‬ ‫لقة جري اث بار الفة والعةمة والعلة وم انة االنحناء ومقاومة‬ ‫حول الطاقة الكامنة فه البنةول إلر طاقة حركية ييقة جةءاً منيا فه‬ ‫من ثلل القياماي‬ ‫االنشغاطية لمعيناي المم ثةمة فه الةرامةت‬ ‫المم حعمة من االجية المثععة لكل قياو‬ ‫كمر العينةت ولنةىا ي م قراء طاقة الكمر ل‬ ‫إختبار الصالدة‬ ‫‪.‬لوحظ من ثلل‬ ‫الفكل ( ‪ ) 4‬والجةول م‪2‬و ن إجياة الفة لنة نقطة الكمر يقل لنة‬ ‫‪Hardness Test‬‬ ‫ةياة طاقة اإلفعاع لمموجاي الةقيقة م‪MW‬و تإ لوحظ فه البوليمراي‬ ‫‪183‬‬ P- ISSN 1991-8941 , E-ISSN 2706-6703 2015 ,( 9), ( 2 ) :181-185 45.5 44.2 42.9 40.5 39.3 39 Poly blend (PS+PMMA) 41.7 40.5 39.9 39 38.1 37.4 43 42.1 41 39.4 38.1 37.5 Poly blend (PVC+PMMA) 40.7 39.8 38.6 37.9 36 35.5 (JUAPS) ‫انصرفة‬ ‫يجهة جايعة االنبار نهعهىو‬ Open Access ‫الم جانمة المم ثةمة فه البحثت ن ةياة طاقة اإلفعاع يقمل من‬ 0w 120w 180w 220w 280w 320w ‫إجياة الفة لن نقطة الكمرت فشلً لن لك فقة لوحظ يشاً ن معةل‬ ‫إجياة الفة فه البوليمراي المف ركة يقل ولكن بنمبة اقل مما ىو لميو‬ ‫فه البوليمراي الم جانمة المكونة لوت حيث عانه المواة البوليمرية من‬ MW ‫وام طال يا من ةون‬ 0w 120w 180w 220w 280w 320w ‫فوه مرن نا ج لن فة الململ البوليمرية‬ ‫حعول كمر فه ازواعرل ولة الفقوق ةاثل الماة البوليمرية ف نمو‬ ‫و جمع مع ةياة اإلجياة مكونة فقوقاً كبر حجماًت و م مر بالنمو مع‬ ‫اإلجياة المممط ح ر يحةث اليفل فه العينةل ما فه حالو البوليمراي‬ ‫المثموطة فمعةل اإلجياة كان قريباً جةاً من معةل إجياة البوليمراي‬ ‫الم جانمة المكونة لول ومما قةم نم ن ج ن البوليمر المف رك يكون‬ ‫المصادر‬ ‫ازمول للم ثةام فه المن جاي ال ه ع مة لمر البوليمراي الم جانمة‬ [1] REFER1. Zhu HM, Jiang XG, Yan JH, Chi Y, Cen KF, Appl Pyrolysis, 82, 1-9, (2008 (. [2] Savrik SA, Erdogan BC, Balkose D, Ulku . J .Appl. Polym Sci, 116, 1811-1822, (2010). [3] Prachaya warakorn J, Khamsri J, Chaochan chaikul K, Sombat sompop N, J Appl.Polym Sci, 102, 598606, (2006). [4]Mojtaba Saeedi ، Ismaeil Ghasemi ،and Mohammad Karrabi ،Iranian Polymer Journal،20 (5),، 423-432, (2011)[5 ] Saburo Moriwaki،Motoi Machida،Hideki Tatsumoto، Yasufumi Otsubo, Applied Thermal Engineering 26،745 – 750 Elsevier Ltd. (2006) [6] Makiko Doi،Kazuhiko Fukatsu، Ryota Shinohara،Masataka Hai kibutu gakkaisi,15، 294 301. (2004) [7] Seung Hwan Lee, Won Gu Lee, Bong Geun Chung ،Ali Khadem hosseiniMacromol .Rapid Commun، 30,1382–1386، (2009) [ 8]Z. Zhao, Z. Li،Q .Xia،H. Xi،Y .Lin، Eur .Poly. J . 44، 1217. (2008) . [9] C. A . Puger، R .L . Carrier, B. Sun, K. S. Ziemer, D. D. Burkey, Macromol. Rapid Commun, 30, 126. (2009). [10] C. Oliver Kappe · Doris Dallinger،Mol Divers 13:71–193،(2009) [11]36. Ondruschka B, Bonrath W Microwaveassisted chemistry astock taking.Chimia 60:326– 329, (2006). [12] Polshettiwar V, Varma RS Aqueous microwave chemistry ،Chem .SocRev37:1546– 1557 (2008) [13] Coquerel Y, Rodriguez J، Microwave-assisted olefin Metathesis .Eur. J. Org. Chem 1125–1132 (2008) . ‫المم ثةمة فه ى ه الةرامةت ي ن البوليمر المف رك لطر مقاومة‬ ‫كبر من البوليمراي‬ ‫كبر لإلفعاع وم المثاليط وال ه كاني‬ ‫ول‬13‫الم جانمةم‬ ‫) قيى اختبار إجهاد انشذ قبم وبعذ انتشعيع (انًتجانسة و‬4( ‫شكم‬ )‫انًشتركة و انًخهىطة‬ ‫) قيى اختبار انشذ‬2( ‫جذول‬ PMMA 42.5 41.1 40.2 39.1 38.4 38.2 Poly (S-CoMMA) 184 PVC 40.3 39 38.5 34 29 28.5 Poly (VC-COMMA) PS 39 37.4 35 32 30.3 30 MW 0w 120w 180w 220w 280w 320w MW P- ISSN 1991-8941 , E-ISSN 2706-6703 2015 ,( 9), ( 2 ) :181-185 (JUAPS) ‫انصرفة‬ ‫يجهة جايعة االنبار نهعهىو‬ Open Access Effect of Microwaves on Some Homo, Co-Polymers and Blended Polymers.. Dhari M. Abdulla & Ahmed k. Mohammed Department of Chemistry ,College of Science, University of Anbar ABSTRACT In this work, the used three main polymers in industry, they are (Poly styrene, Poly vinyl chloride, and Poly methyl metha acrylate) studied under the microwave radiation. The Homo Polymers and Co-Polymers and Blended Polymers using a hand molding casting. Degradation studies were carried out using commercial microwave oven at Different Microwaves power of (120,180,220,280,320W) At a fixed time (5 sec) for all experimental. The specimens were exposed to microwave radiation with multiple time (0.0, 5, 10, 15, 20) second, A degradation were followed via the decreases in average of molecular weights (Mw', Mn) and Increase and decrease of the measurements of the mechanical properties of which have been tested. 181
https://openalex.org/W2126504710
https://europepmc.org/articles/pmc3705267?pdf=render
English
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A Current Overview of Two Viroids That Infect Chrysanthemums: Chrysanthemum stunt viroid and Chrysanthemum chlorotic mottle viroid
Viruses
2,013
cc-by
7,333
Keywords: chrysanthemum; Chrysanthemum stunt viroid (CSVd); Chrysanthemum chlorotic mottle viroid (CChMVd); host-viroid interaction; viroid Viruses 2013, 5, 1099-1113; doi:10.3390/v5041099 Viruses 2013, 5, 1099-1113; doi:10.3390/v5041099 viruses ISSN 1999-4915 www.mdpi.com/journal/viruses OPEN ACCESS viruses ISSN 1999-4915 www.mdpi.com/journal/viruses OPEN ACCESS viruses ISSN 1999-4915 www.mdpi.com/journal/viruses OPEN ACCESS Won Kyong Cho, Yeonhwa Jo, Kyoung-Min Jo and Kook-Hyung Kim * Department of Agricultural Biotechnology, Plant Genomics and Breeding Institute, Institute for Agriculture and Life Sciences, College of Agriculture and Life Sciences, Seoul National University, Seoul 151-921, Korea; E-Mails: wonkyong@gmail.com (W.K.C.); yeonhwajo@gmail.com (Y.J.); jokyoungmin@gmail.com (K.-M.J.) * Author to whom correspondence should be addressed; E-Mail: kookkim@snu.ac.kr; Tel.: +82-2-880-4677; Fax: +82-2-873-2317. Received: 1 March 2013; in revised form: 8 April 2013 / Accepted: 8 April 2013 / Published: 17 April 2013 Received: 1 March 2013; in revised form: 8 April 2013 / Accepted: 8 April 2013 / Published: 17 April 2013 Abstract: The chrysanthemum (Dendranthema X grandiflorum) belongs to the family Asteraceae and it is one of the most popular flowers in the world. Viroids are the smallest known plant pathogens. They consist of a circular, single-stranded RNA, which does not encode a protein. Chrysanthemums are a common host for two different viroids, the Chrysanthemum stunt viroid (CSVd) and the Chrysanthemum chlorotic mottle viroid (CChMVd). These viroids are quite different from each other in structure and function. Here, we reviewed research associated with CSVd and CChMVd that covered disease symptoms, identification, host range, nucleotide sequences, phylogenetic relationships, structures, replication mechanisms, symptom determinants, detection methods, viroid elimination, and development of viroid resistant chrysanthemums, among other studies. We propose that the chrysanthemum and these two viroids represent convenient genetic resources for host–viroid interaction studies. Keywords: chrysanthemum; Chrysanthemum stunt viroid (CSVd); Chrysanthemum chlorotic mottle viroid (CChMVd); host-viroid interaction; viroid Viruses 2013, 5 Viruses 2013, 5 1100 1. Introduction The chrysanthemum (Dendranthema X grandiflorum) is a member of the family Asteraceae, and it is one of the popular flowers in the world. The international market for cut and potted chrysanthemums is increasing, and chrysanthemums in many European and Asian countries are commercially very important for the floral industry [1]. Several pathogens, including viruses, viroids, and phytoplasma, cause serious diseases in chrysanthemums. To date, nine viruses and two viroids are known to infect chrysanthemums [2]. Viroids are the smallest known plant pathogens. They consist of a circular, single-stranded RNA, which does not encode a protein. Viroid RNAs range from 246 to 401 bases [3,4]. Viroids traffic from cell to cell via plasmodesmata [5]. To date, over 30 species of viroids have been reported; these can be divided into two families, the Pospiviroidae and the Avsunviroidae [6]. The family Pospiviroidae includes five genera, such as Apscaviroid, Cocadviroid, Coleviroid, Hostuviroid, and Pospiviroid. So far, ten species including Chrysanthemum stunt viroid (CSVd) and Potato spindle tuber viroid (PSTVd) are members of the genus Pospiviroid [7]. The genomic RNA of members of the family Pospiviroidae, which replicate in the nucleus, assumes rod-like or quasi-rod-like conformation in which, based on local sequence similarity, five domains have been proposed: the left terminal, pathogenicity, central, variable, and right-terminal domains [8]. Moreover, several conserved regions have been identified in the rod-like conformation, including the central conserved region (CCR), likely involved in replication [9–12], and the terminal conserved region (TCR) or the terminal conserved hairpin (TCH), which appear mutually exclusive [13–15]. The family Avsunviroidae is composed of three genera, such as Avsunviroid, Elaviroid, and Pelamoviroid. Peach latent mosaic viroid (PLMVd) and Chrysanthemum chlorotic mottle viroid (CChMVd) are members of the genus Pelamoviroid. They have highly branched structures with self-cleaving ribozymes, which are not present in the family Pospiviroidae. These viroids replicate in the host chloroplast [16]. Interestingly, chrysanthemums are a common host for two different viroids, the CSVd and the CChMVd, which are quite different from each other in structure and function (Table 1). Here, we summarize and discuss current research associated with CSVd and CChMVd, and we suggest that chrysanthemums and viroids would serve as convenient genetic resources for host-viroid interaction studies. 2. Symptoms, Isolation, Nucleotide Sequences, and Structures Chrysanthemum stunt disease was reported in the early 1950s [17]. It causes light green young leaves, stunting, small leaves and flowers, and reduced rooting ability. Later, infectious material was first isolated from leaves of stunted chrysanthemum, after nucleic acid extraction, centrifugation in sucrose density gradient and electrophoresis in polyacrylamide gels demonstrated the presence of a low molecular weight RNA, which was different from the known PSTVd [17]. This novel viroid, associated with stunt disease in chrysanthemums, was distinct from other known viruses, and it was named CSVd [18]. The complete nucleotide sequence and secondary structure of CSVd was first determined in Australia in 1981 [19]. The identified CSVd comprised 356 bases and displayed about 69% sequence identity to PSTVd. Subsequently, another CSVd with 354 bases was sequenced [20]. Viruses 2013, 5 Viruses 2013, 5 1101 Table 1. Characteristics of CSVd and CChMVd viroids. Table 1. Characteristics of CSVd and CChMVd viroids. Characteristics CSVd CChMVd Disease Chrysanthemum stunt Chrysanthemum chlorotic mottle Symptoms Light green young leaves, chlorotic spots, stunting, small leaves and flowers, and decreased rooting ability Yellow-green mottling, chlorosis, and dwarfed size Family and genus Pospiviroidae, Pospiviroid Avsunviroidae, Pelamoviroid Genome size 354–356 nt 398–401 nt Replication method Asymmetric rolling circle mechanism Symmetric rolling circle mechanism with the hammerhead ribozymes Replication localization Nucleus Chloroplast Structure Rod-like structure including central conserved region (CCR) Branched conformation including hammerhead ribozymes Transmission Sap, grafting, and seed Sap, grafting Host Chrysanthemums, Petunia hybrida, tomato, Gynura aurantiaca, Ageratum, dahlia, Senecio, Vinca major, Argyranthemum frutescens and many plants belonging to the families Solanaceae and Asteraceae Restricted to chrysanthemums The structures and structural transitions of various viroids, including CSVd, have been determined with thermodynamic, kinetic, and hydrodynamic methods (Figure 1a) [21]. Recently, the sequences of three different CSVd isolates were determined from the US, China, and Australia. Both the US and Australian isolates were composed by several sequence variants, confirming the quasi-species nature of the viroid, but the Chinese isolate consisted of a single variant indicating a low molecular variability for CSVd [22]. Comparative analyses of the nucleotide sequences of 117 CSVd sequence variants revealed nucleotide variations at 103 sites scattered throughout the CSVd genome. However, it is not known whether these nucleotide changes are related to the species specificity of CSVd infections [23]; in addition, possible relationships between nucleotide changes and variant-specific pathogenicity have been recently questioned [23]. 2. Symptoms, Isolation, Nucleotide Sequences, and Structures Chrysanthemum chlorotic mottle disease was first reported in 1967 in the cultivar “Yellow Delaware” in New York State [24]. This disease could be transmitted by grafting one chrysanthemum to another chrysanthemum cultivar. It causes yellow-green mottling, chlorosis, and dwarf symptoms, but some infected cultivars were asymptomatic [25]. Due to the low abundance of CChMVd in infected plants, compared to the abundance of CSVd, the complete, 398–401 nt sequence of CChMVd was only identified in 1997 (Figure 1b) [26]. Transcripts of CChMVd in vitro were also infectious and caused chlorotic mottle disease [26]. CChMVd can form hammerhead structures in both plus and minus strands, which self-cleave during in vitro and in vivo transcription (Figure 1c) [26]. The hammerhead structures of CChMVd display unique features, including an unpaired A residue after the conserved A9 residue in the plus self-cleaving domain, and an unusually long helix II in the minus one (Figure 1c). The predicted secondary structure of CChMVd is a very stable, branched conformation, which is similar to that of PLMVd. In contrast to viroids with a quasi-rod-like conformation, both Viruses 2013, 5 1102 CChMVd and PLMVd were insoluble in 2 M LiCl [26]. In one recent study, an in vitro transcript of CChMVd was inoculated to measure the mutation rate of CChMVd, which resulted in being the highest one ever reported in any biological entity [27]. CChMVd and PLMVd were insoluble in 2 M LiCl [26]. In one recent study, an in vitro transcript of CChMVd was inoculated to measure the mutation rate of CChMVd, which resulted in being the highest one ever reported in any biological entity [27]. Figure 1. Predicted secondary structures of CSVd and CChMVd. (A) Predicted secondary structure of CSVd was adapted with permission from [22]. The central conserved region (CCR) is indicated by light-red shading and the terminal conserved region is indicated by light-blue shading; (B) Predicted secondary structure of CChMVd was adapted with permission from [28]. Plus and minus self-cleavage domains are delimited by flags, residues conserved in most natural hammerhead structures are boxed, and the self-cleavage sites are indicated by arrows. Light-blue shading and light-red shading in flags, boxes, and arrows refer to plus and minus polarities, respectively. The changes in the tetraloop delimited by positions 82–85 (UUUC to GAAA) that convert a symptomatic variant into non-symptomatic are shown with green-colored boxes [29]. The light-yellow square demarcates the domain that alternatively can form a kissing-loop interaction [30]; (C) Hammerhead structures of the plus and minus strands of CChMVd were adapted with permission from [28]. Residues conserved in most natural hammerhead structures are on light-blue shading and light-red shading in the plus and minus polarities, respectively, and the self-cleavage sites are indicated by arrows. Blue colored A indicates the position of the extra A in the CChMVd secondary structure and in its plus hammerhead structure. Numbering is done based on the previous study [28]. Viruses 2013, 5 Viruses 2013, 5 1103 3. Host Range Due to the limited number of studies on viroids, it was thought that CSVd only infected chrysanthemums [31]. Initial reports showed that chrysanthemum plants could serve as host for both CSVd and CChMVd. Moreover, circular and linear forms of CSVd, purified from infected chrysanthemum plants, were infectious when inoculated into Gynura aurantiaca plants [32]. Also, a linear CSVd RNA, synthesized by in vitro transcription, could infect chrysanthemum plants and other plants that belong to the families Solanaceae and Asteraceae, without causing any disease symptoms [33]. Under natural conditions, infections of CSVd have been reported in Petunia hybrida [34], Ageratum, Dahlia [35], and Senecio (cineraria) [36]. CSVd was also identified in Vinca major with reverse transcriptase (RT)-PCR [37]. An infection of CSVd, identified with RT-PCR, was also reported in asymptomatic Argyranthemum frutescens (marguerite daisy) [38,39]. In contrast to the quite wide range identified for CSVd, the known host range of CChMVd is very restricted. For example, of 51 species and cultivars tested, CChMVd was infectious in only two chrysanthemum species [40]. In general, CSVd and CChMVd are mechanically transmitted through tools, like knives and scissors, used on infected chrysanthemums and then on healthy material for grafting and flower cutting. A previous study also demonstrated seed-borne transmission of CSVd in chrysanthemums; in that case, the ambient temperature during a cross could influence the rate of CSVd transmission to progeny [41]. However, CSVd could not be transmitted from infested soil [42], and no insect vector has been reported to transmit it. Viruses 2013, 5 1104 In Akita Prefecture, Japan, CChMVd infections in chrysanthemums caused distinct yellow leaf mottling and necrosis. These symptoms were caused by CChMVd variants containing a UUUC tetraloop (Figure 1b) [50], a structural domain already identified as a specific CChMVd pathogenic determinant [26]. A previous study had detected CChMVd infections in various chrysanthemums grown for three years in Akita Prefecture, revealing that 20% of chrysanthemums were infected with CChMVd [51]. In natural conditions, chrysanthemums that were only infected with CChMVd variants with the UUUC tetraloop did not show any noticeable disease symptoms [51]. In this case, light and temperature may interfere with symptoms induced by variants containing the symptomatic domain. Furthermore, in Kyoto, CChMVd was also detected in cut chrysanthemums from Japan and the Netherlands; this indicated that CChMVd infections have spread over a wide range in Japan [52]. In Korea, CSVd was first identified in chrysanthemum cv. Chunkwang in 2001. The complete sequences for two isolates (K1 and K2) were determined [53]. Later, a total of 64 commercial chrysanthemum cultivars in Korea were tested for CSVd infections, and the infection rate of each cultivar ranged from 9.7% to 66.8% [54]. Furthermore, the complete sequence was reported for the CChMVd-SSHA6 viroid from Korea; this variant caused yellow spots and growth reduction in some infected cultivars [54]. In China, CChMVd was first reported in 2008. In 2010, 13 samples with mild chlorotic spots were collected, and all were infected with CChMVd [55]. In India, chrysanthemum cultivars were screened for CSVd infections with RT-PCR and DNA-RNA hybridization: 70% of cultivars were infected with CSVd [36]. 4. Identification of the Two Viroids Many large-scale screening studies have been performed to detect CSVd and CChMVd. For instance, a total of 2,480 chrysanthemum samples collected from six Australian states were tested for viroid and virus infections with enzyme-linked, immunosorbent assays (ELISAs) and cDNA probes [43]. As a result, CSVd has been found in mixed infection with other viruses [43]. CSVd is one of several quarantined pathogens in European countries, under order of the European Union’s Plant Health Directive (2000/29/EC) [44]. The European Food Safety Authority (EFSA) has released a Scientific Opinion on the assessment of the risk of solanaceous pospiviroids for the EU territory and the identification and evaluation of risk management options [45]. In Italy, to produce healthy chrysanthemum plants, phytosanitary measures, like RT-PCR and hybridization assays, were carried out to test for CSVd infections in 39 seasonal and 93 year-round chrysanthemums [1]. In Slovenia, 12 out of 200 chrysanthemum plants were found to be infected with CSVd [46]. Recently, a study reported the first identification of CSVd in Turkey; where two infected plants out of 154 chrysanthemums tested positive in an RT-PCR assay [47]. Moreover, CSVd was identified by RT-PCR in infected chrysanthemum plants in Egypt [48]. In Asian countries, CSVd and CChMVd have been reported in Japan, Korea, China, and India. Cultivated and wild chrysanthemums were tested for CSVd infections in Japan with RT-PCR [49]. Out of 89 samples, 80 samples were infected with CSVd. In addition, eight wild chrysanthemum species, which did not exhibit any CSVd symptoms, carried CSVd. Among 21 CSVd isolates, a total of five variants were revealed; these variants showed frequent mutations. Viruses 2013, 5 1105 Viruses 2013, 5 that a kissing-loop interaction is important for in vitro folding and viroid infectivity (Figure 1b) [61]. For instance, the introduction of a single mutation in the kissing loops led to low or no infectivity, but an introduction of second mutation restoring the kissing-loop interaction resulted in an infectious agent [61]. These results indicated that the loop-loop tertiary interactions are important for viroid infectivity and for the folding and catalytic activity of most natural hammerheads [30]. 6. Symptom Determinants Of the two viroids discussed here, CSVd is the more serious pathogen. It leads to reductions in the quality and quantity of chrysanthemum production. However, the sequence regions that confer the different symptoms are currently unknown. In contrast, the functional determinants for pathogenicity have been mapped in CChMVd. A previous study identified a nonpathogenic CChMVd strain that could protect against challenge inoculations with a pathogenic strain. Sequence analysis and site-directed mutagenesis have revealed that a substitution from UUUC to GAAA in the pathogenic CChMVd strain led to a nonpathogenic viroid with normal replication ability (Figure 1b). The identified pathogenicity determinant was located in a tetraloop of the predicted, branched conformation of CChMVd [62]. Later, the tetraloop of CChMVd, was further investigated with site-directed mutagenesis, bioassay, and analyses of the progenies [29]. The authors found that substitution of the tetraloop with a triloop or a pentaloop did not affect the infectivity of CChMVd. However, the thermodynamically stable GAAA tetraloop of the nonpathogenic CChMVd strain could not be replaced with other stable tetraloops of the UNCG family without causing functional changes. These data indicate that the sequence was the major factor that preserved the functional tetraloop motif, rather than the structure. After the introduction of a site-directed modified CChMVd variant into chrysanthemum, this nonpathogenic variant eventually evolved into a pathogenic CChMVd one, which acquired the UUUC tetraloop characteristics. This study provided evidence of the stronger biological fitness of the pathogenic CChMVd with the UUUC tetraloop, compared to that of the nonpathogenic CChMVd with the GAAA tetraloop. 5. Replication Mechanisms Viroids replicate in vivo via a rolling circle mechanism, which requires a specific cleavage procedure that converts multimeric viroid copies into monomeric forms [56,57]. Viroids, including CSVd, in the family Pospiviroidae replicate in the nucleus via an asymmetric rolling circle mechanism [58]. In contrast, CChMVd and members of the family Avsunviroidae replicate via a symmetric rolling circle mechanism in the chloroplast, mediated by the hammerhead ribozyme. The hammerhead ribozyme in the family Avsunviroidae determines self-cleavage of oligomeric RNAs during viroid replication; replication is further catalyzed by RNA polymerase and RNA ligase [59]. The hammerhead ribozyme of CChMVd has been intensively studied. The plus hammerhead ribozyme of CChMVd contains an additional A (A10) residue between the conserved A9 and the quasi-conserved G10.1 residues (Figure 1c) [28]. The additional A10 residue causes a moderate decrease in the trans-cleaving rate, while substitution of A10 to C and A10 to G causes major detrimental effects (Figure 1c). In contrast, the A10 to U substitution increased the trans-cleaving rate by 3–4-fold. Some hammerheads display deviations from the consensus sequence because certain residues might be involved in unknown important functions, other than self-cleavage [28]. In addition, high-pressure experiments identified two different types of CChMVd hammerhead conformation that exhibit fast-cleaving and slow cleaving activities [60]. Interactions between loop 1 and loop 2 of CChMVd hammerheads in both polarity strands (and in most natural hammerheads) have been shown to play major roles in self-clearing activity of these ribozymes [30]. Moreover, site-directed mutagenesis, bioassays, and progeny analysis studies showed 8. Efforts to Eliminate Viroids from Infected Plants Several attempts have been made to generate viroid-free plants. First, a long-term heat treatment at 35 °C for 14–37 weeks was proposed for treating CSVd-infected plants [77]. In contrast, a cold treatment was shown to be effective for eliminating CSVd in infected chrysanthemum plants [78]. Those authors grew CSVd-infected chrysanthemum plants at 5 °C for six months; subsequently, the meristem tips of cold-treated chrysanthemums were used to generate non-infected chrysanthemum plants [78]. Another study found that promoting vegetative propagation during a low-temperature period could eliminate CSVd from the infected chrysanthemums [42]. However, it was also shown that, after reducing the level of CSVd RNA in the plant during a low-temperature period, CSVd replication could again increase when the infected plants were grown at normal or high temperatures [79]. Recently, a Japanese research group established a new approach for obtaining CSVd-free chrysanthemums with leaf primordium-free, shoot apical meristems (LP-free SAMs). The attachment of LP-free SAMs to the root tips of CSVd-free chrysanthemums or cabbage resulted in the production of 14% and 3% CSVd-free plants, respectively [80]. A previous study generated CSVd-free and CSVd-infected plants by culturing different-sized SAMs dissected from the “Piato” plant; the authors reported that CSVd-infected plants flowered, even in the long-day condition [81]. These data suggested that CSVd might induce autonomous flowering in chrysanthemum, which is known to be a qualitative, short-day flowering plant [81]. In the same way, this research group has regenerated CChMVd-free chrysanthemums [52]. To date, the elimination of viroids from infected plants has remained a challenge. Viruses 2013, 5 1106 viroids, including CSVd, belonging to four genera [73]. In addition, a multiplex, direct RT-PCR method was developed to detect CSVd and CChMVd in a small amount of plant tissues [74,75]. Furthermore, a previous study developed real-time PCR (TaqMan®) assays for detecting six viroids, including CSVd, infecting solanaceous hosts [44]. Recently, a multiplex RT-PCR primer set was developed to detect viruses and viroids, including Chrysanthemum virus B, Tomato aspermy virus, CSVd, and CChMVd in chrysanthemums [76]. These assays might be useful for detecting different viroids infecting the same host simultaneously. These methods could be applied to quarantine, certification, and screening purposes. 7. Detection Methods Due to the fact that viroids have very low molecular weights, polyacrylamide gel electrophoresis (PAGE) was first used to detect viroid RNAs [63,64]. Molecular method to detect and quantify CSVd RNA in infected plants based on hybridization assays, where the viroid RNA hybridized to a 32P-labeled cDNA of CSVd, were also developed [65,66]. The 32P-labelled cDNA probe that targeted PSTVd was able to detect both PSTVd and CSVd [67]. In addition, several nonradioactive probes were developed, like photobiotin-labeled DNA probes, [68,69]. Both 32P-labeled and biotin-labeled cDNA probes were used and compared for the detection of CSVd and PSTVd [69]. The most sensitive method for detecting viroids is probably the RT-PCR-based approach. A previous study developed a simple and rapid method for nucleic acid extraction, without tissue homogenization, for detecting CSVd [70]. Typically, two different methods have been applied for detecting CSVd, either a combination of RT-PCR and hybridization, or a combination of RT-PCR and ELISA [39,70]. To identify several viroids or a novel viroid in a single host, several RT-PCR primer sets or digoxigenin-labeled polyprobes have been developed based on consensus sequences identified in all pospiviroids [71,72]. Also, a one-tube/one-step RT-PCR method was developed to detect seven Viruses 2013, 5 10. Other Studies Associated with CSVd and CChMVd Cross-protection can be defined as the interference in symptom expression generated by a previously inoculated viroid against subsequent infection by other strains of the same or a closely related viroid [87]. Five different viroids have been used for cross-protection studies in tomato and chrysanthemum plants [87]. Each viroid displayed different symptoms in chrysanthemum plants and, interestingly, three of them, CSVd, a mild PSTVd strain, and a severe PSTVd strain, could protect against Citrus exocortis viroid (CEVd); in contrast, CChMVd could not protect against the severe PSTV strain, CEVd, or CSVd, even though the challenge-inoculated viroid had replicated. Thus, viroid-infected plants showing attenuated symptoms might induce cross-protection against other viroids. Moreover it has been shown that non-symptomatic strain of CChMVd induces cross-protection against the symptomatic strain of the same viroid species [62]. Virus and viroid infections generate small interfering RNAs that interact with the RNA-induced silencing complex (RISC) of the host. A previous study demonstrated the presence of small RNAs (22 nt) derived from CSVd by northern blot analysis accumulating in similar amounts in different plants, regardless of differences in symptoms [88]. In addition, small interfering RNAs may be involved in the inhibition of enzyme activity; for example, the stunted phenotype of CSVd-infected plants might be associated with decreased levels of the plant growth hormone gibberelic acid (GA), due to disturbed GA 20-oxidase activity [89]. Viruses 2013, 5 1107 were produced. Among the F1 progeny, 14 were not infected with CSVd, thus indicating that CSVd resistance was achieved in the first hybrid generation [82]. CSVd was not present in the SAMs or LPs of resistant chrysanthemum cultivars after grafting. Taking the absence of CSVd in the SAMs and LPs as a marker, 20 cultivars were identified as CSVd resistant out of 85 commercial cultivars tested; however, later grafting experiments showed that CSVd was detected in two of these candidate resistant cultivars [83]. Another previous study examined six cultivars, and identified a cultivar named “Utage,” which exhibited a reduced level of CSVd replication. A total of 67 cultivars, obtained from the self-pollination of “Utage,” were tested for CSVd resistance by RT-PCR. Of those, three plants (C7, A30, and A27) were found to have strong CSVd resistance [84]. Transgenic chrysanthemum lines were generated where a specific double-stranded RNA ribonuclease gene (pac1) of Schizosaccharomyces pombe was introduced with Agrobacterium-mediated transformation [85,86]. The generated transgenic plant, which expressed pac1, exhibited reduced levels of viroid infection and accumulation [85]. In addition, these plants were resistant to Tomato spotted wilt virus (TSWV). When hybrid lines were crossed with the transgenic line or with a wild chrysanthemum species, the progeny displayed either resistance or high susceptibility to TSWV infections. This study demonstrated the utility of pac1 for achieving both viroid and virus resistance. 9. Identification and Development of Viroid-Resistant Chrysanthemum Cultivars Most cultivated chrysanthemums are known to be infected with CSVd, which even if asymptomatic, could transfer CSVd to other susceptible cultivars in fields. Controlling CSVd with chemical treatments and culturing approaches is currently a challenging endeavor. Therefore, the ideal would be to find chrysanthemum cultivars resistant to CSVd, for developing new CSVd-resistant chrysanthemum lines. In a recent study, 35 chrysanthemum lines, including commercial cultivars, wild species, and interspecific hybrids, were screened to identify resistant plants by exposing uninfected plants to CSVd-infected plants with a grafting system [82]. RT-PCR was routinely performed to check for CSVd infections in the upper leaves of the scion every month after grafting. Of the 35 lines, the “Okayamaheiwa” cultivar showed strong resistance against CSVd. After crossing the “Okayamaheiwa” cultivar with two other susceptible cultivars, “Sei-elza” and “Anri,” 76 and 8 F1 progeny individuals Viruses 2013, 5 Viruses 2013, 5 1108 chrysanthemum interactions with CSVd and CChMVd have several advantages. First, the two viroids are quite different from each other in replication mechanisms and structure. They belong to two different viroid families, but they do have a common host: chrysanthemums. Second, several protein- RNA interaction techniques will lead to the identification of chrysanthemum proteins that might function for both viroids, while others might be specific for either CSVd or CChMVd. In addition, next-generation sequencing-based approaches might provide information on the chrysanthemum transcriptome and how it is regulated by the two different viroids. Chrysanthemum plants are easily transformed, and creating transgenic plants is not difficult. However, there are several obstacles to using the chrysanthemum as a host, including the finding of a viroid-free, diploid chrysanthemum, because most chrysanthemums are infected with viroids and many are polyploid. Currently, our research group is working to establish a model system for host-viroid interactions with transcriptomic approaches. We anticipate that host transcript RNA profiling studies will reveal several host candidate genes involved in chrysanthemum-viroid interactions. Characterization of host gene functions will provide important information in understanding viroid disease processes, which may facilitate the development of new methods for their control. Conflict of Interest The authors declare no conflict of interest. The authors declare no conflict of interest. Acknowledgments This research was supported, in part, by grants from the National Research Foundation of Korea (NRF), a grant funded by the Korean government (MEST) (No. 2011-0010027); the Next-Generation BioGreen 21 Program (No. PJ00798402), through the Rural Development Administration; and the Vegetable Breeding Research Center (No. 710001-03), through the Agriculture Research Center program from the Ministry for Food, Agriculture, Forestry, and Fisheries. The authors would like to thank the two anonymous reviewers for their helpful comments. 11. 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Familial multinodular goiter syndrome with papillary thyroid carcinomas: mutational analysis of the associated genes in 5 cases from 1 Chinese family
BMC endocrine disorders
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Liao et al. BMC Endocrine Disorders 2013, 13:48 http://www.biomedcentral.com/1472-6823/13/48 Liao et al. BMC Endocrine Disorders 2013, 13:48 http://www.biomedcentral.com/1472-6823/13/48 Open Access * Correspondence: shunyaol@yahoo.com; wz360@hotmail.com 1Diabetes & Endocrinology Center, Sichuan Academy of Medical Science, Sichuan Provincial People’s Hospital, Chengdu 610072, China 2Department of Thyroid Disease & Nuclear Medicine, Sichuan Academy of Medical Science, Sichuan Provincial People’s Hospital, Chengdu 610072, China F ll l f h f l bl h d f h l Familial multinodular goiter syndrome with papillary thyroid carcinomas: mutational analysis of the associated genes in 5 cases from 1 Chinese family Shunyao Liao1*, Wenzhong Song2*, Yunqiang Liu3, Shaoping Deng1,4, Yaming Liang1, Zhenlin Tang2, Jiyuan Huang2, Dandan Dong5 and Gang Xu5 © 2013 Liao et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Full list of author information is available at the end of the article Background PTC is the most prevalent malignancy of the thyroid gland. There has been an increasing incidence of PTC worldwide for the past few decades. The etiology of PTC is related to environmental, hormonal and genetic factors. About 5-15% of PTC patients show a familial occurrence, and fPTC is recognized as a distinct entity only in recent years [1,2]. Families with accumulation of PTCs show an inherited trait of the disease and patients with fPTC often have early age at disease onset and increased severity in successive generations, also, fPTC patients frequently present more aggressive tumors with increased incidence of multifocality, local invasion, lymph node metastases than the sporadic PTC [2,3]. Generally, fPTC is diagnosed when three or more family members have PTC and in the ab- sence of other known associated syndromes [1,2]. PTC has a significant gender bias with much more women affected than men; it is especially suggestive for the familial predis- position when men or children were diagnosed with PTC [1,4]. While, because families share the same environment and a common genetic background, it is difficult to distin- guish between environmental and genetic contributing fac- tors, and also because the majority of fPTC pedigrees are small in size and may present with a variety of additional benign thyroid nodules, the genetic predisposition to fPTC is unknown and the molecular alterations at the origin of the pathology are only now beginning to emerge [1,5,6]. Sporadic PTC is known to be associated with point mutation of the BRAF genes and chromosomal rear- rangements of RET/PTC. The BRAF encodes a serine/ threonine-protein kinase which plays a role in regulat- ing the MAP kinase/ERKs signaling pathway and affects cell division, differentiation and secretion; point muta- tions in BRAF are found in up to 45% PTC cases [7]. The RET protooncogene is one of the receptor tyrosine kinases, cell-surface molecules that transduce signals for cell growth and differentiation; rearrangements of the RET are found in about 35% of sporadic PTC [7]. Abstract Background: Familial papillary thyroid cancer (fPTC) is recognized as a distinct entity only recently and no fPTC predisposing genes have been identified. Several potential regions and susceptibility loci for sporadic PTC have been reported. We aimed to evaluate the role of the reported susceptibility loci and potential risk genomic region in a Chinese familial multinodular goiter (fMNG) with PTC family. Methods: We sequenced the related risk genomic regions and analyzed the known PTC susceptibility loci in the Chinese family members who consented to join the study. These loci included (1) the point mutations of the BRAF and RET; (2) the possible susceptibility loci to sporadic PTC; and (3) the suggested potential fMNG syndrome with PTC risk region. Results: The members showed no mutations in the common susceptible BRAF and RET genomic region, although contained several different heterozygous alleles in the RET introns. All the members were homozygous for PTC risk alleles of rs966423 (C) at chromosome 2q35, rs2910164 (C) at chromosome 5q24 and rs2439302 (G) at chromosome 8p12; while carried no risk allele of rs4733616 (T) at chromosome 8q24, rs965513 (A) or rs1867277 (A) at chromosome 9q22 which were associated with radiation-related PTC. The frequency of the risk allele of rs944289 (T) but not that of rs116909374 (T) at chromosome 14q13 was increased in the MNG or PTC family members. Conclusions: Our work provided additional evidence to the genetic predisposition to a Chinese familial form of MNG with PTC. The family members carried quite a few risk alleles found in sporadic PTC; particularly, homozygous rs944289 (T) at chromosome 14q13 which was previously shown to be linked to a form of fMNG with PTC. Moreover, the genetic determinants of radiation-related PTC were not presented in this family. Keywords: Familial papillary thyroid carcinomas, Multinodular goiter syndrome, Mutational analysis, Genetic association, Risk alleles Full list of author information is available at the end of the article © 2013 Liao et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Liao et al. BMC Endocrine Disorders 2013, 13:48 http://www.biomedcentral.com/1472-6823/13/48 Liao et al. BMC Endocrine Disorders 2013, 13:48 http://www.biomedcentral.com/1472-6823/13/48 Page 2 of 11 wide studies [11]. Abstract DIRC3 predicted a non coding RNA transcript with unknown function, the first 2 exons of DIRC3 replaced exon 1 of HSPBAP1 and formed a DIRC3-HSPBAP1 fusion transcript, which are associated with chromatin remodeling and stress response; (2) It was reported that the heterozygosity G/C of SNP rs2910164 at 5q24 within the precursor of microRNA-146a pre- disposed to PTC by altering expression of miR146a target genes in the Toll-like receptor and cytokine sig- naling pathway [12,13]; (3) The genome-wide study also identified that chromosomal 8q24 was associated with the risk of various cancers, particularly, rs4733616 at 8q24 was founded to be possibly associated with PTC risk in 26 European families [14-16]; (4) The rs2439302, located in the intron of HRG-beta1c at 8p12,was reported to be associated with neuregulin 1 (NRG1) and confer risk of thyroid cancer [11]. HRG-beta1c is one of the NRG1 isoforms and interacts with tyrosine kinase to increase its phosphorylation on tyrosine residues, playing critical roles in the growth and development of multiple organ system; (5) It was repeatedly observed that the rs965513 at 9q22.33 were the strong association signal for NMTC in European people [16-19] and it was proposed that the rs965513 might linked to the nearest thyroid transcription factor of forkhead family (FOXE1) gene, which likely plays a crucial role in thyroid morpho- genesis; furthermore, some research indicated that rs1867277 within the FOXE1 5′ UTR is also a causal vari- ant in thyroid cancer susceptibility [16,20]; (6) Finally, both rs944289 and rs116909374 on 14q13.3 were ob- served to be strongly associated with NMTC in European people [11,16-19,21]. Nonetheless, all these genetic asso- ciations found by the genome-wide association studies have not been investigated in a family based study. Background For the purpose to improve our understanding of the PTC predisposition, based on the re- cent progresses in genetic studies about PTC, we analyzed in this Chinese family (1) the point mutations of the BRAF and RET; (2) the possible susceptibility loci to sporadic PTC; and (3) the suggested potential fMNG syndrome with PTC risk region. be autosomal dominant. For the purpose to improve our understanding of the PTC predisposition, based on the re- cent progresses in genetic studies about PTC, we analyzed in this Chinese family (1) the point mutations of the BRAF and RET; (2) the possible susceptibility loci to sporadic PTC; and (3) the suggested potential fMNG syndrome with PTC risk region. thyroidectomy specimen pathologic examinations disclosed that the architecture and nuclear features of the neoplasm in his both necks were typical for PTC (Figure 2A) and immunohistochemical staining confirmed the diagnosis (Figure 2B, C, D); After the total thyroidectomy and radio- active iodine treatment, the patient is now doing well. Interestingly, in terms of fMNG with PTC, the patient’s mother is diagnosed with MNG in bilateral thyroid and underwent a total thyroidectomy in Chongqing, China (Figure 1 II2). Both of the patient’s maternal twin aunts and a younger male cousin were diagnosed with MNG and PTC by different hospitals in Beijing and Chongqing, China, respectively; the other two maternal aunts were di- agnosed with MNG by different hospitals in Chengdu and Dazhou, China, respectively (Figure 1 II8&10). Methods The fMNG with PTC pedigree is reported in Figure 1. The clinical and pathological findings are summarized in Table 1. The study protocol was approved by the Review Board of Clinical Research of the Sichuan Provincial hospital, and by the Research & Ethics Committee of Sichuan Medical Research Institution. The blood samples were collected from the proband (III2), proband’s parents (II1 & 2) and maternal aunts (II5, 8, & 10) with their written informed consent. Background Al- though somatic mutations of the genes like BRAF and RET exclusively play a causative role in sporadic thyroid cancer development, germline mutations of single nu- cleotide polymorphisms (SNPs) in these genes were also reported to act as modifiers in the cancer process [8,9], it needs to mention here that in a Chinese population, SNPs of BRAF were shown to be associated with PTC [10], and thus it is intriguing to verify these mutations in fPTC families. In addition, a few potential regions for harboring an fPTC gene have been reported: chromosomal region 1q21 linked to fPTC with papillary renal neoplasia [22], 2q21 linked to familial NMTC type 1 syndrome [23], and the telomere abnormalities and chromosome fragil- ity might display in fPTC family [24]; Specifically, famil- ial NMTC and its relationship with familial MNG are recognized as distinct clinical entities, and the molecular pathophysiology of MNG and PTC is different, indeed MNG1 is located at 14q [25]; however, one study in a kindred with MNG and PTC suggested that 14q32 linked to a form of inherited MNG syndrome with a sig- nificant risk of progression to PTC [26]. In the present report we studied 2 PTCs and 3 MNGs obtained from members of one Chinese family. This fam- ily was ascertained through initial identification of the pro- band, a 35-year-old men (III2, Figure 1). The probands’s mother, 5 maternal aunts and 1 younger first cousin were diagnosed with MNG or PTC by different hospitals in China. The mode of inheritance in the family appeared to Recent studies based on population stratification have made progresses to identify several single nucleotide poly- morphisms (SNPs) associated with PTC risk. For exam- ples, (1) It was discovered that rs966423 at 2q35, locating into the intron region of the disrupted in renal carcinoma 3 gene (DIRC3), was significantly associate with European nonmedullary thyroid cancer (NMTC) by the genome- Liao et al. BMC Endocrine Disorders 2013, 13:48 Page 3 of 11 http://www.biomedcentral.com/1472-6823/13/48 Liao et al. BMC Endocrine Disorders 2013, 13:48 http://www.biomedcentral.com/1472-6823/13/48 Page 3 of 11 Figure 1 Pedigree of the Chinese fPTC. Circles and squares indicate female and male family members, respectively. The proband is indicated by an arrow. be autosomal dominant. DNA extraction h h l bl The whole blood was collected from the medial cubital vein into heparin anticoagulant tubes. The total DNA was purified using the spin protocol of QIAamp DNA Blood Mini Kit according to the manufacturer’s direc- tions (Qiagen, Hilden, Germany). The purified DNA was resuspended in TE buffer and stored at 4 °C. Gel electro- phoresis and spectrophotometric determination were used to DNA quantification and quality analysis. The OD260/OD280 ratio of DNA samples were between 1.8- 2.0 and concentration was more than 100 ng/ml. A 35-year-old man (Figure 1 III2) came to our observa- tion: the man complained both his lymph nodes containing palpable lump for more than 10 days, initial ultrasound ex- aminations revealed an 1.9 × 1.4 cm solid mass with irregu- lar & indefinite border, sand calcification and blood flow in his right neck, and also 2 small nodule goiters in his left neck; The thyroid function tests showed the man was euthyroid; both the fine needle aspiration cytological and Table 1 Clinical and pathological study of the collected samples Members Sex Age at diagnosis Histology sizes for PTCs and MNGs Surgical treatment II1 male 64 normal II2 female 62 bilateral MNG MNG (1.2 cm), suspicious lesion completion thyroidectomy III2 male 35 bilateral MNG with PTC PTC in MNG, PTC (1.6 cm) completion thyroidectomy II5 female 56 bilateral MNG with PTC PTC in MNG, PTC (1.5 cm) completion thyroidectomy II8 female 45 bilateral MNG MNG (0.3 cm) II10 female 41 MNG in right thyroid MNG (0.6 cm) Table 1 Clinical and pathological study of the collected samples Liao et al. BMC Endocrine Disorders 2013, 13:48 http://www.biomedcentral.com/1472-6823/13/48 Page 4 of 11 Figure 2 The histological features of the proband’s papillary carcinoma. A: The cytological feature: crowded oval nuclei, nuclear grooves, clearing, elongation and overlapping (HE × 400). B: Galectin-3 showed predominantly cytoplasmic staining with occasional nuclear staining (×200). C: HBME1 showed positive diffuse membrane (×200). D: Cytokeratin 19 showed strong, predominantly cytoplasmic staining (×200). ma. A: The cytological feature: crowded oval nuclei, nuclear grooves, dominantly cytoplasmic staining with occasional nuclear staining n 19 showed strong, predominantly cytoplasmic staining (×200). Figure 2 The histological features of the proband’s papillary carcinoma. A: The cytological feature: crowded oval nuclei, nuclear grooves, clearing, elongation and overlapping (HE × 400). B: Galectin-3 showed predominantly cytoplasmic staining with occasional nuclear staining (×200). C: HBME1 showed positive diffuse membrane (×200). DNA extraction h h l bl D: Cytokeratin 19 showed strong, predominantly cytoplasmic staining (×200). Genetic mutational analysis gel stained with ethidium bromide and purifed using the QIAquick PCR purification kit (Qiagen). Purified PCR products were sequenced directly in both orientations using standard procedures with an ABI PRISM 3100 Gen- etic Analyzer (ABI, CA). The sequences were confirmed with two independent PCRs from two independent DNA samples. The potential regions and susceptibility loci investigated in the study were listed in Table 2. Sequencing was per- formed on PCR-amplified products using primers (Table 2) according to the published sequences or self-designed with Primer Premier 6.1 (PREMIER Biosoft, Palo Alto CA). The PCR amplifications were performed using ABI GeneAmp PCR System 9700 (Applied Biosystems, Foster City, CA). The PCR reaction system included 2U Pfu DNA polymerase (Thermo Fisher Scientific Inc, USA), 50pmol of each sense and antisense primers, 1 × reaction buffer (20 mM Tris–HCl pH8.8, 10 mM KCl, 10 mM (NH4)2SO4, 1% (v/v) Triton X-100), 250 μM dNTP, 2.0 mM MgCl2 and 200 ng genomic DNA in a total vol- ume of 50 μl. The PCR cycling parameters were followed the recommendations for Pfu DNA polymerase according to the manufacturer. Precautions were taken to prevent PCR contamination, and indeed, in each experiment DNA template negative samples were run in parallel. The PCR products were resolved by electrophoresis in a 2% agarose Chr10: 43,601,341-43,602, 077 (737 bp) intron: 33, 825–34,307&34,504-34,561 exon5: 34,308-34,503 (196 bp) STS: 33,825-34,398 Chr10: 43,607,577-43,608,444 (868 bp) exon8: 40,061-40,155 (96 bp) intron: 40,156-40,784&40,896- 40,928 exon9: 40,785-40,895 (111 bp) Chr10: 43,608,459-43,609,249 (791 bp) intron: 40,943-41,487&41,608-41,557 exon10: 41,488-41,607 (120 bp) STS: 41558-41733 5′-CCAGTGGCCCCGCAGGTT-3′5′- GAAAAGCACGTCTCCCCACAGTCC-3′ Chr14: 36, 5′-TGTAATGGCAGCTCTTGACCTT-3′ 5′- ACCTTTGATTGCCCTTAGTTTGA-3′ Chr14: 36,738,229-36,738,674(446 bp) rs116909374: 36,738,361 rs116909374: 36,738,361 susceptibility loci of 9q22. Noticeably, all the family mem- bers including the proband’s father without thyroid disease were homozygous for the risk alleles of (1) rs966423 (CC) in DIRC3, (2) rs2910164 (CC) in Pre-miR-146a and (3) rs2439302 (GG) in HRG-beta1c; While all these members from the Chinese family contained no risk allele of (4) rs4733616 at 8q24, (5) rs965513 and rs1867277 at 9q22. and III3), 35 and 25 years old respectively, were diag- nosed with MNG and PTC (Figure 1); As the family members resided in different cities and denied radiation exposure, no other neoplasia syndromes or somatic gen- etic alterations in the tumor DNA was observed, according to diagnostic criteria of familial MNG with PTC [6], we considered the Chinese family presented hereditary predisposition to PTC. and III3), 35 and 25 years old respectively, were diag- nosed with MNG and PTC (Figure 1); As the family members resided in different cities and denied radiation exposure, no other neoplasia syndromes or somatic gen- etic alterations in the tumor DNA was observed, according to diagnostic criteria of familial MNG with PTC [6], we considered the Chinese family presented hereditary predisposition to PTC. For the susceptibility loci of 14q13.3, as 14q was reported to be specifically linked with MNG1 and a form of MNG with PTC [25,26], it is worth to mention that the risk T allele of rs944289 was presented in the se- quences of the most family members affected with thy- roid disease (II2 & II8, MNG; II5 & III2, MNG with PTC; Table 1). The sequence result in Table 3 showed that both MNG with PTC family members II5 and III2 were heterozygous (CT) and the 2 MNG family mem- bers II2 and II10 were homozygous (TT) at rs944289 locus (Table 3). While for another susceptibility locus of rs116909674 at 14q13.3 which we checked, none of the studied Chinese family members carried the risk alleles. The comparison of the susceptibility loci In the current study, we investigated the exon 15 of BRAF, since several SNPs in the genomic region were reported to contribute to PTC in a Chinese population [8] and the transversions in exon 15 are the common morphotype-specific mutation in adult sporadic PTC. The results were shown in Table 3: the examined BRAF sequences involved these susceptibility loci carried no risk alleles and were the same as common TT at BRAFT1799A and AA at BRAFA1801G. No any other gen- etic mutation was found in the family members. We also investigated all the known RET susceptibility loci to family thyroid diseases in this Chinese family. Ei- ther, no known RET susceptibility loci was mutational in the family members. However, it needs to mention that in the genomic regions which we sequenced, the RET introns contained certain differences among the family members, such as introns between exon 4 and 5 (rs35800403 & rs2742243), between 11 and 12 (rs2256550), between 14 and 15 (rs11238441 & rs2472737) (Table 3), and also, there was a new C to T heterozygous allele in the upstream of rs111306965 in the genome of memberII8 andII10 by re- peatedly sequencing. Additionally, rs1800863 in exon 15 contained variants of synonymous code substitution in the genome of several family members (II2, II5 & II10). The identification for the fMNG with PTC The histological features of the proband’ papillary car- cinoma were shown in Figure 2. The members of the Chinese family were diagnosed with MNG and PTC by different hospitals in China; the affected individuals showed typical MNG or MNG with PTC, bilateral and multicentric nodes. In this Chinese family, there were 2 first-degree blood relatives were diagnosed with bilateral MNG and PTC, 5 second-degree blood relatives includ- ing a pair of twin sisters were diagnosed with MNG or PTC; Also among these family members, 2 men (III2 Page 5 of 11 Page 5 of 11 Liao et al. BMC Endocrine Disorders 2013, 13:48 http://www.biomedcentral.com/1472-6823/13/48 Localization & product 5′-TGCTTGCTCTGATAGGAAAATG-3′ 5′- CCACAAAATGGATCCAGACA-3′ Chr7:140,453,250-140,453,078 (173 bp) intron: 176,315-176,371 exon15:176,372-176,487 (116 bp) RET at Chr10q11.2: 43,572,517-43,625,799 exon5: 34,308-34,503 (196 bp) R313Q: 34,378(G →A) rs77702891: 43,601,894 R330Q: 34,429(G →A) rs80236571: 43,601,945 Chr10: 43,601,341-43,602, 077 (737 bp) intron: 33, 825–34,307&34,504-34,561 exon5: 34,308-34,503 (196 bp) STS: 33,825-34,398 Chr10: 43,607,577-43,608,444 (868 bp) exon8: 40,061-40,155 (96 bp) intron: 40,156-40,784&40,896- 40,928 exon9: 40,785-40,895 (111 bp) Chr10: 43,608,459-43,609,249 (791 bp) intron: 40,943-41,487&41,608-41,557 exon10: 41,488-41,607 (120 bp) STS: 41558-41733 Chr10: 43,608,459-43,609,249 (791 bp) intron: 40,943-41,487&41,608-41,557 exon10: 41,488-41,607 (120 bp) STS: 41558-41733 5′-GTGGGCCCAATGTGTGGATA −3′ 5′- CTCTTCAGGGTCCCATGCTG-3′ Chr10: 43,611,512-43,612,272 (761 bp) intron: 43,996-44,515&44,664-44,756 exon10: 44,516-44,663 (148 bp) exon12: 44,516-44,663 (148 bp) exon13: 46, 305–46,412 (108 bp) S765P: 46,313(T →C) rs75075748: 43,613,829 E768E: 46,324(G →A/C) rs78014899: 43,613,840 V778I: 46,352(G →A) rs75686697: 43,613,868 L790F: 46,390(G →C) rs75030001: 43,613,906 Y791F: 46,392(A →T) rs77724903: 43,613,908 5′-CGGGGAATTTCTGTGGACGA-3′ 5′- ATGGCAGTGTCACACCAGAG-3′ Chr10: 43,613,496-43,614,200 (705 bp) intron: 45,980-46,304&46,413-46,684 exon13: 46, 305– 46,412 (108 bp) misc_difference: 46,327 Chr10: 43,614,767-43,615,517 (751 bp) intron: 47,251-47, 462&47,678-48,001 exon14: 47, 463– 47,677 (215 bp) Chr10: 43,615,159-43,615,837 (679 bp) exon14: 47,643-47,677 (35 bp) intron: 47,678-48,012&48,136- 48,321 exon15: 48,013-48,135 (123 bp) Chr10: 43,615,159-43,615,837 (679 bp) exon14: 47,643-47,677 (35 bp) intron: 47,678-48,012&48,136- 48,321 exon15: 48,013-48,135 (123 bp) Chr10: 43,617,229-43,617,941 (713 bp) intron: 49,713-49, 877&49, 949–50,425 exon16: 49,878- 49,948 (71 bp) STS: 49,832-50,007 Chr10: 43,618,871-43,619,601 (713 bp) intron: 51,355-51, 602&51,741-52,085 exon17: 51, 603– 51,740 (138 bp) 5′-CTCTGATGGGAGTGGCTTGG-3′5′- CCACTCAGGCACCCCTTAAC-3′ 2q35 DIRC3 (noncoding RNA):218,148,746-218,621,316 (472571 bp)rs966423:218,310,340 5q24 Pre-miR-146a: 159,912,359-159,912,457(99 bp) rs2910164: 159,912,418 8q24 rs4733616: 128,662,095 8p12 NRG1 transcript variant HRG-beta1c: 31,496,820- 32,622,558(1,125,738 bp) rs2439302: 32,432,369 5′-CGGCCTCGACCAACACTTAT-3′ 5′-ACTGGGCGTCTCAACTACAATCTG −3′ Chr2: 218,310,115-218,310,537(423 bp) located in the intron region of DIRC3, 5′-ATTTTACAGGGCTGGGACAG-3′ 5′- TCTTCCAAGCTCTTCAGCAG-3′ Chr5: 159,912,297-159,912,523(227 bp) 5′-CACCGGGGATTGGAAGAGATAAG-3′ 5′- TGAAGCCACAGGGGAGAAAAGT −3′ Chr8:128,661,750-128,662,159(410 bp) 5′-AATGCAAGAATGGCCTAACACAAT-3′ 5′-AACCTGGGGSSSSSTCTGAAGC-3′ Chr8: 32,432,326-32,432,660(334 bp) located in intron of NRG1 Page 6 of 11 Liao et al. BMC Endocrine Disorders 2013, 13:48 http://www.biomedcentral.com/1472-6823/13/48 5′-CCGGCTTGAGTTCAGGTATGTAGT-3′ 5′-CCAGGCTCAGGTTATGTCTTTGTT-3′ Chr9: 100,555,758-100,556,177(420 bp) 5′-CCGGCTTGAGTTCAGGTATGTAGT-3′ 5′-CCAGGCTCAGGTTATGTCTTTGTT-3′ Chr9: 100,555,758-100,556,177(420 bp) 5′-CCGGCTTGAGTTCAGGTATGTAGT-3′ 5′-CCAGGCTCAGGTTATGTCTTTGTT-3′ Chr9: 100,555,758-100,556,177(420 bp) 9q22 FoxE1: 100,615,537-100,618,997(3,460 bp)rs1867277: 100,615,914 14q13.3 rs944289: 36,649,246 5′-AGACCAGCTGCAGCCACCCCAACC-3′ 5′-GTCTCGCCGCGCTCTTCCTTCACG-3′ Chr9: 100,615,806-100,616,270(465 bp)located in the STS of FoxE1 Discussion The Chinese family presented hereditary predisposition to PTC, but currently the genetic incline to fPTC is un- known. With the aim of understanding the involvement of genetic factors underlying fPTC, we analyzed the reported possible PTC susceptibility genetic regions by sequence in the Chinese family members who consented to join the study. First, it is worthy to mention that no risk allele of rs965513 (A) or rs1867277 (A) at 9q22 was observed among the Chinese family members. These susceptibility loci of FOXE1 at 9q22 were related to radiation-induced PTC [19], hence it may be reasonable that the FOXE1 risk alleles were not presented in the fa- milial form of MNG with PTC, as the members denied radiation exposure and resided in quite different envir- onment. Either, the Chinese family members carried no risk allele of rs4733616 (T) at 8q24 which has been shown to be associated with sporadic PTC in Europeans [14-16], but the pathogenic role of the allele is currently unknown. Discussion With respect to the other susceptibility loci identified, as shown in Table 3, all the members from the Chinese fam- ily had equal sequences in the (1) DIRC3 susceptibility locus at 2q35, (2) Pre-miR-146a susceptibility locus at 5q24, (3) NRG1 transcript variant HRG-beta1c susceptibil- ity locus at 8p12, (4) susceptibility loci of 8q24, and (5) Table 3 Sequences of susceptibility loci in the family members Table 3 Sequences of susceptibility loci in the family members Chromosome BRAF at Chr7q34 RET at Chr10q11.2: 43,572,517-43,625,799 ences of susceptibility loci in the family members Table 3 Sequences of susceptibility loci in the family members Chromosome BRAF at Chr7q34 RET at Chr10q11.2: 43,572,517-43,625,799 rs121913364 rs113488022 rs35800403 rs2742243 rs77702891 rs80236571 rs75873440 rs77558292 rs77939446 rs80069458 Locus 140,453,134 exon15 140,453,136 exon15 43,601,415 intron 43,601,749 intron 43,601,894 exon5 43,601,945 exon5 43,607,621 exon8&9 43,609,069 exon10 43,609,070 exon10 43,609,077 exon10 Allele A:germline G: germline somatic A →G missense A:germline; somatic C:somatic T:germline T →A missense G/C T/C A:germline G:germline G →A missense A:germline G:germline G →A missense G:germline T:germline G →T missense T:germline C:germline T →C missense G:germline A: germline G →A missense C:germline G:germline C →G missense II1 normal TT AA GC TC GG GG GG TT GG CC II2 MNG TT AA GG TT GG GG GG TT GG CC III2 PTC TT AA GC TC GG GG GG TT GG CC II5 PTC TT AA GG TT GG GG GG TT GG CC II8 MNG TT AA GC TC GG GG GG TT GG CC II10 MNG TT AA GG TT GG GG GG TT GG CC Chromosome RET at Chr10q11.2: 43,572,517-43,625,799 rs76262710 rs79781594 rs77316810 rs77503355 rs79890926 rs121913313 rs2256550 rs75075748 rs78014899 rs75686697 rs75030001 Locus 43,609,096 exon10 43,609,097 exon10 43,609,102 exon10 43,609,103 exon10 43,609,104 exon10 43,609,104 exon10 43,611,865 exon12 43,613,829 exon13 43,613,840 exon13 43,613,868 exon13 43,613,906 exon13 Allele C:germline G: germline T: germline T → C&T →G missense C:germline G: germline G →C missense C:germline T:germline T →C missense A:germline C:germline G:germline T:germline G →A& G →C& G →T missense C:germline G:germline C →G missense not availiable cds-indel T/C intron C:germline T:germline T →C missense A:unkown C: somatic G: germline G →A& G →C cds-synon A:germline G:germline G →A missense C:unkown G:germline G →C missense II1 normal TT GG TT GG CC no del TC TT GG GG GG II2 MNG TT GG TT GG CC no del TT TT GG GG GG III2 PTC TT GG TT GG CC no del TC TT GG GG GG II5 PTC TT GG TT GG CC no del TT TT GG GG GG II8 MNG TT GG TT GG CC no del TC TT GG GG GG II10 MNG TT GG TT GG CC no del TT TT GG GG GG Chromosome RET at Chr10q11.2: 43,572,517-43,625,799 rs77724903 rs79658334 rs11238441 new rs2472737 rs121913306 rs75234356 rs76087194 rs121913309 rs1800863 rs78347871 Locus 43,613,908 exon13 43,614,996 exon14 43,615,382 intron 43,615,404 intron 43,615,505 intron 43,615,567 exon15 43,615,592 exon15 43,615,611 exon15 43,615,613 exon15 43,615,633 exon15 43,617,398 exon16 Allele A:germline T: germline A →T missense A:unkown G: germline T: germline G →A &G →T missense C/T C/T G/A AGC: germline TTT:somatic cds-indel G:germline T:germline T →G missense A:germline G:germline G →A missense not availiable cds- indel not availiable C/ G cds-synon C:germline G:germline G →C missense Table 3 Sequences of susceptibility loci in the family members (Continued) II1 normal AA GG CC CC GA AGC TT GG no del CC GG II2 MNG AA GG CT CC GG AGC TT GG no del CG GG III2 PTC AA GG CC CC GA AGC TT GG no del CC GG II5 PTC AA GG CT CC GG AGC TT GG no del CG GG II8 MNG AA GG CC CT GA AGC TT GG no del CC GG II10 MNG AA GG CT CT GG AGC TT GG no del CG GG The SNP alleles are shown as the reference/variant, referring to NCBI Build 36.3; the common to mutant is showed by “→”; the risk allele is indicated with an asterisk and outlined if presented in the family members, and the different sequences among the family members are shadowed. Discussion Table 3 Sequences of susceptibility loci in the family members (Continued) y loci in the family members (Continued) CC CC GA AGC TT GG no del CC GG CT CC GG AGC TT GG no del CG GG CC CC GA AGC TT GG no del CC GG CT CC GG AGC TT GG no del CG GG CC CT GA AGC TT GG no del CC GG CT CT GG AGC TT GG no del CG GG iant, referring to NCBI Build 36.3; the common to mutant is showed by “→”; the risk allele is indicated with an asterisk and outlined if presented in the family members, members are shadowed. Liao et al. BMC Endocrine Disorders 2013, 13:48 http://www.biomedcentral.com/1472-6823/13/48 Liao et al. BMC Endocrine Disorders 2013, 13:48 http://www.biomedcentral.com/1472-6823/13/48 Liao et al. BMC Endocrine Disorders 2013, 13:48 http://www.biomedcentral.com/1472-6823/13/48 Page 9 of 11 Liao et al. BMC Endocrine Disorders 2013, 13:48 http://www.biomedcentral.com/1472-6823/13/48 As we failed to detect somatic genetic alterations in the tumor DNA, such as the BRAF and RET proto- oncogene in the Chinese family members, in the current study, we investigated the genomic region containing the BRAF susceptible variants in sporadic PTC, and also all the known RET susceptibility loci to thyroid diseases (Tables 2 and 3). Our sequencing results confirmed that the BRAF and RET mutations were not germline muta- tions or susceptibility genetic events in this Chinese fam- ily. However, we noticed that in the sequenced RET genetic region, several different heterozygous alleles were presented among the Chinese family members, and most alleles were in the intron region. Recently, the chromosomal fragile sites breakage was proposed to cause PTC by forming chromosome rearrangement [26]. The chromosomal fragile sites are regions of the genome with a high susceptibility to forming DNA breaks and are often associated with cancer. Exposure to a variety of external factors such as chemotherapeutic, dietary and environmental compounds can induce and accelerate the fragile site breakage. Several intron regions of RET were identified as DNA breakage region. Hence, we are wondering if it is possible that the polymorphisms of in- trons could link to the structural difference in the RET region and could impact the related chromosome archi- tecture and thyroid gene expression, albeit there was no RET mutation in the cancerous thyroid. Discussion Interestingly, there were 2 related facts to be considered: (1) it was shown that transfecting thyroid cells with RET produced morphological changes in nuclei that mimicked those seen in PTC [27]; (2) it is curious that the RET gene is not expressed in the thyroid follicular cells from which PTC develops, but rearrangements of the RET are found in PTC cases [28]. Hence, we think it will be intriguing to investigate the association between the genomic structural of RET region and the regulation mechanism of RET. Our results verified that, for the predisposition to familial form of PTC and radiation-related PTC, their mechanism of PTC susceptibility did not completely overlap each other, since the genetic determinants associated with radiation-related PTC were not presented in the Chinese family members with PTC and MNG. It is also noticeable that all the family members were homozygous for the risk alleles of rs966423 (CC) at 2q35, rs2910164 (CC) at 5q24 and rs2439302 (GG) at 8p12. All these susceptibility loci have been reported to associate with sporadic PTC [11,13], but currently the pathogenic functions of these alleles are not known well. We think all these risk alleles might contribute jointly to the develop- ment of MNG and PTC in the Chinese family members; while considering the risk alleles also presented in the pro- band’s father with normal thyroid, it is possible that differ- ent pathogenic mechanisms exist to activate the tumor transformation in the family members with thyroid disease. Interestingly, we observed that the frequency of T risk allele of rs944289 at 14q13.3 locus was increased in these MNG and PTC Chinese family members (C: T = 0.4:0.6 vs 0.571:0.429 in normal people). Several studies suggested the possible genetic predisposition of 14q to familial PTC [25] while no association between the radiation-related PTC and 14q13.3 [19]. Also, family nontoxic MNG locus maps to chromosome 14q [24]. Further research suggested that rs944289 was located in a CEBP-alpha/CEBP-beta binding element in the 5-prime UTR of a thyroid-specific lincRNA gene, papillary thyroid carcinoma susceptibility candidate 3 (PTCSC3), PTCSC3 had the characteristics of a tumor suppressor, the rs944289 T risk allele reduced PTCSC3 promoter activation and thereby predisposes to PTC [21]. Nevertheless, the tumor suppression mechan- ism of PTCSC3 is currently unknown. Discussion In addition, the thy- roid transcription factor of NK2 homeobox 1, NKX2-1, is also located in the 14q13.3; NKX2-1 regulates the expres- sion of thyroid-specific genes involved in morphogenesis. But how rs944289 was associated with NKX2-1 remains to be investigated. Also, we investigated PTC susceptibility locus of rs116909374 (T) locating between PTCSC3 and NKX2-1 at the same 14q, the family members carried no risk allele at all. Hence, our current work implied the pos- sible role of rs944289 in familial MNG with PTC. Whereas, it is surprise that heterozygosity as CT rather than homozygosity as TT presented in the fPTC family members; the same phenomenon was once suggested as a possible special form of genetic epistasis in the rs2910164 allele of pre-miR-146a gene [12], which may also contrib- uted to this Chinese fMNG with PTC as shown by the study. Briefly, our results in the Chinese family agreed that rs944289 but not rs116909374 at 14q13.3 locus might be associated with genetic predisposition to familial form of MNG with PTC; it will be intriguing to further analyze the pathogenic link between rs944289 and the disease. Our work may provide additional evidence to the gen- etic predisposition to familial form of MNG with PTC. Due to unavailability of samples and the complex of pathogenesis, the current studied Chinese family was small and limited. Nonetheless, for complex diseases like PTC, there may be many genes influencing risk as well as the effects of environment, also, it is much more diffi- cult to collect pedigrees with multiple affected relatives and there is no guarantee of the same (or any) gene (SNP) segregating in these family. To provide insights into the genetic risk factors for familiar PTC, more re- searches are needed. References 21. Jendrzejewski J, He H, Radomska HS, Li W, Tomsic J, Liyanarachchi S, Davuluri RV, Nagy R, de la Chapelle A: The polymorphism rs944289 predisposes to papillary thyroid carcinoma through a large intergenic noncoding RNA gene of tumor suppressor type. Proc Natl Acad Sci USA 2012, 109(22):8646–8651. 1. Nosé V: Familial thyroid cancer: a review. Mod Pathol 2011, 24(Suppl 2):S19–S 1. Nosé V: Familial thyroid cancer: a review. Mod Pathol 2011, 24(Suppl 2):S19–S33. 2. Khan A, Smellie J, Nutting C, Harrington K, Newbold K: Familial nonmedullary thyroid cancer: a review of the genetics. Thyroid 2010, 20(7):795–801. Review. 3. Bonora E, Tallini G, Romeo G: Genetic predisposition to familial nonmedullary thyroid cancer: an update of molecular findings and state-of-the-Art studies. J Oncol 2010, 2010. 385206. 4. Hemminki K, Eng C, Chen B: Familial risks for nonmedullary thyroid cancer. J Clin Endocrinol Metab 2005, 90(10):5747–5753. 5. Morrison PJ, Atkinson AB: Genetic aspects of familial thyroid cancer. Oncologist 2009, 14(6):571–577. 6. Musholt TJ, Musholt PB, Petrich T, Oetting G, Knapp WH, Klempnauer J: Familial papillary thyroid carcinoma: genetics, criteria for diagnosis, clinical features, and surgical treatment. World J Surg 2000, 24(11):1409–1417. 7. Nikiforov YE, Nikiforova MN: Molecular genetics and diagnosis of thyroid cancer. Nat Rev Endocrinol 2011, 7(10):569–580. y pp 2. Khan A, Smellie J, Nutting C, Harrington K, Newbold K: Familial nonmedullary thyroid cancer: a review of the genetics. Thyroid 2010, 20(7):795–801. Review. 3. Bonora E, Tallini G, Romeo G: Genetic predisposition to familial nonmedullary thyroid cancer: an update of molecular findings and state-of-the-Art studies. J Oncol 2010, 2010. 385206. 4. Hemminki K, Eng C, Chen B: Familial risks for nonmedullary thyroid cancer. J Clin Endocrinol Metab 2005, 90(10):5747–5753. 5. Morrison PJ, Atkinson AB: Genetic aspects of familial thyroid cancer. Oncologist 2009, 14(6):571–577. 6. Musholt TJ, Musholt PB, Petrich T, Oetting G, Knapp WH, Klempnauer J: Familial papillary thyroid carcinoma: genetics, criteria for diagnosis, clinical features, and surgical treatment. World J Surg 2000, 24(11):1409–1417. 7. Nikiforov YE, Nikiforova MN: Molecular genetics and diagnosis of thyroid cancer. Nat Rev Endocrinol 2011, 7(10):569–580. 2. Khan A, Smellie J, Nutting C, Harrington K, Newbold K: Familial nonmedullary thyroid cancer: a review of the genetics. Thyroid 2010, 20(7):795–801. Review. 3. Bonora E, Tallini G, Romeo G: Genetic predisposition to familial nonmedullary thyroid cancer: an update of molecular findings and state-of-the-Art studies. J Oncol 2010, 2010. 385206. 22. Competing interests Competing interests The authors have non-financial competing interests. Funding Th d This study was supported by the research grants (to Wenzhong Song and to Shunyao Liao) from Sichuan Provincial Health Department, China (100450,120074). 17. Gudmundsson J, Sulem P, Gudbjartsson DF, Jonasson JG, Sigurdsson A, Bergthorsson JT, et al: Common variants on 9q22.33 and 14q13.3 predispose to thyroid cancer in European populations. Nat Genet 2009, 41(4):460–464. Received: 4 June 2013 Accepted: 17 October 2013 Published: 21 October 2013 20. Landa I, Ruiz-Llorente S, Montero-Conde C, Inglada-Pérez L, Schiavi F, Leskelä S, et al: The variant rs1867277 in FOXE1 gene confers thyroid cancer susceptibility through the recruitment of USF1/USF2 transcription factors. PLoS Genet 2009, 5(9):e1000637. Authors’ contributions 13. Jazdzewski K, Liyanarachchi S, Swierniak M, Pachucki J, Ringel MD, Jarzab B, de la Chapelle A: Polymorphic mature microRNAs from passenger strand of pre-miR-146a contribute to thyroid cancer. Proc Natl Acad Sci USA 2009, 106(5):1502–1505. SY L, YQ L and WZ S designed the molecular genetic studies, participated in the sequence alignment and drafted the manuscript. DD D and G X carried out the immunohistochemical assay. SP D and YM L have been involved in revising the manuscript critically. ZL T, JY H participated in data acquisition and helped to draft the manuscript. All authors read and approved the final manuscript. 14. Neta G, Yu CL, Brenner A, Gu F, Hutchinson A, Pfeiffer R, Sturgis EM, Xu L, Linet MS, Alexander BH, Chanock S, Sigurdson AJ: Common genetic variants in the 8q24 region and risk of papillary thyroid cancer. Laryngoscope 2012, 122(5):1040–1042. Acknowledgments 15. He H, Nagy R, Liyanarachchi S, Jiao H, Li W, Suster S, Kere J, de la Chapelle A: A susceptibility locus for papillary thyroid carcinoma on chromosome 8q24. Cancer Res 2009, 69(2):625–631. g We thank the members of the Chinese fMNG with PTC family for their essential contribution to scientific research. We thank Dr. Hongji Yang and the colleagues in Department of General Surgery, Sichuan Academy of Medical Science, Sichuan Provincial People’s Hospital, for their support and collaboration. 16. Jones AM, Howarth KM, Martin L, Gorman M, Mihai R, Moss L, Auton A, Lemon C, Mehanna H, Mohan H, Clarke SE, Wadsley J, Macias E, Coatesworth A, Beasley M, Roques T, Martin C, Ryan P, Gerrard G, Power D, Bremmer C, Consortium TCUKIN, Tomlinson I, Carvajal-Carmona LG: Thyroid cancer susceptibility polymorphisms: confirmation of loci on chromosomes 9q22 and 14q13, validation of a recessive 8q24 locus and failure to replicate a locus on 5q24. J Med Genet 2012, 49(3):158–163. the colleagues in Department of General Surgery, Sichuan Academy of Medical Science, Sichuan Provincial People’s Hospital, for their support and collaboration. Author details 1 18. Matsuse M, Takahashi M, Mitsutake N, Nishihara E, Hirokawa M, Kawaguchi T, Rogounovitch T, Saenko V, Bychkov A, Suzuki K, Matsuo K, Tajima K, Miyauchi A, Yamada R, Matsuda F, Yamashita S: The FOXE1 and NKX2-1 loci are associated with susceptibility to papillary thyroid carcinoma in the Japanese population. J Med Genet 2011, 48(9):645–648. 1Diabetes & Endocrinology Center, Sichuan Academy of Medical Science, Sichuan Provincial People’s Hospital, Chengdu 610072, China. 2Department of Thyroid Disease & Nuclear Medicine, Sichuan Academy of Medical Science, Sichuan Provincial People’s Hospital, Chengdu 610072, China. 3Department of Medical Genetics and Division of Morbid Genomics, State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu 610041, China. 4Department of Surgery, Harvard Medical School, Massachusetts General Hospital, Boston, MA, USA. 5Department of Pathology, Sichuan Academy of Medical Science, Sichuan Provincial People’s Hospital, Chengdu 610072, China. 19. Takahashi M, Saenko VA, Rogounovitch TI, Kawaguchi T, Drozd VM, Takigawa-Imamura H, Akulevich NM, Ratanajaraya C, Mitsutake N, Takamura N, Danilova LI, Lushchik ML, Demidchik YE, Heath S, Yamada R, Lathrop M, Matsuda F, Yamashita S: The FOXE1 locus is a major genetic determinant for radiation-related thyroid carcinoma in Chernobyl. Hum Mol Genet 2010, 19(12):2516–2523. Received: 4 June 2013 Accepted: 17 October 2013 Published: 21 October 2013 Conclusions Based on our current investigation in the Chinese fMNG with PTC, the risk allele homozygote of rs966423 (CC) at 2q35, rs2910164 (CC) at 5q24 and rs2439302 (GG) at Page 10 of 11 Liao et al. BMC Endocrine Disorders 2013, 13:48 http://www.biomedcentral.com/1472-6823/13/48 8p12 could contribute to the fMNG with PTC, while the other identified risk alleles for sporadic PTC or radiation-related PTC might not be involved. Also, cor- responding to the previous studies on the association be- tween chromosome 14q and fMNG with PTC, our work approved that rs944289 but not rs116909374 at 14q13 locus might be associated with genetic predisposition to a Chinese family MNG with PTC. Though several differ- ent heterozygous alleles in the RET introns presented, the common BRAF and RET mutations were not suscep- tibility genetic events in this Chinese family. 8. Shifrin AL, Ogilvie JB, Stang MT, Fay AM, Kuo YH, Matulewicz T, Xenachis CZ, Vernick JJ: Single nucleotide polymorphisms act as modifiers and correlate with the development of medullary and simultaneous medullary/papillary thyroid carcinomas in 2 large, non-related families with the RET V804M proto-oncogene mutation. Surgery 2010, 148(6):1274–1280. 8. Shifrin AL, Ogilvie JB, Stang MT, Fay AM, Kuo YH, Matulewicz T, Xenachis CZ, Vernick JJ: Single nucleotide polymorphisms act as modifiers and correlate with the development of medullary and simultaneous medullary/papillary thyroid carcinomas in 2 large, non-related families with the RET V804M proto-oncogene mutation. Surgery 2010, 148(6):1274–1280. 9. Shifrin AL, Fay A, Kuo YH, Ogilvie J: Response to “Single nucleotide polymorphisms and development of hereditary medullary thyroid cancer in V804M RET families: disease modification or linkage disequilibrium? ”. Surgery 2012, 151(6):902–903. y 10. Zhang Q, Song F, Zheng H, Zhu X, Song F, Yao X, Zhang L, Chen K: Association between single-nucleotide polymorphisms of BRAF and papillary thyroid carcinoma in a Chinese population. Thyroid 2013, 23(1):38–44. 11. Gudmundsson J, Sulem P, Gudbjartsson DF, Jonasson JG, Masson G, He H, Jonasdottir A, et al: Discovery of common variants associated with low TSH levels and thyroid cancer risk. Nat Genet 2012, 44(3):319–322. 12. Jazdzewski K, Murray EL, Franssila K, Jarzab B, Schoenberg DR, de la Chapelle A: Common SNP in pre-miR-146a decreases mature miR expression and predisposes to papillary thyroid carcinoma. Proc Natl Acad Sci USA 2008, 105(20):7269–7274. References Bignell GR, Canzian F, Shayeghi M, Stark M, Shugart YY, Biggs P, et al: Familial nontoxic multinodular thyroid goiter locus maps to chromosome 14q but does not account for familial nonmedullary thyroid cancer. Am J Hum Genet 1997, 61(5):1123–1130. 25. Bignell GR, Canzian F, Shayeghi M, Stark M, Shugart YY, Biggs P, et al: Familial nontoxic multinodular thyroid goiter locus maps to chromosome 14q but does not account for familial nonmedullary thyroid cancer. Am J Hum Genet 1997, 61(5):1123–1130. 26. Bakhsh A, Kirov G, Gregory JW, Williams ED, Ludgate M: A new form of familial multi-nodular goiter with progression to differentiated thyroid cancer. Endocr Relat Cancer 2006, 13(2):475–483. 27. Fischer AH, Bond J, Taysavang P, Battles OE, Wynford-Thomas D: Papillary thyroid carcinoma oncogene (RET/PTC) alters the nuclear envelope and chromatin structure. Am J Pathol 1998, 153(5):1443–1450. 28. Kitamura Y, Minobe K, Nakata T, Shimizu K, Tanaka S, Fujimori M, Yokoyama S, Ito K, Onda M, Emi M: Ret/PTC3 is the most frequent form of gene rearrangement in papillary thyroid carcinomas in Japan. J Hum Genet 1999, 44(2):96–102. doi:10.1186/1472-6823-13-48 Cite this article as: Liao et al.: Familial multinodular goiter syndrome with papillary thyroid carcinomas: mutational analysis of the associated genes in 5 cases from 1 Chinese family. BMC Endocrine Disorders 2013 13:48. 26. Bakhsh A, Kirov G, Gregory JW, Williams ED, Ludgate M: A new form of familial multi-nodular goiter with progression to differentiated thyroid cancer. Endocr Relat Cancer 2006, 13(2):475–483. 27. Fischer AH, Bond J, Taysavang P, Battles OE, Wynford-Thomas D: Papillary thyroid carcinoma oncogene (RET/PTC) alters the nuclear envelope and chromatin structure. Am J Pathol 1998, 153(5):1443–1450. 28. Kitamura Y, Minobe K, Nakata T, Shimizu K, Tanaka S, Fujimori M, Yokoyama S, Ito K, Onda M, Emi M: Ret/PTC3 is the most frequent form of gene rearrangement in papillary thyroid carcinomas in Japan. J Hum Genet 1999, 44(2):96–102. 28. Kitamura Y, Minobe K, Nakata T, Shimizu K, Tanaka S, Fujimori M, Yokoyama S, Ito K, Onda M, Emi M: Ret/PTC3 is the most frequent form of gene rearrangement in papillary thyroid carcinomas in Japan. J Hum Genet 1999, 44(2):96–102. doi:10.1186/1472-6823-13-48 Cite this article as: Liao et al.: Familial multinodular goiter syndrome with papillary thyroid carcinomas: mutational analysis of the associated genes in 5 cases from 1 Chinese family. BMC Endocrine Disorders 2013 13:48. References Malchoff CD, Sarfarazi M, Tendler B, Forouhar F, Whalen G, Joshi V, Arnold A, Malchoff DM: Papillary thyroid carcinoma associated with papillary renal neoplasia: genetic linkage analysis of a distinct heritable tumor syndrome. J Clin Endocrinol Metab 2000, 85(5):1758–1764. 4. Hemminki K, Eng C, Chen B: Familial risks for nonmedullary thyroid cancer. J Clin Endocrinol Metab 2005, 90(10):5747–5753. 23. McKay JD, Lesueur F, Jonard L, Pastore A, Williamson J, Hoffman L, et al: Localization of a susceptibility gene for familial nonmedullary thyroid carcinoma to chromosome 2q21. Am J Hum Genet 2001, 69(2):440–446. 6. Musholt TJ, Musholt PB, Petrich T, Oetting G, Knapp WH, Klempnauer J: Familial papillary thyroid carcinoma: genetics, criteria for diagnosis, clinical features, and surgical treatment. World J Surg 2000, 24(11):1409–1417. 24. Cantara S, Pisu M, Frau DV, Caria P, Dettori T, Capezzone M, Capuano S, Vanni R, Pacini F: Telomere abnormalities and chromosome fragility in patients affected by familial papillary thyroid cancer. J Clin Endocrinol Metab 2012, 97(7):E1327–E1331. 7. Nikiforov YE, Nikiforova MN: Molecular genetics and diagnosis of thyroid cancer. Nat Rev Endocrinol 2011, 7(10):569–580. Page 11 of 11 Page 11 of 11 Liao et al. BMC Endocrine Disorders 2013, 13:48 http://www.biomedcentral.com/1472-6823/13/48 25. Bignell GR, Canzian F, Shayeghi M, Stark M, Shugart YY, Biggs P, et al: Familial nontoxic multinodular thyroid goiter locus maps to chromosome 14q but does not account for familial nonmedullary thyroid cancer. Am J Hum Genet 1997, 61(5):1123–1130. 26. Bakhsh A, Kirov G, Gregory JW, Williams ED, Ludgate M: A new form of familial multi-nodular goiter with progression to differentiated thyroid cancer. Endocr Relat Cancer 2006, 13(2):475–483. 27. Fischer AH, Bond J, Taysavang P, Battles OE, Wynford-Thomas D: Papillary thyroid carcinoma oncogene (RET/PTC) alters the nuclear envelope and chromatin structure. Am J Pathol 1998, 153(5):1443–1450. 28. Kitamura Y, Minobe K, Nakata T, Shimizu K, Tanaka S, Fujimori M, Yokoyama S, Ito K, Onda M, Emi M: Ret/PTC3 is the most frequent form of gene rearrangement in papillary thyroid carcinomas in Japan. J Hum Genet 1999, 44(2):96–102. doi:10.1186/1472-6823-13-48 Cite this article as: Liao et al.: Familial multinodular goiter syndrome with papillary thyroid carcinomas: mutational analysis of the associated genes in 5 cases from 1 Chinese family. BMC Endocrine Disorders 2013 13:48. 25. References Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit
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Fingierte Interviews als journalistische Textgattung
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Fingierte Interviews als journalistische Textgattung Eine Einführung Andreas Erb Journalistische Begriffe wie Wirklichkeit oder Wahrheit galten für mich als Mythos. […] Es gab für mich jedenfalls einen klaren Unterschied zwischen einem literarischen Menschen und einem, der sich streng an Tatsachen klammert.1 Im Mai 2000 erhob das Nachrichtenmagazin Focus den Vorwurf, Tom Kummer habe seine Interviews, die er mit Prominenten in den letzten Jahren geführt hatte und die z. B. im Magazin der Süddeutschen Zeitung erschienen waren, erfunden. Im Verlauf des Monats kam es dann zu zahlreichen weiteren Vorwürfen gegen die journalistische Praxis von Tom Kummer, zu Gegendarstellungen, zu Richtigstellungen, schließlich zum Vorwurf, Kummer hätte sich einer kriminellen Handlung schuldig gemacht, kurz: Die Affäre Kummer wuchs sich zu einem veritablen Presse-/Kulturskandal aus. Dabei war Tom Kummer gerade zum Hoffnungsträger eines neuen deutschen Journalismus aufgestiegen, galt als viel gefragter und -gedruckter Star einer durchweg jungen Presseszene, die sich an den Ideen des New Journalism orientierte und, so Kummer in seinem autobiografischen Rückblick, einen Journalismus überwinden wollte, der »sich längst hinter eine fade scheinbare Objektivität zurückgezogen« hatte und »unter Fakten jedes Leben begraben würde«.2 Kummers Interviews, etwa mit Pamela Anderson, Charles Bronson, Johnny Depp, Brad Pitt, Sharon Stone und vielen anderen mehr,3 galten als innovative Meisterwerke, die die überkommenen Formen eines uninspirierten und letztlich wenig unterhaltsamen Feuilletons hinter sich ließen. Was 2000 als Skandal und mit der Entlassung der beiden renommierten Chefredakteure des SZ-Magazins Christian Kämmerling und Ulf Poschardt endete, war mir Anlass, zusammen mit den Studierenden des Seminars Jour- 1 | Tom Kummer: Blow up. München 2007, S. 35. 2 | Ebd., S. 90. 3 | Gesammelt sind ein Teil der Interviews als Buch erschienen: »Gibt es etwas Stärkeres als Verführung, Miss Stone?« Star-Interviews von Tom Kummer. Mit einem Vorwort von Ulf Poschardt. München 1997. 16 | A NDREAS E RB nalistische Textgattungen über Form und Inhalt des Interviews einerseits, über journalistische Ethik andererseits nachzudenken. Dies vor allem vor dem Hintergrund, dass Interviews sich bekanntlich medienübergreifend großer Beliebtheit erfreuen. Die Begegnung zweier Gesprächspartner im Sinne des entrevoir scheint offenbar zumindest quantitativ die Königsdisziplin journalistischer Arbeit zu sein; sie ist wichtiges Mittel der Recherche und Informationsbeschaffung, sie gilt zudem über die Atmosphäre der lebendigen Stimme als Garant einer (wie auch immer bewerteten) Authentizität, sie steht für dokumentarischen Charakter mit Wahrheitsanspruch. Neben der literaturhistorischen Erkundung der sog. Interview-Literatur, unterschiedlichen Modi und Techniken der Fragestellung ging es beim Schreiben um die Erfahrung des doppelten Rollenwechsels, wie es die Fake-Interviews von Tom Kummer nahelegen. Ein solches Gespräch, in dem beide Interviewrollen zusammenfallen, lebt in hohem Maß von der genauen Kenntnis des zu Interviewenden: Dessen Antworten müssen für das Publikum so überraschend wie nachvollziehbar – plausibel – klingen. Die Imitation von Tom Kummer ignoriert damit Grenzen von falsch und wahr, von Erfindung und Zitat, von Enthemmung und Konvention. Heraus kommt ein befreites Schreiben im Rahmen der Textgattung Interview, wobei der selbstgewählte Gegenstand im Spiel der Grenzüberschreitung ausgelotet werden kann. In solcher Rollenprosa begegnen sich literarisches und journalistisches Schreiben, die Erprobung von Stillagen befördert das Gefühl für die Dynamik von Frage und (möglicher) Antwort, die den Lesenden aber immer als wahrscheinliche erscheinen müssen. Die drei ausgewählten Interviews4 offenbaren die Möglichkeiten des Experiments, sie verdeutlichen, wie Tonlagen produziert werden und den jeweiligen Gegenstand bzw. Gesprächspartner charakterisieren – und letztlich sind sie ebenfalls Teil einer Implosion des Realen (so der Titel eines Spiegel-Interviews mit Tom Kummer).5 4 | Ein weiteres Interview mit Hunter S. Thompson erschien in der Literaturzeitschrift schliff ([5] 2016). 5 | »Implosion des Realen«. Tom Kummer über fingierte Interviews mit HollywoodStars und sein Verständnis von »Borderline-Journalismus«. In: Der Spiegel 21 (2000), S. 110 (Interview: Marianne Wellershoff), online unter www.spiegel.de/spiegel/ print/d-16466566.html. Externe Links wurden bis zum Zeitpunkt der Drucklegung des Buches geprüf t. Auf etwaige Änderungen zu einem späteren Zeitpunkt haben der Verlag und die Verfasser keinen Einfluss. Eine Haftung des Verlags und der Verfasser ist daher ausgeschlossen.
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Produção científica em Neurociências da UFRGS indexada na Web of Science: 2000-2009 │ Scientific production in Neuroscience at UFRGS indexed in Web of Science: 2000-2009
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Dirce Maria Santin* Sônia Domingues Santos Brambilla** Ida Regina Chittó Stumpf*** Dirce Maria Santin* Sônia Domingues Santos Brambilla** Ida Regina Chittó Stumpf*** Dirce Maria Santin* Sônia Domingues Santos Brambilla** Ida Regina Chittó Stumpf*** Resumo O artigo analisa a produção científica em Neurociências da Universidade Federal do Rio Grande do Sul, em termos de artigos publicados em periódicos nacionais e internacionais registrados na base de dados Web of Science (WoS). A amostra é composta por 847 artigos publicados no período de 2000 a 2009 e indexados na categoria Neurociências da WoS. São utilizados indicadores de atividade, impacto e colaboração para caracterizar padrões e tendências na produção científica em Neurociências da UFRGS. Os resultados demonstram que a área apresenta produção científica significativa e crescente, com alto impacto e fortemente produzida em colaboração. Palavras-chave Bibliometria, Produção científica, Universidade Federal do Rio Grande do Sul, Neurociências. Neurociências. *** Doutora em Comunicação pela Universidade de São Paulo e Professora titular do Departamento de Ciências da Informação e do Programa de Pós-graduação em Comunicação e Informação da UFRGS. Coordenadora do Grupo de Pesquisa em Comunicação Científica/UFRGS. Endereço: Faculdade de Biblioteconomia e Comunicação- UFRGS, Rua Ramiro Barcellos, 2705, 2º andar, Porto Alegre, Rio Grande do Sul, CEP 90035-007, telefone 51 3308 5434, e-mail irstumpf@ufrgs.br. bibliotecária do Instituto de Ciências Básicas da Saúde/UFRGS. Endereço: Instituto de Ciências Básicas da Saúde, Biblioteca, Rua Sarmento Leite, 500, sala 102, Porto Alegre, Rio Grande do Sul, CEP 90050-003, telefone 51 3308- 3363, e-mail dirsantin@yahoo.com.br. ** Doutora em Comunicação e Informação pela UFRGS e bibliotecária pesquisadora parlamentar da Assembleia Legislativa do Estado do Rio Grande do Sul. Endereço: Assembleia Legislativa do Estado do Rio Grande do Sul, Divisão de Biblioteca, Praça Marechal Deodoro, 101, Porto Alegre, Rio Grande do Sul, CEP 90010-300, telefone 51 3210-1164, e-mail sdbrambilla@yahoo.com.br. *** Doutora em Comunicação pela Universidade de São Paulo e Professora titular do Departamento de Ciências da * Mestranda em Comunicação e Informação pela Universidade Federal do Rio Grande do Sul (UFRGS) e bibliotecária do Instituto de Ciências Básicas da Saúde/UFRGS. Endereço: Instituto de Ciências Básicas da Saúde, Biblioteca, Rua Sarmento Leite, 500, sala 102, Porto Alegre, Rio Grande do Sul, CEP 90050-003, telefone 51 3308- 3363, e-mail dirsantin@yahoo.com.br. Mestranda em Comunicação e Informação pela Universidade Federal do Rio Grande do Sul (UFRGS) e bibliotecária do Instituto de Ciências Básicas da Saúde/UFRGS. Endereço: Instituto de Ciências Básicas da Saúde, Biblioteca, Rua Sarmento Leite, 500, sala 102, Porto Alegre, Rio Grande do Sul, CEP 90050-003, telefone 51 3308- 3363, e-mail dirsantin@yahoo.com.br. ** Doutora em Comunicação e Informação pela UFRGS e bibliotecária pesquisadora parlamentar da Assembleia Legislativa do Estado do Rio Grande do Sul. Endereço: Assembleia Legislativa do Estado do Rio Grande do Sul, Divisão de Biblioteca, Praça Marechal Deodoro, 101, Porto Alegre, Rio Grande do Sul, CEP 90010-300, telefone 51 3210-1164, e-mail sdbrambilla@yahoo.com.br. *** Doutora em Comunicação pela Universidade de São Paulo e Professora titular do Departamento de Ciências da Informação e do Programa de Pós-graduação em Comunicação e Informação da UFRGS. Coordenadora do Grupo de Pesquisa em Comunicação Científica/UFRGS. Endereço: Faculdade de Biblioteconomia e Comunicação- UFRGS, Rua Ramiro Barcellos, 2705, 2º andar, Porto Alegre, Rio Grande do Sul, CEP 90035-007, telefone 51 3308 3363, e mail dirsantin@yahoo.com.br. ** Doutora em Comunicação e Informação pela UFRGS e bibliotecária pesquisadora parlamentar da Assembleia Legislativa do Estado do Rio Grande do Sul. Endereço: Assembleia Legislativa do Estado do Rio Grande do Sul, Divisão de Biblioteca, Praça Marechal Deodoro, 101, Porto Alegre, Rio Grande do Sul, CEP 90010-300, telefone 51 3210-1164, e-mail sdbrambilla@yahoo.com.br. p p RS, de 27 a 29 de agosto de 2012. Os dados foram atualizados, a metodologia incluiu novas técnicas e as análises foram ampliadas para este estudo. * Mestranda em Comunicação e Informação pela Universidade Federal do Rio Grande do Sul (UFRGS) e bibliotecária do Instituto de Ciências Básicas da Saúde/UFRGS. Endereço: Instituto de Ciências Básicas da Saúde, Biblioteca, Rua Sarmento Leite, 500, sala 102, Porto Alegre, Rio Grande do Sul, CEP 90050-003, telefone 51 3308- 3363, e-mail dirsantin@yahoo.com.br. ** Doutora em Comunicação e Informação pela UFRGS e bibliotecária pesquisadora parlamentar da Assembleia Legislativa do Estado do Rio Grande do Sul. Endereço: Assembleia Legislativa do Estado do Rio Grande do Sul, Divisão de Biblioteca, Praça Marechal Deodoro, 101, Porto Alegre, Rio Grande do Sul, CEP 90010-300, telefone 51 3210-1164, e-mail sdbrambilla@yahoo.com.br. *** Doutora em Comunicação pela Universidade de São Paulo e Professora titular do Departamento de Ciências da Informação e do Programa de Pós-graduação em Comunicação e Informação da UFRGS. Coordenadora do Grupo de Pesquisa em Comunicação Científica/UFRGS. Endereço: Faculdade de Biblioteconomia e Comunicação- UFRGS, Rua Ramiro Barcellos, 2705, 2º andar, Porto Alegre, Rio Grande do Sul, CEP 90035-007, telefone 51 3308 5434, e-mail irstumpf@ufrgs.br. Scientific production in Neuroscience at UFRGS indexed in Web of Science: 2000- 2009 Abstract The article discusses the scientific production in Neuroscience of the Universidade Federal do Rio Grande do Sul as regards articles published in national and international journals 1 Versão preliminar apresentada no 3º Encontro Brasileiro de Bibliometria e Cientometria, realizado em Gramado, RS, de 27 a 29 de agosto de 2012. Os dados foram atualizados, a metodologia incluiu novas técnicas e as análises foram ampliadas para este estudo. * Liinc em Revista, Rio de Janeiro, v. 9, n. 1, p. 66-84, maio 2013 - http://www.ibict.br/liinc 66 indexed in the Web of Science database. The sample consists of 847 articles published from 2000 to 2009 and indexed in the database in the Neuroscience category. Activity, impact and collaboration indicators are used to characterize patterns and trends in the scientific production in Neuroscience at UFRGS. The results demonstrate that the field presents significant and growing scientific production, with high impact and strongly produced in collaboration. Keywords Bibliometrics, Scientific production, Universidade Federal do Rio Grande do Sul, Neuroscience. Keywords Bibliometrics, Scientific production, Universidade Federal do Rio Grande do Sul, Neuroscience. Introdução A produção científica brasileira experimentou crescimento relevante nas últimas décadas, especialmente no conjunto de subáreas que compõem as áreas biomédicas (GUIMARÃES, 2004). Além do crescimento observado na produção científica, percebem-se desafios igualmente significativos no que se refere ao desenvolvimento de políticas em CT&I (Ciência, Tecnologia e Inovação), sobretudo em relação às universidades, que são as principais produtoras de informação científica e tecnológica no Brasil. Nesse cenário, a avaliação da produção científica constitui recurso essencial para o planejamento de políticas científicas nacionais e institucionais. Os estudos quantitativos, realizados com base em indicadores bibliométricos, podem revelar padrões e tendências de comportamento das diversas áreas do conhecimento, verificados pela comunicação dos resultados de pesquisa registrados em canais formais de divulgação, contribuindo significativamente para a gestão e o desenvolvimento de um campo científico. Este estudo caracteriza a produção científica em Neurociências realizada no âmbito da Universidade Federal do Rio Grande do Sul (UFRGS), em termos de artigos publicados em periódicos nacionais e internacionais registrados na base de dados referencial e multidisciplinar Web of Science, considerando o período de 2000 a 2009. São utilizadas medidas de atividade, impacto e colaboração para caracterizar a produção científica e as dinâmicas da comunicação de resultados das pesquisas nessa área. Essas medidas são representadas por indicadores bibliométricos, que buscam analisar, de forma quantitativa, a pesquisa em Neurociências da UFRGS, a partir dos dados de publicação e citação, e das relações de colaboração estabelecidas entre autores, instituições e países envolvidos. A análise da produção científica da área de Neurociências da UFRGS justifica-se por sua relevância e representatividade no contexto institucional, além da importância e do desenvolvimento da área no cenário nacional e internacional. Considera, também, a amplitude da área de Neurociências, que congrega disciplinas como Anatomia, Bioquímica, Fisiologia e Farmacologia, entre outras, nos estudos sobre o funcionamento do sistema nervoso em humanos e modelos animais. Liinc em Revista, Rio de Janeiro, v. 9, n. 1, p. 66-84, maio 2013 - http://www.ibict.br/liinc 67 A área de Neurociências apresenta forte expansão no cenário científico internacional, tendo sido eleita como prioritária pelo governo dos Estados Unidos na década de 1990, que ficou conhecida como a “Década do Cérebro”. Os avanços recentes também a colocam no centro das preocupações da comunidade científica internacional no início do século XXI. Introdução No Brasil, seu desenvolvimento ocorre desde meados do século XX, incentivado pela criação de sociedades científicas específicas e grupos de pesquisa em diversos institutos, universidades e centros de pesquisa do País (VENTURA 2010). A temática “Neurociências” (Neuroscience) é considerada, na classificação da Web of Science, uma área independente, que reúne periódicos cobrindo diversos assuntos. Na UFRGS, um programa de pós-graduação específico, ligado ao Instituto de Ciências Básicas da Saúde (ICBS), oferece um leque de disciplinas que cobrem a variedade de tópicos abrigados nas Neurociências, em proposta multidisciplinar, com professores pertencentes a vários departamentos e alunos com formação diversificada. Além disso, o Programa de Pós-graduação em Bioquímica, também ligado ao ICBS, contempla a linha de pesquisa Neuroquímica, Neurofarmacologia e Comportamento, resultando em importantes pesquisas sobre o tema na Universidade. O objetivo do estudo, portanto, é dar visibilidade a esta temática, que representa uma área altamente produtiva na UFRGS em relação à produção científica institucional, principalmente se comparada a outras subáreas das Ciências Biológicas e das Ciências da Saúde. O interesse pelos resultados desta ciência especificamente, que tem expandido suas fronteiras com outros campos, também decorre do fato de que uma das autoras atua como bibliotecária do ICBS, ao qual estão vinculados os programas de pós-graduação que realizam a maior parte das pesquisas em Neurociências na Universidade. Embora o estudo não seja exaustivo, por considerar que nem toda a produção científica em Neurociências da UFRGS está publicada na forma de artigos e indexada na Web of Science, acredita-se que pode revelar padrões e tendências da comunicação científica no período de 2000 a 2009, uma vez que os artigos são a principal forma de comunicação dos resultados das pesquisas na área e a base utilizada possui abrangência internacional. Revisão de literatura A universidade como espaço de produção do conhecimento científico surgiu no início do século XIX, quando a Alemanha introduziu a pesquisa neste ambiente, tornando-se modelo para o desenvolvimento das instituições de ensino superior do mundo todo. A integração entre ensino e pesquisa, viabilizada pelo sistema integrado alemão, tornou a ciência essencialmente academizada e produzida por comunidades científicas, distribuídas em campos definidos por disciplinas. A universidade tornou-se a base para formação e capacitação dos produtores da pesquisa científica, representando um corpo de doutrinas diferentes, gerado ou em geração num meio específico, sofrendo as influências dos fatores que compõem a cultura de que faz parte (MEADOWS, 1999; SCHWARTZMAN, 2001). O Brasil seguiu o modelo alemão na criação das universidades, especialmente as públicas, integrando o ensino à pesquisa. Com o desenvolvimento da pós-graduação a partir dos anos de Liinc em Revista, Rio de Janeiro, v. 9, n. 1, p. 66-84, maio 2013 - http://www.ibict.br/liinc 68 68 1970, a relação entre ensino e pesquisa consolidou-se, apresentando reflexos no incremento da produção científica brasileira. Atualmente, as universidades são as principais produtoras de conhecimento científico no Brasil, concentrando a maior parte dos pesquisadores e o maior volume de investimentos em pesquisa e desenvolvimento (GUIMARÃES, 2004). 1970, a relação entre ensino e pesquisa consolidou-se, apresentando reflexos no incremento da produção científica brasileira. Atualmente, as universidades são as principais produtoras de conhecimento científico no Brasil, concentrando a maior parte dos pesquisadores e o maior volume de investimentos em pesquisa e desenvolvimento (GUIMARÃES, 2004). A crescente produção científica das universidades brasileiras revela sua capacidade de produzir e comunicar os resultados das pesquisas realizadas nas diferentes áreas do conhecimento. Nesse contexto, a comunicação dos resultados permite somar esforços individuais dos membros da comunidade científica e potencializá-los num fluxo contínuo (GARVEY, 1979), além de possibilitar a análise dos métodos e resultados pelos pares e justificar a alocacão dos recursos financeiros (CALLON; COURTIAL; PENAN, 1995). A ciência praticada nas universidades estrutura-se em diversas áreas do conhecimento, que se caracterizam por diferentes formas de produzir e comunicar os resultados das pesquisas. No âmbito da comunicação científica, essas diferenças entre as ciências refletem maneiras diferentes de fazer pesquisa nas Ciências Exatas, Ciências Biológicas, Ciências da Saúde, Ciências Sociais e Humanidades – e, por consequência, na forma como são comunicados os resultados científicos. Revisão de literatura Meadows (1999) discute o foco das pesquisas nas áreas das Ciências Sociais e Humanidades e das Ciências Exatas e Biológicas, apontando os aspectos subjetivos das primeiras e o aparente rigor do enfoque hipotético-dedutivo alcançado pelas ciências ditas exatas ou “duras”. Assim, se os pesquisadores pensam e agem diferentemente, também se comunicam dessa forma, e uma das maneiras de verificar essa hipótese seria pela análise dos trabalhos que publicam e das referências que contêm a outras publicações, constituindo ao mesmo tempo uma análise das frentes de pesquisa, através da rede de citações que traça a colaboração entre os pares na ciência. O incremento da produção científica em nível nacional e internacional contribuiu para o desenvolvimento de novas formas de avaliação da ciência, que se orientam por duas vertentes complementares: a avaliação qualitativa, realizada pelos pares, e a avaliação quantitativa, realizada com base em indicadores bibliométricos (CASTRO, 1986). A literatura sobre avaliação da ciência apresenta certo consenso de que a combinação dos dois métodos constitui a alternativa mais adequada aos processos de avaliação. Assim, a avaliação quantitativa não dispensa a avaliação qualitativa, considerada a forma tradicional de avaliação da ciência pelos pares e feita antes da publicação. A análise da produção científica é um dos critérios utilizados para compreender o crescimento da ciência e as estruturas da pesquisa em diferentes contextos, além de critérios como o número de pesquisadores e o volume de verbas investidas (TARGINO, 2000). A avaliação da informação científica pela bibliometria é considerada uma tendência mundial, configurando-se como recurso essencial à construção de indicadores e à definição de estratégias regionais e institucionais, além de servir à avaliação dos resultados das políticas implementadas (VANZ; STUMPF, 2010). Nessa perspectiva, a avaliação dos resultados da atividade científica pode ser realizada com base em indicadores bibliométricos, que resultam da análise dos dados das publicações científicas. A validade dos indicadores, no entanto, está condicionada à contextualização e à representatividade dos dados, devendo priorizar grandes volumes de informações, correspondentes a períodos significativos (MUGNAINI; POBLACIÓN, 2007). Além disso, a avaliação deve considerar as diferenças existentes entre as disciplinas no que se refere ao comportamento dos pesquisadores em relação à comunicação dos resultados das pesquisas (MEADOWS, 1999). Liinc em Revista, Rio de Janeiro, v. 9, n. 1, p. Revisão de literatura 66-84, maio 2013 - http://www.ibict.br/liinc 69 Em estudos bibliométricos, alguns dos indicadores mais utilizados são os que mensuram a atividade científica, o impacto do uso dessa atividade e os níveis de coautoria existentes nas publicações. Os indicadores de atividade são expressos pelo número de trabalhos publicados em determinado período, pela frequência de autores e instituições, entre outras variáveis (GLÄNZEL, 2003). Os indicadores de impacto permitem verificar a visibilidade e o impacto da produção científica, indicando o desempenho de autores e publicações em determinados campos científicos (MOED, 2009). Referem-se ao impacto dos autores (índice H), artigos (índices de citações) e também dos periódicos (fator de impacto). Os indicadores de colaboração são estabelecidos a partir das relações de colaboração existentes entre os agentes científicos no que se refere à publicação conjunta dos resultados das pesquisas (MALTRÁS BARBA, 2003). Revelam aproximações entre pesquisadores individuais, grupos de pesquisa, setores da sociedade, instituições, regiões e países, a partir dos dados de coautoria das publicações. Procedimentos metodológicos Esta pesquisa consiste num estudo bibliométrico, realizado com base em indicadores de atividade, impacto e colaboração, obtidos a partir da análise dos dados das publicações. O nível de agregação é do tipo médio, por abranger a produção científica de uma instituição, com ênfase em determinada área do conhecimento (GLÄNZEL, 2003). Fazem parte da amostra os artigos de autores vinculados à UFRGS, publicados em periódicos nacionais e internacionais no período de 2000 a 2009 e indexados na categoria Neurociências da Web of Science. O período de dez anos constitui uma série temporal significativa, que busca conferir consistência e estabilidade aos dados analisados. A principal fonte de coleta de dados foi o Science Citation Index da Web of Science, que é considerada a mais importante fonte de dados para estudos bibliométricos na atualidade. A base é produzida pelo Institute for Scientific Information (ISI), pertencente ao grupo Thompson Reuters, sediado na Filadélfia, Estados Unidos. Indexa cerca de 12.000 periódicos regionais e internacionais, classificados em 249 categorias de assunto, divididas entre as disciplinas Life Science & Biomedicine; Physical Sciences; Technology; Arts & Humanities; e Social Sciences. A categorização de assunto adotada pela Web of Science sustenta-se pela classificação dos periódicos em que os artigos foram publicados, podendo contemplar até cinco categorias. Como fontes secundárias, foram utilizados o Journal Citation Report (JCR), o Sistema de Pesquisas da UFRGS, disponível na página da Universidade, a Plataforma Lattes e buscadores da Internet para conferência sobre o vínculo dos autores. A coleta de dados foi feita em março de 2010, sendo os registros importados a partir da principal fonte de coleta e normalizados utilizando o software MSExcel®. Os dados referentes às citações recebidas pelos artigos foram atualizados em dezembro de 2012, a fim de garantir mais consistência à identificação dos artigos mais citados. A análise dos resultados partiu da amostra de 847 artigos de autores vinculados à UFRGS, publicados no período em periódicos nacionais e internacionais, classificados na categoria Neurociências da Web of Science. Os indicadores de atividade são representados pelas variáveis Liinc em Revista, Rio de Janeiro, v. 9, n. 1, p. 66-84, maio 2013 - http://www.ibict.br/liinc 70 70 número de artigos, ano de publicação, idioma, títulos de periódicos e autores mais produtivos. Os indicadores de citação contemplam o número de citações recebidas pelos artigos, com destaque para os artigos mais citados da amostra e seus principais citantes. Procedimentos metodológicos Para examinar estes citantes, foi utilizado o campo Times Cited da WOS, que informa quantas vezes o artigo foi citado. Clicando sobre este número, a base de dados gerou relatórios, que possibilitaram identificar a procedência (países e instituições) dos trabalhos (BRAMBILLA, STUMPF, 2012b). Os indicadores de colaboração, por sua vez, consideram a proporção de trabalhos realizados em colaboração e os principais agentes colaboradores, com ênfase na cooperação interinstitucional e internacional expressa na coautoria das publicações. Resultados Com base na amostra de 847 artigos de autores vinculados à UFRGS, publicados no período de 2000 a 2009 e registrados na Web of Science até o momento da coleta de dados, é possível perceber um aumento expressivo na produção científica da Universidade na área de Neurociências. A tabela a seguir apresenta os dados da produção de artigos por período, acompanhados da taxa de crescimento anual. Tabela 1: Artigos em Neurociências da UFRGS (Web of Science) Fonte: dados da pesquisa. Apesar do crescimento geral no número de artigos, percebe-se que há decréscimo significativo nos anos de 2001 e 2004 (-30,4% e -8,4%, respectivamente). Aparentemente, os dados revelam situações isoladas, uma vez que a produção dos anos precedentes está publicada basicamente nos mesmos periódicos. Entretanto, seria necessária uma análise conjuntural junto à comunidade científica em questão para compreender possíveis fatores que influenciaram o decréscimo no número de publicações nos anos mencionados. Por outro lado, os anos de 2002, 2003 e 2009 apresentaram as maiores taxas de crescimento da produção científica (43,7%, 37,3% e 8,1%, respectivamente). O restante do período apresentou Liinc em Revista, Rio de Janeiro, v. 9, n. 1, p. 66-84, maio 2013 - http://www.ibict.br/liinc 71 crescimento constante, com uma taxa de crescimento média de 7% em relação aos artigos publicados de 2000 a 2009. Em comparação com os resultados de estudo realizado por Brambilla (2011; 2012a), sobre a produção científica da UFRGS na área das Ciências Biológicas, que engloba a subárea Neurociências, verificou-se uma curva de crescimento mais irregular, influenciada pela taxa negativa dos anos de 2001 e 2004 e pelo crescimento positivo em 2002, 2003 e 2009, que difere dos dados apresentados naquele trabalho, onde a análise revelava aumento contínuo na taxa de crescimento, como pode ser observado na tabela a seguir: Tabela 2: Produtividade da UFRGS na WOS por área (2000-2009) Fonte: Brambilla (2011). ( ) Nota: a tabela original expõe a pesquisa da UFRGS representada na WoS, por áreas do conhecimento. Para este estudo foram destacados, em excerto, os resultados da área Ciências Biológicas (CB). Nota: a tabela original expõe a pesquisa da UFRGS representada na WoS, por áreas do conhecimento. Para este estudo foram destacados, em excerto, os resultados da área Ciências Biológicas (CB). Em relação ao idioma dos artigos, os dados demonstram a preferência pelo inglês para a publicação dos resultados das pesquisas na área de Neurociências. A Tabela 2 confirma a forte predominância do inglês (98% dos artigos) como idioma das publicações, enquanto 16 artigos foram publicados em português e apenas um artigo foi publicado em espanhol. O predomínio do inglês é comum nas ciências naturais, onde este é considerado o idioma oficial da comunicação científica (MEADOWS, 1999). Tabela 3: Idioma dos artigos Tabela 3: Idioma dos artigos Fonte: dados da pesquisa. Liinc em Revista, Rio de Janeiro, v. 9, n. 1, p. 66-84, maio 2013 - http://www.ibict.br/liinc 72 No que se refere aos títulos de periódicos mais utilizados para publicação, foram identificados os 15 títulos que representam a maior parte dos artigos do corpus da pesquisa. Os resultados constam na tabela abaixo, acompanhados do número de artigos, do país de publicação e do fator de impacto, segundo a edição de 2011 do Journal Citation Report (JCR). Tabela 4: Principais títulos de periódicos Fonte: dados da pesquisa. Tabela 4: Principais títulos de periódicos Fonte: dados da pesquisa. Fonte: dados da pesquisa. Verifica-se que os títulos de periódicos apresentados concentram 607 artigos (72%) do total de 847 artigos analisados. Dentre os 607 artigos, 287 (47%) foram publicados nos Estados Unidos, 120 (20%) foram publicados na Inglaterra, 118 (19%) foram publicados na Holanda e 39 (5%) foram publicados na Irlanda. Nesta listagem, o Brasil está representado por um título de periódico, que reuniu 58 (9%) artigos publicados no período. Observa-se, também, a preferência dos pesquisadores da área de Neurociências pela publicação dos resultados das pesquisas em periódicos estrangeiros, com circulação internacional, mas fator de impacto variado. O padrão de publicação internacional é adequado para a área, uma vez que as temáticas não estão circunscritas a determinados espaços geográficos, culturais ou históricos, mas interessam a toda a comunidade científica internacional (SANTOS, 2003). Liinc em Revista, Rio de Janeiro, v. 9, n. 1, p. Tabela 2: Produtividade da UFRGS na WOS por área (2000-2009) 66-84, maio 2013 - http://www.ibict.br/liinc 73 Em relação à produtividade dos autores, foram identificados os 15 pesquisadores vinculados à UFRGS que contavam com o maior número de artigos publicados e indexados na categoria Neurociências da Web of Science no período analisado. Os autores mais produtivos estão listados na Tabela 5, acompanhados do número de artigos publicados. Em relação à produtividade dos autores, foram identificados os 15 pesquisadores vinculados à UFRGS que contavam com o maior número de artigos publicados e indexados na categoria Neurociências da Web of Science no período analisado. Os autores mais produtivos estão listados na Tabela 5, acompanhados do número de artigos publicados. Tabela 5: Autores mais produtivos da UFRGS na área de Neurociências Tabela 5: Autores mais produtivos da UFRGS na área de Neurociências Fonte: dados da pesquisa. O autor Ivan Antonio Izquierdo, embora atualmente vinculado à Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS), foi professor do quadro permanente do Departamento de Bioquímica da UFRGS desde 1973 até 2003, quando se aposentou. No entanto, continuou a atuar como colaborador convidado na Universidade: de 2003 a 2005, no Departamento de Bioquímica; a partir de 2005, no Programa de Pós-graduação em Ciências Biológicas: Bioquímica; e de 2011 a 2012, no Programa de Pós-graduação em Medicina: Ciências Médicas. Neste estudo, embora Izquierdo tenha contabilizado 101 artigos, foram considerados somente os 82 submetidos como pesquisador da UFRGS, tendo sido os restantes (19) elaborados com autores da instituição. Em relação às medidas de impacto, verificou-se que dos 847 artigos que compõem o corpus da pesquisa, 823 (97%) foram citados, enquanto 24 (3%) não foram citados até dezembro de 2012, quando foram atualizados os dados relativos às citações recebidas. Considerando os artigos Liinc em Revista, Rio de Janeiro, v. 9, n. 1, p. 66-84, maio 2013 - http://www.ibict.br/liinc 74 citados, verifica-se que 590 receberam de uma a 20 citações (70%), 171 receberam de 21 a 40 citações (20%), 46 receberam de 41 a 70 citações (5%) e 16 receberam mais de 71 citações (2%) no período de 2000 a 2012. Os percentuais da distribuição dos artigos pelo número de citações recebidas são representados no gráfico abaixo. Gráfico 1: Distribuição de artigos pelo número de citações recebidas Fonte: dados da pesquisa. Gráfico 1: Distribuição de artigos pelo número de citações recebidas Fonte: dados da pesquisa. Liinc em Revista, Rio de Janeiro, v. 9, n. 1, p. 66-84, maio 2013 - http://www.ibict.br/liinc Destaca-se que os 16 artigos que receberam mais de 71 citações (Quadro 1) foram publicados em inglês, em 11 títulos de periódicos estrangeiros, reunindo 1.471 citações. 2000 foi o ano de publicação de 7 artigos do conjunto dos 16 mais citados, seguido de 2001, 2002, 2003 e 2007 com 2 artigos e de 2006 com um artigo dentre os mais citados. Tabela 2: Produtividade da UFRGS na WOS por área (2000-2009) Destaca-se que os 16 artigos que receberam mais de 71 citações (Quadro 1) foram publicados em inglês, em 11 títulos de periódicos estrangeiros, reunindo 1.471 citações. 2000 foi o ano de publicação de 7 artigos do conjunto dos 16 mais citados, seguido de 2001, 2002, 2003 e 2007 com 2 artigos e de 2006 com um artigo dentre os mais citados. Destaca-se que os 16 artigos que receberam mais de 71 citações (Quadro 1) foram publicados em inglês, em 11 títulos de periódicos estrangeiros, reunindo 1.471 citações. 2000 foi o ano de publicação de 7 artigos do conjunto dos 16 mais citados, seguido de 2001, 2002, 2003 e 2007 com 2 artigos e de 2006 com um artigo dentre os mais citados. Destaca-se que os 16 artigos que receberam mais de 71 citações (Quadro 1) foram publicados em inglês, em 11 títulos de periódicos estrangeiros, reunindo 1.471 citações. 2000 foi o ano de publicação de 7 artigos do conjunto dos 16 mais citados, seguido de 2001, 2002, 2003 e 2007 com 2 artigos e de 2006 com um artigo dentre os mais citados. Liinc em Revista, Rio de Janeiro, v. 9, n. 1, p. 66-84, maio 2013 - http://www.ibict.br/liinc 75 Quadro 1: Artigos mais citados da UFRGS na área de Neurociências Quadro 1: Artigos mais citados da UFRGS na área de Neurociências Liinc em Revista, Rio de Janeiro, v. 9, n. 1, p. 66-84, maio 2013 - http://www.ibict.br/liinc 76 76 Destes 16 artigos mais citados, nove foram produzidos em colaboração com pesquisadores de outros países, como Argentina, Estados Unidos e Portugal. Esta característica revela a importância da colaboração científica internacional para a ampliação do impacto da produção científica da área de Neurociências da UFRGS no cenário nacional e internacional. Os sete artigos restantes foram produzidos entre autores brasileiros, sendo cinco entre pesquisadores de outras instituições nacionais, destacando-se a Universidade Federal de Santa Maria, no Rio Grande do Sul, com três ocorrências, aparecendo ainda três diferentes instituições paulistas (Universidade de São Paulo, Universidade Federal de São Paulo e Instituto do Câncer de São Paulo). Dois trabalhos foram escritos somente entre autores vinculados ao Departamento de Bioquímica da UFRGS. Os artigos listados no quadro acima têm como principais citantes os seguintes países: Brasil (36%), Estados Unidos (18%), Argentina (8%), Canadá (4%), Alemanha (4%), China (3%), França (3%) e Japão (3%). Outros países totalizaram 21% das citações recebidas. Tabela 2: Produtividade da UFRGS na WOS por área (2000-2009) Os dados revelam o alcance internacional dos artigos da área de Neurociências da UFRGS, uma vez que 64% das citações recebidas provêm da comunidade científica internacional, conforme pode ser observado no gráfico abaixo. Gráfico 2: Principais países citantes Fonte: dados da pesquisa. Fonte: dados da pesquisa. Em estudo sobre a internacionalidade e domesticidade da produção científica mundial publicada no período de 2000 a 2002 e indexada na Web of Science, Glänzel e Schubert (2005) identificaram proporção semelhante de citações nacionais na produção científica brasileira nas áreas de Neurociências e Comportamento. A aproximação com os resultados do estudo mencionado demonstra que a produção científica em Neurociências da UFRGS acompanha o alcance internacional obtido pela produção científica nacional, com uma pequena diferença na proporcionalidade de citações recebidas no cenário internacional. Liinc em Revista, Rio de Janeiro, v. 9, n. 1, p. 66-84, maio 2013 - http://www.ibict.br/liinc 77 77 A análise de citações deste estudo também contemplou a identificação das principais instituições citantes dos 16 artigos mais citados da amostra. A Universidade Federal de Santa Maria apareceu como a principal citante dentre as instituições (22%), seguida da UFRGS (16%), da Universidad de Buenos Aires (6%) e da Pontifícia Universidade Católica do Rio Grande do Sul (3%). A distribuição das citações realizadas aos artigos mais citados pelas instituições de origem dos autores é apresentada na Tabela 5, acompanhada do percentual de colaboração em relação ao total de ocorrência de instituições colaboradoras, que totalizou 2.116. Tabela 6: Principais instituições citantes Fonte: dados da pesquisa Tabela 6: Principais instituições citantes Fonte: dados da pesquisa. Os dados revelam a predominância das instituições brasileiras como as principais citantes dos 16 artigos mais citados da amostra, com destaque para as instituições da Região Sul do Brasil, onde está sediada a UFRGS. A distribuição reforça a importância das citações realizadas por trabalhos produzidos no Brasil e a presença de instituições estrangeiras, dos Estados Unidos e da Argentina, entre os citantes dos artigos de maior impacto na área de Neurociências da Universidade. A análise da colaboração científica revela a forte predominância da coautoria na comunicação dos resultados das pesquisas, constituindo um padrão nos artigos da área de Neurociências da UFRGS. Da amostra de 847 artigos, apenas um foi publicado em autoria única, representando a exceção no conjunto da produção científica analisada. O número máximo de autores por artigo foi 15 e a média de autores por trabalho foi 7. Liinc em Revista, Rio de Janeiro, v. 9, n. 1, p. 66-84, maio 2013 - http://www.ibict.br/liinc Tabela 2: Produtividade da UFRGS na WOS por área (2000-2009) A mediana do número de autores também foi 7, reforçando o padrão de colaboração na área. A distribuição do número de autores por artigo está representada no Gráfico 3. Liinc em Revista, Rio de Janeiro, v. 9, n. 1, p. 66-84, maio 2013 - http://www.ibict.br/liinc 78 Gráfico 3: Número de autores por artigo Fonte: dados da pesquisa. Fonte: dados da pesquisa. Fonte: dados da pesquisa. Meadows (1999) chama a atenção para a preferência pela publicação em co-autoria nos trabalhos científicos, alertando que a necessidade de trabalhar em grupo é maior nas ciências “duras” do que nas ciências sociais, que, por sua vez, é maior do que nas humanidades, apontando como causa o custo das pesquisas nas áreas físicas e biológicas, que fica diluído quando repartido pelos diversos grupos. A coautoria entre indivíduos também foi analisada por Vanz (2009), revelando, para a produção científica brasileira, tendência de crescimento ao longo dos anos (2004-2006), com 96% dos artigos publicados por mais de um autor e média de 6,3 por trabalho. Em relação à colaboração internacional, verifica-se que, dentre os 847 artigos analisados, 147 (17%) foram produzidos em colaboração com pesquisadores de outros países. Glänzel (2003) expôs que a produção científica em colaboração internacional cresceu na maioria dos países e que a densidade das redes (nº de países de procedência dos autores) tem aumentado significativamente, comprovando esta tendência. Neste trabalho, verificou-se que o total de países colaboradores foi 16, com destaque para Argentina (37%), Estados Unidos (26%), Austrália (10%), Espanha (7%) e Uruguai (4%). Pesquisadores de outros países também apareceram como coautores dos artigos dos pesquisadores da UFRGS na área de Neurociências: Alemanha, França e Itália tiveram 4 artigos cada em coautoria; enquanto que Portugal e Suécia tiveram 3 artigos cada; Canadá, Chile e Reino Unido apresentaram 2 artigos cada em coautoria; China e Nigéria apresentaram colaboração em um artigo cada, conforme mostra o Gráfico 4. Liinc em Revista, Rio de Janeiro, v. 9, n. 1, p. 66-84, maio 2013 - http://www.ibict.br/liinc 79 79 Gráfico 4: Principais países colaboradores Fonte: dados da pesquisa. nte: dados da pesquisa. Fonte: dados da pesquisa. Percebe-se uma aproximação entre os principais países colaboradores e os principais países citantes, destacados anteriormente. É o caso, por exemplo, de Argentina e Estados Unidos que aparecem entre os principais países colaboradores e também se destacam como os principais países citantes. Liinc em Revista, Rio de Janeiro, v. 9, n. 1, p. 66-84, maio 2013 - http://www.ibict.br/liinc Tabela 7: Principais instituições colaboradoras Fonte: dados da pesquisa. Fonte: dados da pesquisa. Os dados apresentados acima revelam que mais de 60% dos artigos da amostra foram produzidos em colaboração com as instituições listadas na Tabela 7. Demonstram, também, a prevalência das instituições brasileiras, especialmente da Região Sul do País, como as principais colaboradoras, com destaque para o Hospital de Clínicas de Porto Alegre, empresa pública vinculada academicamente à UFRGS. As instituições estrangeiras com maior índice de colaboração são a Universidad de Buenos Aires (Argentina) e a Newcastle University (Austrália). Tabela 2: Produtividade da UFRGS na WOS por área (2000-2009) Já o Brasil, que é principal país colaborador (83%), também é o primeiro entre os citantes (36%). Os resultados permitem inferir que a UFRGS, na área Neurociências, apesar de produzir os artigos representados na Web of Science preferencialmente com autores nacionais, recebe a maior parte das citações (64%) de países estrangeiros, o que indica a visibilidade da produção e reforça o alcance nacional e internacional destes trabalhos. No que se refere à colaboração interinstitucional, foram identificadas as instituições com maior colaboração com a UFRGS na produção de artigos científicos da área de Neurociências no período de 2000 a 2009, conforme apresentado na tabela abaixo. Liinc em Revista, Rio de Janeiro, v. 9, n. 1, p. 66-84, maio 2013 - http://www.ibict.br/liinc 80 Tabela 7: Principais instituições colaboradoras Considerações finais A avaliação quantitativa dos artigos contemplados na amostra deste estudo, sobre a produção da UFRGS em Neurociências, de 2000 a 2009, indexados na Web of Science, ressaltou padrões e comportamentos de uma área em que a Universidade tem tido produção científica significativa e crescente, tornando possível visualizar núcleos preferenciais para publicação, verificados pelas medidas de atividade, impacto e colaboração. Pelas medidas de atividade, foram analisados 847 artigos nesta temática no período, utilizando indicadores de produtividade, ano de publicação, idioma, títulos de periódicos e autores mais produtivos. Os resultados mostraram que a taxa média de crescimento da produção no período Liinc em Revista, Rio de Janeiro, v. 9, n. 1, p. 66-84, maio 2013 - http://www.ibict.br/liinc 81 81 2000/2009 é de 7%, o idioma mais utilizado nos artigos é o inglês e os periódicos em que a produção foi veiculada são, predominantemente, estrangeiros e de impacto. Esta preferência dos pesquisadores pela publicação dos resultados de pesquisas em periódicos estrangeiros, com circulação internacional e de impacto variado constitui um padrão na produção científica em Neurociências da UFRGS. Já as medidas de citação analisaram o número de citações recebidas pelos artigos, com destaque para os mais citados e os principais citantes. Verificou-se que a maior parte dos artigos (97%) já foi citada por outros autores e publicações e os artigos mais citados da amostra receberam citações de autores de países como Brasil, Estados Unidos, Canadá, Alemanha, entre outros, revelando seu alcance nacional e internacional. Dentre os artigos mais citados, a maior parte foi produzida em colaboração com outros países, o que revela a importância da colaboração internacional para a ampliação do impacto da produção científica da área de Neurociências da UFRGS. Quanto às medidas de colaboração, permitiram analisar a proporção de trabalhos realizados em coautoria e os principais agentes colaboradores. Esta análise revelou forte predominância da coautoria na comunicação dos resultados das pesquisas da área. Apesar do alto índice de colaboração, apenas 17% dos artigos foram produzidos entre pesquisadores de outros países, com destaque para Argentina e Estados Unidos. Em relação à colaboração interinstitucional, verifica-se a prevalência das instituições brasileiras, especialmente da Região Sul do País, além da presença, embora limitada, de instituições estrangeiras. Considerações finais Por fim, reforça-se a importância da avaliação da produção científica de uma área do conhecimento e de uma Universidade, não só por permitir a análise sobre o prestígio e a visibilidade alcançada por essa produção, mas para melhor conhecimento de suas especificidades. Artigo recebido em 06/02/2013 e aprovado em 22/03/2013. Artigo recebido em 06/02/2013 e aprovado em 22/03/2013. Referências Liinc em Revista, Rio de Janeiro, v. 9, n. 1, p. 66-84, maio 2013 - http://www.ibict.br/liinc BRAMBILLA, Sônia Domingues Santos. Produção científica da Universidade Federal do Rio Grande do Sul representada na Web of Science: 2000-2009. 2011. Tese (Doutorado em Comunicação e Informação)- Universidade Federal do Rio Grande do Sul. Faculdade de Biblioteconomia e Comunicação, Porto Alegre, 2001. Disponível em: <http://hdl.handle.net/10183/28922>. Acesso em: 21 nov. 2011. ________; STUMPF, Ida Regina Chittó. Produção científica da UFRGS representada na WoS (2000-2009). Perspectivas em Ciência da Informação, v. 17, n. 3, p. 34-50, jul./set. 2012a. BRAMBILLA, Sônia Domingues Santos. Produção científica da Universidade Federal do Rio Grande do Sul representada na Web of Science: 2000-2009. 2011. Tese (Doutorado em Comunicação e Informação)- Universidade Federal do Rio Grande do Sul. Faculdade de Biblioteconomia e Comunicação, Porto Alegre, 2001. Disponível em: <http://hdl.handle.net/10183/28922>. Acesso em: 21 nov. 2011. ________; STUMPF, Ida Regina Chittó. Produção científica da UFRGS representada na WoS (2000-2009). Perspectivas em Ciência da Informação, v. 17, n. 3, p. 34-50, jul./set. 2012a. ________; STUMPF, Ida Regina Chittó. Produção científica da UFRGS representada na WoS (2000-2009). Perspectivas em Ciência da Informação, v. 17, n. 3, p. 34-50, jul./set. 2012a. Liinc em Revista, Rio de Janeiro, v. 9, n. 1, p. 66-84, maio 2013 - http://www.ibict.br/liinc 82 ________. Artigos da UFRGS representados na Web of Science: os mais citados e seus citantes. In: ENCONTRO BRASILEIRO DE BIBLIOMETRIA E CIENTOMETRIA, 3., 2012. Anais... Porto Alegre, 2012b. ________. Artigos da UFRGS representados na Web of Science: os mais citados e seus citantes. In: ENCONTRO BRASILEIRO DE BIBLIOMETRIA E CIENTOMETRIA, 3., 2012. Anais... Porto Alegre, 2012b. CASTRO, Cláudio Moura Ciência e universidade. Rio de Janeiro: Zahar, 1986. CALLON, Michel; COURTIAL, Jean-Pierre; PENAN, Hervé. Cienciometría: la medición de la actividad científica - de la bibliometría a la vigilancia tecnológica. Gijón: Trea, 1995. GARVEY, William D. Communication: the essence of the science. New York: Pergamon Press, 1979. GLÄNZEL, Wolfgang. Bibliometrics as a research field: a course on theory and application of bibliometric indicators. 2003. Disponível em: <http://nsdl.niscair.res.in/bitstream/123456789/968/1/Bib_Module_KUL.pdf>. Acesso em: 10 dez 2012 GLÄNZEL, Wolfgang. Bibliometrics as a research field: a course on theory and app bibliometric indicators. 2003. Disponível g g f y pp bibliometric indicators. 2003. Disponível em: <http://nsdl.niscair.res.in/bitstream/123456789/968/1/Bib_Module_KUL.pdf>. Acesso em: 10 dez. 2012. ________; SCHUBERT, András. Domesticity and internacionality in co-authorship, references and citations. Scientometrics, v. 65, n. 3, p. 323-342, dez. 2005. GUIMARÃES, Jorge A. Liinc em Revista, Rio de Janeiro, v. 9, n. 1, p. 66-84, maio 2013 - http://www.ibict.br/liinc Referências A pesquisa médica e biomédica no Brasil: comparações com o desempenho científico brasileiro e mundial. Ciência e Saúde Coletiva, v. 9, n. 2, p. 303-327, 2004. MALTRÁS BARBA, Bruno. Los indicadores bibliométricos: fundamentos y aplicación al análisis de la ciencia. Gijón: Ediciones Trea, 2003. MEADOWS, Arthur. Jack. A comunicação científica. Brasília: Briquet de Lemos, 1999. MOED, Henk F. New developments in the use of citation analysis in research evaluation. Archivum Immunologiae et Therapiae Experimentalis, v. 57, n. 1, p. 13-18, 2009. MUGNAINI, Rogério; POBLACIÓN, Dinah Aparecida de Aguiar. Impacto de documentos citados em revistas científicas brasileiras de diferentes áreas. In: ENCONTRO NACIONAL DE PESQUISA EM CIÊNCIA DA INFORMAÇÃO, 8., 2007. Anais... Salvador, 2007. SANTIN, Dirce Maria; BRAMBILLA, Sônia Domingues Santos. Produção científica em Neurociências da UFRGS registrada na Web of Science: 2000 a 2009. In: ENCONTRO BRASILEIRO DE BIBLIOMETRIA E CIENTOMETRIA, 3., 2012. Anais... Porto Alegre: [s.n.], 2012. SANTOS, Cássio Miranda dos. Tradições e contradições da pós-graduação no Brasil. Educação e Sociedade, v. 24, n. 83, p. 627-641, ago. 2003. Disponível em: <http://www.scielo.br/pdf/es/v24n83/a16v2483.pdf>. Acesso em: 18 dez. 2012. SCHWARTZMAN, Simon. Um espaço para a ciência: a formação da comunidade científica no Brasil. Brasília: Ministério da Ciência e Tecnologia, 2001. Liinc em Revista, Rio de Janeiro, v. 9, n. 1, p. 66-84, maio 2013 - http://www.ibict.br/liinc 83 83 TARGINO, Maria das Graças. Comunicação científica: uma revisão de seus elementos básicos. Informação & Sociedade, v. 10, n. 2, p. 1-27, 2000. Disponível em: <http://www.ies.ufpb.br/ojs2/index.php/ies/article/view/326/248>. Acesso em: 15 dez. 2012. TARGINO, Maria das Graças. Comunicação científica: uma revisão de seus elementos básicos. Informação & Sociedade, v. 10, n. 2, p. 1-27, 2000. Disponível em: <http://www.ies.ufpb.br/ojs2/index.php/ies/article/view/326/248>. Acesso em: 15 dez. 2012. VANZ, Samile Andréa de Souza. As redes de colaboração científica no Brasil: (2004 a 2006). 2009. Tese (Doutorado em Comunicação e Informação)- Universidade Federal do Rio Grande do Sul. Faculdade de Biblioteconomia e Comunicação. Programa de Pós-Graduação em Comunicação e Informação, Porto Alegre, 2009. Disponível em: <http://hdl.handle.net/10183/17169>. Acesso em: 17 dez. 2012. _________; STUMPF, Ida Regina Chittó Stumpf. Procedimentos e ferramentas aplicados aos estudos bibliométricos. Informação & Sociedade, v. 20, n. 2, p. 67-75, maio/ago. 2010. Disponível em: <http://www.ies.ufpb.br/ojs/index.php/ies/article/view/4817/4358>. Acesso em: 18 nov. 2012. VENTURA, Dora Fix. Um retrato da área de Neurociência e Comportamento no Brasil. Psicologia, v. 26, n. esp., p. 123-129, 2010. Disponível em: <http://www.scielo.br/pdf/ptp/v26nspe/a11v26ns.pdf>. Acesso em: 16 dez. 2012. VENTURA, Dora Fix. Referências Um retrato da área de Neurociência e Comportamento no Brasil. Psicologia, v. 26, n. esp., p. 123-129, 2010. Disponível em: <http://www.scielo.br/pdf/ptp/v26nspe/a11v26ns.pdf>. Acesso em: 16 dez. 2012. 84 84
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Allogeneic Mesothelioma Tumor Lysate-pulsed Autologous Dendritic Cell Vaccine
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Open Peer Review on Qeios Open Peer Review on Qeios Allogeneic Mesothelioma Tumor Lysate- pulsed Autologous Dendritic Cell Vaccine National Cancer Institute Qeios · Definition, February 2, 2020 Open Peer Review on Qeios Qeios ID: 58B76U · https://doi.org/10.32388/58B76U Source National Cancer Institute. Allogeneic Mesothelioma Tumor Lysate-pulsed Autologous Dendritic Cell Vaccine. NCI Thesaurus. Code C121640. A cell-based cancer vaccine composed of autologous dendritic cells (DCs) pulsed with a mixture of lysates from five allogeneic mesothelioma tumor cell lines, with potential immunostimulatory and antineoplastic activities. Upon leukapheresis, DCs are loaded with allogeneic mesothelioma tumor cell lysates. Upon re-administration of the allogeneic mesothelioma tumor lysate-pulsed autologous DC vaccine, the immune system is exposed to an undefined amount of mesothelioma-associated antigens, which stimulates the induction of a specific cytotoxic T-lymphocyte (CTL) response against mesothelioma tumor cells and leads to tumor cell lysis. Qeios ID: 58B76U · https://doi.org/10.32388/58B76U 1/1
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Optimization of Backpropagation for Early Detection of Diabetes Mellitu
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International Journal of Electrical and Computer Engineering (IJECE) Vol. 8, No. 5, October 2018, pp. 3232~3237 ISSN: 2088-8708, DOI: 10.11591/ijece.v8i5.pp3232-3237 International Journal of Electrical and Computer Engineering (IJECE) Vol. 8, No. 5, October 2018, pp. 3232~3237 ISSN: 2088-8708, DOI: 10.11591/ijece.v8i5.pp3232-3237 International Journal of Electrical and Computer Engineering (IJECE) Vol. 8, No. 5, October 2018, pp. 3232~3237 ISSN: 2088-8708, DOI: 10.11591/ijece.v8i5.pp3232-3237  3232  3232 Optimization of Backpropagation for Early Detection of Diabetes Mellitus Rosita Sofiana1, Sutikno2 1Department of Computer Science/Informatics, Faculty of Science and Mathematics, Diponegoro University, Indonesia 2Computer Science from the Gadjah Mada University, Indonesia Article Info Article history: Received Jan 4, 2017 Revised Jan 9, 2018 Accepted Sep 21, 2018 Corresponding Author: Sutikno, Department of Computer Science/Informatics, Diponegoro University, Prof. Soedarto Street, Tembalang, Semarang, Indonesia. Email: tik@undip.ac.id Sutikno, Department of Computer Science/Informatics, Diponegoro University, Prof. Soedarto Street, Tembalang, Semarang, Indonesia. Email: tik@undip.ac.id Sutikno, Department of Computer Science/Informatics, Diponegoro University, Prof. Soedarto Street, Tembalang, Semarang, Indonesia. Email: tik@undip.ac.id Sutikno, Department of Computer Science/Informatics, Diponegoro University, Prof. Soedarto Street, Tembalang, Semarang, Indonesia. Email: tik@undip.ac.id Keywords: Adaptive learning rate Backpropagation Momentum coefficient Nguyen Widrow Optimization Journal homepage: http://iaescore.com/journals/index.php/IJECE Copyright © 2018 Institute of Advanced Engineering and Science. All rights reserved. ABSTRAC Diabetes mellitus is one of the urgent health problems in the world. Diabetes is a condition primarily defined by the level of hyperglycemia giving rise to risk of micro vascular damage. Those who suffer from this disease generally do not realize and tend to overlook the early symptoms. Late recognition of these early symptoms may drive the disease to a more concerning level. One solution to solve this problem is to create an application that may perform early detection of diabetes mellitus so that it does not grow larger. In this article, a new method in performing early detection of diabetes mellitus is suggested. This method is backpropagation with three optimization namely early initialization with Nguyen-Widrow algorithm, learning rate adaptive determination, and determination of weight change by applying momentum coefficient. The observation is conducted by collecting 150 data consisting of 79 diabetes mellitus patient and 71 non diabetes mellitus patient data. The result of this study is the suggested algorithm succeeds in detecting diabetes mellitus with accuracy rate of 99.33%. Optimized backpropagation algorithm may allow the training process goes 12.4 times faster than standard backpropagation. Received Jan 4, 2017 Revised Jan 9, 2018 Accepted Sep 21, 2018 1. INTRODUCTION Diabetes mellitus is one of the world's urgent health problems [1]. According to the data obtained from Word Health Organization (WHO), there were 1.5 million people died from diabetes mellitus in 2012 [1]. It is predicted that the number of people suffering from diabetes mellitus will reach 366 million in 2030 [2]. Diabetes is a condition primarily defined by the level of hyperglycemia giving rise to risk of micro vascular damage [2]. The early symptoms of this disease include polydipsia (excessive thirst), polyphagia (excessive hunger) and polyuria (excessive urination volume). Diabetes mellitus may cause organ dysfunction and failure [3]. y The patients hardly recognize and tend to overlook these early symptoms indicating the risks the disease brings. Medical and laboratory examination are only enforced when the patients suffer from severe pain. Late recognition of the early symptoms results in the increase of the number of diabetes mellitus case. Another aftereffect of late examination and treatment is that they may develop the disease to be more dangerous. One solution to solve this problem is to create an application that may perform early detection of diabetes mellitus so that it does not grow larger. g g Studies concerning this matter have been carried out in several methods, such as combining Regression Tree and Random Forest (RF) [4], Fuzzy Hierarchical Model [5], Genetic Programming [6], Support Vector Machines (SVM), Naïve Bayes [7] and artificial neural network [8],[9]. Input data to be in Journal homepage: http://iaescore.com/journals/index.php/IJECE 3233  Int J Elec & Comp Eng ISSN: 2088-8708 though on these methods can be a digital image, voice, electrocardiogram (ECG) signal and numeric. Image data input includes human body parts. Some examples are iris image [10], [11], face area [12], [13] and magnetic resonance imaging of the brain [14]. Voice data may also be included as a data input based on several parameters that consist of absolute jitter, shimmer, amplitude perturbation quotient, noise-to- harmonic ratio, smoothed amplitude perturbation quotient and relative average perturbation [15]. Artificial neural network is an excellent method to diagnose disease [8], [9], [16-20]. Jayalaksmi and Sansthakumaran point out that artificial neural network may be implanted in diagnosing diabetes mellitus and classifying the early detection of gestational diabetes mellitus [8]. The active parameters involve the number of pregnant times, plasma glucose concentration, blood pressure, triceps skin fold thickness, insulin serum, body mass index, diabetic pedigree function and age. 1. INTRODUCTION In another study, backpropagation was employed to classify the early detection of gestational diabetes mellitus [9]. This study observed 110 data and promoted 10 parameter inputs: family history of diabetes, pre-pregnancy body mass index, history of gestational diabetes, delivery of a large infant, history of miscarriage, abnormal baby in previous pregnancy, history of stillbirth, infections, and history of polycystic ovary syndrome. The weakness of applying backpropagation neural network is it has slower convergence and longer training times [21], [22]. There are several actions taken to recondition this weakness, such as selecting or adjusting the activation function used [22], [23], preparing the data before the training starts [24], refining the weight change of the network with momentum coefficient [21], [25], mending the initialization of early weights [26], rectifying the learning rate [21], [27], and reviving the initialization of the network's early weights [28]. In this article, a new method in performing early detection of diabetes mellitus is proposed. This method is backpropagation with three optimization namely initialization of early weights with Nguyen-Widrow algorithm, learning rate adaptive, and determination of weight change by applying momentum coefficient. The result of this study may give contributions in the new algorithm, the optimized backpropagation algorithm. In addition, the proposed algorithm can be used for early detection of diabetes mellitus disease, so the number of deaths caused by this disease can be reduced. a. Training Data a. Training Data g The data which used in this research originated from the medical records of Dr. H. Suwondo Kendal Hospital's patients in 2016. There are 150 data which covers 79 data of mellitus diabetes patients and 71 data of non-mellitus diabetes patients. The selection method for the training and testing is hold-out method which divides the data randomly into two sets that are training data and testing data. The data composition is 2/3 of training data and 1/3 of testing data. b. Initialization of network weight On the standard backpropagation algorithm initialization of network weight is done by generating random small number, meanwhile in this article, Nguyen-Widrow technique will be used. This technique is introduced by Nguyen and Widrow on two layers neutral network [28]. The process of network training will be stopped if the condition had already been fulfilled. There are two requirements of stopping condition that are: if the value of Mean Squared Error (MSE) resulted by the network is smaller than the specified error value or the epoch of training process is equal to the epoch that has been specified. p d. Feed forward Feed forward process is used to count all the output value on hidden layer and output layer. B k i d. Feed forward Feed forward process is used to count all the output value on hidden layer and output layer. e. Backpropagation d. Feed forward Feed forward process is used to count all the output value on hidden layer and output layer. e Backpropagation p p g Backpropagation process is used to calculate the rate of weight changes on all networks. This calculation includes the value of learning rate and value of network input and also output on every hidden layer. f N t k i ht h p p g Backpropagation process is used to calculate the rate of weight changes on all networks. This calculation includes the value of learning rate and value of network input and also output on every hidden layer. f. Network weight changes 2.1. Backpropagation Optimization 2.1. Backpropagation Optimization Method implemented in this study of early detection of diabetes mellitus is the optimized backpropagation algorithm. Optimization is performed in three approaches namely initialization of early weights with Nguyen-Widrow algorithm, learning rate adaptive, and determination of weight change by applying momentum coefficient. The complete suggested algorithm is depicted in Figure 1. The explanations for each process are as follow: f. Network weight changes f. Network weight changes The network weight changes on standard backpropagation algorithm are calculated by adding the previous network weight and weight changes rate. In this proposed algorithm in addition to adding the weight f. Network weight changes The network weight changes on standard backpropagation algorithm are calculated by adding the previous network weight and weight changes rate. In this proposed algorithm in addition to adding the weight g g The network weight changes on standard backpropagation algorithm are calculated by adding the previous network weight and weight changes rate. In this proposed algorithm in addition to adding the weight Optimization of Backpropagation for Early Detection of... (Rosita Sofiana)  3234 ISSN: 2088-8708  change rate also adds the momentum coefficient. This technique had been applied by Yu and Li to optimize backpropagation algorithm [21]. change rate also adds the momentum coefficient. This technique had been applied by Yu and Li to optimize backpropagation algorithm [21]. p p g g Calculating the Mean Squared Error (MSE) g. Calculating the Mean Squared Error (MSE) g g q MSE calculation process is practiced to count the average value in every epoch on the training process. On the next step, MSE is compared to the specified error value to end the training process. h. Calculating the learning rate value g g In standard backpropagation algorithm, learning rate is not changed in its every epoch. On this algorithm, learning rate value is changed during the training process to maintain the stability of the algorithm. This technique is suggested by Yu and Li [21]. i. Saving the network weight The last step of the network training process is producing the network weight. The weight is saved and used on the testing process. Figure 1. The proposed method in this research Figure 1. The proposed method in this research Figure 1. The proposed method in this research 2.2. Artificial Neutral Network Architecture 2.2. Artificial Neutral Network Architecture Artificial neural network architecture used in this research consists of 12 input unit based on the amount of indicator variable of diabetes mellitus, one hidden layer, and one output unit shown in Figure 2. The input used in this architecture are 12 parameters: age (x1), heredity (x2), Polyuria (x3), Polydipsia (x4), Polyphagia (x5), blood sugar (x6), Infection (x7), weight loss (x8), tingling (x9), fatigue and drowsiness (x10), nearsightedness (x11), and wound (x12). 3.1. Experiment 1 The purpose of this experiment is to determine the effect of momentum values on the accuracy of the optimized backpropagation algorithm to identify diabetes mellitus. The given momentum value variation starts from the range of 0.1 up to 0.9. Meanwhile, other variables are learning rate (α) of 0.6, momentum parameter (µ) of 0.9, maximum epoch of 1000, target error of 0.0001, ratio of learning rate increase (lr_inc) of 1.05, ratio of learning rate decrease (lr_dec) of 0.6 and maximal increase of performance (max_perf) of 0.6. Result of this test is in Table 1. From table 1, it can be seen that the highest epoch number is 64 when the momentum is 0.1 and the lowest epoch number is 30 when the momentum value 0.9 is given. Based on these results can be concluded that the provision of momentum can accelerate the learning process. The cause is the addition of momentum value will reduce the error so that the learning process time is smaller. From table 1, it can also be seen that if the momentum value is raised, then the accuracy level tends to increase. This is proven when giving the momentum value 0.1 to 0.6 yields the same level of accuracy and the momentum value 0.7 to 0.9 increases from the previous value. The greatest accuracy is 99.33% on the momentum coefficient of 0.9. This result is better than the standard backpropagation method proposed by Durairaj and Kalaiselvi with an accuracy of 91% [16]. Table 1. Momentum Value Effect towards Resulted Accuracy Level Momentum Epoch Accuracy (%) MSE 0.1 64 97.33 0.000099808 0.2 62 97.33 0.000097647 0.3 59 97.33 0.000099704 0.4 57 97.33 0.000099456 0.5 63 97.33 0.000099224 0.6 54 97.33 0.000098252 0.7 53 98.67 0.000097597 0.8 58 98.67 0.000097122 0.9 30 99.33 0.000094920 Table 1. Momentum Value Effect towards Resulted Accuracy Level Momentum Epoch Accuracy (%) MSE 3. RESULTS AND ANALYSIS The experiment on this research is classified into two experiments below: f. Network weight changes The unit of output is the status of diabetes millitus (y). 2.2. Artificial Neutral Network Architecture Artificial neural network architecture used in this research consists of 12 input unit based on the amount of indicator variable of diabetes mellitus, one hidden layer, and one output unit shown in Figure 2. The input used in this architecture are 12 parameters: age (x1), heredity (x2), Polyuria (x3), Polydipsia (x4), Polyphagia (x5), blood sugar (x6), Infection (x7), weight loss (x8), tingling (x9), fatigue and drowsiness (x10), nearsightedness (x11), and wound (x12). The unit of output is the status of diabetes millitus (y). Int J Elec & Comp Eng, Vol. 8, No. 5, October 2018 : 3232 - 3237  3235  ISSN: 2088-8708 Int J Elec & Comp Eng Figure 2. Artificial neutral network architecture used in the research Figure 2. Artificial neutral network architecture used in the research Optimization of Backpropagation for Early Detection of... (Rosita Sofiana) 3.2. Experiment 2 p The purpose of this second experiment is to compare the performance between standard backpropagation algorithm and optimized backpropagation algorithm for early detection of diabetes mellitus disease. Tests used several variations of α value of 0.1 to 0.9, maximum epoch of 1000 and error target of 0.0001. The parameters value of optimized backropagation are momentum coefficient of 0.9, ratio of learning rate increase (lr_inc) of 1.05, ratio of learning rate decrease (lr_dec) of 0.6 and maximal increase of performance (max_perf) of 1.06. The results of this experiment are shown in Table 2.  3236  ISSN: 2088-8708 Based on table 2, it can be seen that the increase of learning rate value in backpropagation standard gives decrease of epoch number in range 354 up to 1000. The decrease of epoch number also happened at optimized backpropagation. If the value of learning rate is 0.1 to 0.7 in optimized backpropagation then the epoch number will tend to fall in the range 38 to 62 and if the value of learning rate is given 0.8 and 0.9 then the epoch number increased to 49. The cause is the value of learning rate is too small resulted the learning process takes a long time to reach convergent, but if the value of learning rate is too large process, then the learning will become unstable. From the test results in Table 2, it is seen that optimized backpropagation can decrease the epoch number significantly by 12.4 times compared to standard backpropagation. It can be concluded that optimized backpropagation requires a much faster learning process than the standard backpropagation proposed by [16] and [17] in the same case. The value of learning rate 0.7 resulted in the number of epoch 471 on the backpropagation standard and the epoch number of 38 in the optimized backpropagatiion. Table 2. Performance Comparison between Standard Backpropagation and Optimized Backpropagation Learning Rate (α) Standard Backpropagation Optimized Backpropagation Epoch MSE Epoch MSE 0.1 1000 0.000504265 62 0.0000992774 0.2 1000 0.000197236 61 0.0000971700 0.3 1000 0.000118091 51 0.0000981289 0.4 858 0.000099902 45 0.0000975993 0.5 681 0.000099890 44 0.0000974009 0.6 579 0.000099859 38 0.0000967791 0.7 471 0.000099868 38 0.0000977896 0.8 412 0.000099834 49 0.0000984445 0.9 354 0.000099829 49 0.0000990080 Table 2. Performance Comparison between Standard Backpropagation and Optimized Backpropagation Learning Rate (α) Standard Backpropagation Optimized Backpropagation 4. CONCLUSION Optimized backpropagation algorithm with initialization of early weights with Nguyen-Widrow algorithm, learning rate adaptive, and determination of weight change by applying momentum coefficient for early detection of diabetes mellitus that produced the best accuracy level of 99.33%. Optimized backpropagation is able to accelerate the training process by 12.4 times compare to standard backpropagation. Int J Elec & Comp Eng, Vol. 8, No. 5, October 2018 : 3232 - 3237 Int J Elec & Comp Eng classifier,” IEEE Transactions on Biomedical Engineering , vol. 61, pp. 1027–1033, 2014. [13] S. Ting and B. Zhang, “Diabetes Mellitus Detection Based on Facial Block Texture Features Using the Gabor Filter,” Computational Science and Engineering (CSE), 2014 IEEE 17th International Conference on, IEEE, pp. 1- 6, 2014. [14] G. Khan, et al., “Detection of Cerebral Atrophy in Type-II Diabetes Mellitus by Magnetic Resonance Imaging of Brain,” Journal of Ayub Medical College Abbottabad, vol. 22, pp. 67–70, 2010. [15] D. Chitkara and R. K. Sharma, “Voice Based Detection of Type 2 Diabetes Mellitus,” Advances in Electrical, Electronics, Information, Communication and Bio-Informatics (AEEICB), 2016 2nd International Conference on, IEEE, 2016. [16] M. 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Abdar, et al., “Comparing Performance of Data Mining Algorithms in Prediction Heart Diseases,” International Journal of Electrical and Computer Engineering (IJECE), vol. 5, pp. 1569-1576, 2015. [21] C.-C. Yu and B.-D. Liu, “A backpropagation algorithm with adaptive learning rate and momentum coefficient,” Neural Networks, 2002. IJCNN. Proceedings of the 2002 International Joint Conference on, IEEE, pp. 1218–1223, 2002. 2] K. Eom, et al., “Performance improvement of backpropagation algorithm by automatic activation function ga tuning using fuzzy logic,” Neurocomputing, vol. 50, pp. 439–460, 2003. g g y g p g pp [23] K. V. N. Babu and D. R. Edla, “New Algebraic Activation Function for Multi- Layered Feed Forward Neural Networks,” IETE Journal of Research, vol. 2063, pp. 71–79, 2016. 4] R. Asadi, et al., “New Supervised Multi Layer Feed Forward Neural Network Model to Accelerate Classificati with High Accuracy,” European Journal of Scientific Research, vol. 33, pp. 163–178, 2009. [25] G. P. Drago, M. Morando, and S. Ridella, “An Adaptive Momentum Back Propagation (AMBP),” Neural Computing & Application, vol. 3, pp. 213–221, 1995. REFERENCES [1] World Health Organization, “Global Report on Diabetes,” World Health Organization (WHO), 2016. [2] World Health Organization, “Definition and Diagnosis of Diabetes Mellitus and Intermediate Hyperglyc World Health Organization (WHO), 2006. [3] V. Jakhmola and P. Tangri, “Diabetes Mellitus a Silent Killer: Role of DPP4 Inhibitors in Treatment,” Journal of Pharmaceutical Science And Bioscientific Research (JPSBR), vol. 2, pp. 49–53, 2012. [4] M. T. M. K. Sabariah, et al., “Early Detection of Type II Diabetes Mellitus with Random Forest and Classification and Regression Tree (CART),” Advanced Informatics: Concept, Theory and Application (ICAICTA), IEEE, pp. 238-242, 2014. [5] R. B. Lumanto and E. Irwansyah, “The Early Detection of Diabetes Mellitus (DM) Using Fuzzy Hierarchical Model,” Procedia Computer Science, vol. 59, pp. 312–319, 2015. [6] M. A. Pradhan, et al., “Design of Classifier for Detection of Diabetes Mellitus Using Genetic Programming,” International Conference on Computer Science and Information Technology (ICCSIT’2011), IEEE, pp. 125–130, 2011. [7] B. A. Tama and F. S. Rodiyatul, “An Early Detection Method of Type-2 Diabetes Mellitus in Public Hospital,” Telkomnika, vol. 9, pp. 287–294, 2011. [8] T. Jayalakshmi and A. Santhakumaran, “A Novel Classification Method for Diagnosis of Diabetes Mellitus Using Artificial Neural Networks,” 2010 International Conference on Data Storage and Data Engineering, IEEE, pp. 159-153, 2010. [9] P. S. Muller, et al., “Application of Computational Technique in Design of Classifier for Early Detection of Gestational Diabetes Mellitus,” Applied Mathematical Sciences, vol. 9, pp. 3327–3336, 2015. [10] I. P. D. Lesmana, et al., “Abnormal Condition Detection of Pancreatic Beta-Cells as The Cause of Diabetes Mellitus based on Iris Image,” Instrumentation, Communications, Information Technology, and Biomedical Engineering (ICICI-BME), 2011 2nd International Conference, IEEE, 2011. [11] A. D. Wibawa and M. H. Purnomo, “Early detection on the condition of Pancreas organ as the cause of diabetes mellitus by real time iris image processing,” Circuits and Systems, 2006. APCCAS 2006. IEEE Asia Pacific Conference on, IEEE, pp. 1008-1010, 2006. f pp 2] B. Zhang, et al., “Noninvasive diabetes mellitus detection using facial block color with a sparse representati Int J Elec & Comp Eng, Vol. 8, No. 5, October 2018 : 3232 - 3237  3237  3237  Int J Elec & Comp Eng ISSN: 2088-8708 Int J Elec & Comp Eng p g pp pp 6] R. Asadi and S. Abdul, “Review of Feed Forward Neural Network classification preprocessing techniques Proceedings of the 3rd International Conference on Mathematical Sciences, AIP, pp. 567–573, 2014. [27] S. Iranmanesh, “A Differential Adaptive Learning Rate Method for Back-Propagation Neural Networks,” Proceedings of the 10th WSEAS International Conference on Neural Networks, ACM, pp. 30–34, 2010. f f pp [28] D. Nguyen and B. Widrow, “Improving the Learning Speed of 2-Layer Neural Networks by Choosing Initial Values of the Adaptive Weights,” Neural Networks, 1990 IJCNN International Joint Conference on, IEEE, pp.III- 21-III-26, 1990. Optimization of Backpropagation for Early Detection of... (Rosita Sofiana)
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https://link.springer.com/content/pdf/10.1007/JHEP04(2012)049.pdf
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New physics models of direct CP violation in charm decays
˜The œJournal of high energy physics/˜The œjournal of high energy physics
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Published for SISSA by Springer Published for SISSA by Springer Received: February 20, 2012 Revised: March 22, 2012 Accepted: March 24, 2012 Published: April 11, 2012 Received: February 20, 2012 Revised: March 22, 2012 Accepted: March 24, 2012 Published: April 11, 2012 New physics models of direct CP violation in charm decays JHEP04(2012)049 Wolfgang Altmannshofer,a Reinard Primulando,a,b Chiu-Tien Yua,c and Felix Yua aFermi National Accelerator Laboratory, P.O. Box 500, Batavia, IL 60510, U.S.A. bDepartment of Physics, College of William and Mary, Williamsburg, VA 23187, U.S.A. cDepartment of Physics, University of Wisconsin, Madison, WI 53706, U.S.A. E-mail: waltmann@fnal.gov, rprimulando@email.wm.edu, cyu27@wisc.edu, felixyu@fnal.gov Wolfgang Altmannshofer,a Reinard Primulando,a,b Chiu-Tien Yua,c and Felix Yua aFermi National Accelerator Laboratory, P.O. Box 500, Batavia, IL 60510, U.S.A. bDepartment of Physics, College of William and Mary, Williamsburg, VA 23187, U.S.A. cDepartment of Physics, University of Wisconsin, Madison, WI 53706, U.S.A. E-mail: waltmann@fnal.gov, rprimulando@email.wm.edu, cyu27@wisc.edu, felixyu@fnal.gov Abstract: In view of the recent LHCb measurement of ∆ACP, the difference between the time-integrated CP asymmetries in D →K+K−and D →π+π−decays, we perform a comparative study of the possible impact of New Physics degrees of freedom on the direct CP asymmetries in singly Cabibbo suppressed D meson decays. We systematically discuss scenarios with a minimal set of new degrees of freedom that have renormalizable couplings to the SM particles and that are heavy enough such that their effects on the D meson decays can be described by local operators. We take into account both constraints from low energy flavor observables, in particular D0 −¯D0 mixing, and from direct searches. While models that explain the large measured value for ∆ACP with chirally enhanced chromomagnetic penguins are least constrained, we identify a few viable models that contribute to the D meson decays at tree level or through loop induced QCD penguins. We emphasize that such models motivate direct searches at the LHC. Keywords: Beyond Standard Model, CP violation Keywords: Beyond Standard Model, CP violation ArXiv ePrint: 1202.2866 doi:10.1007/JHEP04(2012)049 Open Access Contents 1 Introduction 2 2 CP asymmetries in neutral D meson decays 4 3 Effective Hamiltonian approach 6 3.1 ∆F = 1 effective Hamiltonian 6 3.2 ∆F = 2 effective Hamiltonian 8 4 New physics contributions at tree level 9 4.1 Flavor changing Z 9 4.2 Flavor changing Z′ 11 4.3 Flavor changing heavy gluon 14 4.4 Charged vector boson 16 4.5 Two Higgs doublet model 16 4.6 Scalar octet 21 4.7 Scalar diquarks 25 5 New physics contributions to gluon penguins 27 2 CP asymmetries in neutral D meson decays JHEP04(2012)049 4 New physics contributions at tree level 4.1 Flavor changing Z 4.2 Flavor changing Z′ 4.3 Flavor changing heavy gluon 4.4 Charged vector boson 4.5 Two Higgs doublet model 4.6 Scalar octet 4.7 Scalar diquarks 1 Introduction Recently, the LHCb collaboration presented the first evidence for CP violation (CPV) in charm quark decays [1]. In particular, a difference between the time-integrated CP asymmetries in D →K+K−and D →π+π− Recently, the LHCb collaboration presented the first evidence for CP violation (CPV) in charm quark decays [1]. In particular, a difference between the time-integrated CP asymmetries in D →K+K−and D →π+π− ∆ACP, LHCb = ACP(K+K−) −ACP(π+π−) = (−0.82 ± 0.21 ± 0.11)% (1.1) (1.1) has been reported, which is non-zero at 3.5σ. This measurement is consistent at about the 1σ level with the previous measurement from CDF [2], and the previous world average from the Heavy Flavor Averaging Group [3]. The new world average, combining the LHCb result with previous measurements of ACP(K+K−) and ACP(π+π−) at BaBar [4], Belle [5] and CDF [2], is [3] JHEP04(2012)049 ∆ACP, World Average = (−0.645 ± 0.180)% . (1.2) (1.2) The interpretation of this measurement as a sign of New Physics (NP) requires a well- understood Standard Model (SM) calculation of this observable. Simple arguments dictate that the SM contribution to direct CPV in D0 decays must be both CKM suppressed and loop suppressed. Concretely, the tree level decays D →K+K−and D →π+π−(we implicitly include both D0 and ¯D0 when discussing neutral D decay modes) only involve the first two quark generations, which cannot access the CP violating Kobayashi-Maskawa (KM) phase. The KM phase does enter into the loop-induced gluon penguin diagram for singly-Cabibbo suppressed D0 decays that thus can provide both the required weak and strong phase difference relative to the leading SM tree amplitude. This implies that the SM prediction is loop suppressed as well as CKM suppressed, and the na¨ıve expec- tation for direct CPV in singly-Cabibbo suppressed D0 decays is parametrically given as O((αs/π)(VubV ∗ cb)/(VusV ∗ cs)) ∼10−4. This leads to the conclusion that the LHCb evidence of CPV at about the percent level is a sign of New Physics. A precise SM calculation, however, is difficult to accomplish. Although tree level and loop level SM contributions to the quark level processes c →us¯s and c →ud ¯d are read- ily calculated, the evaluation of the hadronic matrix elements ⟨K+K−|(¯uΓ1s)(¯sΓ2c)|D0⟩, for example, is not easily performed. In the simplest approach, na¨ıve factorization, the hadronic matrix elements are “factorized” into ⟨K+|(¯uΓ1s|0⟩⟨K−|(¯sΓ2c)|D0⟩which is for- mally the leading term in the heavy charm quark limit. 5 New physics contributions to gluon penguins 6 Conclusions A Hadronic matrix elements in na¨ıve factorization B Anomalous dimensions C Loop functions 37 – 1 – 1 Introduction JHEP04(2012)049 Even though there is large uncertainty in the SM value of ∆ACP, it is nevertheless important and exciting to consider the possibility that we are seeing evidence of NP. Lit- erature prior to the LHCb result emphasized the continued fact that CPV in the charm sector is considered an excellent probe of NP beyond the SM [10–12]. Among the most promising probes of CPV in the charm sector are observables in D0 −¯D0 mixing [13–17] and singly Cabibbo suppressed D decays [18–20]. In fact, since flavor physics observables can probe energy scales much higher than those directly measured, we could potentially expect that NP at the LHC would first be seen from its flavor effects at low energies and only later accessed directly. Our goal is to investigate the possibility that NP is indeed responsible for the large ∆ACP measurement and to outline the corresponding NP parameter space consistent with all experimental constraints for a variety of NP models. Some recent work has discussed the NP possibility both model independently [21] and in the context of various concrete NP scenarios, including up-type flavor changing neutral currents (FCNCs), fourth-generation fermions, R-parity violating supersymmetry, and the MSSM with nonstandard sources of flavor violation [22–26]. Our work differs from these previous analyses since we consider a much broader range of new NP possibilities and apply a full gamut of experimental constraints, both from low energy experiments and collider searches. In particular, we systematically discuss models with a minimal set of new degrees of freedom with renormalizable couplings to the SM particles and are heavy enough such that their effects on the D meson decays can be described by local operators. Specifically, we consider models with new massive neutral gauge bosons that have flavor changing tree level couplings to quarks, models with extended scalar sectors, and models where the D →K+K−and D →π+π−decays are modified at the loop level by gluon penguins. As discussed in [18, 21, 26], the loop induced ∆F = 1 chromomagnetic dipole operator (here and throughout, F refers to charm number except where noted) is, on general grounds, expected to be the least constrained approach for generating large nonstandard effects in D0 meson decays. On the other hand, the effects of four fermion operators that are, for example, induced by tree level exchange of flavor changing NP degrees of freedom, are highly constrained by D0 −¯D0 mixing data. 1 Introduction As the charm mass is close to ΛQCD, however, this approach suffers from large 1/mc power corrections. In particular, so-called annihilation diagrams are ignored, where quarks are pair-produced from the vac- uum to complete the K or π mesons, as are long-range QCD effects such as final state rescattering, where constituent s quarks of a D →K+K−decay rescatter into d quarks of a π+π−final state. Alternative techniques such as the topological diagram approach orga- nize decay and annihilation amplitudes according to weak current insertions and SU(3)F light quark flavor symmetry, and then try to extract amplitudes and phases directly from D0 branching ratio data. Several recent papers have dicussed improved estimates for ∆ACP in the SM. In [6], a NLO QCD factorization calculation is amended by an estimate of the effect of certain – 2 – 1/mc suppressed penguin amplitudes using D0 branching ratio data. Assuming an O(1) strong phase, the authors find the SM can potentially give |∆ACP| ∼0.4%. As their result admittedly neglects several effects which could alternatively reduce or enhance this estimate, they conclude that the measured value of ∆ACP could be reproduced in the SM. Studies that inform magnitudes and phases of D0 meson decay amplitudes directly from data were performed in [7–9] following a topological diagram approach. The authors of [7] arrive at a slightly smaller ∆ACP ∼−0.25% estimate, which remains, they highlight, more than 2σ away from the world average. In [8, 9], the correlation between direct CPV in D →K+K−and π+π−and other D meson decays is emphasized as an important cross-check of the LHCb result. 1/mc suppressed penguin amplitudes using D0 branching ratio data. Assuming an O(1) strong phase, the authors find the SM can potentially give |∆ACP| ∼0.4%. As their result admittedly neglects several effects which could alternatively reduce or enhance this estimate, they conclude that the measured value of ∆ACP could be reproduced in the SM. Studies that inform magnitudes and phases of D0 meson decay amplitudes directly from data were performed in [7–9] following a topological diagram approach. The authors of [7] arrive at a slightly smaller ∆ACP ∼−0.25% estimate, which remains, they highlight, more than 2σ away from the world average. In [8, 9], the correlation between direct CPV in D →K+K−and π+π−and other D meson decays is emphasized as an important cross-check of the LHCb result. 1 Introduction As is well known, the D0 −¯D0 constraints become more effective with heavier NP degrees of freedom [18], leading to the expectation that almost no NP parameter space remains in models where four fermion operators are responsible for nonstandard direct CPV in – 3 – D →K+K−and D →π+π−. Our work shows to what extent this na¨ıve expectation holds true and identifies a few exceptions. We emphasize that each of the viable NP possibilities that can accomodate a large ∆ACP motivates a further phenomenological study focusing on the allowed parameter space identified in this work, which we leave for a future study. In section 2, we review aspects of CPV in neutral D meson decays that are most relevant for our analysis. In section 3, we present the ∆F = 1 and ∆F = 2 effective Hamiltonians that can describe NP contributions to the D →K+K−and D →π+π− decays and to D0 −¯D0 mixing, respectively. The various NP models that contribute to CPV at tree level are discussed in section 4, while the NP models that contribute at loop level are discussed in section 5. We conclude in section 6. Technical details about hadronic matrix elements and renormalization group running, as well as a collection of loop functions can be found in the appendices. D →K+K−and D →π+π−. Our work shows to what extent this na¨ıve expectation holds true and identifies a few exceptions. We emphasize that each of the viable NP possibilities that can accomodate a large ∆ACP motivates a further phenomenological study focusing on the allowed parameter space identified in this work, which we leave for a future study. In section 2, we review aspects of CPV in neutral D meson decays that are most relevant for our analysis. In section 3, we present the ∆F = 1 and ∆F = 2 effective Hamiltonians that can describe NP contributions to the D →K+K−and D →π+π− decays and to D0 −¯D0 mixing, respectively. The various NP models that contribute to CPV at tree level are discussed in section 4, while the NP models that contribute at loop level are discussed in section 5. We conclude in section 6. Technical details about hadronic matrix elements and renormalization group running, as well as a collection of loop functions can be found in the appendices. JHEP04(2012)049 2 CP asymmetries in neutral D meson decays The neutral D meson mass eigenstates D1 and D2 are linear combinations of the strong interaction eigenstates, D0 and ¯D0 |D1,2⟩= p|D0⟩± q| ¯D0⟩. (2.1) (2.1) The factors q and p are given by q p = s M∗ 12 −i 2Γ∗ 12 M12 −i 2Γ12 , (2.2) (2.2) where M12 and Γ12 are the dispersive and absorptive part of the D meson mixing amplitude. CP violation in D meson mixing is signaled by |q/p| ̸= 1 or φ = Arg(q/p) ̸= 0. The normalized mass and width differences, x and y, in the neutral D meson system are given by x = ∆MD Γ = 2τRe q p  M12 −i 2Γ12  , y = ∆ΓD 2Γ = −2τIm q p  M12 −i 2Γ12  , (2.3) (2.3) where the lifetime of the D0 mesons τ = 1/Γ = 0.41 ps [10]. where the lifetime of the D0 mesons τ = 1/Γ = 0.41 ps [10]. where the lifetime of the D0 mesons τ = 1/Γ = 0.41 ps [10]. The time integrated CP asymmetry in the decay of neutral D mesons to a final CP eigenstate f = K+K−, π+π−is defined as The time integrated CP asymmetry in the decay of neutral D mesons to a final CP eigenstate f = K+K−, π+π−is defined as ACP(f) = Γ(D0 →f) −Γ( ¯D0 →f) Γ(D0 →f) + Γ( ¯D0 →f) = Am + Ai + Ad f . (2.4) (2.4) The time integrated CP asymmetry receives contributions from CPV in mixing Am, CPV in interference of decays with and without mixing Ai, and from CPV in the decay itself – 4 – Ad f. The “indirect” CP asymmetries Am and Ai are approximately independent of the final state and depend only on D0 −¯D0 mixing parameters Am = ηf CP y 2  p q − q p  cos φ , (2.5) Ai = ηf CP x 2  p q + q p  sin φ , (2.6) (2.5) (2.6) where ηf CP is the CP parity of the final state. The “direct” CP asymmetry Ad f is instead sensitive to the final state. 2 CP asymmetries in neutral D meson decays The decay amplitudes of singly Cabibbo suppressed D meson decays A(D0 →f) = Af and A( ¯D0 →f) = ¯Af can be written as [18] JHEP04(2012)049 Af = AT f  1 + rfei(δf+φf) , (2.7) ¯Af = ηf CPAT f  1 + rfei(δf−φf) , (2.8) (2.7) (2.8) where AT f is the dominant singly Cabibbo suppressed tree level amplitude, which can be taken real by convention, and rf is the relative size of subleading (“penguin”) amplitudes. With respect to the tree amplitude, the penguin amplitudes can have a relative weak phase φf and a relative strong phase δf. Under the assumption that rf is small, one arrives at the following expression for the direct CP asymmetry Ad f = 2rf sin δf sin φf . (2.9) (2.9) The difference between the time-integrated CP asymmetries in D →K+K−and D → π+π−measured by LHCb is given by [1] The difference between the time-integrated CP asymmetries in D →K+K−and D → π+π−measured by LHCb is given by [1] ∆ACP = Ad K+K−−Ad π+π−+ ∆⟨t⟩ τ (Am + Ai) , (2.10) (2.10) where ∆⟨t⟩/τ = (9.8 ± 0.9)% is a small difference in the average decay times of the D0 mesons in the K+K−and π+π−sample [1]. Given the existing bounds on the indirect CP asymmetries [3], the LHCb measurement of ∆ACP is an excellent approximation of the difference in the direct CP asymmetries. As already mentioned in the Introduction, charm CPV in the SM is strongly Cabibbo suppressed. Furthermore, in the SM, direct CP violation in D →K+K−and D →π+π− decays comes from the interference of the tree level contribution with a loop suppressed penguin amplitude and correspondingly, rf ∼O(αs/π)(VubV ∗ cb)/(VusV ∗ cs) ∼10−4. Even though the weak phase of the SM penguin is large (γ ∼70◦) and assuming a maximal strong phase, a na¨ıve SM estimate for ∆ACP is therefore smaller than the global average by at least an order of magnitude. Sizable direct CP asymmetries in the D →K+K−and D →π+π−decays are only possible in the SM if the relevant hadronic matrix elements are strongly enhanced [27]. Despite several recent studies [6–8], it remains unclear to what extent such an enhance- ment is present and whether the value of ∆ACP measured by LHCb can be explained within the SM. In the following we investigate the possibility that the measured ∆ACP is due to New Physics. – 5 – 3.1 ∆F = 1 effective Hamiltonian In the New Physics frameworks discussed below, contributions to the singly Cabibbo sup- pressed D →K+K−and D →π+π−decays can be described by the following effective Hamiltonian Heff=  X p λp 2 X i=1  C(1)p i O(1)p i + ˜C(1)p i ˜O(1)p i  + X i  C(1) i O(1) i + ˜C(1) i ˜O(1) i   + h.c. , ( ) JHEP04(2012)049 (3.1) where λp = VcpV ∗ up, and the operators O(1) i are given by where λp = VcpV ∗ up, and the operators O(1) i are given by O(1)p 1 = (¯up)V −A(¯pc)V −A , (3.2a) O(1)p 2 = (¯uαpβ)V −A(¯pβcα)V −A , (3.2b) O(1) 3 = (¯uc)V −A X q (¯qq)V −A , (3.2c) O(1) 4 = (¯uαcβ)V −A X q (¯qβqα)V −A , (3.2d) O(1) 5 = (¯uc)V −A X q (¯qq)V +A , (3.2e) O(1) 6 = (¯uαcβ)V −A X q (¯qβqα)V +A , (3.2f) O(1) 7 = 3 2(¯uc)V −A X q eq(¯qq)V +A , (3.2g) O(1) 8 = 3 2(¯uαcβ)V −A X q eq(¯qβqα)V +A , (3.2h) O(1) 9 = 3 2(¯uc)V −A X q eq(¯qq)V −A , (3.2i) O(1) 10 = 3 2(¯uαcβ)V −A X q eq(¯qβqα)V −A , (3.2j) O(1) 8g = gs 8π2 mc¯uσµν(1 + γ5)cβta αβGa µν , (3.2k) O(1) S1 = (¯uPLs)(¯sPLc) , (3.2l) O(1) S2 = (¯uαPLsβ)(¯sβPLcα) , (3.2m) O(1) T1 = (¯uσµνPLs)(¯sσµνPLc) , (3.2n) O(1) T2 = (¯uασµνPLsβ)(¯sβσµνPLcα) . (3.2o) O(1)p 1 = (¯up)V −A(¯pc)V −A , (3.2a) O(1)p 2 = (¯uαpβ)V −A(¯pβcα)V −A , (3.2b) O(1) 3 = (¯uc)V −A X q (¯qq)V −A , (3.2c) O(1) 4 = (¯uαcβ)V −A X q (¯qβqα)V −A , (3.2d) O(1) 5 = (¯uc)V −A X q (¯qq)V +A , (3.2e) O(1) 6 = (¯uαcβ)V −A X q (¯qβqα)V +A , (3.2f) O(1) 7 = 3 2(¯uc)V −A X q eq(¯qq)V +A , (3.2g) O(1) 8 = 3 2(¯uαcβ)V −A X q eq(¯qβqα)V +A , (3.2h) O(1) 9 = 3 2(¯uc)V −A X q eq(¯qq)V −A , (3.2i) O(1) 10 = 3 2(¯uαcβ)V −A X q eq(¯qβqα)V −A , (3.2j) O(1) 8g = gs 8π2 mc¯uσµν(1 + γ5)cβta αβGa µν , (3.2k) O(1) S1 = (¯uPLs)(¯sPLc) , (3.2l) O(1) S2 = (¯uαPLsβ)(¯sβPLcα) , (3.2m) O(1) T1 = (¯uσµνPLs)(¯sσµνPLc) , (3.2n) O(1) T2 = (¯uασµνPLsβ)(¯sβσµνPLcα) . 3.1 ∆F = 1 effective Hamiltonian (3.2o) O(1)p 1 = (¯up)V −A(¯pc)V −A , (3.2a) O(1)p 2 = (¯uαpβ)V −A(¯pβcα)V −A , (3.2b) O(1) 3 = (¯uc)V −A X q (¯qq)V −A , (3.2c) O(1) 4 = (¯uαcβ)V −A X q (¯qβqα)V −A , (3.2d) O(1) 5 = (¯uc)V −A X q (¯qq)V +A , (3.2e) O(1) 6 = (¯uαcβ)V −A X q (¯qβqα)V +A , (3.2f) O(1) 7 = 3 2(¯uc)V −A X q eq(¯qq)V +A , (3.2g) O(1) 8 = 3 2(¯uαcβ)V −A X q eq(¯qβqα)V +A , (3.2h) O(1) 9 = 3 2(¯uc)V −A X q eq(¯qq)V −A , (3.2i) O(1) 10 = 3 2(¯uαcβ)V −A X q eq(¯qβqα)V −A , (3.2j) O(1) 8g = gs 8π2 mc¯uσµν(1 + γ5)cβta αβGa µν , (3.2k) O(1) S1 = (¯uPLs)(¯sPLc) , (3.2l) O(1) S2 = (¯uαPLsβ)(¯sβPLcα) , (3.2m) O(1) T1 = (¯uσµνPLs)(¯sσµνPLc) , (3.2n) O(1) T2 = (¯uασµνPLsβ)(¯sβσµνPLcα) . (3.2o) (3.2o) The index q runs over all active quark flavors, the index p runs over all active down type quark flavors, α and β are color indices (that are implicitly summed over), eq is the electric – 6 – charge of the quark q, (V ± A) refers to the Dirac structures γµ(1 ± γ5), PR,L = 1 2(1 ± γ5) and σµν = i 2(γµγν −γνγµ). The operators ˜O(1){p} i are obtained from O(1){p} i by replacing γ5 →−γ5. ( ) The operators O(1)p 1,2 are the so-called current-current operators. In the SM, tree level W exchange generates at the matching scale the Wilson coefficient C(1)p 1 ≃GF / √ 2. The QCD penguin operators O(1) 3,4,5,6 and the chromomagnetic operator O(1) 8g are first generated at O(αs) and proportional to VubV ∗ cb. The chromomagnetic operator is proportional to the charm quark mass but can be chirally enhanced by v/mc from NP. The QED penguin operators O(1) 7,8,9,10 are also proportional to VubV ∗ cb. They are of O(α) and negligible in the D →K+K−and D →π+π−decays in the SM. In the NP models discussed below that have tree level contributions to the D →K+K−and D →π+π−decay amplitudes, however, the QED penguin operators can be relevant. The scalar operators O(1) S1,S2 become important in the context of the 2HDM discussed in section 4.5 and the scalar octet discussed in section 4.6. The tensor operators O(1) T1,T2 do not contribute to D →K+K−and D → π+π−decays in na¨ıve factorization. 3.1 ∆F = 1 effective Hamiltonian We consider them nonetheless, because they mix with the scalar operators under renormalization group running. JHEP04(2012)049 The ratio rf that enters the expression for the direct CP asymmetry eq. (2.9) can be written as a function of the Wilson coefficients appearing in eq. (3.1). We use the results from [18] for the hadronic matrix elements that are obtained using na¨ıve factorization for O(1) 1,...,6 and QCD factorization [28, 29] for O(1) 8g . The matrix elements obtained in na¨ıve factorization are formally the leading terms in an expansion in αs and ΛQCD/mc [28, 29]. In the case of D meson decays, however, and as mentioned in the Introduction, it is known that power corrections, in particular annihilation contributions, which are formally suppressed by 1/mc, can be equally important [6, 8, 18]. The na¨ıve factorization results can therefore only be considered as rough estimates and in our numerical analysis, we will allow for enhancements up to a plausible factor of 3 [6, 18]. For our analysis, we extend the results for the hadronic matrix elements given in [18] by including the QED penguin and scalar operators (see appendix A for details). We find rfeiφf ≃1 λp C(1)p 1 + C(1)p 2 Nc !−1 λp(C(1)p 2 )NP Nc +C(1) 4 + C(1) 3 Nc −C(1) 10 2 −C(1) 9 2Nc −3αs 4π N2 c −1 N2c C(1) 8g +χf C(1) 6 + C(1) 5 Nc −C(1) 8 2 −C(1) 7 2Nc −C(1) S1 8 −C(1) S2 8Nc −αs 4π N2 c −1 N2c C(1) 8g ! +(C(1) i ↔˜C(1) i ) ! , (3.3) ! (3.3) where Nc = 3 is the number of colors and p = s, f = K+K−for the D →K+K−decay and p = d, f = π+π−for the D →π+π−decay. The chiral factors χf are approximately given by χK+K−≃2m2 K mcms , χπ+π−≃ 2m2 π mc(md + mu) , (3.4) (3.4) with all quark masses evaluated at the scale of the D meson µ ≃mD ≃1.8 GeV. All the Wilson coefficients in eq. (3.3) are evaluated at this scale. We use LO renormalization group – 7 – running for C(1) 1,...,10 and C(1) S1,S2,T1,T2 as well as for C(1) 8g to evolve the Wilson coefficients from the high matching scale, where NP degrees of freedom are integrated out, down to µ ≃mD. The corresponding anomalous dimensions are collected in appendix B. 1The absorptive part of the mixing amplitude Γ12 is not sensitive to new short distance dynamics. 3.1 ∆F = 1 effective Hamiltonian We do not include 2-loop mixing between C(1) 8g and the other Wilson coefficients. In view of the large uncertainties in the evaluation of the hadronic matrix elements, we consider this approximate approach to be fully justified. While there are no strong phase differences between the several operator matrix ele- ments in the na¨ıve factorization approach, they can be generated by large power corrections or final state interactions. Throughout this work, we will assume O(1) strong phase differ- ences, following [6, 18]. JHEP04(2012)049 3.2 ∆F = 2 effective Hamiltonian We apply the most recent averages and 1σ errors of the experimental results on the D0 −¯D0 mixing parameters [3] allow the long distance contributions to vary in the ranges MLD 12 ∈[−0.02, 0.02] ps−1 and ΓLD 12 ∈[−0.04, 0.04] ps−1 [37], such that by themselves they can saturate the experimental values. We apply the most recent averages and 1σ errors of the experimental results on the D0 −¯D0 mixing parameters [3] x = (0.63+0.19 −0.20)% , y = (0.75 ± 0.12)% , q/p| = 0.89+0.17 −0.15 , φ = (−10.1+9.4 −8.8)◦, (3.8) (3.8) at the 2σ level throughout our analysis. We note that in many of our minimal field content scenarios, the NP vertices used in the ∆F = 1 operators are also used for the ∆F = 2 operators, leading to a phase relation 2φF=1 = φF=2 between the CPV for D0 decays and the CPV for D0 −¯D0 mixing. This relation implies that the D0 −¯D0 mixing constraint is best satisfied by eliminating the CPV in D0 −¯D0 mixing and saturating the D0 −¯D0 mixing transition amplitude. On the other hand, in non-minimal constructions, this phase relation could be different, possibly making the null observation of CPV in D0 −¯D0 mixing the more restrictive constraint. JHEP04(2012)049 4 New physics contributions at tree level We concentrate on New Phyiscs models where the new degrees of freedom are heavy enough such that their effects in low energy observables can be reliably described by the local operators introduced in section 3. We do not consider scenarios with very light mediators, which is beyond the scope of this work. Moreover, we focus on models where the new degrees of freedom have renormalizable couplings to SM degrees of freedom. In this section, we analyze models where at most one new field is added to the SM. We first discuss extensions of the SM in which a massive neutral gauge boson leads to tree level contributions to the D →K+K−and D →π+π−decay ampitudes. We consider a flavor changing coupling of the SM Z boson in section 4.1, a flavor changing Z′ in section 4.2 and a flavor changing heavy gluon in section 4.3. We also comment on the possible effects of a new charged gauge boson in section 4.4. Then, we analyze models with extended scalar sectors, namely a 2 Higgs doublet model with Minimal Flavor Violation in section 4.5, a model with a scalar octet in section 4.6 and a model with a scalar diquark in section 4.7. Models that contain more than one non-SM particle and where NP contributions to the D meson decays are first generated at the one loop level are discussed in section 5. 3.2 ∆F = 2 effective Hamiltonian In the models discussed below, the most important flavor constraints come often from D0 −¯D0 and K −¯K mixing. New Physics contributions to meson mixing can be described by the effective Hamiltonian Heff= 5 X i=1 C(2) i O(2) i + 3 X i=1 ˜C(2) i ˜O(2) i + h.c. . (3.5) (3.5) In the case of D0 −¯D0 mixing, the most important operators for our analysis are given by In the case of D0 −¯D0 mixing, the most important operators for our analysis are given by O(2)D 1 = (¯uαγµPLcα)(¯uβγµPLcβ) , ˜O(2)D 1 = (¯uαγµPRcα)(¯uβγµPRcβ) , ˜O(2)D 2 = (¯uαPRcα)(¯uβPRcβ) . (3.6) 1 ( γµ )( βγ β) ˜O(2)D 2 = (¯uαPRcα)(¯uβPRcβ) . (3.6) (3.6) In the case of K −¯K mixing, the operators most relevant for our analysis are In the case of K −¯K mixing, the operators most relevant for our analysis are O(2)K 1 = ( ¯dαγµPLsα)( ¯dβγµPLsβ) , ˜O(2)K 1 = ( ¯dαγµPRsα)( ¯dβγµPRsβ) , O(2)K 4 = ( ¯dαPLsα)( ¯dβPRsβ) , O(2)K 5 = ( ¯dαPLsβ)( ¯dβPRsα) . (3.7) (3.7) In the above expressions, PR,L = 1 2(1 ± γ5) and α, β are color indices (that are implicitly summed over). ( ) The Wilson coefficients C(2) i are again obtained by integrating out the NP degrees of freedom at a scale of the order of the mass of the new particles. Using renormalization group evolution [30, 31], these coefficients are subsequently run down to the low scale where the hadronic matrix elements [32–34] are given. Combining Wilson coefficients with the hadronic matrix elements gives the NP contribution to the dispersive part of the mixing amplitude M12.1 In the case of D0 −¯D0 mixing, the SM contributions to neither the dispersive part nor the absorptive part of the mixing amplitude can be predicted reliably as they are dominated by long distance effects [35, 36]. In our numerical analysis, we 1The absorptive part of the mixing amplitude Γ12 is not sensitive to new short distance dynamics. – 8 – allow the long distance contributions to vary in the ranges MLD 12 ∈[−0.02, 0.02] ps−1 and ΓLD 12 ∈[−0.04, 0.04] ps−1 [37], such that by themselves they can saturate the experimental values. 4.1 Flavor changing Z We consider a flavor changing coupling of the SM Z boson to the right-handed charm and up quark We consider a flavor changing coupling of the SM Z boson to the right-handed charm and up quark (4.1) Lint = Xcu¯cRγµuRZµ + h.c. , (4.1) where Xcu is a complex parameter. A complementary setup, where flavor changing cou- plings involving the top quark generate an effective c →u transition at the loop level by a double flavor flip c →t →u, is discussed in [26]. Flavor changing Z couplings can appear in various scenarios [38], for example in models with non-sequential generations of quarks [39] – 9 – (a) (b) Figure 1. Example tree level Feynman diagrams that contribute (a) to the D →K+K−and D →π+π−decay amplitudes and (b) to D0 −¯D0 mixing in the cases of a flavor changing Z, Z′ and heavy gluon G′. (a) (b) (a) (b) Figure 1. Example tree level Feynman diagrams that contribute (a) to the D →K+K−and D →π+π−decay amplitudes and (b) to D0 −¯D0 mixing in the cases of a flavor changing Z, Z′ and heavy gluon G′. and also in models with extra U(1) gauge symmetries [40], or can be loop induced as in SUSY models. In the absence of SU(2)L breaking sources, the ¯cRuRZ coupling has the form of a charge radius interaction and vanishes for q2 →0, where q is the momentum of the Z boson. The dominant contribution to the coupling Xcu is therefore in general expected to be proportional to v2/Λ2 NP, where v is the Higgs vacuum expectation value (vev) and ΛNP is the NP scale where the flavor changing Z coupling is generated. JHEP04(2012)049 As shown in diagram (a) of figure 1, the Xcu coupling leads to tree level contributions to the Wilson coefficients ˜C(1) 5 , ˜C(1) 7 and ˜C(1) 9 ˜C(1) 5 = −1 3 g 2cW X∗ cu 4M2 Z , ˜C(1) 7 = 2 3gcW X∗ cu 4M2 Z , ˜C(1) 9 = −2 3 gs2 W cW X∗ cu 4M2 Z . (4.2) (4.2) The flavor changing ¯cuZ coupling also inevitably generates tree level contributions to D0 − ¯D0 mixing ˜C(2)D 1 = (X∗ cu)2 2M2 Z . (4.3) (4.3) If the Z boson has flavor changing couplings to left-handed quarks, 1-loop contributions to ϵ′/ϵ would also be generated. 4.1 Flavor changing Z In order to avoid this constraint, we restrict ourselves to the ¯cRuRZ coupling. In figure 2, we show the regions in the |Xcu| — Arg(Xcu) plane that are compatible with the range for ∆ACP in eq. (1.2) at the 1σ level. The green (solid) band is obtained using the expressions for the decay amplitude in na¨ıve factorization. The blue (dashed) band assumes an enhancement of the hadronic matrix elements by a factor of 3. The red (dash-dotted) region is excluded by the constraints from D0 −¯D0 mixing. The D0 −¯D0 constraints are minimized for Arg(Xcu) = 0, π/2, π, 3π/2, where constraints from CPV in D0 −¯D0 are not effective and the dominant constraint comes from the normalized mass difference x. Indeed, if no enhancement of the hadronic matrix elements is assumed, sizeable NP effects in ∆ACP are only compatible with D0 −¯D0 mixing in a small corner of parameter space with Arg(Xcu) ≃π/2, 3π/2. Still, barring the finetuned situations – 10 – Figure 2. Regions in the |Xcu| — Arg(Xcu) plane compatible with the data on ∆ACP, WA at the 1σ level in the model with a flavor changing Z. The green (solid) band corresponds to the expressions for the decay amplitude in na¨ıve factorization, the blue (dashed) band assumes an enhancement of the hadronic matrix elements by a factor of 3. The red (dash-dotted) region is excluded by the D0 −¯D0 mixing constraints. JHEP04(2012)049 Figure 2. Regions in the |Xcu| — Arg(Xcu) plane compatible with the data on ∆ACP, WA at the 1σ level in the model with a flavor changing Z. The green (solid) band corresponds to the expressions for the decay amplitude in na¨ıve factorization, the blue (dashed) band assumes an enhancement of the hadronic matrix elements by a factor of 3. The red (dash-dotted) region is excluded by the D0 −¯D0 mixing constraints. Arg(Xcu) = π/2, 3π/2, sizeable NP effects in ∆ACP also imply indirect CPV in D0 −¯D0 mixing close to the current experimental bounds. The required size of the flavor changing coupling v2/Λ2 NP ∝|Xcu| ≃10−4 points towards a NP scale of ΛNP ≲few × 10 TeV, where this coupling is generated and not necessarily within the immediate reach of direct searches. 4.2 Flavor changing Z′ Next, we consider a leptophobic massive Z′ gauge boson with tree level flavor chang- ing couplings to right-handed up and charm quarks. Models of this type can be easily constructed if the Z′ couples with SM degrees of freedom through higher dimensional op- erators [41]. Flavor changing couplings of a Z′ can also arise, for example, in models with family non-universal couplings [40, 42]. We parameterize the interactions of the Z′ in the following way Lint = gL¯ui Lγµui LZ′ µ + gu¯ui Rγµui RZ′ µ +gL ¯di Lγµdi LZ′ µ + gd ¯di Rγµdi RZ′ µ +Xcu¯cRγµuRZ′ µ + h.c. , (4.4) (4.4) where the flavor universal couplings gu, gd and gL are free real parameters and the (small) flavor changing coupling Xcu is a free, complex parameter. We restrict ourselves to a tree level c →u coupling and do not consider t →c and t →u couplings that could induce the c →u transition at the loop level. – 11 – Figure 3. The MZ′ −|Xcu| plane, setting Arg(Xcu) = π/2. In the left plot, ∆ACP is evaluated in na¨ıve factorization and in the right plot, we allow for an enhancement by a factor of 3. Along the solid green and blue lines the NP contributions to ∆ACP match the world average. The different green and blue lines correspond to different common choices of the flavor conserving couplings gu = gd = gL as indicated. The black dashed line shows the constraint on the flavor conserving coupling from dijet searches. The red (dash-dotted) region is excluded by D0 −¯D0 mixing constraints. JHEP04(2012)049 Figure 3. The MZ′ −|Xcu| plane, setting Arg(Xcu) = π/2. In the left plot, ∆ACP is evaluated in na¨ıve factorization and in the right plot, we allow for an enhancement by a factor of 3. Along the solid green and blue lines the NP contributions to ∆ACP match the world average. The different green and blue lines correspond to different common choices of the flavor conserving couplings gu = gd = gL as indicated. The black dashed line shows the constraint on the flavor conserving coupling from dijet searches. The red (dash-dotted) region is excluded by D0 −¯D0 mixing constraints. 4.2 Flavor changing Z′ Depending on whether the flavor conserving couplings of the Z′ are to left-handed or right-handed quarks, the flavor changing ¯cuZ′ coupling can induce tree level contributions to the Wilson coefficients ˜C(1) 5 , ˜C(1) 3 , and ˜C(1) 9 Depending on whether the flavor conserving couplings of the Z′ are to left-handed or right-handed quarks, the flavor changing ¯cuZ′ coupling can induce tree level contributions to the Wilson coefficients ˜C(1) 5 , ˜C(1) 3 , and ˜C(1) 9 ˜C(1) 3 = (gu + 2gd) 3 X∗ cu 4M2 Z′ , ˜C(1) 9 = 2(gu −gd) 3 X∗ cu 4M2 Z′ , ˜C(1) 5 = gLX∗ cu 4M2 Z′ . (4.5) (4.5) If we assume gu = gd, then only contributions to ˜C(1) 3 and ˜C(1) 5 are generated. The con- tributions from ˜C(1) 3,5,9 to the D →K+K−and D →π+π−decay amplitudes are color suppressed. The contribution from ˜C(1) 5 is helicity enhanced. The flavor changing ¯cuZ′ coupling also induces tree level contributions to D0 −¯D0 mixing ( )2 ˜C(2)D 1 = (X∗ cu)2 2M2 Z′ . (4.6) (4.6) We highlight the following point: as both the NP contributions to the ∆F = 2 mixing amplitude and the ∆F = 1 decay amplitudes are described by dimension 6 operators, they decouple with the NP mass squared. Yet while the ∆F = 2 amplitude is obviously proportional to the square of the flavor changing coupling, the ∆F = 1 amplitude is linearly proportional in this coupling. Correspondingly, the constraint from D0−¯D0 mixing – 12 – becomes more effective with heavier NP mass. Analogous arguments hold in all the other NP scenarios discussed in this work. If the Z′ boson has flavor changing couplings to left-handed quarks, tree level contri- butions to K −¯K mixing would be unavoidably generated since the ¯cLuLZ′ and ¯sLdLZ′ couplings are related by the CKM matrix due to SU(2)L invariance. Since constraints coming from K −¯K mixing are considerably stronger than those coming from D0 −¯D0 mixing, we restrict ourselves to the ¯cRuRZ coupling. As a result, 1-loop contributions to ϵ′/ϵ that can lead to constraints are also absent. The dijet searches at hadron colliders set additional constraints on the model. In this paper, we consider the searches at UA2 [43], CDF [44] and CMS [45]. 4.2 Flavor changing Z′ The UA2 collaboration probed the light dijet mass region, from 130 GeV to 300 GeV, while the CDF search covers a dijet mass range from 260 GeV to 1.4 TeV. A higher mass range, 1.0 −4.1 TeV, is probed by the CMS experiment. There are other dijet searches from the DØ [46] and ATLAS experiments [47]. The DØ collaboration analyzed 109 pb−1 of data, however, while CDF analyzed 1.13 fb−1, indicating the DØ bound is less competitive than the one from CDF. The ATLAS bound is expected to be comparable with the CMS bound since both of the experiments analyzed 1 fb−1 of data. JHEP04(2012)049 We simulate the Z′ production using MadGraph 5 [48], and the width of the Z′ is calculated with CompHEP [49], varying gu = gd = gL. We compare the simulated cross section with the limit on dijet production from UA2 (figure 2 of [43]), CDF (table I of [44]) and CMS (table 1 of [45]). In calculating the bound, we ignore Xcu since it is at least one order of magnitude smaller than gu, gd and gL. The plots in figure 3 show the MZ′ −|Xcu| plane, setting Arg(Xcu) = π/2. In the left plot, ∆ACP is evaluated in the na¨ıve factorization approach, while in the right plot we allow for an enhancement of the hadronic matrix elements by a factor of 3. Along the green (solid, left plot) and blue (solid, right plot) lines, the NP contributions to ∆ACP match the world average. The different green or blue lines correspond to different choices of the flavor conserving couplings gu = gd = gL = 0.1, 0.2, or 0.5. The region below the black (dashed) line requires a flavor conserving coupling that is excluded by dijet searches. The red (dash-dotted) region is excluded by the constraints from D0 −¯D0 mixing. The choice Arg(Xcu) = π/2 corresponds to a maximal phase for the NP contributions to the D →K+K−and D →π+π−decays while simultaneously minimizing the constraint from D0 −¯D0 mixing. Choosing Arg(Xcu) = π/3 would lead to O(1) phases both in the decays and in D0 −¯D0 mixing, and the corresponding constraint would be more stringent by a factor of ∼2. As expected, the D0 −¯D0 constraint becomes more effective with larger Z′ mass. 4.2 Flavor changing Z′ We observe that even allowing for an enhancement in ∆ACP by a factor of three, the D0 −¯D0 mixing constraint in combination with dijet searches rules out a Z′ as a tree level NP explanation for the measured ∆ACP. We do not consider Z′ masses below 100 GeV, which would be constrained from Z −Z′ mixing [50], but the exact constraints would be model dependent and are beyond the scope of this work. – 13 – Figure 4. As figure 3 but in the model with flavor changing heavy gluon. The shaded vertical bands are excluded by dijet pair searches at ATLAS (left band) and CMS (right band). JHEP04(2012)049 Figure 4. As figure 3 but in the model with flavor changing heavy gluon. The shaded vertical bands are excluded by dijet pair searches at ATLAS (left band) and CMS (right band). 4.3 Flavor changing heavy gluon (4.9) (4.9) Compared to the Z′ case, the contribution to D0 −¯D0 mixing is suppressed by a factor |(1−Nc)/(2Nc)| = 1/3. As discussed for the Z′ case, we do not consider flavor changing cou- plings to left-handed quarks to avoid the stringent constraints from K −¯K mixing and ϵ′/ϵ. Collider constraints come again from dijet searches at the hadron colliders, and we evaluate them in a similar fashion to the Z′ case. Additional constraints arise from recent results on four jet searches at the LHC, searching for pair production of dijet resonances. Pair production of the heavy gluon is fixed by QCD to good approximation and the pro- duction cross section depends only on the G′ mass [53]. The mass range from 100 GeV to 200 GeV is covered by an ATLAS search using 34 pb−1 [54] while a CMS search using 2.2 fb−1 [55] starts at 320 GeV. The intermediate region from 200 GeV to 320 GeV is not considered in the CMS search because of the multijet trigger turn-on effects on the QCD background fit curve, which well models the QCD multijet background above 320 GeV. We simulate pair production of the G′ resonance using Madgraph 5. We find that a G′ mass in the ranges 100 GeV < MG′ < 200 GeV is excluded. Assuming the signal acceptance to be 3% (13%), which corresponds to the lowest (highest) signal efficiency found in [55], we find that a G′ mass from 320 GeV up to 720 GeV (1000 GeV) is excluded. We conservatively use the stronger bound derived from an acceptance of 13% in the following discussion. JHEP04(2012)049 The plots in figure 4 show the MG′ −|Xcu| plane with Arg(Xcu) = π/2 in order to minimize the constraint from D0 −¯D0 mixing. In the left plot, ∆ACP is evaluated in the na¨ıve factorization approach, while in the right plot we allow for an enhancement by a factor of 3. The NP contributions to ∆ACP match the world average along the green (solid, left plot) and blue (solid, right plot) lines. The different green or blue lines correspond to different choices of the flavor conserving couplings gu = gd = gL = 0.1, 0.2, or 0.5. The region below the black (dashed) line requires a flavor conserving coupling that is excluded by dijet resonance searches. The red (dash-dotted) region is excluded by the constraints from D0−¯D0 mixing. 4.3 Flavor changing heavy gluon Heavy color octet vector bosons that have flavor changing couplings to SM quarks can arise, for example, in models with warped extra dimensions [51] and also in models of axigluons with family non-universal couplings [52]. We concentrate again on a direct tree level coupling between right-handed up and charm quarks Lint = gL¯ui LγµT aui L(G′)a µ + gu¯ui RγµT aui R(G′)a µ +gL ¯di LγµT adi L(G′)a µ + gd ¯di RγµT adi R(G′)a µ +Xcu¯cRγµT auR(G′)a µ + h.c. . (4.7) (4.7) The flavor universal couplings gu, gd and gL are free real parameters: the (small) flavor violating coupling Xcu is a free, complex parameter. The heavy gluon can generate tree level contributions to ˜C(1) 3,4,5,6 and ˜C(1) 9,10 ˜C(1) 4 = (gu + 2gd) 3 X∗ cu 8M2 G′ , ˜C(1) 3 = −1 Nc ˜C(1) 4 , ˜C(1) 10 = 2(gu −gd) gu + 2gd ˜C(1) 4 , ˜C(1) 9 = −1 Nc ˜C(1) 10 , ˜C(1) 6 = gLX∗ cu 8M2 G′ , ˜C(1) 5 = −1 Nc ˜C(1) 6 . (4.8) (4.8) To generate ˜C(1) 3,4 and ˜C(1) 9,10, the RH flavor conserving coupling is required, while for ˜C(1) 5,6 the LH flavor conserving coupling is required. If we assume gu = gd, ˜C(1) 9,10 are absent. The Wilson coefficients ˜C(1) 3,5,9 are color suppressed, and furthermore, their contributions to the decay amplitudes are color suppressed. The contributions to the decay amplitudes from ˜C(1) 5 and ˜C(1) 6 are helicity enhanced. Due to the presence of ˜C(1) 6 , we expect slightly larger NP contributions to ∆ACP than the Z′ scenario. To generate ˜C(1) 3,4 and ˜C(1) 9,10, the RH flavor conserving coupling is required, while for ˜C(1) 5,6 the LH flavor conserving coupling is required. If we assume gu = gd, ˜C(1) 9,10 are absent. The Wilson coefficients ˜C(1) 3,5,9 are color suppressed, and furthermore, their contributions to the decay amplitudes are color suppressed. The contributions to the decay amplitudes from ˜C(1) 5 and ˜C(1) 6 are helicity enhanced. Due to the presence of ˜C(1) 6 , we expect slightly larger NP contributions to ∆ACP than the Z′ scenario. – 14 – Similar to the models discussed before, the flavor changing ¯cuG′ coupling also leads to tree level contributions to D0 −¯D0 mixing ˜C(2)D 1 = 1 −Nc 2Nc (X∗ cu)2 2M2 G′ . 4.3 Flavor changing heavy gluon The vertical bands are excluded by the dijet pair searches at ATLAS (left band) and CMS (right band). Because of different O(1) factors in the D →K+K−and D →π+π−decay amplitudes and D0 −¯D0 mixing compared to the Z′, a heavy gluon appears slightly better suited to generate nonstandard effects in ∆ACP. Yet only after allowing for an enhancement in ∆ACP by a factor of 3 can the combined constraints from D0 −¯D0 mixing and dijet searches be made compatible with the measured ∆ACP. The corresponding corner of parameter space is characterized by light G′ masses MG′ ≲300 GeV. As the currently available results for dijet pair searches do not exclude the range between 200 GeV and 320 GeV, a heavy gluon cannot be ruled out as a possible NP explanation of the observed ∆ACP. If a heavy gluon is indeed responsible for the large value of ∆ACP, indirect CPV in D0 −¯D0 mixing is also expected to be close to the current experimental bounds. – 15 – (a) (b) (c) (d) (e) Figure 5. Example Feynman diagrams that contribute to (a) the D →K+K−and D →π+π− decay amplitudes, (b) D0 −¯D0 mixing, (c) K −¯K mixing, (d) the B+ →τν decay and (e) the Bd →Xsγ decay in the discussed 2HDM with MFV. (c) (b) (a) (a) (d) (e) JHEP04(2012)049 (d) (e) Figure 5. Example Feynman diagrams that contribute to (a) the D →K+K−and D →π+π− decay amplitudes, (b) D0 −¯D0 mixing, (c) K −¯K mixing, (d) the B+ →τν decay and (e) the Bd →Xsγ decay in the discussed 2HDM with MFV. 4.4 Charged vector boson We consider a new vector boson with charge ±1 that couples to right-handed up and down type quarks We consider a new vector boson with charge ±1 that couples to right-handed up and down type quarks Lint = gRV R ij ¯di Ruj RW ′−+ h.c. , (4.10) (4.10) where V R ij is a unitary mixing matrix, the analog of the CKM matrix in the right-handed sector. One possibility to introduce such a W ′ gauge boson is through an additional SU(2)R gauge group [56]. Yet as long as the coupling structure in eq. (4.10) is realized, the exact implementation of the W ′ is of no relevance for the following discussion. Tree level exchange of the W ′ gives contributions to the current-current Wilson co- efficient ˜C(1)p 1 . The corresponding operator ˜O(1)p 1 has the same hadronic matrix ele- ment as the SM operator O(1)p 1 . Therefore, tree level exchange of the W ′ cannot gen- erate a direct CP asymmetry because there is no strong phase difference with respect to the LO SM contribution. Loop level contributions, i.e. gluon penguins with W ′ loops, have a structure that is analogous to the SM penguin contribution. If we assume that the mass of the W ′ is larger than the mass of the SM W boson, then the couplings gRV R cb V R∗ ub have to be considerably larger than the SM couplings gVcbV ∗ ub in order to generate a sizable ∆ACP. Such a W ′ would then lead to unacceptably large NP contributions to B →D and B →π transitions, and thus we will not consider this scenario any further. 4.5 Two Higgs doublet model One of the simplest extensions of the SM scalar sector is the 2 Higgs doublet model (2HDM) (see [57] for a recent review). The most general couplings of the 2 Higgs bosons to SM fermions read Lint = Yu ¯QUHu + Yd ¯QDHd + Yℓ¯LEHd (4.11) +Xu ¯QUH† d + Xd ¯QDH† u + Xℓ¯LEH† u + h.c. , (4.11) – 16 – where we suppress the flavor indices on the quark fields and the coupling matrices X and Y . Generically, the neutral components of both Higgs bosons acquire a vev ⟨H0 u,d⟩= vu,d and the fermion mass matrices receive contributions from both Y and X. As Y and X are independent matrices in flavor space, the couplings in eq. (4.11) lead to flavor changing neutral Higgs vertices at tree level and are strongly constrained from meson mixing observables. The most effective way to control flavor changing effects in the 2HDM is the Minimal Flavor Violation (MFV) ansatz [58, 59]. Applied to the 2HDM, the MFV assumption states that the “wrong” Higgs couplings X can be expanded in powers of the Yukawa couplings Y JHEP04(2012)049 Xu = ϵuYu + ϵ′ uYuY † u Yu + ϵ′′ uYdY † d Yu + . . . , Xd = ϵdYd + ϵ′ dYuY † u Yd + ϵ′′ dYdY † d Yd + . . . , (4.12) (4.12) where the ϵi are free complex parameters. For simplicity, we will also assume Xℓ= ϵℓYℓ We stress that in this particular 2HDM, there exists no preferred basis for the 2 Higgs doublets. The ϵi parameters as well as tan β = vu/vd are basis dependent and none of them separately are actually physical parameters (see [60] for a detailed discussion). In the following analysis, we fix a basis by setting ϵd = 0. In this basis, a large tan β can be approximately identified with the basis invariant enhancement of the coupling of the right handed strange quark to the charged Higgs with respect to its SM Yukawa coupling. We now investigate the parameter space of this 2HDM with MFV in a basis with ϵd = 0. We work in the regime of large tan β and assume ϵ′′ u, ϵ′ d ≪1 in order to ensure that tree level FCNCs are under control. Furthermore, we allow the parameters ϵu, ϵ′ u and ϵ′′ d as well as ϵℓto be O(1). 2Note that this expression as well as the ones given below are not basis invariant. They only hold in bases where the same conditions on the ϵi and tan β hold, under which they were derived. (i) Direct searches at LEP for a charged Higgs give the bound MH± ≳80 GeV [65]. 4.5 Two Higgs doublet model As we will see below, in this region of parameter space, sizable nonstandard contributions to the D →K+K−amplitude can arise, while the most important constraints can be kept under control. In this scenario, tree level charged Higgs exchange, as shown in diagram (a) of figure 5, gives the dominant NP contribution to the D →K+K−decay. For large values of tan β and assuming ϵu ∼O(1) we find2 ˜C(1) S1 = mcms v2 ϵu tan β 1 + ˜ϵs tan β VusV ∗ cs M2 H± , (4.13) (4.13) with v2 = v2 u + v2 d = 1742 GeV2, and ˜ϵs ≡ϵ′′ dy2 s. For large tan β and ϵ′′ d ∼O(1), we find ˜ϵs ∼10−3, such that ˜ϵs only becomes relevant for extremely large tan β. The parameter ϵu simultaneously lifts the 1/ tan β suppression of the ¯cRsLH+ vertex and provides a source of CPV. The 2HDM with MFV have been thoroughly studied in the literature (see [59, 61–64]) and various constraints have been identified. In the following, we discuss the most impor- tant constraints: (i) Direct searches at LEP for a charged Higgs give the bound MH± ≳80 GeV [65]. 2Note that this expression as well as the ones given below are not basis invariant. They only hold in bases where the same conditions on the ϵi and tan β hold, under which they were derived. – 17 – (ii) The B+ →τ +ν and K+ →µ+ν decays are known to be important low energy probes of extended Higgs sectors. Combining the experimental results from Belle [66, 67] and BaBar [68, 69] on Br(B →τν) with a conservative SM prediction based on |Vub| = (3.89 ± 0.44) × 10−3 [10] and fB+ = 196.9 ± 8.9 MeV [70] (ii) The B+ →τ +ν and K+ →µ+ν decays are known to be important low energy probes of extended Higgs sectors. Combining the experimental results from Belle [66, 67] and BaBar [68, 69] on Br(B →τν) with a conservative SM prediction based on |Vub| = (3.89 ± 0.44) × 10−3 [10] and fB+ = 196.9 ± 8.9 MeV [70] Br(B →τν)SM = (1.04 ± 0.25) × 10−4 , (4.14) (4.14) we find we find RBτν = Br(B →τν)exp Br(B →τν)SM = 1.58 ± 0.32 . 4.5 Two Higgs doublet model (4.15) (4.15) In our 2HDM, tree level exchange of a charged Higgs (see diagram (d) in figure 5) results in 2 JHEP04(2012)049 RBτν = 1 −m2 B M2 H± tan β 1 + ˜ϵb tan β 1 ϵℓ 2 , (4.16) (4.16) where we defined ˜ϵb ≡ϵ′′ dy2 b. For large tan β and ϵ′′ d ∼O(1), one typically has ˜ϵb ∼10−2−10−1. The factors ˜ϵb and ϵℓkeep the ¯bRuLH± and ¯τRνLH± couplings small for large tan β and the experimental constraint from B+ →τ +ν is easily avoided, provided sign(ϵℓ) = −1. In fact, for such choices of parameters, the ∼2σ discrepancy between the SM prediction and the experimental result for Br(B →τν) is reduced. For the observable Rℓ23 [71], which is sensitive to charged Higgs contributions to the K →µν decay, we find Rℓ23 = 1 −m2 K M2 H± tan β 1 + ˜ϵs tan β 1 ϵℓ . (4.17) (4.17) From the experimental side, one has [71] From the experimental side, one has [71] Rℓ23 = 0.999 ± 0.007 , (4.18) (4.18) which, in our framework, leads only to constraints for extremely large tan β. which, in our framework, leads only to constraints for extremely large tan β. which, in our framework, leads only to constraints for extremely large tan β. (iii) Charged Higgs loops lead to contributions to D0 −¯D0 and K −¯K mixing (see di- agrams (b) and (c) of figure 5). In the considered scenario, we find the following dominant NP contributions (iii) Charged Higgs loops lead to contributions to D0 −¯D0 and K −¯K mixing (see di- agrams (b) and (c) of figure 5). In the considered scenario, we find the following dominant NP contributions C(2)D 1 ≃ −1 16π2 m4 s v4 (VcsV ∗ us)2 8M2 H± tan4 β |1 + ˜ϵs tan β|4 , (4.19) C(2)K 1 ≃ −1 16π2 m4 t v4 (VtsV ∗ td)2 4M2 H±  4 2  (4.19) H  |˜ϵt|4h1(xt) + |˜ϵt|2h2(xt, xW )  , (4.20) (4.20) with xt = m2 t /M 2 H±, xW = M2 W /M 2 H±, ˜ϵt ≡ϵu + ϵ′ uy2 t , and the analytical expressions for the loop functions h1 and h2 are given in appendix C. 3The corresponding contribution from a bottom quark loop is strongly Cabibbo suppressed and turns t to be much smaller. 4.5 Two Higgs doublet model The expression for C(2)D 1 is suppressed by four powers of the strange quark mass and is only relevant for very large tan β.3 The Wilson coefficient C(2)K 1 arises from box diagrams including both 3The corresponding contribution from a bottom quark loop is strongly Cabibbo suppressed and turns out to be much smaller. – 18 – one W ± and one H± insertion as well as diagrams with two H± insertions. Its contribution to kaon mixing can be relevant for ˜ϵt ≃O(1). Yet even for |˜ϵt| ≃0.5, the constraint on the charged Higgs mass is as low as the bound from direct searches MH± ≳80 GeV. one W ± and one H± insertion as well as diagrams with two H± insertions. Its contribution to kaon mixing can be relevant for ˜ϵt ≃O(1). Yet even for |˜ϵt| ≃0.5, the constraint on the charged Higgs mass is as low as the bound from direct searches MH± ≳80 GeV. (iv) As shown in diagram (e) of figure 5, the Bd →Xsγ decay also receives 1-loop charged Higgs contributions. As is well known, the good agreement of the experimental data and the SM prediction of its branching ratio leads to the constraint MH± ≳300 GeV in a 2HDM of type II [72]. This bound, however, does not apply in the model considered here. For the ratio of the b →sγ amplitudes in our 2HDM with MFV model to the 2HDM of type II, we find, to a good approximation, (iv) As shown in diagram (e) of figure 5, the Bd →Xsγ decay also receives 1-loop charged Higgs contributions. As is well known, the good agreement of the experimental data and the SM prediction of its branching ratio leads to the constraint MH± ≳300 GeV in a 2HDM of type II [72]. This bound, however, does not apply in the model considered here. For the ratio of the b →sγ amplitudes in our 2HDM with MFV model to the 2HDM of type II, we find, to a good approximation, JHEP04(2012)049 A(b →sγ)MFV A(b →sγ)II = ˜ϵt tan β 1 + ˜ϵb tan β + |˜ϵt|2y(xt) , (4.21) (4.21) where y(xt) is a function of xt = m2 t /MH± and is O(1). We point out that because of the ˜ϵt and ˜ϵb factors, the b →sγ amplitude can be complex, which relaxes the Bd → Xsγ constraint considerably [73]. 4.5 Two Higgs doublet model Still, charged Higgs masses as low as the constraint from direct searches require ˜ϵt = ϵu + ϵ′ uy2 t ≪1, which implies a considerable amount of fine tuning. where y(xt) is a function of xt = m2 t /MH± and is O(1). We point out that because of the ˜ϵt and ˜ϵb factors, the b →sγ amplitude can be complex, which relaxes the Bd → Xsγ constraint considerably [73]. Still, charged Higgs masses as low as the constraint from direct searches require ˜ϵt = ϵu + ϵ′ uy2 t ≪1, which implies a considerable amount of fine tuning. (v) Complex ϵi parameters also lead to contributions to electric dipole moments (EDMs) (see [61, 62]). While a detailed study of EDMs is beyond the scope of this work, we mention that in the studied framework, the contributions to the EDMs depend on the parameters ˜ϵt and ˜ϵb. Therefore, by allowing for a certain amount of fine tuning, EDM constraints do not exclude sizable CP violating effects in the D →K+K− decay that depend mainly on ϵu. (vi) Charged Higgses are also constrained by bounds on the branching ratio of the top quark decay t →H+b. ATLAS and CMS obtained bounds at the level of Br(t → H+b) ≲5% under the assumption Br(H−→τν) = 1 [74]. CDF and DØ also give bounds considering the H →cs final state. These bounds are at the level of Br(t →H+b) ≲10% −20% [75, 76]. In our setup, the ratio of the t →H+b and t →Wb branching ratios is given by (vi) Charged Higgses are also constrained by bounds on the branching ratio of the top quark decay t →H+b. ATLAS and CMS obtained bounds at the level of Br(t → H+b) ≲5% under the assumption Br(H−→τν) = 1 [74]. CDF and DØ also give bounds considering the H →cs final state. These bounds are at the level of Br(t →H+b) ≲10% −20% [75, 76]. In our setup, the ratio of the t →H+b and t →Wb branching ratios is given by (vi) Charged Higgses are also constrained by bounds on the branching ratio of the top quark decay t →H+b. ATLAS and CMS obtained bounds at the level of Br(t → H+b) ≲5% under the assumption Br(H−→τν) = 1 [74]. CDF and DØ also give bounds considering the H →cs final state. 4.5 Two Higgs doublet model These bounds are at the level of Br(t →H+b) ≲10% −20% [75, 76]. In our setup, the ratio of the t →H+b and t →Wb branching ratios is given by ΓtHb ΓtWb = 1 −(mH/mt)2 1 −(mW /mt)2 1 m2 W m2 t + m4 t −2m4 W ×  m2 t |˜ϵt|2 + m2 b tan2 β |1 + ˜ϵb tan β|2  (m2 t −m2 H) +4Re  ˜ϵt tan β 1 + ˜ϵb tan β  m2 t m2 b  . (4.22) (4.22) For the parameter choices detailed below, we find the charged Higgs branching ratio into τν does not exceed 10% due to the strongly enhanced couplings to strange quarks. Also, since Br(t →H±b) ≲10%, top decays do not lead to constraints for our choice of parameters. For the parameter choices detailed below, we find the charged Higgs branching ratio into τν does not exceed 10% due to the strongly enhanced couplings to strange quarks. Also, since Br(t →H±b) ≲10%, top decays do not lead to constraints for our choice of parameters. – 19 – Figure 6. Regions in the MH± - tan β plane compatible with the data on ∆ACP, WA at the 1σ level in the discussed 2HDM. In a basis with ϵd = 0, we set ϵu = i, ˜ϵt = 0.05i, ϵℓ= −1 and chose ϵ′′ d such that ˜ϵs = 10−3. All other ϵi are set to zero. The green (solid) band corresponds to the expressions for the decay amplitude in na¨ıve factorization, blue (dashed) band assumes an enhancement of the hadronic matrix elements by a factor of 3. The red (dash-dotted) region is excluded by D0 −¯D0 mixing constraints. JHEP04(2012)049 Figure 6. Regions in the MH± - tan β plane compatible with the data on ∆ACP, WA at the 1σ level in the discussed 2HDM. In a basis with ϵd = 0, we set ϵu = i, ˜ϵt = 0.05i, ϵℓ= −1 and chose ϵ′′ d such that ˜ϵs = 10−3. All other ϵi are set to zero. The green (solid) band corresponds to the expressions for the decay amplitude in na¨ıve factorization, blue (dashed) band assumes an enhancement of the hadronic matrix elements by a factor of 3. The red (dash-dotted) region is excluded by D0 −¯D0 mixing constraints. (vii) Additional constraints on the 2HDM can come from direct searches for neutral Higgs bosons. 4.5 Two Higgs doublet model The relation between the charged and the neutral Higgs bosons, however, depends on the details of the Higgs potential. For simplicity we assume a Higgs potential such that there is one neutral Higgs boson h that has SM-like couplings to gauge bosons and fermions. Consequently, the other two bosons H and A do not couple to gauge bosons and have couplings to bottom quarks that are enhanced by tan β/(1 + ˜ϵb tan β). While the masses of the Higgs bosons are in principle free parameters, we will assume MH ≃MA ≃MH± to avoid constraints from electroweak precision observables. Constraints can arise from neutral Higgs boson searches in the H →ττ final state at ATLAS and CMS [77], as well as from searches at DØ and CDF in the H →bb final state [78, 79]. In the following, we discuss a benchmark scenario that avoids all considered constraints but allows for nonstandard values for ∆ACP. We set ϵu = i, ˜ϵt = 0.05i, ϵℓ= −1, and chose ϵ′′ d such that ˜ϵs = 10−3. All other ϵi are set to zero. Figure 6 shows the regions in the MH± — tan β plane compatible with the data for ∆ACP, WA at the 1σ level. The green (solid) band is obtained using the expressions for the decay amplitude in na¨ıve factorization. The blue (dashed) band assumes an enhancement of the hadronic matrix elements by a factor of 3. The red (dash-dotted) region in the upper left corner is excluded by D0 −¯D0 mixing constraints. We observe that sizable NP contributions to the direct CP asymmetry are only possible for small charged Higgs masses and large values of tan β ∼O(100) and larger. It is important to note that such large values for tan β are not in conflict with a requirement – 20 – (a) (b) Figure 7. Example Feynman diagrams that contribute to (a) the D →K+K−and D →π+π− decay amplitudes and (b) D0 −¯D0 mixing in the scalar octet model. (b) (a) (b) (a) Figure 7. Example Feynman diagrams that contribute to (a) the D →K+K−and D →π+π− decay amplitudes and (b) D0 −¯D0 mixing in the scalar octet model. of perturbative Yukawa couplings. 4.5 Two Higgs doublet model Indeed, due to the ˜ϵb (we find 0.03 < ˜ϵb < 0.09 in the considered region of tan β) and ϵℓfactors, the bottom and tau Yukawa remain well below 1.4 Extremely large values of tan β are not stable under radiative corrections. Generically, 1-loop corrections to the Higgs potential lead to 1/ tan β ∼1/(4π)2. Values of tan β ≃ few × 100, however, seem a reasonable possibility. JHEP04(2012)049 The shown region of parameter space is not significantly constrained by any of the considered bounds. In particular, the bound from Br(Bd →Xsγ) is avoided because of the small ˜ϵt. As stated before, the branching ratio of the charged Higgs into τν does not exceed 10% due to the strongly enhanced couplings to strange quarks, and in addition, constraints from top decays are satisfied since Br(t →H±b) ≲10%. Similarly, we find that the branch- ing ratios of the neutral Higgs bosons to ττ are tiny, O(0.1%), and the stringent bounds from ATLAS and CMS [77] are easily avoided. Finally, due to the ˜ϵb factor that controls the size of the ¯bbH and ¯bbA couplings, the H →bb searches at Tevatron [78, 79] do not lead to constraints. We expect that the collider phenomenology of this large parameter space will focus on a combination of complementary probes, including tests for non-SM Yukawa couplings of the light Higgs boson and heavy flavor searches for pairs of dijet resonances. A complete phenomenological study of this 2HDM with MFV is left for future work. We stress that the 2HDM with MFV can only significantly affect the direct CP asym- metry in D →K+K−. New Physics effects in ACP(π+π−) are strongly suppressed by md/ms and negligibly small. The considered model also does not lead to large nonstan- dard effects in indirect CPV in D0 −¯D0 mixing. These statements are not necessarily true if the couplings of the Higgses radically departure from the MFV ansatz (see [24] for a re- lated study). Even though such models will generically have flavor changing neutral Higgs interactions, they can in principle be made compatible with low energy flavor constraints. A detailed study of such setups, however, is beyond the scope of this work. 4.6 Scalar octet Couplings analogous to the ones in eq. (4.11) are also possible if the second scalar SU(2)L doublet is a color octet instead of a color singlet [82]. We write Lint = Yu ¯QUH + Yd ¯QDH† (4.23) +Xu ¯QT aUφa 8 + Xd ¯QT aDφa† 8 + h.c. (4.23) 4The phenomenology of generating appropriate bottom and tau masses in the very large tan β regime in the context of supersymmetric models was studied in [80, 81]. We remark however that the considered scenario cannot be realized in the MSSM where the ϵi factors are only loop induced and not much larger than 10−2. – 21 – where H is the SM Higgs and φa 8, a = 1, . . . 8, is the color octet. Since a color octet must not aquire a vev, the quark masses are entirely provided by the SM Higgs, and the Yukawa couplings Y are given by their SM values. As in the case of the 2HDM, we will first assume that the couplings X have the MFV structure Xu = ζuYu + ζ′ uYuY † u Yu , Xd = ζdYd + ζ′ dYdY † u Yu . (4.24) (4.24) We can neglect higher powers of the down-type Yukawa couplings in the expansion because in the considered scenario they are fixed to their small SM values. This automatically ensures that there are no tree level flavor changing interactions of the neutral component of the scalar octet with up quarks. Furthermore, the interactions of the charged component of the scalar octet cannot lead to tree level contributions to the D →K+K−amplitude in na¨ıve factorization, as the two quarks that couple to the color octet cannot hadronize into a kaon or pion, which are color singlets. Contributions to the D →K+K−decay can, however, come from annihilation topologies involving the charged octet state. Even though such contributions are formally suppressed by 1/mc, they can be sizable in D meson decays. Nonetheless, we now consider departures from the MFV ansatz for the couplings and come back to the MFV framework at the end of the section. In particular, we consider the following (small) non-MFV entry in the Xu coupling matrix in the basis where the quark Yukawa couplings are diagonal JHEP04(2012)049 [Xu]12 = ζuycXcu , (4.25) (4.25) with Xcu ∼VcsV ∗ us ∼λ ∼0.2. 4.6 Scalar octet JHEP04(2012)049 Constraints from electroweak precision observables are under control, provided all components of the scalar octet have approximately the same mass and are heavier than 100 GeV [83]. There has been extensive work done on collider constraints on scalar octets [84, 85]. Because of the small couplings to light quarks in the considered framework, single pro- duction of scalar octets is strongly suppressed, and dijet resonance searches at the hadron colliders give no significant constraints in the considered region of parameter space. In- stead, the main collider constraints on the scalar octet doublet come from the dijet pair searches at the LHC. We simulate the production of scalar octet pairs (including pro- duction of neutral pairs and charged pairs) using MadGraph 5 and compare the obtained cross sections with the bounds set by ATLAS [54] and CMS [55]. We find that the region from 100 GeV to 200 GeV that is covered by the ATLAS search is fully excluded. The CMS search excludes scalar octet masses from 320 GeV until approximately 550 GeV for 13% acceptance (see section 4.3 for comments regarding the 200-320 GeV region). As our scalar octets decay predominantly to b quarks, searches for final states with multiple b jets at Tevatron can potentially lead to constraints. Using the CDF result [86], the authors of [87] find that scalar octet masses of Mφ8 ≲200 GeV are excluded. Updated results from CDF [79] and DØ [78] do not give significantly improved bounds on the corresponding cross sections. In figure 8, we show the regions in the Mφ8 - ζu plane that are compatible with the data for ∆ACP, WA at the 1σ level, assuming ζu = ζd/2 real and setting Xcu = |VcsV ∗ us|eiπ/2. The green (solid) band corresponds to the expressions for the decay amplitude obtained in na¨ıve factorization, and the blue (dashed) band assumes an enhancement of the hadronic matrix elements by a factor of 3. The red (dash-dotted) region is excluded by D0 −¯D0 mixing constraints and the vertical bands are excluded by the dijet pair searches at ATLAS (left band) and CMS (right band). As expected, constraints from D0 −¯D0 mixing allow for a sizable ∆ACP only for small scalar masses. 4.6 Scalar octet In contrast to the MFV interactions, the above term leads to a flavor changing neutral current uLcRφ0 8 coupling and, due to SU(2)L invariance, to a O(1) correction of the dLcRφ± 8 coupling Lint = ζuycXcu ¯uLT acR φ0a 8 +ζuyc(V ∗ cd + XcuV ∗ ud) ¯dLT acR φ−a 8 + h.c. . (4.26) (4.26) The tree level exchange of the neutral component of the scalar octet, as shown in diagram (a) of figure 7, then leads to the following contributions to the Wilson coefficients, The tree level exchange of the neutral component of the scalar octet, as shown in diagram (a) of figure 7, then leads to the following contributions to the Wilson coefficients, ˜C(1) S1 = mcms v2 ζuζd Xcu 4M2 φ8 , (4.27) ˜C(1) S2 = −4 Nc ˜C(1) T1 = 4 ˜C(1) T2 = −1 Nc ˜C(1) S1 . (4.27) Even though the tensor operators do not contribute to the D →K+K−decay, we include the corresponding Wilson coefficients here because they mix with the scalar operators under renormalization. Even though the tensor operators do not contribute to the D →K+K−decay, we include the corresponding Wilson coefficients here because they mix with the scalar operators under renormalization. Tree level contributions to D0 −¯D0 mixing from the exchange of the neutral complex scalar φ0 8 can only arise if both the flavor changing couplings ¯uLcRφ0 8 and ¯cLuRφ0 8 are present simultaneously. Thus, in our setup, contributions to D0 −¯D0 mixing first arise at – 22 – the loop level. The dominant contribution comes from a charm quark loop, as shown in diagram (b) of figure 7, C(2)D 1 = 1 16π2 N3 c −2Nc + 1 4N2c m4 c v4 X2 cu 8M2 φ8 |ζu|4 . (4.28) (4.28) Similar to the situation discussed in the context of the 2HDM above, other flavor changing processes like kaon mixing or Bd →Xsγ do not directly constrain ζu or ζd as they involve couplings to right-handed top and bottom quarks that are proportional to ζt = ζu + ζ′ uy2 t and ζb = ζd + ζ′ dy2 t , respectively. Keeping these couplings small to avoid constraints from perturbativity and, in particular from Bd →Xsγ, requires a considerable amount of fine tuning. On the other hand, since color octets cannot couple to leptons, the constraints from B →τν and K →µν are automatically avoided. 4.6 Scalar octet Given the constraints from the dijet pair searches at LHC, only a small window with octet masses between 200 GeV and 320 GeV and large values for ζu,d ≳20 remains where significant values for ∆ACP can be generated by the scalar octet with couplings close to the MFV ansatz without assuming any enhancement of the hadronic matrix elements. Scalar octets heavier than the CMS bound of Mφ8 ≃ – 23 – Figure 8. Regions in the Mφ8 - ζu plane compatible with the data on ∆ACP, WA at the 1σ level in the scalar octet model, assuming ζu = ζd/2 real and setting Xuc = |VcsV ∗ us|eiπ/2. The green (solid) band corresponds to the expressions for the decay amplitude obtained in na¨ıve factorization, the blue (dashed) band assumes an enhancement of the hadronic matrix elements by a factor of 3. The red (dash-dotted) region is excluded by D0 −¯D0 mixing constraints. The vertical shaded bands are excluded by dijet pair searches at LHC. JHEP04(2012)049 Figure 8. Regions in the Mφ8 - ζu plane compatible with the data on ∆ACP, WA at the 1σ level in the scalar octet model, assuming ζu = ζd/2 real and setting Xuc = |VcsV ∗ us|eiπ/2. The green (solid) band corresponds to the expressions for the decay amplitude obtained in na¨ıve factorization, the blue (dashed) band assumes an enhancement of the hadronic matrix elements by a factor of 3. The red (dash-dotted) region is excluded by D0 −¯D0 mixing constraints. The vertical shaded bands are excluded by dijet pair searches at LHC. (a) (b) (c) Figure 9. Example Feynman diagrams that contribute to (a) the D →K+K−and D →π+π− decay amplitudes, (b) D0 −¯D0 mixing and (c) K −¯K mixing in the discussed diquark model. (b) (a) (c) (a) (b) (c) Figure 9. Example Feynman diagrams that contribute to (a) the D →K+K−and D →π+π− decay amplitudes, (b) D0 −¯D0 mixing and (c) K −¯K mixing in the discussed diquark model. 550 GeV can lead to nonstandard values for ∆ACP only for extremely large ζu,d ≳60 and assuming the hadronic matrix elements to be 3 times the na¨ıve factorization estimate. The same conclusion holds for the pure MFV setup discussed at the beginning of the section, provided that the annihilation contribution with the charged scalar exchange is of similar size as the na¨ıve factorization contribution from the flavor changing neutral scalar. 4.7 Scalar diquarks Numerous variations of scalar diquarks can have renormalizable couplings to the SM quarks and lead to an interesting flavor phenomenology [88, 89]. Here, we consider a diquark that has quantum numbers such that it leads to contributions to meson mixing only at the loop level, but contributes to the D →K+K−and D →π+π−decay amplitudes at tree level in na¨ıve factorization. One such possibility is a scalar that transforms as ¯6 under SU(3), as a singlet under SU(2)L, and has hypercharge −1/3.5 Such a diquark can couple to right-handed up and down type quarks, Lint = Xij ¯U cα i Dβ j φαβ 6 + h.c. , (4.29) (4.29) JHEP04(2012)049 where i, j are flavor indices and the diquark φ6 is symmetric in the color indices φαβ 6 = φβα 6 . The couplings Xij are a source of flavor violation and in general free complex parameters. We remark that the considered diquark can also couple to left-handed quarks. Considering couplings to left- and right-handed quarks, however, simultaneously induces left-right mix- ing ∆F = 2 operators, resulting in very severe constraints from D and K meson mixing. Therefore, we concentrate on couplings to the right-handed quarks only. The tree level contributions of the diquark to the D →K+K−and D →π+π−decay amplitudes are shown in diagram (a) of figure 9 and read ˜C(1)p 1 = ˜C(1)p 2 = XcpX∗ up 16M2 φ6 . (4.30) (4.30) Since QCD is parity conserving, the matrix element of the operators ˜O(1)p 1 are the same as the SM operators O(1)p 1 , and no strong phase difference occurs between them. Corre- spondingly, ˜C(1)p 1 does not contribute to the direct CP asymmetries (see eq. (3.3)). Due to the different color structure of the operators ˜O(1)p 2 , however, a different strong phase can be expected in their matrix elements and a non-zero contribution to the direct CP asymmetries can be generated by the weak phase in ˜C(1)p 2 ∝XcpX∗ up. p Contributions to D0 −¯D0 mixing are first induced at the loop level (see diagram (b) in figure 9) ˜C(2)D 1 ≃− 1 16π2 Nc + 3 32M2 φ6  X p=d,s X∗ upXcp   2 , (4.31) (4.31) where, for simplicity, we restrict ourselves to couplings of the diquark to the first two generations of quarks and expand the result in small quark masses. 5All other possibilities are scalars that can also have lepton number violating couplings. One example is a SU(3) triplet, SU(2)L singlet with hypercharge −1/3 which corresponds to a right-handed down squark with R-parity violating couplings. 4.6 Scalar octet Both in the MFV and the quasi-MFV setup, the scalar octet can only significantly affect the direct CP asymmetry in D →K+K−. New Physics effects in ACP(π+π−) are strongly suppressed by md/ms and negligibly small. As in the case of the 2HDM, this is not necessarily the case if the octet couplings are allowed to deviate more radically from MFV. We expect collider searches in the multi-b jet final state, as discussed in [84, 85], to provide the best sensitivity to this model. – 24 – 4.7 Scalar diquarks We note that even with small couplings to quarks ≲0.1, sizable effects in flavor ob- servables can be generated. Consequently, dijet searches at hadron colliders do not lead to relevant constraints. Important collider constraints on diquarks, however, come from the – 25 – Figure 10. Regions in the Mφ6 - |XcsX∗ us| plane compatible with the data on ∆ACP, WA at the 1σ level in the diquark model, setting Arg(XcsX∗ us) = π/2. The green (solid) band corresponds to the expressions for the decay amplitude obtained in na¨ıve factorization, the blue (dashed) band assumes an enhancement of the hadronic matrix elements by a factor of 3. The red (dash-dotted) region is excluded by D0 −¯D0 constraints. The vertical shaded bands are excluded by dijet pair searches at LHC. JHEP04(2012)049 Figure 10. Regions in the Mφ6 - |XcsX∗ us| plane compatible with the data on ∆ACP, WA at the 1σ level in the diquark model, setting Arg(XcsX∗ us) = π/2. The green (solid) band corresponds to the expressions for the decay amplitude obtained in na¨ıve factorization, the blue (dashed) band assumes an enhancement of the hadronic matrix elements by a factor of 3. The red (dash-dotted) region is excluded by D0 −¯D0 constraints. The vertical shaded bands are excluded by dijet pair searches at LHC. dijet pair searches at LHC. Using MadGraph 5 to simulate diquark pair production, we find that the ATLAS search excludes the region from 100 GeV to 200 GeV. The CMS search ex- cludes diquarks with masses from 320 GeV until 1000 GeV assuming an acceptance of 13%. In figure 10, we show regions in the Mφ6 — |XcsX∗ us| plane compatible with the data for ∆ACP, WA at the 1σ level, considering only Xcs and Xus to be non-zero and setting Arg(XcsX∗ us) = π/2. The green (solid) band corresponds to the expression for the decay amplitude in na¨ıve factorization. The blue (dashed) band assumes an enhancement of the hadronic matrix elements by a factor of 3. The red (dash-dotted) region is excluded by the D0 −¯D0 mixing constraints. The vertical bands are excluded by the dijet pair searches at ATLAS (left band) and CMS (right band). Since only strange quark couplings are considered, ∆ACP is entirely generated by ACP(K+K−) in this setup. 4.7 Scalar diquarks Despite the fact that the D →K+K−decay amplitude arises already at the tree level while D0−¯D0 mixing is only induced at the loop level, constraints from D0 −¯D0 mixing combined with the dijet pair searches exclude a sizable ∆ACP unless the hadronic matrix elements are enhanced by a factor more than 3. This is due to the fact that the contributions to ∆ACP coming from the operator ˜O(1)s 2 are color suppressed and neither enhanced by RG effects nor by chiral factors. Considering only the couplings Xcd and Xud leads to an analogous situation, but in this case ∆ACP entirely stems from ACP(π+π−). If all 4 couplings Xcs, Xus, Xcd and Xud are present, the D0−¯D0 mixing constraint can be strongly reduced by assuming a GIM-like mechanism, i.e. assuming the coupling matrix X to be (approximately) unitary. This happens, for example, if X is (approximately) proportional to unity in the weak eigenstate basis for the quarks. In that case, however, ACP(π+π−) ≃ACP(K+K−) and the difference between the two, ∆ACP, is very small. – 26 – (a) (b) (c) Figure 11. Example Feynman diagrams that contribute to (a) the D →K+K−and D →π+π− decay amplitudes and (b and c) D0−¯D0 mixing in the discussed models with fermion + scalar loops. (b) (a) (c) (c) (b) (a) Figure 11. Example Feynman diagrams that contribute to (a) the D →K+K−and D →π+π− decay amplitudes and (b and c) D0−¯D0 mixing in the discussed models with fermion + scalar loops. JHEP04(2012)049 In addition, also loop contributions to kaon mixing are induced if all four couplings are present. Apart from a contribution analogous to eq. (4.31), mixed φ6 — W loops (shown in diagram (c) of figure 9) can become very important. For the latter, we find C(2)K 4 = C(2)K 5 = g2 16π2 (VcsV ∗ cd) (XcsX∗ cd) × 1 2M2 φ6 m2 c M2 W log m2 c M2 φ6 ! , (4.32) (4.32) where we expand the result in the charm quark mass and keep only the leading term that is enhanced by a large logarithm. Despite the suppression by m2 c/M 2 W , these contributions are very relevant because the matrix elements of O(2)K 4 and O(2)K 5 are strongly chirally enhanced and enhanced by renormalization group effects.6 Furthermore, the simultaneous presence of Xus and Xud also leads to tree level contributions to ϵ′/ϵ. 4.7 Scalar diquarks Even after tuning phases in order to minimize constraints from CPV in kaon mixing and ϵ′/ϵ, we do not find any region in parameter space of the considered diquark model in which a sizable ∆ACP is compatible with all constraints. 5 New physics contributions to gluon penguins We now consider possibilities for new physics in the gluon penguin diagrams at the 1-loop level. We consider models with new fermion and scalar fields, where the fermion is Dirac or Majorana and with or without GIM suppression in section 5.1 and section 5.2, respectively. A scenario with chirally enhanced magnetic penguins is considered in section 5.3. As discussed in the Introduction, the chiral enhancement of the decay amplitudes allows such a scenario to be the least constrained by D0 −¯D0 mixing. 6The analogous contribution to D0 −¯D0 mixing is suppressed by the strange quark mass and has a much smaller chiral enhancement from the matrix elements: it is therefore negligible. 5.1 Fermion + scalar loop without GIM mechanism We add one heavy fermion χ and one heavy scalar φ to the SM that couple with right- handed up type quarks Lint = Xu¯uRχφ + Xc¯cRχφ + Xt¯tRχφ + h.c. . (5.1) (5.1) 6The analogous contribution to D0 −¯D0 mixing is suppressed by the strange quark mass and has a much smaller chiral enhancement from the matrix elements: it is therefore negligible. – 27 – The couplings Xi necessarily violate flavor and are free complex parameters. In the fol- lowing we consider the case where the new particles are SU(2)L singlets. We assume the fermion is electrically neutral and a SU(3) singlet and the scalar is a SU(3) triplet. Different quantum number assignments do not lead to qualitatively different results concerning the D →K+K−and D →π+π−decay amplitudes. We do not consider new fields with flavor breaking couplings to left handed quarks to avoid constraints from kaon mixing and ϵ′/ϵ. The Lagrangian considered in this framework incorporates an accidental Z2 symmetry where χ and φ are odd under the symmetry while the SM fields are even. In the considered minimal framework, the lightest of χ and φ is therefore stable and becomes the dark matter. If the colored scalar is lighter than the fermion, additional interactions have to be introduced to prevent the scalar from being absolutely stable. If these additional interactions are weak enough such that the scalar remains stable on detector scales, bounds from searches for long-lived particles apply [90, 91]. If instead the scalar decays promptly, then it is highly model dependent whether or not the considered scenario is excluded by current collider searches. JHEP04(2012)049 In the case where the neutral fermion is the lighter particle, SUSY searches with jets + /ET at the Tevatron [92, 93] and LHC [94, 95] provide constraints to the model. In particular, both ATLAS [94] and CMS [95] give bounds on the production cross section for a simplified model that contains the first and second squark generations and a neutralino lightest supersymmetry particle (LSP). We simulate the production cross section using Prospino 2.1 [96] and set all the superpartner masses to be 4.5 TeV except the relevant squarks and the LSP, following [94]. The obtained cross section is then scaled by 1/8 since in our minimal scenario there is only one colored scalar instead of eight. 5.1 Fermion + scalar loop without GIM mechanism We find that neither the ATLAS nor the CMS searches currently put bounds on our model. The discussion of the collider constraints is the same regardless of whether the neutral fermion is a Majorana or a Dirac particle. On the other hand, the nature of the fermion plays a crucial role in the low energy phenomenology of the considered model. 5.1.1 Dirac fermion The interactions specified in eq. (5.1) lead to 1-loop gluon penguin contributions to the Wilson coefficients of the ∆F = 1 effective Hamiltonian that are shown in diagram (a) of figure 11 and lead to ˜C(1) 6 = αs 4πXuX∗ c 1 8M2 φ p(z) , ˜C(1) 3 = ˜C(1) 5 = −1 Nc ˜C(1) 4 = −1 Nc ˜C(1) 6 , ˜C(1) 8g = XuX∗ c 1 4M2 φ g(z) . (5.2) (5.2) The loop functions depend on the ratio of the fermion and scalar masses z = M2 χ/M 2 φ. We find p(1) = −1/24, g(1) = 1/48. Their full analytical expressions can be found in ap- pendix C. The relation between the Wilson coefficients C(1) 3,4,5,6 of the QCD penguin oper- ators is universal for all models where they are induced by gluon penguins. – 28 – Figure 12. The Mφ - Mχ plane in the models where a gluon penguin is induced by a Majorana fermion - scalar loop without GIM mechanism (left) and with GIM mechanism (right). The blue contours indicate the (left) |XuX∗ c | = 0.06, . . . 0.14 or (right) |δcu| = 0.05, . . . 0.25 values in agree- ment with the measured ∆ACP, WA, assuming maximal phases of π/2. An enhancement of the hadronic matrix elements by a factor of 3 is assumed in both plots. The red (dash-dotted) region is excluded by the D0 −¯D0 mixing constraints. In the region above the black (solid diagonal) line, the colored scalar is lighter than the fermion. JHEP04(2012)049 Figure 12. The Mφ - Mχ plane in the models where a gluon penguin is induced by a Majorana fermion - scalar loop without GIM mechanism (left) and with GIM mechanism (right). The blue contours indicate the (left) |XuX∗ c | = 0.06, . . . 0.14 or (right) |δcu| = 0.05, . . . 0.25 values in agree- ment with the measured ∆ACP, WA, assuming maximal phases of π/2. An enhancement of the hadronic matrix elements by a factor of 3 is assumed in both plots. The red (dash-dotted) region is excluded by the D0 −¯D0 mixing constraints. In the region above the black (solid diagonal) line, the colored scalar is lighter than the fermion. The couplings in eq. (5.1) also lead, in principle, to 1-loop box contributions to the ∆F = 1 effective Hamiltonian. 5.1.1 Dirac fermion These contributions, however, are strongly suppressed by an additional factor of |Xu|2 and are therefore negligible. Finally, 1-loop box contributions to D0 −¯D0 mixing are also induced (see diagram (b) of figure 11) of figure 11) ˜C(2)D 1 = (XuX∗ c )2 16π2 1 M2 φ 1 8f(z) , (5.3) (5.3) with f(1) = −1/3. The analytical expression for f is given in appendix C. with f(1) = −1/3. The analytical expression for f is given in appendix C. We find that D0 −¯D0 mixing constraints exclude NP contributions to ∆ACP as large as the world average even if we allow for an enhancement factor of 3 in the hadronic matrix elements. 5.1.2 Majorana fermion We find qualitatively different results for the Majorana fermion. While the gluon penguin contributions to the decay amplitudes are identical for Majorana and Dirac fermions, the box contributions to D0 −¯D0 mixing differ. In the case of a Majorana fermion, “crossed” box diagrams also exist (see diagram (c) of figure 11). One finds ˜C(2)D 1 = (XuX∗ c )2 16π2 1 M2 φ 1 8f(z) + 1 4 ˜f(z)  , (5.4) (5.4) with ˜f(1) = 1/6. The analytical expression for ˜f can be found in appendix C. with ˜f(1) = 1/6. The analytical expression for ˜f can be found in appendix C. – 29 – The left plot in figure 12 shows the Mφ — Mχ plane in the considered scenario. The blue contours labeled with 0.06, . . . 0.14 show the |XuX∗ c | values that are required to be in agreement with the measured ∆ACP, WA at the 1σ level, assuming Arg(XuX∗ c ) = π/2. An enhancement of the hadronic matrix elements by a factor of 3 is assumed. The red (dash-dotted) region is excluded by the D0 −¯D0 mixing constraints. In the region above the black (solid diagonal) line shaded in dark gray, the colored scalar is lighter than the fermion and would be a stable colored particle: for this region of parameter space, the model must be extended to be viable. We observe that if the Majorana and scalar masses are equal, Mφ = Mχ, the con- straint from D0 −¯D0 mixing can be avoided. Indeed, for such a ratio of masses, the box and crossed box contributions to the mixing amplitude cancel. The exact mass ratio where such a cancellation occurs depends on the quantum numbers of the scalar and the Majo- rana fermion. For example, in the well know SUSY case of squark — gluino boxes, the cancellation occurs for M˜g ≃1.6 × M˜q (see [97] for a recent discussion.). Because of this cancellation, there exists a narrow region of parameter space where the NP contribution to ∆ACP can explain the measured value. The same is in principle true without assuming any enhancement of the hadronic matrix elements. In such a case, however the D0 −¯D0 mixing constraints hardly allow any deviation from the Mφ = Mχ line. JHEP04(2012)049 5.2 Fermion + scalar loop with GIM mechanism We consider now a framework that allows the incorporation of a GIM-like mechanism. In addition to the heavy fermion, we introduce 3 scalars, φu,c,t, which are scalar partners of the 3 right-handed up-type quarks. If the scalars couple universally to quarks and are approximately degenerate in mass, summing over the three scalar loops strongly suppresses flavor changing neutral current processes. As discussed in [98] for this setup, ∆F = 2 processes are more strongly GIM suppressed with respect to ∆F = 1 processes and we expect more room for NP in the decay amplitudes. We consider the following flavor universal couplings between the quarks and scalars (5.5) Lint = gφ¯uRχφu + gφ¯cRχφc + gφ¯tRχφt + h.c. . (5.5) In the basis where the quark masses are diagonal, we write the mass matrix for the scalars as ˆ M2 φ = M2 φI + M2 φδ , (5.6) (5.6) where δij ≪1 are a source of flavor violation. Such a setup is, for example, realized in the MSSM by gluinos and right-handed up squarks. Here, we focus on the case where the new particles are SU(2)L singlets, the scalars are SU(3) triplets, and the fermion is a SU(3) singlet. Different quantum number assignments do not lead to qualitatively different results concerning the D →K+K−and D →π+π−decay amplitudes. Similar to the scenario without the GIM mechanism discussed above, the framework considered here is only mildly constrained by collider searches. The difference with respect to the case without GIM mechanism is that now there are three scalars, and the production cross section for scalar pair production is therefore three times larger than in section 5.1. On – 30 – the other hand, one of the scalars decays predominantly into top quarks and the constraint from squark pair production with direct decays into light quarks and the lightest neutralino from [94, 95] cannot be applied for this scalar. Analogously to the previous scenario without GIM, we scale the production cross section obtained by Prospino 2.1 by the ratio of the number of scalars in the model, i.e. a 2/8 scaling. We find that the ATLAS search [94] does not put any bound on our model. The CMS search [95] on the other hand excludes a small corner of parameter space with scalar masses between 300 GeV and 350 GeV and fermion masses below ≃100 GeV. 7They do contribute in QCD factorization via annihilation diagrams that we do not consider here. 5.2 Fermion + scalar loop with GIM mechanism This holds regardless of whether the fermion is Majorana or Dirac. The flavor phenomenology is, however, qualitatively different for the two cases. JHEP04(2012)049 5.2.1 Dirac fermion The 1-loop gluon penguin contributions to the decay amplitudes are ˜C(1) 6 = αs 4πδuc g2 φ 8M2 φ P(z) , ˜C(1) 3 = ˜C(1) 5 = −1 Nc ˜C(1) 4 = −1 Nc ˜C(1) 6 , ˜C(1) 8g = δuc g2 φ 4M2 φ G(z) , (5.7) ˜C(1) 8g = δuc g2 φ 4M2 φ G(z) , (5.7) (5.7) where δuc = δ∗ cu is a complex dimensionless parameter of flavor violation as defined in eq. (5.6). For the loop functions, we find P(1) = 1/30, G(1) = −1/80. Their analytical expressions can be found in the appendix. Now, 1-loop box contributions to the ∆F = 1 effective Hamiltonian are not suppressed by additional small mixing angles. For the minimal set of couplings defined in eq. (5.5), we find 4 ˜C(1) 3 = 1 2 ˜C(1) 9 = g4 φ 16π2 δuc 12M2 φ B(z) , (5.8) (5.8) where B(1) = 1/48. Its analytical expression can be found in appendix C. We remark that in na¨ıve factorization, this combination of Wilson coefficients does not contribute directly to the D →K+K−and D →π+π−decays that have either strange or down quarks in the final state.7 Through renormalization group running, however, the other QCD penguin operators (in particular ˜O(1) 6 ) are induced and lead to non-zero contributions. Although such contributions are subleading, they are included in our numerical analysis. Finally, the 1-loop box contributions to D0 −¯D0 mixing are given by ˜C(2)D 1 = δ2 uc 16π2 g4 φ M2 φ 1 8F(z) , (5.9) (5.9) with F(1) = −1/30. The analytical expression for F can be found in appendix C. with F(1) = −1/30. The analytical expression for F can be found in appendix C. Similar to the situation without a GIM mechanism, we find that the gluon penguins that are induced by a Dirac fermion loop cannot viably account for the measured ∆ACP, WA, even if an enhancement of the hadronic matrix elements by a factor of 3 is assumed. 7They do contribute in QCD factorization via annihilation diagrams that we do not consider here. – 31 – Figure 13. Regions in the Mφ — gugq|Acu|/Mφ plane compatible with the data for ∆ACP, WA at the 1σ level in the model with chirally enhanced gluon penguins, setting Arg(Acu) = π/3. In the left (right) plot Mχ = 100 (250) GeV. 5.2.2 Majorana fermion The gluon penguin contributions to the decay amplitudes, that are induced by a Majorana fermion — scalar loop, are identical to the Dirac case, but the box contributions to the decay amplitudes and to D0 −¯D0 mixing differ. Adding the “crossed” boxes, we find The gluon penguin contributions to the decay amplitudes, that are induced by a Majorana fermion — scalar loop, are identical to the Dirac case, but the box contributions to the decay amplitudes and to D0 −¯D0 mixing differ. Adding the “crossed” boxes, we find ˜C(1) 3 = 1 2 ˜C(1) 9 = g4 φ 16π2 δuc 12M2 φ  B(z) −1 2 ˜B(z)  , ˜C(2)D 1 = δ2 uc 16π2 g4 φ M2 φ 1 8F(z) + 1 4 ˜F(z)  . (5.10) (5.10) th ˜B(1) = −1/12, ˜F(1) = 1/20. Their analytical expressions can be found in appendix C. The corresponding situation in the Mφ — Mχ plane is shown in the right plot in figure 12, assuming an enhancement of the hadronic matrix elements by a factor of 3. The blue contours labeled with 0.05, . . . 0.25 show the |δuc| that are required for agreement with the measured ∆ACP, WA at the 1σ level, setting Arg(δuc) = π/2. Now, the ratio of the scalar to Majorana mass where the box contributions to meson mixing cancel is Mφ ≃2.3 × Mχ. We observe that constraints from D0 −¯D0 mixing leave more regions of parameter space open, as expected. Still, the measured value for ∆ACP can be explained by NP contributions only in a narrow region along the Mφ ≃2.3 × Mχ line. 5.2.1 Dirac fermion The green (solid) band corresponds to the expression for the decay amplitude in na¨ıve factorization, the blue (dashed) band assumes an enhancement by a factor of 3. The red (dash-dotted) region in the upper left corners is excluded by the D0 −¯D0 mixing constraint. The dark gray vertical region is excluded by jets + /ET searches at LHC. JHEP04(2012)049 Figure 13. Regions in the Mφ — gugq|Acu|/Mφ plane compatible with the data for ∆ACP, WA at the 1σ level in the model with chirally enhanced gluon penguins, setting Arg(Acu) = π/3. In the left (right) plot Mχ = 100 (250) GeV. The green (solid) band corresponds to the expression for the decay amplitude in na¨ıve factorization, the blue (dashed) band assumes an enhancement by a factor of 3. The red (dash-dotted) region in the upper left corners is excluded by the D0 −¯D0 mixing constraint. The dark gray vertical region is excluded by jets + /ET searches at LHC. 5.3 Chirally enhanced magnetic penguins Finally, we discuss a setup that leads to chirally enhanced chromomagnetic penguins. We introduce a Majorana fermion χ that is singlet under the SM gauge group, as well as – 32 – scalar partners, φq and φu, to the left-handed quark doublets Q and the right-handed up- type quarks U, respectively. We consider the following interactions among those degrees of freedom Lint, mass = Xu ¯Uχφu + Xq ¯Qχφq + Aφ∗ uφqH + h.c. +m2 u|φu|2 + m2 q|φq|2 + Mχχχ . (5.11) (5.11) We set the coupling matrices Xu and Xq to be universal in flavor space: Xq = gqI, Xu = guI. We further assume the mass matrices for the scalars m2 u and m2 q are universal and, for simplicity, also equal: m2 q = m2 u = M2 φI. The only new source of flavor violation is then the trilinear coupling A. After electroweak symmetry breaking, the trilinear coupling A leads to mixing between the φu and the isospin +1/2 component of φq. This setup resembles to a large extent the MSSM with flavor changing trilinear couplings in the up-squark sector as discussed in [18, 26]. We set the coupling matrices Xu and Xq to be universal in flavor space: Xq = gqI, Xu = guI. We further assume the mass matrices for the scalars m2 u and m2 q are universal and, for simplicity, also equal: m2 q = m2 u = M2 φI. The only new source of flavor violation is then the trilinear coupling A. After electroweak symmetry breaking, the trilinear coupling A leads to mixing between the φu and the isospin +1/2 component of φq. This setup resembles to a large extent the MSSM with flavor changing trilinear couplings in the up-squark sector as discussed in [18, 26]. JHEP04(2012)049 Both the elements Acu and Auc can lead to c →u transitions. Expanding the contri- bution to the Wilson coefficients in Acuv/M2 φ ≪1, we find C(1) 8g = gqgu M2 φ v mc AcuMχ 4M2 φ ˜G(z) , ˜C(1) 8g = gqgu M2 φ v mc A∗ ucMχ 4M2 φ ˜G(z) , (5.12) (5.12) where z = M2 χ/M 2 φ and ˜G(1) = −1/24. The full analytical expression for the loop function ˜G can be found in appendix C. We highlight the expected chiral enhancement of the magnetic penguins by a factor of v/mc. 6 Conclusions The LHCb measurement of the difference in the time dependent CP asymmetries in the singly Cabibbo suppressed D →K+K−and D →π+π−decays, ∆ACP, is the first evidence for charm CP violation. Although there are large uncertainties in the SM prediction, the measurement could indicate New Physics, and NP interpretations are nevertheless motivated and exciting. In this paper, we studied the effect of NP degrees of freedom for nonstandard direct CPV in the D →K+K−and D →π+π−decays while also considering constraints both from low and high energy experiments. JHEP04(2012)049 As is shown in [21], models that give rise to chirally enhanced chromomagnetic c →u penguin operators are the least constrained by low energy data and can easily accommodate the large ∆ACP value measured by LHCb. The most prominent examples for such models are supersymmetric scenarios as discussed in [18] and very recently in [26]. We studied the chirally enhanced chromomagnetic penguins in the framework of a simplified model that contains scalar partners of the left- and right-handed up-type quarks as well as a Majorana fermion. We confirm that low energy observables, in particular D0 −¯D0 mixing, as well as collider searches do not significantly constrain the model’s parameter space that leads to a sizable ∆ACP. Models that contribute to the D meson decays through four fermion operators are generically expected to be strongly constrained by D0 −¯D0 mixing data [21]. Nonstandard effects in the decays are only possible if the new degrees of freedom mediating the c →u transition are very light. In this work, we quantified this statement through a systematic study of models with a minimal set of new degrees of freedom giving rise to four fermion operators both at the tree and the loop level. In summary, we find: • Flavor changing couplings of the the SM Z boson can induce a ∆ACP as large as the observed value if the NP phase is moderately tuned to avoid constraints from indirect CP violation in D0 −¯D0 mixing. • A Z′ that mediates the c →u transition at tree level cannot account for the observed ∆ACP due to the combined constraints from D0 −¯D0 mixing and dijet searches. 5.3 Chirally enhanced magnetic penguins Box diagrams with the new fermion and the scalars also lead to contributions to D0−¯D0 mixing. Considering only the Acu coupling, we find ˜C(2)D 2 = g2 qg2 u 16π2 1 M2 φ (Acuv)2 M4 φ 1 2 ˜F(z) , (5.13) (5.13) where the loop function ˜F is the same as in section 5.2. The Auc coupling leads to the anal- ogous contribution to the coefficient C(2)D 2 . If both couplings are present simultaneously, contributions to C(2)D 4 and C(2)D 5 are also generated. In figure 13, we show regions in the Mφ — gugq|Acu|/Mφ plane compatible with the data on ∆ACP, WA at the 1σ level, considering Acu as the only source of flavor violation and setting Arg(Acu) = π/3. In the plot on the left (right) we set Mχ = 100 (250) GeV. The green (solid) band corresponds to the expression for the decay amplitude in na¨ıve factorization. The blue (dashed) band assumes an enhancement of the hadronic matrix elements by a factor of 3. The red (dash dotted) region is excluded by the D0 −¯D0 mixing constraints. The gray shaded regions are excluded by SUSY searches with jets + /ET . Results from ATLAS [94] and CMS [95] indicate that scalar masses up to 675 GeV are excluded for a fermion mass of 100 GeV (see left plot). On the other hand, for a fermion mass of 250 GeV, only a small region around 500 GeV is excluded by CMS data alone. For heavier fermion masses, the full range of scalar masses is allowed by present collider constraints. – 33 – Because of the chiral enhancement factor v/mc in the decay amplitudes, D0 −¯D0 mixing constraints allow for a large ∆ACP in the considered setup. Even for scalar masses of 1 TeV and larger, NP contributions to D0 −¯D0 mixing are more than an order of magnitude below the experimental constraints for NP contributions to ∆ACP that agree with the experimental value. 6 Conclusions • A heavy gluon with a flavor changing tree level c →u coupling and with a mass of 200 GeV ≲MG′ ≲320 GeV cannot fully be excluded as NP explanation for the mea- sured ∆ACP if a moderate enhancement of the hadronic matrix elements is allowed. • In a 2HDM with MFV, there exist regions of parameter space that can lead to a sizable ∆ACP. They are characterized by light charged Higgs masses and strongly enhanced couplings of the right handed strange quark to the charged Higgs, with – 34 – respect to its SM Yukawa coupling. Avoiding constraints from perturbativity and B →Xsγ, however, requires a considerable amount of fine tuning. respect to its SM Yukawa coupling. Avoiding constraints from perturbativity and B →Xsγ, however, requires a considerable amount of fine tuning. respect to its SM Yukawa coupling. Avoiding constraints from perturbativity and B →Xsγ, however, requires a considerable amount of fine tuning. • Scalar octets can also induce large nonstandard effects in a mass window 200 GeV ≲Mφ8 ≲320 GeV that is left open by current collider searches. The viable parameter space is analogous to the 2HDM with MFV model and appears to be, to some extent, fine tuned. • The scalar diquark model we consider is ruled out by D0 −¯D0 mixing as a NP explanation of ∆ACP. JHEP04(2012)049 • The minimal models that induce NP effects in the D meson decays through loops with Dirac fermions and scalars are strongly constrained by D0 −¯D0 mixing data and cannot give rise to a sizable ∆ACP. • If Majorana fermions and scalars appear in the loops, then D0−¯D0 mixing constraints can be avoided for a particular ratio of Majorana and scalar masses that depends on the exact quantum number assignment for the particles. Correspondingly, in such models there exist regions of parameter space that lead to large ∆ACP in agreement with the data. We note that our results are robust, since changes to the central value of ∆ACP, new direct search constraints, and the enhancement or suppression of flavor bounds coming from additional field content can be readily applied to our minimal models and our derived Wilson coefficients. As we showed, the New Physics parameter space favored for an explanation of the LHCb evidence for charm CP violation is largely within the current reach of various direct searches at the LHC. 6 Conclusions Thus the well-known complementarity between low energy flavor measurements and high energy direct probes may prove fruitful as we continue to search for New Physics. Our work emphasizes this synergy by presenting a broad study of minimal New Physics models, discussing both their effects on low energy flavor observables as well as their high energy collider signatures. We have demonstrated that a number of intriguing New Physics models can viably explain the large ∆ACP measurement, and we have concretely isolated the interesting parameter spaces of such models which must now be searched directly. A Hadronic matrix elements in na¨ıve factorization To evaluate the hadronic matrix elements of the operators in the ∆F = 1 effective Hamil- tonian in eq. (3.1), we use na¨ıve factorization ⟨K+K−|(¯uΓ1s)(¯sΓ2c)|D0⟩ ≃⟨K+|(¯uΓ1s)|0⟩⟨K−|(¯sΓ2c)|D0⟩, (A.1) (A.1) where Γi represent the various Dirac and color structures. In this approximation, it is straightforward to evaluate the hadronic matrix elements ⟨Oi⟩≡⟨K+K−|Oi|D0⟩ where Γi represent the various Dirac and color structures. In this approximation, it is straightforward to evaluate the hadronic matrix elements ⟨Oi⟩≡⟨K+K−|Oi|D0⟩ JHEP04(2012)049 ⟨O(1) 1 ⟩= Nc⟨O(1) 2 ⟩= Nc⟨O(1) 3 ⟩= ⟨O(1) 4 ⟩ (A.2) = −2Nc⟨O(1) 9 ⟩= −2⟨O(1) 10 ⟩, 1 χf ⟨O(1) 1 ⟩= Nc⟨O(1) 5 ⟩= ⟨O(1) 6 ⟩= −2Nc⟨O(1) 7 ⟩ = −2⟨O(1) 8 ⟩= −8⟨O(1) S1 ⟩= −8Nc⟨O(1) S2 ⟩, ⟨O(1) T1⟩= ⟨O(1) T2⟩= 0 , (A.2) where Nc = 3 is the number of colors and χf is the appropriate chiral factor from eq. (3.4). Using QCD factorization methods, the matrix element of the chromomagnetic operator is [18] N2 1 where Nc = 3 is the number of colors and χf is the appropriate chiral factor from eq. (3.4). Using QCD factorization methods, the matrix element of the chromomagnetic operator is [18] ⟨O(1) 8g ⟩= −αs 4π N2 c −1 N2c (3 + χ)⟨O(1) 1 ⟩. (A.3) (A.3) As QCD conserves parity, the matrix elements of the chirality flipped operators ˜O(1) i are identical to the ones shown above. As QCD conserves parity, the matrix elements of the chirality flipped operators ˜O(1) i are identical to the ones shown above. Acknowledgments Fermilab is operated by Fermi Research Alliance, LLC under Contract No. De-AC02- 07CH11359 with the United States Department of Energy. R.P. thanks the National Science Foundation for support under Grants PHY-0757481 and PHY-1068008. R.P. and C.-T.Y are supported by the Fermilab Fellowship in Theoretical Physics. The authors would like to thank T. J. Khoo for useful information regarding the ATLAS jets+/ET search, Andy Cohen and Martin Schmaltz for useful comments, and Daniele Alves, Jonathan Arnold, Bogdan Dobrescu, Stefania Gori and David Straub for useful discussions. – 35 – B Anomalous dimensions For completeness we collect here all the anomalous dimensions of the ∆F = 1 operators that are required for our analysis. The LO anomalous dimension matrix that governs the running and mixing of the current-current operators O(1)p 1,2 , the QCD penguin operators O(1) 3,...,6 and the QED penguin operators O(1) 7,...,10 is given by (see e.g. [99]) γ0 1,...,10 =                    −6 Nc 6 −2 3Nc 2 3 −2 3Nc 2 3 0 0 0 0 6 −6 Nc 0 0 0 0 0 0 0 0 0 0 −22 3Nc 22 3 −4 3Nc 4 3 0 0 0 0 0 0 6 − 2f 3Nc −6 Nc + 2f 3 −2f 3Nc 2f 3 0 0 0 0 0 0 0 0 6 Nc −6 0 0 0 0 0 0 −2f 3Nc 2f 3 −2f 3Nc 6(1−N2 c ) Nc + 2f 3 0 0 0 0 0 0 0 0 0 0 6 Nc −6 0 0 0 0 d−2u 3Nc 2u−d 3 d−2u 3Nc 2u−d 3 0 6(1−N2 c ) Nc 0 0 0 0 2 3Nc −2 3 2 3Nc −2 3 0 0 −6 Nc 6 0 0 d−2u 3Nc 2u−d 3 d−2u 3Nc 2u−d 3 0 0 6 −6 Nc                    , (B.1) (B.1) – 36 – where Nc = 3 is the number of colors, f is the number of active quark flavors, and u and d are the numbers of active up- and down-type quarks, respectively. ( ) where Nc = 3 is the number of colors, f is the number of active quark flavors, and u and d are the numbers of active up- and down-type quarks, respectively. (1) Leading order running of the chromomagnetic operator O(1) 8g is given by γ0 8g = 4N2 c −8 Nc . B Anomalous dimensions (B.2) (B.2) For the LO anomalous dimension matrix responsible for the running and mixing of the scalar and tensor operators O(1) S1,S2 and O(1) T1,T2, we find For the LO anomalous dimension matrix responsible for the running and mixing of the scalar and tensor operators O(1) S1,S2 and O(1) T1,T2, we find γ0 ST =       6−6N2 c Nc 0 1 Nc −1 −6 6 Nc −1 2 2−N2 c 2Nc 48 Nc −48 2N2 c −2 Nc 0 −24 48−24N2 c Nc 6 4N2 c +2 −Nc       , (B.3) JHEP04(2012)049 (B.3) which agrees with [31] once the different conventions for the operators and the σµν matrix are taken into account. At leading order and in the limit of massless down and strange quarks, the scalar and tensor operators do not mix into other operators. which agrees with [31] once the different conventions for the operators and the σµν matrix are taken into account. At leading order and in the limit of massless down and strange quarks, the scalar and tensor operators do not mix into other operators. As QCD conserves parity, the anomalous dimensions for the chirality flipped operators ˜O(1) i are identical to the ones shown above. Loop functions C Loop functions The functions B and ˜B appear in the box and crossed box contributions to the D meson decay amplitudes of that framework: P(z) = 1 −5z + 13z2 + 3z3 18(1 −z)4 + 2z3 3(1 −z)5 log(z) , G(z) = −1 + 8z + 17z2 24(1 −z)4 + z2(3 + z) 4(1 −z)5 log(z) , B(z) = 1 + 5z 8(1 −z)3 + z(2 + z) 4(1 −z)4 log(z) , ˜B(z) = z(5 + z) 2(1 −z)3 + z(1 + 2z) (1 −z)4 log(z) , F(z) = −1 + 10z + z2 3(1 −z)4 −2z(1 + z) (1 −z)5 log(z) , ˜F(z) = −z(17 + 8z −z2) 6(1 −z)4 −z(1 + 3z) (1 −z)5 log(z) . P(z) = 1 −5z + 13z2 + 3z3 18(1 −z)4 + 2z3 3(1 −z)5 log(z) , G(z) = −1 + 8z + 17z2 24(1 −z)4 + z2(3 + z) 4(1 −z)5 log(z) , B(z) = 1 + 5z 8(1 −z)3 + z(2 + z) 4(1 −z)4 log(z) , ˜B(z) = z(5 + z) 2(1 −z)3 + z(1 + 2z) (1 −z)4 log(z) , JHEP04(2012)049 F(z) = −1 + 10z + z2 3(1 −z)4 −2z(1 + z) (1 −z)5 log(z) , ˜F(z) = −z(17 + 8z −z2) 6(1 −z)4 −z(1 + 3z) (1 −z)5 log(z) . Finally, the loop function ˜G appears in the expression for the chromomagnetic penguin loop in section 5.3 ˜G(z) = −1 + 5z 4(1 −z)3 −z(2 + z) 2(1 −z)4 log(z) . 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 source are credited. C Loop functions The loop functions h1 and h2 appear in the charged Higgs contributions to kaon mixing in the 2HDM in section 4.5 h1(x) = 1 + x 2(1 −x)2 + x (1 −x)3 log(x) , h2(x, y) = x −4y (1 −x)(y −x) + 3y2 log(y) (1 −y)(x −y)2 +2xy −4y2 + x2(3y −1) (1 −x)2(y −x)2 log(x) . h1(x) = 1 + x 2(1 −x)2 + x (1 −x)3 log(x) , h2(x, y) = x −4y (1 −x)(y −x) + 3y2 log(y) (1 −y)(x −y)2 +2xy −4y2 + x2(3y −1) (1 −x)2(y −x)2 log(x) . h2(x, y) = x −4y (1 −x)(y −x) + 3y2 log(y) (1 −y)(x −y)2 +2xy −4y2 + x2(3y −1) (1 −x)2(y −x)2 log(x) . The loop functions p, g, f and ˜f appear in the discussion of the model with fermion — scalar loops without a GIM mechanism in section 5.1. The functions p and g occur in the expressions for the gluon penguin contributions to the D meson decay amplitudes. The function f comes from the evaluation of a box diagram contributing to D0 −¯D0 mixing and ˜f comes from the corresponding crossed box diagram p(z) = −2 −7z + 11z2 36(1 −z)3 − z3 6(1 −z)4 log(z) , g(z) = 1 −5z −2z2 24(1 −z)3 − z2 4(1 −z)4 log(z) , f(z) = −1 + z (1 −z)2 − 2z (1 −z)3 log(z) , ˜f(z) = − 2z (1 −z)2 −z(1 + z) (1 −z)3 log(z) . (C.1) ˜f(z) = − 2z (1 −z)2 −z(1 + z) (1 −z)3 log(z) . (C.1) (C.1) – 37 – – 37 – The loop functions P, G, F and ˜F are the analogues to p, g, f and ˜f in the fermion — scalar model with a GIM mechanism discussed in section 5.2. References [1] LHCb collaboration, R. Aaij et al., A search for time-integrated CP-violation in D0 →h−h+ decays, LHCb-CONF-2011-061 [arXiv:1112.0938]. 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https://openalex.org/W3155266168
https://hal.inrae.fr/hal-03287276/file/viruses-13-00673-v2.pdf
English
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Household Cases Suggest That Cats Belonging to Owners with COVID-19 Have a Limited Role in Virus Transmission
Viruses
2,021
cc-by
6,087
To cite this version: Pierre Bessière, Maxime Fusade-Boyer, Mathilda Walch, Laetitia Lèbre, Jessie Brun, et al.. House- hold Cases Suggest That Cats Belonging to Owners with COVID-19 Have a Limited Role in Virus Transmission. Viruses, 2021, 13 (4), pp.673. ￿10.3390/v13040673￿. ￿hal-03287276￿ Distributed under a Creative Commons Attribution 4.0 International License   Citation: Bessière, P.; Fusade-Boyer, M.; Walch, M.; Lèbre, L.; Brun, J.; Croville, G.; Boullier, S.; Cadiergues, M.-C.; Guérin, J.-L. Household Cases Suggest That Cats Belonging to Owners with COVID-19 Have a Limited Role in Virus Transmission. Viruses 2021, 13, 673. https:// doi.org/10.3390/v13040673 Keywords: COVID-19; cats; reverse-zoonosis HAL Id: hal-03287276 https://hal.inrae.fr/hal-03287276v1 Submitted on 15 Jul 2021 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License viruses viruses viruses Communication Household Cases Suggest That Cats Belonging to Owners with COVID-19 Have a Limited Role in Virus Transmission Pierre Bessière 1,* , Maxime Fusade-Boyer 1, Mathilda Walch 1, Laetitia Lèbre 1, Jessie Brun 2, Guillaume Croville 1 , Séverine Boullier 3, Marie-Christine Cadiergues 2,4 and Jean-Luc Guérin 1 1 IHAP, Université de Toulouse, INRAE, ENVT, 31300 Toulouse, France; maxime.fusade-boyer@envt.fr (M.F.-B.); mathilda.walch@envt.fr (M.W.); laetitia.lebre@envt.fr (L.L.); guillaume.croville@envt.fr (G.C.); jean-luc.guerin@envt.fr (J.-L.G.) 2 Small Animal Clinic Université de Toulouse ENVT 31300 Toulouse France; jessie brun@envt fr (J B ); 1 IHAP, Université de Toulouse, INRAE, ENVT, 31300 Toulouse, France; maxime.fusade-boyer@envt.fr (M.F.-B.); mathilda.walch@envt.fr (M.W.); laetitia.lebre@envt.fr (L.L.); guillaume.croville@envt.fr (G.C.); jean-luc.guerin@envt.fr (J.-L.G.) g j g 2 Small Animal Clinic, Université de Toulouse, ENVT, 31300 Toulouse, France; jessie.brun@envt.fr (J.B.); marie-christine.cadiergues@envt.fr (M.-C.C.) 3 InTheRes, Université de Toulouse, INRAE, ENVT, 31300 Toulouse, France; severine.boullier@envt.f 4 Infinity Université de Toulouse INSERM CNRS UT3 ENVT 31300 Toulouse France 3 InTheRes, Université de Toulouse, INRAE, ENVT, 31300 Toulouse, France; severine.boullier@envt.fr 4 Infinity, Université de Toulouse, INSERM, CNRS, UT3, ENVT, 31300 Toulouse, France * Correspondence: pierre.bessiere@envt.fr * Correspondence: pierre.bessiere@envt.fr * Correspondence: pierre.bessiere@envt.fr Abstract: Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is responsible for COVID- 19 and spread rapidly following its emergence in Wuhan in 2019. Although cats are, among other domestic animals, susceptible to SARS-CoV-2 infection, little is known about their epidemiological role in the dynamics of a household infection. In this study, we monitored five cats for viral shedding daily. Each cat was confined with its COVID-19 positive owners in separate households. Low loads of viral nucleic acid were found in two cats, but only one developed anti-SARS-CoV-2 antibodies, which suggests that cats have a limited role in COVID-19 epidemiology.   Citation: Bessière, P.; Fusade-Boyer, M.; Walch, M.; Lèbre, L.; Brun, J.; Croville, G.; Boullier, S.; Cadiergues, M.-C.; Guérin, J.-L. Household Cases Suggest That Cats Belonging to Owners with COVID-19 Have a Limited Role in Virus Transmission. Viruses 2021, 13, 673. https:// doi.org/10.3390/v13040673 Academic Editor: Julia A. Beatty Received: 3 March 2021 Accepted: 12 April 2021 Published: 14 April 2021   Citation: Bessière, P.; Fusade-Boyer, M.; Walch, M.; Lèbre, L.; Brun, J.; Croville, G.; Boullier, S.; Cadiergues, M.-C.; Guérin, J.-L. Household Cases Suggest That Cats Belonging to Owners with COVID-19 Have a Limited Role in Virus Transmission. Viruses 2021, 13, 673. https:// doi.org/10.3390/v13040673 Academic Editor: Julia A. Beatty Received: 3 March 2021 Accepted: 12 April 2021 Published: 14 April 2021 Keywords: COVID-19; cats; reverse-zoonosis 1. Introduction Since its emergence in late 2019, SARS-CoV-2, the etiological agent of COVID-19, has infected millions of people throughout the world. The virus spread from an animal reservoir, probably of bats, and might have involved an intermediate host. This highlights the importance of animals and the One Health perspective in the control of a zoonotic and emerging disease [1]. In the past few months, spillover events have resulted in several countries reporting a viral transmission from humans to animals. These events mostly involved cats [2–6], but also dogs [6,7] and minks [8]. Since other domestic animals, such as ferrets [9] and hamsters [10], are also susceptible to SARS-CoV-2 infection, reverse zoonosis events involving these animals will likely occur. In most case reports involving cats, animals are sampled only once and few studies perform daily monitoring of viral shedding. Human-to-cat transmission might be underestimated and there is a strong need to better understand the role of cats in COVID-19 epidemiology [11]. In this study, we monitored viral shedding in five different cats living in five different households with their quarantined and COVID-19-positive owners. Received: 3 March 2021 Accepted: 12 April 2021 Published: 14 April 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. 2.4. ELISA The serological status of the cats was assessed using a commercial SARS-CoV-2 N double antigen enzyme-linked immunosorbent assay (ELISA) kit in accordance with the manufacturer’s instructions (ID Screen; ID-Vet, Montpellier, France). 2.2. RT-qPCR from Feline and Environmental Swabs Swabs were briefly vortexed in 500 µL PBS and viral RNA was extracted from 140 µL in accordance with the manufacturer’s instructions (QIamp viral RNA; Qiagen, Toronto, ON, Canada). RNA extractions were performed within the 48 h following sampling (swabs were kept at 4 ◦C in the meantime). RT-qPCR was performed on 96-well plates with a final volume of 20 µL in a Light Cycler system (Roche, Penzberg, Germany). The mixes were prepared in accordance with the manufacturer’s instructions (QuantiNova Probe; Qiagen, Toronto, ON, Canada) with 2 µL of RNA and primers that targeted the E-gene. To obtain human-derived viral RNA and perform phylogenetic analysis, the owner from case 3 self-performed a nasal swab which was treated as a feline one. To check whether the material in the oropharyngeal and rectal swabs originated from felines, RT-qPCR targeting the feline 40S ribosomal protein S7 (RPS7) gene was also performed. SARS-CoV-2-positive swabs were further tested for human ribosomal protein L30 (RPL30) mRNA expression, to rule out human contamination. RPS7 and RPL30 RT-qPCR were performed on 96-well plates in a final volume of 10 µL in a Light Cycler System (Roche, Penzberg, Germany). Mixes were prepared according to the manufacturer’s instructions (QuantiFast; Qiagen, Toronto, ON, Canada). The primers’ sequences are listed in Supplementary Table S2. 2.3. Virus Isolation Vero E6 cells were plated in a 12-well plate and cultured in Dulbecco’s modified Eagle medium (DMEM) and complemented with 10% of heat-inactivated fetal bovine serum and 1% of penicillin-streptomycin at 37 ◦C and with 5% of CO2. Cells at 90% confluence were incubated with 100 µL of each RT-qPCR positive viral sample in DMEM complemented the 2% of heat-inactivated fetal bovine serum with 1% of penicillin-streptomycin, 2 µg/mL of ciprofloxacin, 10 µg/mL of BM-cyclin 1, and 1 µg/mL of amphotericin B (Merck, Darmstadt, Germany) for 72 h. Infectivity was assessed by screening cells for cytopathic effects and, at the end of the 72-h waiting period, the presence of SARS-CoV-2 nucleic acids was confirmed by RT-qPCR. Experiments were carried out in a biosafety level 3 facility at the National Veterinary School of Toulouse. 2.1. Owners and Cats 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/). All of the owners developed COVID-19-like symptoms (fever, cough, headache, fa- tigue, and a loss of taste or smell) and subsequently tested positive for SARS-CoV-2 infection by means of RT-qPCR performed on nasopharyngeal swabs in a medical facility in accordance with the French governmental guidelines [12]. They all belonged to clusters involving friends and relatives who were never in contact with the cats. Four owners were veterinary students, while a cat from case 5 was owned by a veterinarian’s relative. Viruses 2021, 13, 673. https://doi.org/10.3390/v13040673 https://www.mdpi.com/journal/viruses Viruses 2021, 13, 673 2 of 8 All of the cats lived strictly indoors. For one week during their quarantine, the owners performed daily oropharyngeal and rectal swabs on their cats and the cats’ food bowls (environmental swabs) following veterinary instructions (EC approval SSA_2020_0010). These instructions were given by phone and through a document, available in Supplemen- tary File S1. Swabs were collected daily and stored dry at 4 ◦C in the meantime. The delay between the onset of the owners’ symptoms and the first cat swabbing ranged from four to eight days. Blood samples were taken from all of the cats at least six weeks after their owners’ recovery to assess the cats’ serological status. For more details about the owners, see Supplementary Table S1. 2.5. Serum Neutralization Assay Vero-E6 cells were plated in 96-plates and cultured DMEM complemented with 10% of heat-inactivated fetal bovine serum and 1% of penicillin-streptomycin at 37 ◦C and with 5% of CO2. The samples and controls were heat-inactivated at 56 ◦C for 30 min, serially diluted in DMEM starting at 1:10, mixed with 100 TCID50 of SARS-CoV-2 (previously amplified and titrated on Vero-E6 cells), incubated for 2 h at 37 ◦C, and transferred to a culture plate. Following a 1-h incubation at 37 ◦C, the virus-serum dilutions were removed, and the cells were washed with PBS. Cells were incubated in a growth medium (DMEM complemented with 2% of heat-inactivated fetal bovine serum and 1% of penicillin-streptomycin) for 72 h Viruses 2021, 13, 673 3 of 8 at 37 ◦C and with 5% of CO2 and were screened for cytopathic effects. Serum from a SARS-CoV-2 immunized mouse (kindly provided by JC. Guéry) was used as a positive control. PBS was used as a negative control. Experiments were carried out in a biosafety level 3 facility at the National Veterinary School of Toulouse. at 37 ◦C and with 5% of CO2 and were screened for cytopathic effects. Serum from a SARS-CoV-2 immunized mouse (kindly provided by JC. Guéry) was used as a positive control. PBS was used as a negative control. Experiments were carried out in a biosafety level 3 facility at the National Veterinary School of Toulouse. 2.6. Whole-Genome Sequencing A SARS-CoV-2 whole-genome amplification was performed according to the ARTIC amplicon sequencing protocol for MinION for nCoV-2019 [13]. Briefly, reverse transcription of viral RNA was performed using an SSIV Reverse Transcriptase (Thermo Fisher Scientific, Inc., Mississauga, ON, Canada) and random hexamers followed by a PCR with the Q5 Hot Start DNA Polymerase (New England Biolabs, Ipswich, MA, USA) and the hCoV- 2019/nCoV-2019 Version 3 Amplicon Set. Samples were submitted to this protocol and the amplification was visually checked by running 5 µL aliquots on 1% agarose gel. DNA libraries were prepared using an SQK-LSK109 Ligation sequencing kit supplied by ONT (Oxford Nanopore Technologies, Oxford, UK) associated with an EXP-NBD104 (ONT) native barcoding kit for the multiplexing of 3 samples. In total, 30 fmol DNA libraries were loaded on a FLO-FLG001 flongle and were run on a MinION Mk1C device (ONT) for 10 h. Fast base-calling was performed in real-time with Guppy (v3.5) embedded in the MK1C software (v19.12.12) with the ‘Trim Barcode’ option on (ONT). Fastq files were mapped on the MW420003.1 reference genome using bwa-sw (v0.7.15) and consensus genomes were produced with bcftools mpileup (v1.6). Consensus sequences were then checked using the Geneious (v.2019.03) software (Biomatters, Ltd., Auckland, New Zealand). The owner- and feline-derived viral sequences were submitted to GenBank (accession numbers MW505982.1 and MW513511.1 respectively). 3.1. Case Description Five neutered European cats (two females and three males) belonging to owners with COVID-19 were enrolled in this study. The animals ranged in age from 1 to 10 years old. None of the cats developed any symptoms. Age, breed, gender, and samples are recapitulated in Table 1. 2.7. Phylogenetic Analysis Whole-genome sequences of the 50 strains most closely related to hCoV-19/cat/France/ LHOOQ/2020 and hCoV-19/human/FRA/66JQ-O/2020 (the strains isolated from case 3 and its owner, respectively) were downloaded from NCBI using a BLASTn search. All cat-isolated SARS-CoV-2 sequences also were loaded, as well as one strain representative of each clade according to GISAID classification. All of the sequences were aligned using the MAFFT online server and tested for recombination events using the Recombination Detection Program 4 (RDP4) [14]. When several of the collected sequences in the same host and place were similar, only one was kept for the following steps. Phylogenetic analysis was performed using a generalized time-reversible (GTR) model with discrete gamma distribution and four categories implemented in MegaX (MEGA X: Molecular Evolutionary Genetics Analysis across computing platforms; Kumar, Stecher, Li, Knyaz, and Tamura 2018) performing a 1000-bootstrap resampling analysis—a process visualized with Figtree (v.1.4.2). 3.3. One Cat Out of Five Developed Anti-SARS-CoV-2 Antibodies 3.3. One Cat Out of Five Developed Anti-SARS-CoV-2 Antibodies Antibodies were found in case 3 only and the other samples were negative. Serum from case 3 yielded positive ELISA titers (OD value: 1.088; % positivity: 121%), which was confirmed by the detection of neutralizing antibodies (neutralization titer > 1:1280). These results suggest that SARS-CoV-2 was effectively transmitted in only this case, despite the proximity of the other owners with their cats. 3.2. Two Cats Out of Five Shed SARS-CoV-2 Cases 3 and 5 tested positive for SARS-CoV-2 RNA. For case 3, low quantities of viral nucleic acids were detected in a rectal swab (Ct 37.0) and an oropharyngeal swab (Ct 35.6) three and four days, respectively, after the owner’s diagnosis. Viral nucleic acids were also detected in the cat’s feeding bowl on days 5 and 6 after the owner’s diagnosis (Ct 33.6 and 34.2 respectively). For case 5, viral nucleic acid was detected once on an oropharyngeal swab (Ct 36.7) and once on an environmental swab (Ct 37.5) (Table 1). All Viruses 2021, 13, 673 4 of 8 cat and environmental swabs were tested for feline 40S ribosomal protein S7 (RPS7) mRNA expression to check whether swabbing was performed appropriately: feline RPS7 mRNA expression was detected in all feline swabs and most environmental swabs (including SARS-CoV-2-positive ones). SARS-CoV-2-positive samples were further tested for human ribosomal protein L30 (RPL30) mRNA expression, which was not detected, suggesting that the presence of viral RNA was not due to human contamination. Virus isolation from positive swabs on Vero E6 cells were unsuccessful. All other swabs were negative at the other time points. Table 1. Summary of the cases, including breed, age, gender, the time between the onset of owner’s symptoms and cat’s first swab, number, and RT-qPCR Ct values of positive swabs (Day 1 corresponding to the first day the cat was enrolled in the study) and serological status determined by ELISA and serum neutralization assays. Case Breed Age (Years) Gender Days Between Owners’ Symptoms Onset and Cat Swabbing Positive Swabs ELISA/ SN (Days 1) OP R Env 1 DSH 1 Female 4 None None None Neg/Neg (54) 2 DSH 10 Male 6 None None None Neg/Neg (62) 3 DSH 3 Female 5 Day 2: 35.6 Day 1: 35.7 Day 1: 33.6 Day 2: 34.2 Pos/Pos (47) 4 DSH 1 Male 8 None None None Neg/Neg (51) 5 DSH 6 Male 7 Day 3: 36.7 None Day 6: 37.5 Neg/Neg (174) DSH: domestic shorthair cat; OP: oropharyngeal; R: rectal; Env: environment; Neg: negative; Pos: positive; SN: serum neutralization assay. 1: time between the onset of owners’ symptoms and the ELISA test. Table 1. 3.2. Two Cats Out of Five Shed SARS-CoV-2 Summary of the cases, including breed, age, gender, the time between the onset of owner’s symptoms and cat’s first swab, number, and RT-qPCR Ct values of positive swabs (Day 1 corresponding to the first day the cat was enrolled in the study) and serological status determined by ELISA and serum neutralization assays. Table 1. Summary of the cases, including breed, age, gender, the time between the onset of owner’s symptoms and cat’s first swab, number, and RT-qPCR Ct values of positive swabs (Day 1 corresponding to the first day the cat was enrolled in the study) and serological status determined by ELISA and serum neutralization assays. DSH: domestic shorthair cat; OP: oropharyngeal; R: rectal; Env: environment; Neg: negative; Pos: positive; SN: serum n 1: time between the onset of owners’ symptoms and the ELISA test. at; OP: oropharyngeal; R: rectal; Env: environment; Neg: negative; Pos: positive; SN: serum neutralization assay. of owners’ symptoms and the ELISA test. cussion 4. Discussion scussio Cats belonging to COVID-19-positive owners were monitored for viral shedding al signs. None of the cats developed symptoms. SARS-CoV-2 nucleic acids w d by RT-qPCR in two cats, for two consecutive days in cat 3, and only once in c both had high Cts values. They were also found on the dinner bowls of both esting that the virus was shed in the house, although it could have emanated f wners as well as from the cats. We detected RPS7 mRNA using RT-qPCR in all o haryngeal and rectal swabs, demonstrating that the owners managed to swab als correctly. Furthermore, human RPL30 mRNA was not detected in any SA 2-positive feline or environmental swabs, thus decreasing the probability that presence was due to human contamination. Case 3 had the only cat that tested for anti-SARS-CoV-2 antibodies using both ELISA and serum virus neutraliza s. The other cats tested negative by means of RT-qPCR for SARS-CoV-2 and, acc o serological tests made a few weeks later, the cats also tested negative for antibo Cats belonging to COVID-19-positive owners were monitored for viral shedding and clinical signs. None of the cats developed symptoms. SARS-CoV-2 nucleic acids were found by RT-qPCR in two cats, for two consecutive days in cat 3, and only once in cat 5, and both had high Cts values. They were also found on the dinner bowls of both cats, suggesting that the virus was shed in the house, although it could have emanated from the owners as well as from the cats. We detected RPS7 mRNA using RT-qPCR in all of the oropharyngeal and rectal swabs, demonstrating that the owners managed to swab their animals correctly. Furthermore, human RPL30 mRNA was not detected in any SARS-CoV-2-positive feline or environmental swabs, thus decreasing the probability that viral RNA presence was due to human contamination. Case 3 had the only cat that tested positive for anti-SARS-CoV-2 antibodies using both ELISA and serum virus neutralization assays. The other cats tested negative by means of RT-qPCR for SARS-CoV-2 and, according to serological tests made a few weeks later, the cats also tested negative for antibodies. This suggests that the cats were never infected, rather than that the shedding window was missed when swabbing the animals. Since viral shedding was weak and transient, cats may not be efficient at shedding SARS-CoV-2. 3.4. Phylogenetic Analysis The viruses in Table 1, sequence from case 3, and the owner-derived sequence are both labelled in red. Other feline-derived viral sequences are labeled in purple. Bootstrap supports >60% are indicated next to the nodes; the scale bar indicates the numbers of nucleotide substitutions per site. The strain hCoV-19/Wuhan/WIV04/2019 was used as the outgroup. Whole-genome maximum likelihood phylogenetic tree of SARS-CoV-2. The viruses in Table 1, sequence f d the owner-derived sequence are both labelled in red. Other feline-derived viral sequences are labeled in pur supports >60% are indicated next to the nodes; the scale bar indicates the numbers of nucleotide substituti he strain hCoV-19/Wuhan/WIV04/2019 was used as the outgroup. Figure 1. Whole-genome maximum likelihood phylogenetic tree of SARS-CoV-2. The viruses in Table 1, sequence from case 3, and the owner-derived sequence are both labelled in red. Other feline-derived viral sequences are labeled in purple. Bootstrap supports >60% are indicated next to the nodes; the scale bar indicates the numbers of nucleotide substitutions per site. The strain hCoV-19/Wuhan/WIV04/2019 was used as the outgroup. Whole-genome maximum likelihood phylogenetic tree of SARS-CoV-2. The viruses in Table 1, sequence f d the owner-derived sequence are both labelled in red. Other feline-derived viral sequences are labeled in pur p supports >60% are indicated next to the nodes; the scale bar indicates the numbers of nucleotide substitut The strain hCoV-19/Wuhan/WIV04/2019 was used as the outgroup. Figure 1. Whole-genome maximum likelihood phylogenetic tree of SARS-CoV-2. The viruses in Table 1, sequence from case 3, and the owner-derived sequence are both labelled in red. Other feline-derived viral sequences are labeled in purple. Bootstrap supports >60% are indicated next to the nodes; the scale bar indicates the numbers of nucleotide substitutions per site. The strain hCoV-19/Wuhan/WIV04/2019 was used as the outgroup. 3.4. Phylogenetic Analysis A partial viral genome (accession number MW513511.1) was obtained from the posi- tive oropharyngeal swab of case 3, while a whole genome (accession number MW505982.1) was obtained from the owner’s nasopharyngeal swab. Phylogenetic analysis performed on the whole genome showed that both viruses in our study had clustered with viruses from Switzerland and belonged to the clade GH according to GISAID nomenclature. This clade is characterized by the nucleotide substitutions C241T, C3037T, A23403G, G25563T, the amino acid substitution Q57H in the NS3 protein, and the substitution D614G in the spike protein, which is known to enhance infectivity and viral fitness [15]. Phylogenetic analysis also showed that both sequences from case 3 were very closely related, sharing more than 99.99% genetic identity (Figure 1). Both sequences differed only by the synonymous G26031A mutation and an amino acid substitution F843L in the ORF1ab protein that was not described in the literature. Nevertheless, since only a partial genome was obtained from the cat sample, two regions (from amino acid (aa) 262 to aa 318, and from aa 448 to aa 520) were not successfully sequenced and other differences may have been missed. Viruses 2021, 13, 673 5 of 8 ences 1. Whole-genome maximum likelihood phylogenetic tree of SARS-CoV-2. The viruses in Table 1, sequence fr nd the owner-derived sequence are both labelled in red. Other feline-derived viral sequences are labeled in purp p supports >60% are indicated next to the nodes; the scale bar indicates the numbers of nucleotide substitutio The strain hCoV-19/Wuhan/WIV04/2019 was used as the outgroup. Figure 1. Whole-genome maximum likelihood phylogenetic tree of SARS-CoV-2. The viruses in Table 1, sequence from case 3, and the owner-derived sequence are both labelled in red. Other feline-derived viral sequences are labeled in purple. Bootstrap supports >60% are indicated next to the nodes; the scale bar indicates the numbers of nucleotide substitutions per site. The strain hCoV-19/Wuhan/WIV04/2019 was used as the outgroup. Whole-genome maximum likelihood phylogenetic tree of SARS-CoV-2. The viruses in Table 1, sequence f d the owner-derived sequence are both labelled in red. Other feline-derived viral sequences are labeled in pu p supports >60% are indicated next to the nodes; the scale bar indicates the numbers of nucleotide substitut The strain hCoV-19/Wuhan/WIV04/2019 was used as the outgroup. Figure 1. Whole-genome maximum likelihood phylogenetic tree of SARS-CoV-2. cussion 4. Discussion suggests that the cats were never infected, rather than that the shedding window ed when swabbing the animals. Since viral shedding was weak and transient, not be efficient at shedding SARS-CoV-2. In experimental studies, cats inoculated intranasally or intratracheally with SA 2 shed viruses up to 10 days post-infection [16,17] and were able to infect non-i d cats through direct or indirect contact [18]. However, the inoculum dose and g In experimental studies, cats inoculated intranasally or intratracheally with SARS-CoV- 2 shed viruses up to 10 days post-infection [16,17] and were able to infect non-inoculated cats through direct or indirect contact [18]. However, the inoculum dose and the infection route may not reflect the natural circumstances in which a cat could be naturally infected. In a more recent study, three cats living together in one household with COVID-19-positive owners were monitored for viral shedding and the SARS-CoV-2 RNA was sequentially detected in all three cats—whether this was due to a cat-to-cat transmission remains Viruses 2021, 13, 673 6 of 8 unknown. Interestingly, viral RNA was detected up to 11 days after the onset of the owners’ symptoms [19]. unknown. Interestingly, viral RNA was detected up to 11 days after the onset of the owners’ symptoms [19]. In our study, the cats lived in small to moderate-sized households and the owners did not take particular hygiene measures while interacting with their pets. Despite these elements, in only two cats was viral RNA detected. Cat 5 did not develop anti-SARS-CoV-2 antibodies and a viral genome was found only once in an oropharyngeal swab and once in an environmental swab. It remains unknown whether the swabs were contaminated with viral RNA from the owner. In addition, human cases with transient SARS-CoV- 2 shedding and an absence of antibody responses have been reported [20,21] and the same phenomenon could occur in cats. Notably, Temman and colleagues failed to detect anti-SARS-CoV-2 antibodies in nine cats belonging to COVID-19 positive students [22]. The infected cats did not display any clinical signs, which is consistent with previous experimental studies [16–18]. However, subclinical manifestations cannot be excluded and it is worth noting there have been some reports of symptomatic cats in households with COVID-19 patients [2–4,19]. Age and comorbidities are likely involved in these differences and further studies are required to better understand SARS-CoV-2 pathogenesis in cats. cussion 4. Discussion q p g In addition to the epidemiological context, phylogenetic analysis supports the human- to-cat transmission hypothesis since the strains detected in the owners and the cats shared a high genetic identity. The feline viral genome differed only by one amino-acid and one nucleotide substitution. A sequence comparison with the other available feline-derived SARS-CoV-2 genomes did not reveal any shared cat-specific mutation. None of the available viral sequences from cats harbored this substitution, which suggests a random event rather than an adaptation to cats despite the low number of available sequences. Our study has a number of limitations. The sample size was limited due to the con- straining nature of the study’s protocol, which renders it difficult to generalize our findings to a larger cat population. Due to the strict lockdown measures at the time of this study, the owners performed the swabbing unsupervised. They received detailed instructions, but we cannot exclude the possibility that the samples were not taken thoroughly enough, despite the RPS7 mRNA presence, or that they were contaminated by the owners’ hands or fomites, despite the human RPL30 mRNA absence. In addition, there is a lack of evidence regarding the sensitivity of oropharyngeal swabbing compared to nasal or nasopharyngeal swabbing in cats. In conclusion, the SARS-CoV-2 genome was detected by RT-qPCR in two cats out of five, which suggests that human-to-cat transmission is not an infrequent event. Given the variability of clinical manifestations, COVID-19 in cats is likely under-diagnosed. However, viral shedding was weak and transient, which hints that, even when infected, cats probably play a limited role in COVID-19 epidemiology. Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/v13040673/s1, Table S1: Households descriptions, Table S2: primers and probe for RT- qPCR [23–25], file S1: swabbing instructions given to the owners. 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Available online: https://solidarites-sante.gouv.fr/IMG/ pdf/covid-19__rt-Pcr-Ambulatoire-Fiche-preleveurs.Pdf (accessed on 20 May 2020). p y oV-2019 Sequencing Protocol (single Sample) V1 (protocols.io.Bdbfi2jn). Protocols.io 2020, 13. [CrossRef] 13. Quick, J. NCoV-2019 Sequencing Protocol (single Sample) V1 (protocols.io.Bdbfi2jn). Protocols.io 2020, 14. Martin, D.P.; Murrell, B.; Golden, M.; Khoosal, A.; Muhire, B. RDP4: Detection and Analysis of Recombination Patterns in Virus Genomes. Virus Evol. 2015, 1, vev003. [CrossRef] 15. Hou, Y.J.; Chiba, S.; Halfmann, P.; Here, C.; Kuroda, M.; Dinnon, K.H., III; Leist, S.R.; Schafer, A.; Nakajima, N.; Takahashi, K.; et al. SARS-CoV-2 D614G Variant Exhibits Efficient Replication Ex Vivo and Transmission In Vivo. Science 2020, 370, 1464–1468. [CrossRef] 16. Gaudreault, N.N.; Trujillo, J.D.; Carossino, M.; Meekins, D.A.; Morozov, I.; Madden, D.W.; Indran, S.V.; Bold, D.; Balaraman, V.; Kwon, T.; et al. SARS-CoV-2 Infection, Disease and Transmission in Domestic Cats. Emerg. Microbes Infect. 2020, 9, 2322–2332. [CrossRef] [PubMed] 17. Shi, J.; Wen, Z.; Zhong, G.; Yang, H.; Wang, C.; Huang, B.; Liu, R.; He, X.; Shuai, L.; Sun, Z.; et al. Susc Dogs, and Other Do-Mesticated Animals to SARS–coronavirus 2. Science 2020, 368, 1016–1020. cussion 4. Discussion Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Ethics Committee Sciences et Santé Animale n◦115 (protocol code COVIFEL approved on 1 September 2020, registered under SSA_2020_010). Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Ethics Committee Sciences et Santé Animale n◦115 (protocol code COVIFEL approved on 1 September 2020, registered under SSA_2020_010). Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Data Availability Statement: The data presented in this study are available on request from the corre- sponding author. The data are not publicly available due to the interests of retaining patient confidentiality. Data Availability Statement: The data presented in this study are available on request from the corre- sponding author. The data are not publicly available due to the interests of retaining patient confidentiality. 7 of 8 Viruses 2021, 13, 673 Acknowledgments: We warmly thank the cat owners for their involvement in the study. We are very thankful to J.-C. Guéry for providing serum from a SARS-CoV-2 immunized mouse. Acknowledgments: We warmly thank the cat owners for their involvement in the study. We are very thankful to J.-C. Guéry for providing serum from a SARS-CoV-2 immunized mouse. 21. Lin, Y.-C.; Cheng, C.-Y.; Chen, C.-P.; Cheng, S.-H.; Chang, S.-Y.; Hsueh, P.-R. A Case of Transient Existence of SARS-CoV-2 RNA in the Respiratory Tract with the Absence of Anti-SARS-CoV-2 Antibody Response. Int. J. Infect. Dis. 2020, 96, 464–466. [CrossRef] [PubMed] 22. Temmam, S.; Barbarino, A.; Maso, D.; Behillil, S.; Enouf, V.; Huon, C.; Jarud, A.; Chevallier, L.; Backovic, M.; Perot, P.; et al. Absence of SARS-CoV-2 Infection in Cats and Dogs in Close Contact with a Cluster of COVID-19 Patients in a Veterinary Campus. One Health 2020, 10, 100164. [CrossRef] [PubMed] 25. De Jonge, H.J.M.; Fehrmann, R.S.N.; De Bont, E.S.J.M.; Hofstra, R.M.W.; Gerbens, F.; Kamps, W.A.; de Vries, E.G.E.; van der Zee, A.G.J.; te Meerman, G.J.; ter Elst, A. Evidence Based Selection of Housekeeping Genes. PLoS ONE 2007, 2, e898. [CrossRef] ( ) y 24. Penning, L.C.; Vrieling, H.E.; Brinkhof, B.; Riemers, F.M.; Rothuizen, J.; Rutteman, G.R.; Hazewinkel, H.A. A Validation of 10 Feline Reference Genes for Gene Expression Measurements in Snap-Frozen Tissues. Veter. Immunol. Immunopathol. 2007, 120, 212–222. [CrossRef] [PubMed] , , [ ] [ ] 23. Corman, V.M.; Landt, O.; Kaiser, M.; Molenkamp, R.; Meijer, A.; Chu, D.K.; Drosten, C. Detection of 2 (2019-NCoV) by Real-Time RT-PCR. Eurosurveillance 2020, 25, 2000045. [CrossRef] [PubMed] [ ] [ ] dt, O.; Kaiser, M.; Molenkamp, R.; Meijer, A.; Chu, D.K.; Drosten, C. Detection of 2019 Novel Corona-Virus al-Time RT-PCR. Eurosurveillance 2020, 25, 2000045. [CrossRef] [PubMed] References [CrossR 18. Halfmann, P.J.; Hatta, M.; Chiba, S.; Maemura, T.; Fan, S.; Takeda, M.; Kinoshita, N.; Hattori, S.-i.; Sagai-Tagava, Y.; Iwatsuki- Horimoto, K.; et al. Transmission of SARS-CoV-2 in Do-Mestic Cats. N. Engl. J. Med. 2020, 383, 592–594. [CrossRef] [PubMed] g 19. Neira, V.; Brito, B.; Agüero, B.; Berrios, F.; Valdés, V.; Gutierrez, A.; Aryama, N.; Espinoza, P.; Retamal, P.; Holmes, E.C.; et al. A Household Case Evidences Shorter Shed-Ding of SARS-CoV-2 in Naturally Infected Cats Compared to Their Human Owners. Emerg. Microbes Infect. 2020, 10, 1–22. 20. Alonso, F.O.M.; Sabino, B.D.; Guimarães, M.A.A.M.; Varella, R.B. Recurrence of SARS-CoV-2 Infection with a More Severe Case After Mild COVID-19, Reversion of RT-QPCR for Positive and Late Antibody Response: Case Report. J. Med. Virol. 2021, 93, 655–656. [CrossRef] [PubMed] 21. Lin, Y.-C.; Cheng, C.-Y.; Chen, C.-P.; Cheng, S.-H.; Chang, S.-Y.; Hsueh, P.-R. A Case of Transient Existence of SARS-CoV-2 RNA in the Respiratory Tract with the Absence of Anti-SARS-CoV-2 Antibody Response. Int. J. Infect. Dis. 2020, 96, 464–466. [CrossRef] [PubMed] p [CrossRef] [PubMed] 8 of 8 Viruses 2021, 13, 673 y 24. Penning, L.C.; Vrieling, H.E.; Brinkhof, B.; Riemers, F.M.; Rothuizen, J.; Rutteman, G.R.; Hazewinkel, H.A. A Validation of 10 Feline Reference Genes for Gene Expression Measurements in Snap-Frozen Tissues. Veter. Immunol. Immunopathol. 2007, 120, 212–222. [CrossRef] [PubMed] 25. De Jonge, H.J.M.; Fehrmann, R.S.N.; De Bont, E.S.J.M.; Hofstra, R.M.W.; Gerbens, F.; Kamps, W.A.; de Vries, E.G.E.; van der Zee, A.G.J.; te Meerman, G.J.; ter Elst, A. Evidence Based Selection of Housekeeping Genes. PLoS ONE 2007, 2, e898. [CrossRef]
https://openalex.org/W2169186489
https://angeo.copernicus.org/articles/25/1141/2007/angeo-25-1141-2007.pdf
English
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A new method to estimate ionospheric electric fields and currents using data from a local ground magnetometer network
Annales geophysicae
2,007
cc-by
11,002
1 Introduction Abstract. In this study we present a new method to esti- mate ionospheric electric fields and currents using ground magnetic recordings and measured or modeled ionospheric electric conductivity as the input data. This problem has been studied extensively in the past, and the standard anal- ysis technique for such a set of input parameters is known as the KRM method (Kamide et al., 1981). The new method presented in this study makes use of the same input data as the traditional KRM method, but differs significantly from it in the mathematical approach that is used. In the KRM method one tries to find such a potential electric field, that the resulting current system has the same curl as the iono- spheric equivalent currents. In the new method we take a different approach, so that we determine such a curl-free cur- rent system that, together with the equivalent currents, it is consistent with a potential electric field. This approach re- sults in a slightly different equation, that makes better use of the information contained in the equivalent currents. In this paper we concentrate on regional studies, where the (un- known) boundary conditions at the borders of the analysis area play a significant role in the KRM solution. In order to overcome this complication, we formulate a novel numer- ical algorithm to be used with our new calculation method. This algorithm is based on the Cartesian elementary current systems (CECS). With CECS the boundary conditions are implemented in a natural way, making regional studies less prone to errors. We compare the traditional KRM method and our new CECS-based formulation using several realis- tic models of typical meso-scale phenomena in the auroral ionosphere, including a uniform electrojet, the -bands and the westward traveling surge. It is found that the error in the CECS results is typically about 20%–40%, whereas the errors in the KRM results are significantly larger. Determination of ionospheric electrodynamic parameters from direct or indirect measurements is a fundamental task in ionospheric physics. Over the years several methods have been developed to estimate various parameters or their combinations from different sets of measured or modeled data (see e.g. Untiedt and Baumjohann, 1993; or Amm et al., 2003, for references). In this paper we present a new method to estimate ionospheric electric fields and currents using ground magnetic recordings and ionospheric electric conductivity as the input data. A new method to estimate ionospheric electric fields and currents using data from a local ground magnetometer network H. Vanham¨aki and O. Amm Finnish Meteorological Institute, Space Research Unit, P.O. Box 503, 00101 Helsinki, Finland Received: 8 February 2007 – Revised: 26 April 2007 – Accepted: 7 May 2007 – Published: 4 June 2007 H. Vanham¨aki and O. Amm Finnish Meteorological Institute, Space Research Unit, P.O. Box 503, 00101 Helsinki, Finland Received: 8 February 2007 – Revised: 26 April 2007 – Accepted: 7 May 2007 – Published: 4 June 2007 Ann. Geophys., 25, 1141–1156, 2007 www.ann-geophys.net/25/1141/2007/ © European Geosciences Union 2007 Ann. Geophys., 25, 1141–1156, 2007 www.ann-geophys.net/25/1141/2007/ © European Geosciences Union 2007 Annales Geophysicae Published by Copernicus GmbH on behalf of the European Geosciences Union. 1 Introduction This problem has been studied extensively in the past, especially by Kamide and co-workers (Kamide et al., 1981; Murison et al., 1985, and references therein). The standard analysis technique for this set of in- put parameters is known as the KRM method developed by Kamide et al. (1981). The ground magnetic data is most conveniently used in form of ionospheric equivalent currents. By definition, they are divergence-free horizontal sheet currents, that produce the same magnetic field below the ionosphere as the real (un- known) 3-dimensional current system. Equivalent currents can be calculated using standard techniques, like spherical harmonic analysis (Chapman and Bartels, 1940) in global scales and spherical cap harmonic analysis (Haines, 1985) in regional studies, or elementary current method (Amm and Viljanen, 1999; Pulkkinen et al., 2003) in all scales. The other input parameter, distribution of ionospheric Pedersen and Hall conductivities, is more difficult to estimate reliably. Conductivities may be obtained from satellite or all-sky cam- era images (e.g. Lummerzheim et al., 1991; Janhunen, 2001; Aksnes et al., 2005), but the temporal and spatial coverage is often insufficient and a number of unknown model parame- ters or empirical relations need to be assumed. Also statisti- cal models like Fuller-Rowell and Evans (1987) may be used, but they are not very accurate during disturbed conditions. If no other measurement are available, a rough estimate of the ionospheric conductances may be obtained from the ground magnetic data, as done by Ahn et al. (1998). Keywords. Ionosphere (Auroral ionosphere; Electric fields and currents; Instruments and techniques) Correspondence to: H. Vanham¨aki (heikki.vanhamaki@fmi.fi) Correspondence to: H. Vanham¨aki (heikki.vanhamaki@fmi.fi) Published by Copernicus GmbH on behalf of the European Geosciences Union. 1142 H. Vanham¨aki and O. Amm: Ionospheric currents from local magnetic data to the magnetic field is zero due to the high conductivity in this direction. The new method presented in this article makes use of the same input data as the traditional KRM method, but dif- fers significantly from it in the mathematical approach. In the KRM method one tries to find such a potential electric field, that the resulting current system has the same curl as the ionospheric equivalent currents. Once this electric field is obtained, also the field aligned currents (FAC) and corre- sponding curl-free horizontal currents (which are magneti- cally invisible below the ionosphere) may be calculated. 2 Theory If Eq. (4) is solved globally, we only have to fix the zero level of potential. Also in semi-global studies, that cover either the northern or southern auroral regions, boundary conditions of the electric potential are not problematic, as they have to be specified only at the mid-latitudes, where the electric field is rather small in any case. However, in meso-scale studies, covering areas of few hundred or at most few thousand km across, boundary conditions play an impor- tant role. This was demonstrated by Murison et al. (1985), who studied a situation where the Harang discontinuity was present over northern Scandinavia. They found that in the regional analysis the electric field, and consequently also the currents, depend strongly on the boundary conditions. This severely limits the use of the KRM method in regional stud- ies. Recently Kamide et al. (2003) have developed a lo- cal variant of the KRM method, where the KRM is used in If Eq. (4) is solved globally, we only have to fix the zero level of potential. Also in semi-global studies, that cover either the northern or southern auroral regions, boundary conditions of the electric potential are not problematic, as they have to be specified only at the mid-latitudes, where the electric field is rather small in any case. However, in meso-scale studies, covering areas of few hundred or at most few thousand km across, boundary conditions play an impor- tant role. This was demonstrated by Murison et al. (1985), who studied a situation where the Harang discontinuity was present over northern Scandinavia. They found that in the regional analysis the electric field, and consequently also the currents, depend strongly on the boundary conditions. This severely limits the use of the KRM method in regional stud- ies. Recently Kamide et al. (2003) have developed a lo- cal variant of the KRM method, where the KRM is used in In this section we first give a short review of the KRM method and then introduce our own, somewhat different ap- proach to solving the same problem. We obtain a partial differential equation that we solve numerically using the Cartesian elementary current systems (CECS), introduced in Sect. 3.1. In this study we use the thin-sheet approximation, i.e. we assume that ionospheric horizontal currents flow at a thin spherical layer at about 100 km altitude. 2.1 The KRM method Kamide et al. (1981) developed the KRM method for deter- mining ionospheric electric field and currents in situations where estimates of height-integrated ionospheric Pedersen and Hall conductances, 6P and 6H, together with ground magnetic measurements are available. From the ground mag- netic measurements one can obtain the ionospheric equiv- alent current density J eq using standard techniques (e.g. Chapman and Bartels, 1940; Haines, 1985; Amm and Vil- janen, 1999). For a vertical background magnetic field, J eq is equal to the divergence-free part of the true ionospheric current den- sity J (see e.g. Untiedt and Baumjohann, 1993), so that ∇× J eq = ∇× J. (1) In the past the KRM method has been used mostly in global or semi-global scales, but in this study we concentrate on regional analysis. In these smaller scales the (unknown) boundary conditions at the borders of the analysis area affect the KRM solution significantly, as was shown by Murison et al. (1985) and discussed further in Sect. 2.1. In Sect. 3 we formulate a novel numerical algorithm to be used with our new calculation method. This algorithm is based on the Cartesian elementary current systems (CECS), that were in- troduced by Amm (1997). With the use of CECS the bound- ary conditions can be implemented in a very convenient and natural way, which makes regional studies less prone to er- rors. We compare the traditional KRM method and our new CECS based formulation first in a simple electrojet situation in Sect. 4. In Sect. 5 we continue the comparisons using two realistic data based models of typical meso-scale phenom- ena in the auroral ionosphere, namely the -bands and the westward traveling surge (WTS). Section 6 is summary and conclusions. (1) The ionospheric electric field E is assumed to be given by a potential φ as E = −∇φ. (2) (2) E = −∇φ. In the KRM method Eqs. (1) and (2) are used together with ionospheric Ohm’s law In the KRM method Eqs. (1) and (2) are used together with ionospheric Ohm’s law J = 6P E + 6H ˆez × E (3) (3) to obtain a differential equation for the electric potential, (4) 6H∇2φ+∇6H·∇φ+(∇6P ×∇φ)z=−(∇×J eq)z. (4) If ionospheric conductances and equivalent currents are know, the electric potential can be solved. This gives a com- plete solution of the ionospheric electric properties, for the electric field is given by Eq. j∥= ∇· J. j∥= ∇· J. (5) j∥= ∇· J. 1 Introduction In the new method this approach is reversed, so that we de- termine such a curl-free current system that, together with the equivalent currents, it is consistent with a potential elec- tric field. This approach results in a slightly different equa- tion, that makes better use of the information contained in the equivalent currents. The mathematical formulation of both the KRM method and our new approach are presented in more detail in Sect. 2. 2.1 The KRM method (2), horizontal currents are ob- tained from Ohm’s law and FAC are given by current conti- nuity, (5) 2 Theory We concentrate on local scale studies, where we use a Cartesian coordinate sys- tem with x-axis pointing North, y-axis East and z-axis down. The Earth’s magnetic field is assumed to be parallel to the z-axis, which is a reasonable approximation near the auroral oval. Furthermore, we assume that the electric field parallel Ann. Geophys., 25, 1141–1156, 2007 www.ann-geophys.net/25/1141/2007/ 1143 H. Vanham¨aki and O. Amm: Ionospheric currents from local magnetic data The equation we obtained is similar in structure to Eq. (4). One significant difference is that in the KRM equation only ∇×J eq appears, whereas now also the vector J eq itself is needed. In a limited area J eq may have a Laplacian part that has zero curl inside the analysis area. This part of J eq does not contribute to the KRM solution, but it is included in Eq. (8). areas of good data coverage and required boundary condi- tions are obtained using the AMIE technique (Assimilative Mapping of Ionospheric Electrodynamics, Richmond and Kamide, 1988). This allows one to use the local KRM in a rather straightforward manner. However, it should be kept in mind that in absence of global data coverage AMIE gives results that are mostly based on statistical models, and there- fore the obtained boundary conditions may not be very accu- rate. When solving Eq. (8) we have to specify the conductances, so the same problems arise as in the KRM method. Further- more, if Eq. (8) is solved in some limited area, we have to specify some boundary conditions for the curl-free part of the ionospheric current density. In Sect. 3 we present an al- gorithm based on the CECS, where the boundary conditions are handled in a natural and convenient manner. Probably the greatest uncertainties in the KRM results are caused by uncertainties in the input conductance dis- tributions (Murison et al., 1985). Two-dimensional iono- spheric conductance distributions are quite difficult to obtain from direct measurements. Large scale conductance distribu- tions may be derived from satellite or all-sky camera images (e.g. Lummerzheim et al., 1991; Janhunen, 2001; Aksnes et al., 2005), statistical models (e.g., Fuller-Rowell and Evans, 1987) or the ground magnetic data (Ahn et al., 1998), as dis- cussed in the Introduction. It should be noted that in both methods, KRM and the new formulation, the electric field is assumed to be an irrotational potential field. Vanham¨aki et al. 2 Theory (2007) have shown that in some very dynamical situations ionospheric self-induction creates significant induced rotational electric fields, that drive large horizontal currents and FAC. The induced rotational part of the electric field may be estimated from the time derivative of the equivalent currents (in a rather approximate way), as done in Vanham¨aki et al. (2007). Consequently, we can estimate that part of the equivalent currents that is asso- ciated with the induced electric field, and subtract it from the total J eq if necessary. This way we can obtain a more reli- able estimate for the potential part of the electric field even in those cases where induction is important. 2.2 Different approach In the KRM method one tries to find such a potential elec- tric field that the curl of the corresponding current density is equal to the curl of the equivalent currents. Another possi- ble approach is to try to find such a curl-free current system that together with the equivalent currents it is consistent with a non-rotational potential electric field. This latter approach will now be developed. As stated above, for a vertical background magnetic field, J eq is equal to the divergence-free part of the true iono- spheric current density J. In this case the curl-free part of J, together with associated FAC, is magnetically invisi- ble below the ionosphere. These are good approximations even with moderate (χ≈75◦) inclinations of the main mag- netic field (Untiedt and Baumjohann, 1993). Consequently we may write J as a sum of the equivalent currents and a potential part of the current, 3.1 CECS We can represent the ionospheric electric fields and cur- rents by using special non-local vector basis functions, Carte- sian Elementary Current Systems (CECS). CECS were intro- duced by Amm (1997) and although the name “CECS” refers to current systems, they can be used to represent any smooth enough 2-dimensional vector field in planar geometry. There are two different types of CECS, one is divergence-free (DF) and the other curl-free (CF). Together they form a complete set of basis functions. The elementary systems, illustrated in Fig. 1, are defined as J = J eq −∇8. (6) J = J eq −∇8. (6) We can solve the electric field from Ohm’s law as We can solve the electric field from Ohm’s law as E = (6P J −6H ˆez × J)/62, (7) where E = (6P J −6H ˆez × J)/62, (7) (7) where where 62 = 62 P + 62 H. Edf = V df 2πρ′ ˆeφp (9) Ecf = V cf 2πρ′ ˆeρp. (10) 62 = 62 P + 62 H. (9) The condition ∇×E=0 leads to a differential equation for the potential part of the current density, Ecf = V cf 2πρ′ ˆeρp. (10) (10) 626H∇28 + β1 · ∇8 −(β2 × ∇8)z = = −626P (∇× J eq)z + β1 · J eq −(β2 × J eq)z, (8) (8) Here ρ′= q (x−xp)2+(y−yp)2 is the distance between the observation point (x, y) and the CECS pole located at (xp, yp). Unit vectors ˆeφp and ˆeρp are given in the cylindri- cal coordinate system centered at the CECS pole. Constants where β1 = 62∇6H −6H∇62 β2 = 62∇6P −6P ∇62. www.ann-geophys.net/25/1141/2007/ Ann. Geophys., 25, 1141–1156, 2007 Ann. Geophys., 25, 1141–1156, 2007 1144 H. Vanham Fig. 1. Schematic presentation of curl-free (upper) and divergence- free (lower) Cartesian elementary current systems (CECS). H. Vanham¨aki and O. Amm: Ionospheric currents from local magnetic data H. Vanham¨aki and O. Amm: Ionospheric currents from local magnetic data 1144 Here Ex(rn) is the x-component of E at the grid point rn, and so on. In a similar fashion the collection of the CECS scaling factors representing the field E is indicated using fraktur style, V, and is defined as V= h V cf (rp1), V df (rp1), V cf (rp2), . . . 3.1 CECS , V df (rpM) iT .(12) Here V cf (rpm) is the scaling factor of the curl-free CECS located at grid point rpm. There is a linear relation between the vector components and the CECS scaling factors, E = M · V. (13) E = M · V. (13) The matrix M depends only on the geometry of the vector and CECS grids, and can be calculated using Eqs. (9) and (10). The matrix M depends only on the geometry of the vector and CECS grids, and can be calculated using Eqs. (9) and (10). 3.2 Algorithm for numerical calculations With the elementary systems we can formulate the approach of Sect. 2.2 in a somewhat different way. The goal is to find such a curl-free current system J cf that the sum J=J eq+J cf is consistent with a potential electric field. As before, we assume that the equivalent currents are the same as the divergence-free part of the total currents, J eq=J df , which is a good approximation at high geomagnetic latitudes. Fig. 1. Schematic presentation of curl-free (upper) and divergence- free (lower) Cartesian elementary current systems (CECS). V cf and V df are called the scaling factors of the CF and DF CECS, respectively. g pp g g g We begin by calculating the electric field E1 that is con- sistent with the equivalent currents. This can be written in form (cf. Eq. 7) The elementary systems are defined in such a way, that the CF CECS has a Dirac δ-function divergence and the DF CECS a δ-function curl at its pole, E1 = (6P J eq −6H ˆez × J eq)/62, (14) (14) ∇· Ecf = V cf δ(x −xp) δ(y −yp) (∇× Edf )z = V df δ(x −xp) δ(y −yp). where, as before, 62=62 P +62 H. In general, the electric field E1 is not curl-free and it may be very different from the real electric field E. It should also be noted that there may be some regions in the analysis area where 6P ≈6H≈0, but J eq̸=0. The easiest way to deal with these regions is to simply exclude them from the analysis, as the electric field cannot be determined in such regions. where, as before, 62=62 P +62 H. In general, the electric field E1 is not curl-free and it may be very different from the real electric field E. It should also be noted that there may be some regions in the analysis area where 6P ≈6H≈0, but J eq̸=0. The easiest way to deal with these regions is to simply exclude them from the analysis, as the electric field cannot be determined in such regions. By placing a sufficient number of CF and DF CECS at different locations of the plane, one can construct any 2- dimensional vector field from its sources and curls, in ac- cordance with Helmholtz’s theorem. (17) where we have defined a new matrix K2. Both the electric field E2 and the curl-free currents J cf are unknown, but the matrix K2 relating them depends just on the structure of the calculation grids and on the conductances, and it can be cal- culated using Eqs. (14), (9) and (10). – Calculate the electric field E1 that is consistent with Ohm’s law and the equivalent currents. – Divide E1 into curl- and divergence-free parts. – Construct a relation between the unknown curl-free part of the current J cf and the electric field E2 consistent with it. We may further construct a matrix relation similar to Eq. (15), that divides E2 into curl- and divergence-free parts using CECS, – Solve for J cf using the condition that the total electric field E1+E2 is curl-free. E2 = M2 · V2. (18) (18) E2 = M2 · V2. Also in this case the matrix M2 depends only on he geometry of the calculation grids. Now we can use Eqs. (17) and (18) together, and write a relation between the still unknow curl- free currents and CECS representation of the electric field E2, This CECS-based calculation algorithm is slightly different from the method presented in Sect. 2.2. For example, in the CECS method we do not have to calculate the gradients of conductances or the curl of the equivalent currents. However, the basic approach of solving the curl-free currents instead of electric potential is the same. In the CECS algorithm we do not have to provide any explicit boundary conditions. The CECS represent the curl and divergence of the vector fields, so the natural and automatically included boundary condition is to assume that the curl and divergence vanish outside the analysis region. V2 = inv(M2) · K2 · Icf . (19) (19) In the following calculations we need only the divergence- free part of E2, which is given by the divergence-free CECS Vdf 2 . This part of the scaling factors may be singled out by picking appropiate rows of the matrix L≡inv(M2)·K2. Equa- tion (19) can be written out as In the following calculations we need only the divergence- free part of E2, which is given by the divergence-free CECS Vdf 2 . This part of the scaling factors may be singled out by picking appropiate rows of the matrix L≡inv(M2)·K2. (17) Equa- tion (19) can be written out as In this article we concentrate on regional studies, but it should be mentioned that the new calculation method can also be used in global scales. The theory presented in Sect. 2.2 does not depend on the specific geometry that is used, and also the numerical algorithm presented in this sec- tion can be used in spherical geometry. The necessary mod- ification is to simply use Spherical elementary current sys- tems (SECS, introduced by Amm, 1997) instead of CECS.   V cf 2 (rp1) V df 2 (rp1) V cf 2 (rp2) ...   =   L11 L12 . . . L21 L22 . . . ... ... ...  ·   I cf (rp1) I cf (rp2) I cf (rp3) ...  . (20) (20) We see that in this case the divergence-free scaling factors V df 2 correspond to the odd rows of the matrix L. By defining a new matrix Ldf consisting of the odd rows, we may write a matrix relation between the divergence-free part of the CECS representation of E2 and the curl-free current system, 3.2 Algorithm for numerical calculations When CECS are used to represent ionospheric currents, the divergence of the CF CECS at its pole is interpreted as a vertically flowing FAC. By placing a sufficient number of CF and DF CECS at different locations of the plane, one can construct any 2- dimensional vector field from its sources and curls, in ac- cordance with Helmholtz’s theorem. When CECS are used to represent ionospheric currents, the divergence of the CF CECS at its pole is interpreted as a vertically flowing FAC. The next step is to divide the field E1 into curl- and divergence-free parts. As in Eq. (13), there is a linear relation between the vector components and CECS representation of E1, In practical numerical calculations vector components are given at some discrete grid points and the CECS systems are placed at another grid. The elementary systems defined in Eqs. (9) and (10) are singular at the origin, where ρ′→0. This means that some care must be used, so that the vectors components are not evaluated in the immediate vicinity of the CECS poles. In practice we use interleaved grids, so that vector components are evaluated only at the corners of the CECS grid cells. Our notation is such that the 2-dimensional vector fields (in this example the electric field) are indicated using italics, E, as done already in the previous sections. The collection of the x- and y-components of the field E at all grid points is written in script style, as E, and can be written out as E1 = M1 · V1. (15) (15) E1 = M1 · V1. The CECS representation, which also gives the division into curl- and divergence-free parts, is easily obtained by in- verting the equation. In the following, we need only the divergence-free part of E1. The corresponding scaling fac- tors of the DF CECS are denoted by Vdf 1 . The unknown curl-free part of the ionospheric currents, J cf =−∇8 in Eq. (6), can be constructed using just CF CECS, as J cf = Ncf · Icf , (16) (16) J cf = Ncf · Icf , where vector Icf contains the CF CECS scaling factors and matrix Ncf depends only on the geometry of the calculation E =  Ex(r1), Ey(r1), Ex(r2), . . . , Ey(rN) T . (11) (11) Ann. Geophys., 25, 1141–1156, 2007 www.ann-geophys.net/25/1141/2007/ 1145 H. Vanham¨aki and O. 3.2 Algorithm for numerical calculations Amm: Ionospheric currents from local magnetic data grids. The electric field E2 that is consistent with J cf can be calculated as in Eq. (14), just replacing J eq by J cf . Because also this inverted Ohm’s law is linear, together with Eq. (16) it results in Ldf can be contructed through the steps taken in Eqs. (16– 21). After solving Eq. (23) we know the true ionospheric cur- rent density J=J eq+J cf , and consequently also the electric field can be solved using Eq. (7). grids. The electric field E2 that is consistent with J cf can be calculated as in Eq. (14), just replacing J eq by J cf . Because also this inverted Ohm’s law is linear, together with Eq. (16) it results in In summary, the calculation algorithm based on the ele- mentary systems is following: E2 = K2 · Icf , (17) E2 = K2 · Icf , (17) 4 Results for a simple electrojet 0 5 10 15 Pedersen conductance 1/Ω 0 10 20 30 40 Hall conductance 1/Ω J0 Pedersen conductance 0 −800 −600 −400 −200 0 J0 A/km J0x J0y −20 −15 −10 −5 0 E0 mV/m E0x E0y −400 −200 0 200 400 −0.1 0 0.1 div(E0) X (km) V/km2 −400 −200 0 200 400 −2 −1 0 1 2 FAC0 X (km) A/km2 A/km2 Fig. 2. The electrojet model. Electrojet is uniform in the y-direction. This figure shows the profiles of Pedersen and Hall conductances, electric field E0, ionospheric current density J 0, divergence of the electric field and FAC in the x-direction. Calculations are done in a 25×49 grid, with 35 km and 50 km spacing in x- and y-directions, respectively. The KRM equation Eq. (4) is solved using a finite difference scheme with successive overrelaxation (Press et al., 1992, chapter 19). We use a boundary condition φ=0 for the elec- tric potential in the KRM solution. The new CECS based solution is calculated as outlined in Sect. 3.2. The required matrix inversions are calculated using singular value decom- position (Press et al., 1992, chapter 2). the electrojet. For both methods four quantities are shown: the calculated electric field E, its divergence ∇·E, the iono- spheric sheet current density J and the FAC. For each vari- able also the difference between the calculated result and the original model is shown. The electric field at the central electrojet region is repro- duced relatively well by the KRM method, but the φ=0 boundary condition causes some errors near the northern and southern boundaries. Because all boundaries have the same potential, the line integral of the electric field from the north- ern to southern boundary must vanish. This is the reason why the KRM electric field in Fig. 3 shows oppositely di- rected side lobes at the both northern and southern edges of the main jet. Consequently, also the divergence of the elec- tric field shows a similar behavior. In the central part of the electrojet the KRM method gives a good estimate for ∇·E, The results of the traditional KRM and the new CECS methods are shown in Figs. 3 and 4, respectively. Both calcu- lation methods produce results that show quite strong bound- ary effects at the eastern and western sides, where the elec- trojet is artificially truncated by the boundaries of the calcu- lation area. 4 Results for a simple electrojet In this section we apply both the traditional KRM method of Sect. 2.1 and the new CECS algorithm presented in Sect. 3.2 to a simple one-dimensional electrojet. The generic electro- jet model, illustrated in Fig. 2, is uniform in the y-direction. In the x-direction the electric field, Pedersen conductance and Hall/Pedersen conductance ratio all have a Gaussian pro- file with a half-width of ∼260 km on top of a uniform back- ground. As explained in Sect. 2, the input quantities of the KRM and CECS methods are the Pedersen and Hall conduc- tances together with the equivalent currents. In this model the electric field and the conductance gradients are parallel, which means that the equivalent currents are simply the Hall currents. We also use the correct conductance distributions given in the model, although in real situations accurate esti- mates are quite hard to obtain. Vdf 2 = Ldf · Icf . (21) (21) If we assume that the total electric field E=E1+E2 is curl- free, then the rotational parts of E1 and E2 must cancel each other, so that Vdf 2 = −Vdf 1 . (22) (22) With this condition we can solve the unknown curl-free part of the ionospheric currents as With this condition we can solve the unknown curl-free part of the ionospheric currents as Icf = −inv(Ldf ) · Vdf 1 . (23) (23) As explained above, the vector Vdf 1 is obtained from the equivalent currents using Eqs. (14) and (15) and the matrix Ann. Geophys., 25, 1141–1156, 2007 www.ann-geophys.net/25/1141/2007/ H. Vanham¨aki and O. Amm: Ionospheric currents from local magnetic data H. Vanham¨aki and O. Amm: Ionospheric currents from local magnetic data 1146 0 5 10 15 Pedersen conductance 1/Ω 0 10 20 30 40 Hall conductance 1/Ω −20 −15 −10 −5 0 E0 mV/m E0x E0y −800 −600 −400 −200 0 J0 A/km J0x J0y −400 −200 0 200 400 −0.1 0 0.1 div(E0) X (km) V/km2 −400 −200 0 200 400 −2 −1 0 1 2 FAC0 X (km) A/km2 Fig. 2. The electrojet model. Electrojet is uniform in the y-direction. This figure shows the profiles of Pedersen and Hall conductances, electric field E0, ionospheric current density J 0, divergence of the electric field and FAC in the x-direction. H. Vanham¨aki and O. Amm: Ionospheric currents from local magnetic data 1147 H. Vanham¨aki and O. Amm: Ionospheric currents from local magnetic data −200 0 200 E, max = 18.1 mV/m X (km) E−E0, max = 15.9 mV/m −0.1 0 0.1 −200 0 200 X (km) div(E), V/km2 −0.05 0 0.05 div(E−E0), V/km2 −200 0 200 J, max = 729 A/km X (km) J−J0, max = 99 A/km −2 0 2 −800 −400 0 400 800 −200 0 200 Y (km) X (km) FAC, A/km2 −0.2 0 0.2 −800 −400 0 400 800 Y (km) FAC−FAC0, A/km2 Fig. 3. KRM results for the electrojet model. At each row the KRM result is shown on the left side and the difference between the KRM result and the original model is on the right side. Rows from top to bottom are: electric field, divergence of the electric field, horizontal currents and FAC. Note the different scales of the vector plots. E−E0, max = 15.9 mV/m div(E−E0), V/km2 E−E0, max = 15.9 mV/m div(E), V/km2 div(E−E0), V/km2 −0.05 0 0.05 div(E−E0), V/km2 0 J−J0, max = 99 A/km FAC−FAC0, A/km2 FAC−FAC0, A/km2 −2 0 2 −800 −400 0 400 800 −200 0 200 Y (km) X (km) FAC, A/km2 −0.2 0 0.2 −800 −400 0 400 800 Y (km) FAC−FAC0, A/km2 Fig. 3. KRM results for the electrojet model. At each row the KRM result is shown on the left side and the difference between the KRM result and the original model is on the right side. Rows from top to bottom are: electric field, divergence of the electric field, horizontal currents and FAC. Note the different scales of the vector plots. the main electrojet. The FAC distribution given by the KRM method is slightly too wide and the peak amplitude is about 10% smaller than in the original model, but on the whole the result is good. but the calculated distribution is much wider than the origi- nal model, leaking outside the actual electrojet. This exam- ple highlights the importance of boundary conditions, when the KRM method is used in local studies. Further examples have been given by Murison et al. (1985). In contrast to the electric field, the horizontal current J and vertical FAC are generated very well by the KRM method. This is understand- able, since the currents are concentrated in a narrow strip of enhanced conductivity, where also the electric field was reconstructed accurately. 4 Results for a simple electrojet For that reason we show the results at a smaller area, omitting 300 km of the calculation grid at both ends of Ann. Geophys., 25, 1141–1156, 2007 www.ann-geophys.net/25/1141/2007/ H. Vanham¨aki and O. Amm: Ionospheric currents from local magnetic data −200 0 200 E, max = 18.9 mV/m X (km) di (E) V/k 2 E−E0, max = 4.3 mV/m div(E−E0), V/km2 E, max = 18.9 mV/m E−E0, max = 4.3 mV/m div(E−E0), V/km2 div(E), V/km2 −0.01 0 0.01 div(E−E0), V/km2 J−J0, max = 139 A/km −0.1 0 0.1 −200 0 200 X (km) div(E), V/km2 −200 0 200 J, max = 765 A/km X (km) FAC A/km2 J−J0, max = 139 A/km J−J0, max = 139 A/km FAC FAC0 A/km2 FAC−FAC0, A/km2 FAC, A/km2 −0.5 0 0.5 −800 −400 0 400 800 Y (km) FAC−FAC0, A/km2 −1 0 1 −800 −400 0 400 800 −200 0 200 Y (km) X (km) FAC, A/km Fig. 4. Results of the CECS method for the electrojet model. Layout is similar to Fig. 3. Note the different scales of the vector plots. Fig. 4. Results of the CECS method for the electrojet model. Layout is similar to Fig. 3. Note the different s there is also a clear asymmetry in the East-West direction, which results from the sudden termination of the electrojet at these boundaries. However, the current system seems to be reproduced more poorly than with the KRM method. There is about 15% error in the main electrojet current. Main part of the electrojet consists of the Hall currents, which in this case are the same as J eq. The Pedersen currents are curl-free and are connected to the FAC system. The CECS method underestimates the divergent Pedersen currents, so that the difference J−J 0 points almost directly northward and the FAC given by the CECS method are consistently too small. It should be noted that the CECS basis functions used in this paper are intrinsically 2-dimensional and thus, regardless of the specific application, are not optimally suited for repre- senting 1-dimensional vector fields in a bounded domain. There exists also a 1-dimensional variant of the elementary systems, used by Vanham¨aki et al. (2003) and Juusola et al. (2006), which offer a much more suitable set of basis func- tions for modeling 1-dimensional structures. However, this approach is not pursued further in this study. From Figs. 3 and 4 it seems that in this simple example the KRM method does produce somewhat better results for J and FAC, while the new CECS method is able to generate E and ∇·E more accurately. H. Vanham¨aki and O. Amm: Ionospheric currents from local magnetic data The difference between the KRM result and the original model, J−J 0, is an almost uniform North-East directed current, that has magnitude of ∼14% of but the calculated distribution is much wider than the origi- nal model, leaking outside the actual electrojet. This exam- ple highlights the importance of boundary conditions, when the KRM method is used in local studies. Further examples have been given by Murison et al. (1985). In contrast to the electric field, the horizontal current J and vertical FAC are generated very well by the KRM method. This is understand- able, since the currents are concentrated in a narrow strip of enhanced conductivity, where also the electric field was reconstructed accurately. The difference between the KRM result and the original model, J−J 0, is an almost uniform North-East directed current, that has magnitude of ∼14% of The new CECS method is able to reproduce the electric field much more accurately than the KRM method, as can be seen in Fig. 4. The difference to the original model is a rela- tively uniform, roughly North-West directed component that has a magnitude of ∼20% of the main electrojet field. The divergence of the electric field is also generated very well in the main electrojet area. The largest errors appear at the northern and southern boundaries of the calculation area, and Ann. Geophys., 25, 1141–1156, 2007 www.ann-geophys.net/25/1141/2007/ 1148 H. Vanham¨aki and O. Amm: Ionospheric currents from local magnetic data H. Vanham¨aki and O. Amm: Ionospheric currents from local magnetic data 1148 H. Vanhamaki and O. Amm: Ionospheric currents from local magnetic data −200 0 200 E, max = 18.9 mV/m X (km) E−E0, max = 4.3 mV/m −0.1 0 0.1 −200 0 200 X (km) div(E), V/km2 −0.01 0 0.01 div(E−E0), V/km2 −200 0 200 J, max = 765 A/km X (km) J−J0, max = 139 A/km −1 0 1 −800 −400 0 400 800 −200 0 200 Y (km) X (km) FAC, A/km2 −0.5 0 0.5 −800 −400 0 400 800 Y (km) FAC−FAC0, A/km2 Fig. 4. Results of the CECS method for the electrojet model. Layout is similar to Fig. 3. Note the different scales of the vector plots. 5.1 -band The -band model is illustrated in Fig. 5. The model consists of the Pedersen and Hall conductances, the potential electric field E0, the divergence of the electric field and correspond- ing sheet currents J 0 together with the FAC. Numerical cal- culations are done in the same way as in Sect. 4. The results obtained using the two methods are given in Figs. 6 and 7. The results are also compared in Table 2, where the error numbers from Eq. (24) are given. In this example the KRM method fails almost completely in generating either the electric field or the horizontal currents, which is again due to the wrong boundary condition. If we want to obtain better results, we must have some additional a priori infor- mation about the structure of the electric field, so that better boundary conditions can be chosen. Although the error num- bers given in Table 2 for ∇·E and FAC are very large, these quantities seem to be produced somewhat better than the vec- tor fields. Especially the divergence of the electric field has reasonable resemblance to the original model, at least qual- itatively. However, the details are not generated correctly, as the area of negative ∇·E at the middle of the calculation grid is too weak and slightly misplaced, and the positive di- vergences at both sides are overestimated. The FAC in the KRM result are mostly concentrated in two small regions, in the same way as in the original model, although neither the exact position nor the magnitude are correct. In the KRM results there are also some weaker and more spread upward and downward FAC areas, that are not present in the original model. H. Vanham¨aki and O. Amm: Ionospheric currents from local magnetic data More quantitative estimate for the accuracy of the two methods can be obtained by calculating the mean error in the results, as error(a) = ⟨|a −a0|⟩ ⟨|a0|⟩ · 100% (24) (24) Ann. Geophys., 25, 1141–1156, 2007 www.ann-geophys.net/25/1141/2007/ www.ann-geophys.net/25/1141/2007/ 1149 H. Vanham¨aki and O. Amm: Ionospheric currents from local magnetic data H. Vanham¨aki and O. Amm: Ionospheric currents from local magnetic data Table 1. Errors in the electrojet results calculated by the KRM and CECS methods. Error is calculated using Eq. (24) and data presented in Figs. 3 and 4. Table 2. Errors in the -band results calculated by the KRM and CECS methods. Error is calculated using Eq. (24) and data pre- sented in Figs. 6 and 7. E ∇·E J FAC KRM 97% 90% 99% 172% CECS 43% 45% 43% 104% E ∇·E J FAC KRM 108% 74% 29% 7.5% CECS 42% 9.1% 24% 40% aration of the total model current into divergence- and curl- free parts we use 42 km separation for the CECS, but in the actual calculations the original 50 km separation is used. Here <> denotes average over the calculation area, a is ei- ther E, ∇·E, J or FAC and a0 is the corresponding model value. The error estimates given by Eq. (24) for the electrojet case are given in Table 1. Before discussing these estimates, some important properties of Eq. (24) should be noted. It is easy to see that only the correct solution has 0% error, and a zero solution (e.g. E=0) has 100% error. However, some solu- tions may have errors >100%, but it is questionable whether they are worse than the zero solution. For example a solu- tion that is very close to the correct one, but spatially dis- placed by just few grid cells may have a very large error as calculated from Eq. (24). With these precautions in mind, Table 1 confirms the previous conclusion that in this exam- ple the new CECS method gives more accurate results for the electric field, whereas the current system (especially FAC) is calculated better with the KRM method. The two methods have almost the same percentual errors in the horizontal cur- rent J, although the plots of J−J 0 in Figs. 3 and 4 are quite different. 5.1 -band 5 Results for realistic data-based models In this section we use the KRM and CECS methods with two realistic data-based models, namely the -bands and the westward traveling surge (WTS). These models have been published by Amm (1995) and Amm (1996). They are based on observational data obtained at northern Scandinavia by the Scandinavian Magnetometer Array, the EISCAT radar and the EISCAT magnetometer cross, and the STARE radar. In these more complicated models we cannot identify J eq with the Hall currents. Instead we calculate the equivalent currents as the divergence-free part of the total ionospheric current density. We divide the total current into divergence- and curl-free parts using the CECS method, as done for the electric field in Sect. 3.2. In order to avoid committing an inverse crime, i.e. using exactly the same numerical process both in preparing the input data and then solving the inverse problem, we use different grid spacings in the separation and in the actual calculations and also add 2% of normally dis- tributed noise to the resulting equivalent (or divergence-free) currents. The original models are given in a regular grids with 50 km resolution in both x- and y-directions. In the sep- The new CECS based method gives clearly better results, as can be seen in Fig. 7 and in Table 2. The basic shapes of E and J are produced well, although the magnitude and direction of the fields are not quite the same as in the original model. Also ∇·E and FAC are produced better than with the KRM method, and especially the divergence of the electric field is in quite a good qualitative agreement with the model. The FAC given by the CECS method are less accurate, but also in this case the CECS method seems to give a better result than the KRM method. Ann. Geophys., 25, 1141–1156, 2007 www.ann-geophys.net/25/1141/2007/ H. Vanham¨aki and O. Amm: Ionospheric currents from local magnetic data 1150 p g 5 10 15 20 −300 −150 0 150 300 X (km) Pedersen conductance, 1/Ω 20 40 60 80 100 120 Hall Conductance, 1/Ω −300 −150 0 150 300 E0, max = 186.6 mV/m X (km) J0, max = 2930 A/km −2 −1.5 −1 −0.5 0 −150 100 350 600 −300 −150 0 150 300 Y (km) X (km) div(E0), V/km2 −20 −10 0 10 −150 100 350 600 Y (km) FAC0, A/km2 Fig. 5. The -band model. 5 Results for realistic data-based models Pedersen and Hall Conductances, electric field E0, ionospheric current density J 0, divergence of the electric field and FAC. Note the different scales of the vector plots. 5 10 15 20 −300 −150 0 150 300 X (km) Pedersen conductance, 1/Ω 20 40 60 80 100 120 Hall Conductance, 1/Ω 5 10 15 20 −300 −150 0 150 300 X (km) Pedersen conductance, 1/Ω 20 40 60 80 100 120 Hall Conductance, 1/Ω Hall Conductance, 1/Ω −300 −150 0 150 300 E0, max = 186.6 mV/m X (km) J0, max = 2930 A/km −2 −1.5 −1 −0.5 0 −150 100 350 600 −300 −150 0 150 300 Y (km) X (km) div(E0), V/km2 −20 −10 0 10 −150 100 350 600 Y (km) FAC0, A/km2 Fig. 5. The -band model. Pedersen and Hall Conductances, electric field E0, ionospheric current density J 0, divergence of the electric field and FAC. Note the different scales of the vector plots. −20 −10 0 10 −150 100 350 600 Y (km) FAC0, A/km2 −2 −1.5 −1 −0.5 0 −150 100 350 600 −300 −150 0 150 300 Y (km) X (km) div(E0), V/km2 FAC0, A/km2 div(E0), V/km2 Fig. 5. The -band model. Pedersen and Hall Conductances, electric field E0, ionospheric current density J 0, divergence of the electric field and FAC. Note the different scales of the vector plots. H. Vanham¨aki and O. Amm: Ionospheric currents from local magnetic data 1151 H. Vanham¨aki and O. Amm: Ionospheric currents from local magnetic data p g −300 −150 0 150 300 E, max = 47.3 mV/m X (km) E−E0, max = 169.6 mV/m −0.5 0 0.5 −300 −150 0 150 300 X (km) div(E), V/km2 0 0.5 1 1.5 div(E−E0), V/km2 −300 −150 0 150 300 J, max = 3403 A/km X (km) J−J0, max = 2458 A/km −20 0 20 −150 100 350 600 −300 −150 0 150 300 Y (km) X (km) FAC, A/km2 −10 0 10 20 −150 100 350 600 Y (km) FAC−FAC0, A/km2 Fig. 6. KRM results for the -band model. Layout is similar to Fig. 3. Note the different scales of the vector plots. −300 −150 0 150 300 E, max = 47.3 mV/m X (km) div(E) V/km2 E−E0, max = 169.6 mV/m div(E E0) V/km2 E−E0, max = 169.6 mV/m E, max = 47.3 mV/m −0.5 0 0.5 −300 −150 0 150 300 X (km) div(E), V/km2 div(E−E0), V/km2 0 0.5 1 1.5 div(E−E0), V/km2 div(E), V/km2 −300 −150 0 150 300 J, max = 3403 A/km X (km) FAC, A/km2 J−J0, max = 2458 A/km FAC−FAC0, A/km2 J, max = 3403 A/km J−J0, max = 2458 A/km FAC−FAC0, A/km2 FAC, A/km2 −20 0 20 −150 100 350 600 −300 −150 0 150 300 Y (km) X (km) FAC, A/km2 −10 0 10 20 −150 100 350 600 Y (km) FAC−FAC0, A/km . KRM results for the -band model. Layout is similar to Fig. 3. Note the different scales of the vector plots. The WTS is a very dynamical phenomenon and inductive effects may play a significant role in it, as was reported by Vanham¨aki et al. (2007). In these situations the ionospheric electric field is not a pure potential field, as is assumed both in the KRM and CECS methods, but there is also a significant rotational part. This may be taken into account in an approx- imate way by estimating the rotational electric field using the The solution obtained using the new CECS-based method is shown in Fig. 10. Apart from some deviations at the east- ern boundary and at the North-West corner the CECS method gives very accurate results. It is clear from Figs. 9 and 10 that the CECS method is able to generate all the parameters more accurately than the KRM method. 5.2 WTS field seems to be reproduced more poorly than the other pa- rameters, at least when judging qualitatively from Fig. 9. The largest errors in E are concentrated near the boundaries and are apparently caused by the incorrect boundary conditions. However, in order to make a better guess at the boundary conditions for the electric potential we would need some ad- ditional information about E. In this case the largest errors in E are concentrated in areas where conductances are rather small, so the horizontal currents are not affected as much. The input WTS model is shown in Fig. 8 and the KRM and CECS results are illustrated in Figs. 9 and 10, respectively. The KRM method is able to reproduce the most prominent large scale patterns of ∇·E, J and FAC with some accuracy. However, there are also significant deviations from the orig- inal model at some areas and the detailed structure of the WTS system is distorted in the KRM solution. The electric The input WTS model is shown in Fig. 8 and the KRM and CECS results are illustrated in Figs. 9 and 10, respectively. The KRM method is able to reproduce the most prominent large scale patterns of ∇·E, J and FAC with some accuracy. However, there are also significant deviations from the orig- inal model at some areas and the detailed structure of the WTS system is distorted in the KRM solution. The electric Ann. Geophys., 25, 1141–1156, 2007 www.ann-geophys.net/25/1141/2007/ H. Vanham¨aki and O. Amm: Ionospheric currents from local magnetic data This is confirmed in Ta- ble 3 where the errors calculated using Eq. (24) are given. Ann. Geophys., 25, 1141–1156, 2007 www.ann-geophys.net/25/1141/2007/ 1152 H. Vanham¨aki and O. Amm: Ionospheric currents from local magnetic data H. Vanham¨aki and O. Amm: Ionospheric currents from local magnetic data −300 −150 0 150 300 E, max = 116.3 mV/m X (km) E−E0, max = 81.8 mV/m −1.5 −1 −0.5 0 −300 −150 0 150 300 X (km) div(E), V/km2 −0.2 0 0.2 0.4 0.6 0.8 div(E−E0), V/km2 −300 −150 0 150 300 J, max = 2842 A/km X (km) J−J0, max = 1032 A/km −10 0 10 −150 100 350 600 −300 −150 0 150 300 Y (km) X (km) FAC, A/km2 −5 0 5 −150 100 350 600 Y (km) FAC−FAC0, A/km2 Fig. 7. Results of the CECS method for the -band model. Layout is similar to Fig. 3. Note the different scales of the vector plots. −300 −150 0 150 300 E, max = 116.3 mV/m X (km) div(E) V/km2 E−E0, max = 81.8 mV/m div(E E0) V/km2 E, max = 116.3 mV/m −1.5 −1 −0.5 0 −300 −150 0 150 300 X (km) div(E), V/km2 −0.2 0 0.2 0.4 0.6 0.8 div(E−E0), V/km2 div(E), V/km2 div(E−E0), V/km2 J−J0, max = 1032 A/km FAC−FAC0, A/km2 −300 −150 0 150 300 J, max = 2842 A/km X (km) FAC A/km2 J−J0, max = 1032 A/km J, max = 2842 A/km FAC−FAC0, A/km2 FAC, A/km2 −10 0 10 −150 100 350 600 −300 −150 0 150 300 Y (km) X (km) FAC, A/km2 −5 0 5 −150 100 350 600 Y (km) Y (km) e CECS method for the -band model. Layout is similar to Fig. 3. Note the different scales of the vector plots Fig. 7. Results of the CECS method for the -band model. Layout is similar to Fig. 3. Note the different sca time derivative of the equivalent currents and then subtract- ing the inductive part from J eq, as discussed in Sect. 2.2 and in Vanham¨aki et al. (2007). time derivative of the equivalent currents and then subtract- ing the inductive part from J eq, as discussed in Sect. 2.2 and in Vanham¨aki et al. (2007). H. Vanham¨aki and O. Amm: Ionospheric currents from local magnetic data H. Vanham¨aki and O. Amm: Ionospheric currents from local magnetic data 1153 p g 0 5 10 15 20 −300 −150 0 150 300 X (km) Pedersen conductance, 1/Ω 20 40 60 80 Hall Conductance, 1/Ω −300 −150 0 150 300 E0, max = 30.9 mV/m X (km) J0, max = 756 A/km −0.2 −0.1 0 −600 −300 0 300 600 −300 −150 0 150 300 Y (km) X (km) div(E0), V/km2 −6 −4 −2 0 2 −600 −300 0 300 600 Y (km) FAC0, A/km2 Fig. 8. The WTS model. Layout is similar to Fig. 5. Note the different scales of the vector plots. 20 40 60 80 Hall Conductance, 1/Ω 0 5 10 15 20 −300 −150 0 150 300 X (km) Pedersen conductance, 1/Ω Pedersen conductance, 1/Ω Hall Conductance, 1/Ω −300 −150 0 150 300 E0, max = 30.9 mV/m X (km) J0, max = 756 A/km −0.2 −0.1 0 −600 −300 0 300 600 −300 −150 0 150 300 Y (km) X (km) div(E0), V/km2 −6 −4 −2 0 2 −600 −300 0 300 600 Y (km) FAC0, A/km2 FAC0, A/km2 0 Fig. 8. The WTS model. Layout is similar to Fig. 5. Note the different scales of the vector plots. et al. (1981). The new method introduced here differs from the KRM method in two important ways. Firstly, the primary unknown to be solved in the new method is the curl-free part of the ionospheric current system, whereas the KRM method formulates the problem in terms of an electric potential. Sec- ondly, in the numerical implementation we use the Carte- sian elementary current systems (CECS), that offer a con- venient way to represent 2-dimensional vector fields, espe- cially when the vector fields have to be divided into curl- and divergence-free parts. These new features lead to a different formulation of the problem, as explained in detail in Sects. 2 and 3. Table 3. Errors in the WTS results calculated by the KRM and CECS methods. Error is calculated using Eq. (24) and data pre- sented in Figs. 9 and 10. E ∇·E J FAC KRM 80% 169% 51% 52% CECS 19% 46% 8.8% 17% works well on global and semiglobal scales, at smaller areas the unknown boundary conditions for the electric potential play a significant role in the KRM solution, as was pointed out by Murison et al. (1985). 6 Summary and conclusions We have presented a new method for estimating ionospheric electric fields and currents using ground magnetic recordings and ionospheric electric conductances as input data. This problem, using the same set of input data, has traditionally been analyzed using the KRM method introduced by Kamide Ann. Geophys., 25, 1141–1156, 2007 www.ann-geophys.net/25/1141/2007/ H. Vanham¨aki and O. Amm: Ionospheric currents from local magnetic data 1153 ki and O. Amm: Ionospheric currents from local magnetic data H. Vanham¨aki and O. Amm: Ionospheric currents from local magnetic data Our approach of solving the curl-free part of the ionospheric current results in an equation In this article we concentrated on regional studies, where magnetic measurements and estimates of the ionospheric conductances are available only at a limited region of few hundred or thousand km across. While the KRM method Ann. Geophys., 25, 1141–1156, 2007 www.ann-geophys.net/25/1141/2007/ H. Vanham¨aki and O. Amm: Ionospheric currents from local magnetic data 1154 H. Vanham¨aki and O. Amm: Ionospheric currents from local magnetic data −300 −150 0 150 300 E, max = 33.8 mV/m X (km) E−E0, max = 56.7 mV/m −0.2 0 0.2 0.4 −300 −150 0 150 300 X (km) div(E), V/km2 0 0.2 0.4 div(E−E0), V/km2 −300 −150 0 150 300 J, max = 626 A/km X (km) J−J0, max = 425 A/km −4 −2 0 2 −600 −300 0 300 600 −300 −150 0 150 300 Y (km) X (km) FAC, A/km2 −1 0 1 2 −600 −300 0 300 600 Y (km) FAC−FAC0, A/km2 Fig. 9. KRM results for the WTS model. Layout is similar to Fig. 3. Note the different scales of the vector plots. −300 −150 0 150 300 E, max = 33.8 mV/m X (km) E−E0, max = 56.7 mV/m div(E E0) V/km2 div(E−E0), V/km2 −0.2 0 0.2 0.4 −300 −150 0 150 300 X (km) div(E), V/km2 0 0.2 0.4 div(E−E0), V/km2 −300 −150 0 150 300 J, max = 626 A/km X (km) FAC A/km2 J−J0, max = 425 A/km FAC FAC0 A/km2 J−J0, max = 425 A/km FAC−FAC0, A/km2 −4 −2 0 2 −600 −300 0 300 600 −300 −150 0 150 300 Y (km) X (km) FAC, A/km2 −1 0 1 2 −600 −300 0 300 600 Y (km) FAC−FAC0, A/km2 g. 9. KRM results for the WTS model. Layout is similar to Fig. 3. Note the different scales of the vector plots In Sect. 4 we compared the KRM and CECS methods by analyzing a simple 1-dimensional electrojet model. In Sect. 5 we further applied the two methods to two realistic models of typical meso-scale phenomena in the auroral iono- sphere, namely the -bands and the westward traveling surge (WTS). In the electrojet case the result was quite even, as the CECS method produced better estimates for the electric field while the KRM method was able to generate the FAC more accurately. H. Vanham¨aki and O. Amm: Ionospheric currents from local magnetic data 1155 H. Vanham¨aki and O. Amm: Ionospheric currents from local magnetic data p g −300 −150 0 150 300 E, max = 29.9 mV/m X (km) E−E0, max = 20.3 mV/m −0.2 0 0.2 −300 −150 0 150 300 X (km) div(E), V/km2 0 0.1 0.2 div(E−E0), V/km2 −300 −150 0 150 300 J, max = 729 A/km X (km) J−J0, max = 83 A/km −6 −4 −2 0 2 −600 −300 0 300 600 −300 −150 0 150 300 Y (km) X (km) FAC, A/km2 0 1 2 −600 −300 0 300 600 Y (km) FAC−FAC0, A/km2 Fig. 10. Results of the CECS method for the WTS model. Layout is similar to Fig. 3. Note the different scales of the vector plots. E−E0, max = 20.3 mV/m 2 −300 −150 0 150 300 E, max = 29.9 mV/m X (km) E−E0, max = 20.3 mV/m −0.2 0 0.2 −300 −150 0 150 300 X (km) div(E), V/km2 0 0.1 0.2 div(E−E0), V/km2 div(E−E0), V/km2 div(E), V/km2 −300 −150 0 150 300 J, max = 729 A/km X (km) FAC A/km2 J−J0, max = 83 A/km FAC FAC0 A/km2 J, max = 729 A/km J−J0, max = 83 A/km FAC−FAC0, A/km2 −6 −4 −2 0 2 −600 −300 0 300 600 −300 −150 0 150 300 Y (km) X (km) FAC, A/km2 0 1 2 −600 −300 0 300 600 Y (km) FAC−FAC0, A/km2 he CECS method for the WTS model. Layout is similar to Fig. 3. Note the different scales of the vector plots. Fig. 10. Results of the CECS method for the WTS model. Layout is similar to Fig. 3. Note the different scal CECS basis functions are not optimal for representing es- sentially 1-dimensional structures, as discussed in Sect. 4. However, in the three data-based test cases, which show full 2-dimensional variability, the new CECS method was clearly superior to the traditional KRM method. The error estimates calculated using Eq. (24) show that the errors in the CECS results are around 20%–40% in the model cases, whereas the errors in the KRM results are significantly larger. However, it should be mentioned that in these examples we used the correct Pedersen and Hall conductance distributions in the calculations. In real situations there may be large uncertain- ties in the conductance estimates, as discussed in Sect. 2.1. H. Vanham¨aki and O. Amm: Ionospheric currents from local magnetic data This may be partly explained by the fact that the (Eq. 8) that makes better use of the information contained in the input equivalent currents than the KRM formulation. An- other advantage is that with CECS the boundary conditions are implemented in a natural way, without having to specify any explicit values for the vector fields or potentials at the boundaries. Thus we expect the new method to be more suit- able for regional studies than the traditional KRM method. The new calculation method may also be used in global stud- ies, as mentioned in Sect. 3. Ann. Geophys., 25, 1141–1156, 2007 www.ann-geophys.net/25/1141/2007/ References Lummerzheim, D., Rees, M. H., Craven, J. D., and Frank, L. A.: Ionosheric conductances derived from DE-1 auroral images, J. Atmos. Terr. Phys., 53, 281–289, 1991. Ahn, B.-H., Richmond, A. D., Kamide, Y., et al.: An ionospheric conductance model based on ground magnetic disturbance data, J. Geophys. Res., 103, 14 769–14 780, 1998. Murison, M., Richmond, A. D., Matsushita, S., and Baumjohann, W.: Estimation of Ionospheric Electric Fields and Currents From a Regional Magnetometer Array, J. Geophys. Res., 90, 3525– 3530, 1985. Aksnes, A., Amm, O., Stadsnes, J., et al.: Ionospheric conduc- tances derived from satellite measurements of auroral UV and X-ray emissions, and ground-based data: A comparison, Ann. Geophys., 23, 343–358, 2005, http://www.ann-geophys.net/23/343/2005/. Press W. H., Teukolsky, S. A., Vetterling, W. T., and Flannery, B. P.: Numerical Recipes in Fortran 77, 973 pp., Campridge University Press, Cambridge, 2nd ed., 1992. Amm O.: Direct determination of the local ionospheric Hall con- ductance distribution from two-dimensional electric and mag- netic field data: Application of the method using models of typi- cal ionospheric electrodynamic situations, J. Geophys. Res., 100, 21 473–21 488, 1995. Pulkkinen, A., Amm, O., Viljanen, A., and BEAR Working Group: Ionospheric equivalent current distributions determined with the method of spherical elementary current systems, J. Geophys. Res., 108(A2), 1053, doi:10.1029/2001JA005085, 2003. Amm, O.: Improved electrodynamic modeling of an omega band and analysis of its current system, J. Geophys. Res., 101, 2677– 2683, 1996. Untiedt, J. and Baumjohann, W.: Studies of polar current systems using the IMS Scandinavian magnetometer array, Space Sci. Rev., 63, 245–390, 1993. Amm, O.: Ionospheric elementary current systems in spherical co- ordinates and their application, J. Geomagnetism and Geoelec- tricity, 49, 947–955, 1997. Richmond, A. D. and Kamide, Y.: Mapping electrodynamic fea- tures of the high-latitude ionosphere from localized observations: Technique, J. Geophys. Res., 93, 5741–5759, 1988. Amm, O. and Viljanen, A.: Ionospheric disturbance magnetic field continuation from the ground to the ionosphere using spherical elementary current systems, Earth, Planets Space, 51, 431–440, 1999. Vanham¨aki H., Amm, O., and Viljanen, A.: One-dimensional up- ward continuation of the ground magnetic field disturbance us- ing spherical elementary current systems, Earth, Planets Space, 2003. Amm, O., Viljanen, A., Pulkkinen, A., Sillanp¨a¨a, I., and Van- ham¨aki, H.: Methods for combined ground-based and space- based analysis of ionospheric current systems, Proc. Fourth Oer- sted International Science Team Conference, Copenhagen, Den- mark, 23–27 September 2002, p. 181–184, 2003. H. Vanham¨aki and O. Amm: Ionospheric currents from local magnetic data Additionally, in the studied examples the input models had about the same resolution as was used in the calculations, but in reality also much smaller scale variations would be present. These could not be reproduced by neither the KRM nor the new CECS method, nor any by any other method that is based on ground magnetometer input data. Ann. Geophys., 25, 1141–1156, 2007 www.ann-geophys.net/25/1141/2007/ 1156 H. Vanham¨aki and O. Amm: Ionospheric currents from local magnetic data We conclude that the new CECS-based calculation method is well suitable for regional studies and seems to produce more accurate results than the traditional KRM method. One possible topic for a future study is a more thorough compar- ison between the CECS method and the local AMIE-KRM code mentioned in Sect. 2.1. Also a systematic evaluation of the uncertainties caused by inaccurate conductance estimates would be useful when interpreting the results. Fuller-Rowell, T. J. and Evans, D. S.: Height-integrated Pedersen and Hall conductivity patterns inferred from the TIROS-NOAA satellite data, J. Geophys. Res., 92, 7606–7618, 1987. Haines, G. V.: Spherical cap harmonic analysis, J. Geophys. Res., 90, 2583–2591, 1985. Janhunen, P.: Reconstruction of electron precipitation characteris- tics from a set of multiwavelength digital all-sky auroral images, J. Geophys. Res., 106, 18 505–18 516, 2001. Juusola, L., Amm, O., and Viljanen, A.: One-dimensional elemen- tary current systems and their use for determining ionospheric currents from satellite measurements, Earth, Planets Space, 58, 667–678, 2006. Acknowledgements. The authors would like to thank A. Viljanen for his valuable comments on the manuscript. The work of H. Van- ham¨aki was supported by the Finnish Graduate School in Astron- omy and Space Physics. Kamide, Y., Richmond, A. D., and Matsushita, S.: Estimation of ionospheric electric fields, ionospheric currents, and field- aligned currents from ground magnetic records, J. Geophys. Res., 86, 801–813, 1981. Topical Editor M. Pinnock thanks H. Wang and A. Aikio for their help in evaluating this paper. Kamide, Y., Kihn, E. A., Ridley, A. J., Cliver, E. W., and Kadowaki, Y.: Real-time specifications of the geospace environment, Space Sci. Rev., 107, 307–316, 2003. References Vanham¨aki H., Amm, O., and Viljanen, A.: Role of inductive elec- tric fields and currents in dynamical ionospheric situations, Ann. Geophys., 25, 437–455, 2007, http://www.ann-geophys.net/25/437/2007/. Chapman, S. and Bartels, J.: Geomagnetism, vol. II, pp. 1049, Ox- ford University Press, New York, 1940. Ann. Geophys., 25, 1141–1156, 2007 www.ann-geophys.net/25/1141/2007/
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In memoriam - Prof. Dr. Manuel Molina Ortega (1931-1999)
Eclética química
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Prof. Dr. Manuel Molina Ortega (1931 - 1999)1 Prof. Dr. Manuel Molina Ortega (1931 - 1999)1 Eclética Química Print version ISSN 0100-4670On-line version ISSN 1678-4618 Eclet. Quím. vol.27 no.special São Paulo 2002 http://dx.doi.org/10.1590/S0100-46702002000200001 Cristo Bladimiros Melios A partir de Março de 1965 foi contratado como Instrutor, passando a fazer parte do quadro da Faculdade, lotado no Departamento de Química, Área de Química Analítica. A partir de 1965, foi lentamente montando, a duríssimas penas, o que culminou como Departamento de Química Analítica, em Abril de 1977, pouco tempo após a fundação da UNESP, ocorrida em Janeiro de 1976. O Prof. Molina defendeu a sua Tese de Doutoramento em Julho de 1970, na extinta FFCL Araraquara. A sua Tese versou sobre Complexos do Íon Prata (I) com Homólogos Piridínicos. O seu Concurso de Livre-Docência realizou-se em meados de 1980, no IQ/UNESP. A Tese correspondente versou sobre "Uma Proposta de Algoritmo para a Investigação de Complexos em Solução a Partir de Dados Espectrofotométricos". Após concurso público foi investido no cargo de Prof. Titular, a partir de 1984. No 1º Semestre de 1987 estagiou no Departamento de Química Analítica da Universidade de Córdoba onde realizou trabalhos em colaboração com o internacionalmente conceituado Prof. Miguel Valcarcel. Participou de numerosos Congressos no País e no exterior. Sua obra científica é atestada por mais de 90 publicações, incluindo periódicos nacionais, internacionais e trabalhos completos publicados em Anais, além de cerca de 140 comunicações em conclaves, nacionais e internacionais. Exerceu por diversas vezes, o cargo de Chefe do Departamento de Química Analítica; foi Diretor do Instituto de Química da UNESP, de Novembro de 1980 a Novembro de 1984. Foi coordenador da Comissão Organizadora do VI ENQA, realizado em Araraquara, em Setembro de 1991. Atuou como assessor e consultor científico das principais Agências de Fomento à Pesquisa, em nível nacional. Pertenceu aos corpos editoriais de periódicos nacionais e estrangeiros, recebeu numerosas homenagens e fez parte de grande número de bancas examinadoras. Aposentou-se do IQ/UNESP em Março de 1995. Nesse mesmo ano, juntamente com o Prof. Oswaldo Godinho, então recém aposentado do IQ/UNICAMP, dirigiu-se a São Luís do Maranhão, a convite do Departamento de Química da Universidade Federal localizada naquele Estado, com o objetivo precípuo de contribuir para implementação do curso de Pós-Graduação em Química Analítica daquela Universidade. Infelizmente, no segundo semestre de 1996, o Prof. Molina foi acometido de derrame cerebral, o que fez reduzir substancialmente a sua atividade científica. Não desanimou, entretanto. Cristo Bladimiros Melios Departamento de Química Analítica - Instituto de Química - UNESP - 14.801-970 - Araraquara - SP - Brasil Departamento de Química Analítica - Instituto de Química - UNESP - 14.801-970 - Ar Durante o ano de 1999, dois ex-docentes do Instituto de Química da UNESP, de grande proeminência, faleceram. Um deles foi o Prof. Dr. Waldemar Saffioti, falecido em Abril. O Prof. Saffioti foi homenageado naquele mesmo ano, na Sessão Inaugural do 12º Encontro Regional de Química, realizado em Outubro, em Ribeirão Preto, SP. Coube a nós a honra, na ocasião, de invocar o obituário do Prof. Saffioti, anteriormente publicado (Massabni, A.C., Melios, C.B., Franco, D.W., Quím. Nova, 22, 630, 1999; An. Assoc. Bras. Quím., 48 (3), iiii, 1999), além de fornecer outras informações, oriundas do longo convívio com o referido Mestre. Surge agora a oportunidade, talvez ímpar, de homenagear o segundo docente falecido em 1999, que é o Prof. Dr. Manuel Molina Ortega. É uma feliz coincidência caber a nós, novamente, render homenagem a mais um estimado docente do antigo quadro do nosso Instituto. Tentaremos trazer à tona a sua imagem aos seus ex-alunos, colegas e amigos aqui presentes. Aos que não o conheceram, temos a imensa satisfação de proporcionar-lhes esta apresentação póstuma. O Prof. Molina (como era mais conhecido) nasceu em Granada (Espanha), em 15 de Julho de 1931, tendo falecido em São Luís do Maranhão, em 16 de Julho de 1999, um dia, portanto, após completar 68 anos de idade. O Prof. Molina graduou-se Bacharel em Química pela Faculdade de Ciências da Universidade de Zaragoza, Espanha, em 1959. De 1960 a 1961 foi Professor Assistente da Cadeira de Físicoquímica, nessa mesma Faculdade. Em 1961 emigrou para o Brasil e nesse mesmo ano foi contratado pela Pirelli S/A, Seção de Pneumáticos, situada em Capuava-SP, onde permaneceu até o final de 1964, tendo alcançado o cargo de Vice-Chefe do Serviço Tecnológico. tendo alcançado o cargo de Vice-Chefe do Serviço Tecnológico. No final de 1964 transferiu-se para Araraquara, sendo inicialmente contratado pela então Faculdade de Filosofia, Ciências e Letras como Professor Visitante. A partir de Março de 1965 foi contratado como Instrutor, passando a fazer parte do quadro da Faculdade, lotado no Departamento de Química, Área de Química Analítica. No final de 1964 transferiu-se para Araraquara, sendo inicialmente contratado pela então Faculdade de Filosofia, Ciências e Letras como Professor Visitante. Cristo Bladimiros Melios Com o auxílio, principalmente, de intensa fisioterapia, quando já estava quase retornando às atividades normais, sofreu, em Setembro de 1997, novo derrame, bem mais grave que o anterior; este afastou- o, definitivamente, da militância científica. Parece, entretanto, oportuno salientar que, apesar do restrito tempo de trabalho dos Profs. Molina e Godinho na Universidade Federal do Maranhão, o desempenho dos mesmos foi de tal envergadura que, em Setembro de 1997, o corpo docente dessa Instituição reivindicava, para São Luís, o Encontro Nacional de Química Analítica previsto para 2001. Com a enfermidade do Prof. Molina e posterior desistência do Prof. Godinho, o entusiasmo do pessoal de São Luís provavelmente arrefeceu e o Encontro Nacional de 2001 acabou realizando-se na UNICAMP, ficando para 2003 o conclave a efetivar-se em São Luís. pessoal de São Luís provavelmente arrefeceu e o Encontro Nacional de 2001 acabou realizando-se na UNICAMP, ficando para 2003 o conclave a efetivar-se em São Luís. Inesquecíveis no Prof. Molina são a excelência de sua didática, a inteligência, a inspiração científica, o abraço afetuoso e a palavra amiga, quando mais necessários, o refinado senso de humor. A sua retidão de caráter ainda é paradigma para a grande maioria daqueles que privaram de sua convivência e amizade. Foram suas características inquestionáveis a inegável habilidade experimental, a compulsão (e mesmo obsessão) no sentido de obter dados com a maior exatidão e precisão possíveis e a tentativa de automatização dos processos. Estabelece o dito popular que "ninguém é insubstituível". Nós comungamos, em grande parte, com a essência desse dito. Entretanto, é forçoso reconhecer também que certas pessoas são mais difíceis de substituir do que outras. Acreditamos que o Prof. Molina é uma dessas pessoas. A sua inspiração, ensinamentos e magnanimidade ainda permeiam e transpiram em muitos de nós que com ele convivemos por décadas. À guisa desta homenagem ao Prof. Molina, a palestra de abertura que proferimos versou sobre um dos temas de sua preferência, manifestado ainda na década de 60, antes da conclusão do seu Doutoramento. O Prof. Molina acreditava no desenvolvimento experimental de procedimentos que permitissem conduzir a estimativas, ainda que bastante aproximadas, dos coeficientes de atividade individuais de íons metálicos, ligantes e, principalmente, dos complexos oriundos das interações entre metais e ligantes, em solução. O sonho por ele acalentado durante muitos anos pôde ser concretizado próximo ao final de sua carreira no IQ/UNESP, quando condições favoráveis de diversas naturezas foram-lhe proporcionadas. 1 - Parte do conteúdo da Palestra de Abertura do 13º Encontro Regional de Química (21-26/10/01, Araraquara, SP) proferida em 21/10/01. Cristo Bladimiros Melios Conseguiu, antes de seu falecimento, apesar de gravemente enfermo, ver os resultados de seus esforços, juntamente com os de seus colaboradores e orientados, na linha de pesquisas supra mencionada, publicados em periódicos de ampla circulação internacional, com a crítica de assessores especializados (consultar, por exemplo, Talanta, 43, 1689, 1697, 1996; Internat. J. Environ. Anal. Chem., 63, 295, 1997). Cerca de três meses após o seu falecimento, uma obra clássica da Química Analítica (Harris, D.C., "Quantitative Chemical Analysis", 5th. Ed., Freeman, New York, 1999, p. 183) conferia crédito ao sonho concretizado do Prof. Molina, exibindo e registrando resultados de um de seus trabalhos recentes. Em 2001, na segunda edição de obra mais avançada do mesmo autor ("Exploring Chemical Analysis", 2nd. Ed., Freeman, New York, 2001, p. 240), os mesmos resultados voltaram a ser estampados e reconhecidos. Nós, que estivemos associados ao Prof. Molina em numerosas atividades acadêmicas, estamos profundamente gratos pela oportunidade que tivemos de conhecê-lo e de poder apreciar e testemunhar seus elevados ideais e padrões de qualidade em todas as fases de sua vida pessoal e profissional. Casou-se, em primeiras núpcias, com a Sra. Carmen Calvo Añon e, em segundas núpcias, com a Sra. Elaine C.M. Teixeira. Do primeiro casamento originaram-se quatro filhos e, do segundo, três.
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High mortality rates in men initiated on anti-retroviral treatment in KwaZulu-Natal, South Africa
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OPEN ACCESS In attaining UNAIDS targets of 90-90-90 to achieve epidemic control, understanding who the current utilizers of HIV treatment services are will inform efforts aimed at reaching those not being reached. A retrospective chart review of CAPRISA AIDS Treatment Program (CAT) patients between 2004 and 2013 was undertaken. Of the 4043 HIV-infected patients initiated on ART, 2586 (64.0%) were women. At ART initiation, men, compared to women, had significantly lower median CD4+ cell counts (113 vs 131 cells/mm3, p <0.001), lower median body mass index (BMI) (21.0 vs 24.2 kg/m2, p<0.001), higher mean log viral load (5.0 vs 4.9 copies/ml, p<0.001) and were significantly older (median age: 35 vs. 32 years, p<0.001). Men had higher mortality rates compared to women, 6.7 per 100 person-years (p-y), (95% CI: 5.8–7.8) vs. 4.4 per 100 p-y, (95% CI: 3.8–5.0); mortality rate ratio: 1.54, (95% CI: 1.27–1.87), p <0.001. Age-standardised mortality rate was 7.9 per 100 p-y (95% CI: 4.1–11.7) for men and 5.7 per 100 p-y (95% CI: 2.7 to 8.6) for women (standardised mor- tality ratio: 1.38 (1.15 to 1.70)). Mean CD4+ cell count increases post-ART initiation were lower in men at all follow-up time points. Men presented later in the course of their HIV dis- ease for ART initiation with more advanced disease and experienced a higher mortality rate compared to women. Citation: Naidoo K, Hassan-Moosa R, Yende-Zuma N, Govender D, Padayatchi N, Dawood H, et al. (2017) High mortality rates in men initiated on anti- retroviral treatment in KwaZulu-Natal, South Africa. PLoS ONE 12(9): e0184124. https://doi.org/ 10.1371/journal.pone.0184124 Editor: Cristian Apetrei, University of Pittsburgh Centre for Vaccine Research, UNITED STATES Received: June 14, 2017 Accepted: August 18, 2017 Published: September 13, 2017 Published: September 13, 2017 Copyright: © 2017 Naidoo et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. RESEARCH ARTICLE Data Availability Statement: All relevant data are within the paper. Funding: This work was supported by the US President’s Emergency Plan for AIDS Relief (PEPFAR), the Global Fund to fight AIDS, Tuberculosis and Malaria and the National Institutes of Health Comprehensive International Program of Research on AIDS (UM1AI069469). K.N. and N.P. were supported by the Columbia University-South Africa Fogarty AIDS International Training and Research Program (AITRP, Grant D43 High mortality rates in men initiated on anti- retroviral treatment in KwaZulu-Natal, South Africa Kogieleum Naidoo1,2*, Razia Hassan-Moosa1*, Nonhlanhla Yende-Zuma1,2, Dhineshree Govender1, Nesri Padayatchi1,2, Halima Dawood1, Rochelle Nicola Adams1, Aveshen Govender1, Tilagavathy Chinappa1, Salim Abdool-Karim1,2,3, Quarraisha Abdool- Karim1,3 1 Centre for the AIDS Programme of Research in South Africa (CAPRISA), Durban, KwaZulu-Natal, South Africa, 2 MRC-CAPRISA HIV-TB Pathogenesis and Treatment Research Unit, Doris Duke Medical Research Institute, University of KwaZulu-Natal, Durban, KwaZulu-Natal, South Africa, 3 Mailman School of Public Health, Department of Epidemiology, Columbia University, New York, New York, United States of America a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 * Kogie.Naidoo@caprisa.org (KN); Razia.Hassan-Moosa@caprisa.org (RHM) * Kogie.Naidoo@caprisa.org (KN); Razia.Hassan-Moosa@caprisa.org (RHM) High mortality rates in men initiated on ART TW000231). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. In order to achieve the UNAIDS 90-90-90 goals for epidemic control and enhance individ- ual and population level impact of public access anti-retroviral therapy (ART), detailed under- standing of gaps in access to treatment services and factors that contribute to poor HIV In order to achieve the UNAIDS 90-90-90 goals for epidemic control and enhance individ- ual and population level impact of public access anti-retroviral therapy (ART), detailed under- standing of gaps in access to treatment services and factors that contribute to poor HIV treatment outcomes including ongoing mortality, is imperative. Data from other countries are inconsistent and contradictory, with some studies demonstrating gender imbalances in access to HIV treatment services and in clinical outcomes among patients on ART [4,5], and others demonstrating poorer clinical outcomes in women [6–8] or no gender difference in immuno- logical response or mortality [9]. TW000231). 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 the following competing interests to declare: 1) Money to Institution for payment/support of work submitted: • PEPFAR • National Institute of Health/ NAID • Howard Hughes Medical Institute • PHE 2) Board Membership: • Executive Director, Board of Control, Centre for the AIDS Program of Research in South Africa (CAPRISA) • Medscape Infectious Disease & HIV/AIDS Editorial Advisory Boards 3) Public access funding: Research Councils: Yes. 4) Grants: • Study: The CAPRISA Regional Center for Advanced Clinical Management. Duration of funding: 04/01/2014-03/31/2019. • Study: Addressing challenges in scaling up TB and HIV treatment integration in public health settings in SA. Duration of funding: 10/01/2014-12/31/2016. • Newton Fund (Naidoo K) South African Medical Research Council 01/01/2016-12/31/2018. Addressing challenges in scaling up TB and HIV treatment integration in public health settings in South Africa. Description: The major goal on this study is to develop, implement and evaluate an integrated TB-HIV primary care model of service delivery that can improve HIV and TB outcomes in co-infected patients. Role: Principal Investigator. • BR-C 13/0056 (Naidoo K) BroadReach/USAID 10/ 01/2014-12/31/2016. Addressing challenges in scaling up TB and HIV treatment integration in public health settings in SA. Description: The major goal on this study is to develop, implement and evaluate an integrated TB-HIV primary care model of service delivery that can improve HIV and TB outcomes in co-infected patients. Role: Principal Investigator • OPP1137034 (Hatherill M) UCT/Bill & Melinda Gates Foundation 10/05/2015 – 02/28/ 2019. A Clinical Trial of a Correlate of Risk Targeted Screen and Treat Strategy to Impact TB Control. Description: The major goal of this study is to control the global TB epidemic by testing a strategy to identify people with undiagnosed TB and people at high risk of developing TB disease using correlates of risk, in order to treat them before they transmit TB to others. Role: Subcontract Principal Investigator. • Extramural Unit (Abdool Karim SS). South African Medical Research Council 01/01/ 2016-12/31/2018. MRC-CAPRISA HIV-TB Pathogenesis and Treatment Research Unit. Description: The major goal on this MRC extramural unit is to undertake research to reduce morbidity and mortality from HIV-TB co-infection. Role: Co - Principal Investigator. • Medical Research Council of South Africa (MRC) SHIP Program (Scriba T) South African Medical Research Council 08/13/2014 – 07/31/2017. Notwithstanding the higher burden in women and 5–7 year earlier acquisition of HIV infection in adolescent girls and young women between the ages of 15–24 years [10], men are known to be poor utilizers of health care services [11] and characteristically present at a late stage of disease [12]. Women, are the predominant users of public sector ART [13], with reports from South Africa indicating that up to 60% of eligible women received ART com- pared to 41% of eligible men by mid-2011 [14]. This reflects the reality that infected women are linked to care through routinely offered HIV testing when utilizing antenatal and other sexual reproductive health services; an option that is not available for men. A South African study assessing the sustainability of task shifting in a rural primary care clinic demonstrated that 68.8% of those accessing ART services between 2004 and 2010 (p < 0.05) were women [15], and those men that access ART are less likely to be retained in care [16]. In a country with the largest ART rollout program, it is important to understand who the current utilizers of HIV treatment services are, as this will help inform efforts aimed at reach- ing those currently not being reached. Introduction In South Africa, an estimated 6.2 million people are currently living with HIV infection [1]. South Africa has been successful in implementing the largest anti-retroviral programme in the world with more than 3 million HIV infected people currently on treatment [2]. Despite this, high HIV incidence rates continue to occur, and HIV related morbidity and mortality domi- nates as the main cause of hospitalization and premature death for the last 2 decades [3]. PLOS ONE | https://doi.org/10.1371/journal.pone.0184124 September 13, 2017 1 / 12 This study sought to investigate whether there were gen- der and age differences in those accessing HIV care services and clinical outcomes in those initiated on ART among urban and rural patients utilizing a free HIV treatment program in KwaZulu-Natal, South Africa. Patient baseline clinical characteristics A total of 4043 ambulant HIV infected patients, 1457 (36%) men and 2586 women (64%), 14 years and older, were initiated on ART between June 2004 and August 2013. Baseline clinical characteristics are presented in Table 1. Women comprised 59.4% and 40.6%, (p <0.001) of the study cohort at the urban and rural sites, respectively. Men were older, (median age of 35 vs 32 years), p<0.001, presented more often with WHO stage 4 disease (14.3% vs 9.7%), p<0.001 and were more likely to have had a past history of tuberculosis (TB) (34.4% vs 26.3%), p <0.001, (Table 1). At ART initiation, men had lower median CD4+ cell counts (113 vs131 cells/mm3), p <0.001, a lower median body mass index (BMI) (21.0 vs 24.2 kg/m2), p<0.001 and higher mean log viral loads (5.0 vs 4.9 copies/ml), p<0.001 (Table 1). The overall retention rate at 72 months was 91.8%, with 89.9% of men compared to 91.8% of women (p = 007) retained in care. Men and women were loss to follow-up at a median (IQR) of 9.7 (2.7–23.7) and 12.6 (4.4–27.2) months respectively (p = 0.118). Rates of viral suppression, (89.5% vs 96%, p = 0.094) and proportion diagnosed with TB after ART initiation, (3.8% vs 4.2%, p = 0.620), did not differ significantly between men and women. In contrast, ART regi- men change; 71% vs 9.5% p = 0.012 was significantly different between men and women (Table 1). High mortality rates in men initiated on ART Data analysis Systems immunology of ID93 vaccine-induced protection against recurrent TB disease. The aim of this consortium is to understand biomarkers of TB recurrence in multiple cohorts. Role: Co-Principle Investigator. This does not alter the authors’ adherence to PLOS ONE policies on sharing data and materials. There are no restrictions on sharing of data. We used unpaired t-test, and Wilcoxon rank sum test for continuous data and Fisher’s exact test for categorical data to compare demographics and clinical characteristics between men and women. Data was censored at 72 months of follow-up. Kaplan-Meier was used to con- struct survival curves and compare them using the log-rank test. Poisson approximations and F-tests were used to construct 95% confidence intervals (CI) for mortality rate, TB incidence rate and rate ratios, respectively. We calculated age-standardised mortality rates using 5-year age band. The province of KwaZulu-Natal was used as a standard population. Predictors of mortality were assessed through both univariate and multivariate proportional hazards regres- sion. Proportionality was assessed by fitting time dependent covariates in a model created by interacting baseline variables with survival time. The association between gender and CD4+ cell count was determined through linear mixed models. All models were adjusted for baseline covariates such as clinic site, age, tubercu- losis status at baseline, past history of TB, WHO stage, CD4+ cell count and viral load. We con- ducted sensitivity analysis evaluating the impact of ART programme maturity on the effect of gender on mortality during ART initiation periods of 2004–2008 and 2009–2013. We calculated attributable risk in order to assess the relative contribution and impact of specific covariates on mortality. The effect of loss to follow-up on mortality was determined through sensitivity analysis. P values less than 0.05 were considered statistically significant. All analyses were conducted using SAS, version 9.4 (SAS Institute INC., Cary) and R version 3.2.2. Role of the funding source. The funders of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. The corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication. Methods Study design We undertook a retrospective chart review of ambulant HIV infected, ART naïve adult patients enrolled into the PEPFAR-funded CAPRISA AIDS Treatment Programme (CAT) between June 2004 and August 2013. Patients were enrolled from two different catchment populations in KwaZulu-Natal; a TB clinic in the urban eThekwini district and a rural primary health care clinic in Vulindlela, rural KwaZulu-Natal. ART eligibility criteria, and definitions of immunologic and virological response to ART, defined as <400 copies/ml was as per cur- rent South African Government HIV/AIDS treatment guidelines [17]. Following ART initia- tion, patients presented for clinical review and adherence assessment weekly for the first two weeks, then monthly for the first 6 months, and every 3 months thereafter unless clinically indicated. Routine demographic and clinical data were recorded at baseline and at follow-up visits. Laboratory safety assessments and CD4+ cell counts and viral loads were conducted at baseline and every 6 months or as clinically indicated. Patients were regarded as lost to follow-up if they missed 3 consecutive scheduled visits and if all attempts to track them telephonically and physically had failed. No lost to follow-up patients re-engaged in care. Patients provided writ- ten informed consent for participation in the CAT programme and for the use of their medical records. Approval for data collection and analysis was obtained from the University of Kwa- Zulu-Natal, Biomedical Research Ethics Committee (Ref: E248/05). 2 / 12 PLOS ONE | https://doi.org/10.1371/journal.pone.0184124 September 13, 2017 High mortality rates in men initiated on ART Table 1. Baseline and follow-up characteristics of men and women initiated on ART. Variable Men (N = 1457) Women (N = 2586) p-value Characteristics at ART initiation Overall age (years), median(IQR) 35 (30–41) 32 (28–38) <0.001 Age group: n (%) <24 45 (3.1) 232 (9.0) <0.001 24–34 655 (45.0) 1350 (52.3) 35+ 757 (52.0) 1001 (38.8) BMI(kg/m2), median(IQR) 21 (19.0–3.2) 24.2 (21.1–28.1) <0.001 CD4+ cell count (cells/mm3), median(IQR)a 113 (47–177) 131 (68–189) <0.001 CD4+ cell count, cells/mm3: n (%) <50 344 (25.9) 423 (18.3) <0.001 50–200 783 (58.9) 1428 (61.6) >200 203 (153) 466 (20.1) Log VL, mean(SD)b 50 (0.9) 4.9 (0.9) <0.001 Rural site, n (%) 692 (47.5) 1536 (59.4) <0.001 Urban site, n (%) 765 (52.5) 1050 (40.6) WHO stage 1–3, n (%)c 1244 (85.7) 2315 (90.3) <0.001 WHO stage 4, n (%)c 208 (14.3) 250 (9.7) Previous history of TB, n (%)d 483 (34.4) 661 (26.3) <0.001 Prevalence of TB, n (%) 467 (32.1) 505 (19.5) <0.001 Follow-up Number initiated on second line ART, n (%) 104 (7.1) 245 (9.5) 0.012 Lost to follow-up 161 (11.1) 239 (9.2) 0.070 a396 t t h d i i b li CD4 t https://doi.org/10.1371/journal.pone.0184124.t001 https://doi.org/10.1371/journal.pone.0184124.t001 were 5.1 per 100 person-years (p-y) (95% CI: 4.6–5.6) and 6.7 per 100 p-y (95% CI: 5.8–7.8) in men and 4.4 per 100 p-y (95% CI: 3.8–5.0) among women (mortality rate ratio: 1.54 (95% CI: 1.29–1.91, p< 0.001), (Fig 1). Age-standardised mortality rate was 7.9 per 100 p-y (95% CI: 4.1–11.7) for men and 5.7 per 100 p-y (95% CI: 2.7 to 8.6) for women (standardised mortality ratio: 1.38 (1.15 to 1.70). Mortality rates observed for men and women were significantly dif- ferent at all follow-up time points up to 72 months; men had higher mortality rates throughout follow-up; peaking in the first 6 months post ART initiation at 15.2 (95% CI: 12.3–18.4) and 10.2 (95% CI: 8.5–12.2), between men and women respectively, p = 0.004 (Fig 1). Mortality rates were highest in young men between the ages of 20 and 24 years at 11.8 per 100 p-y, (95% CI: 5.7–21.8) vs 3.9 per 100 p-y (95% CI: 2.4–5.9) in women of the same age, mortality rate ratio: 3.06 (95% CI: 1.45–6.46), p = 0.003 (Table 2). Mortality Deaths occurred among 173 men and 239 women, over 8033.71 person-years of follow-up, (median of 16.3 months [interquartile range (IQR), 8.1 to 35]), (Fig 1). Overall mortality rates 3 / 12 PLOS ONE | https://doi.org/10.1371/journal.pone.0184124 September 13, 2017 PLOS ONE | https://doi.org/10.1371/journal.pone.0184124 September 13, 2017 Among those  25 years, mortality rates among men were significantly different compared to women: 6.6 per 100 p-y (95% CI 5.6–7.7) vs 4.4 per 100 p-y (95% CI 3.9–5.1), mortality rate ratio: 1.49 (95% CI: 1.21– 1.83), p <0.001 (Table 2). The mortality rate between men and women stratified by baseline CD4+ cell count was 10.7 (95% CI 8.3–13.6) and 8.2 (95% CI 6.5–10.1) for patients with base- line CD4+ cell counts <50 cells/mm3, p = 0.098; 5.4 (95% CI 4.3–6.7) and 3.8 (95% CI 3.2–4.6) for baseline CD4+ cell counts of 50–200 cells/mm3, p = 0.012 and 5.2 (95% CI 2.8–8.9) and 2.1 (95% CI 1.2–3.3). Mortality rate ratio stratified by baseline CD4+ cell count comparing men 4 / 12 PLOS ONE | https://doi.org/10.1371/journal.pone.0184124 September 13, 2017 High mortality rates in men initiated on ART Fig 1. Kaplan-Meier estimates of cumulative annualised mortality rates between men and women i ART Fig 1. Kaplan-Meier estimates of cumulative annualised mortality rates between men and women accessing ART. Fig 1. Kaplan-Meier estimates of cumulative annualised mortality rates between men and women accessing ART. Fig 1. Kaplan-Meier estimates of cumulative annualised mortality rates between men and women accessing ART. https://doi.org/10.1371/journal.pone.0184124.g001 https://doi.org/10.1371/journal.pone.0184124.g001 and women was 1.31 (95% CI 0.95–1.8) for baseline CD4+ cell counts < 50 cells/mm3, p = 0.098; 1.42 (95% CI 1.08–1.87) for baseline CD4+ cell counts of 50–200 cells/mm3, p = 0.012 and 2.1 (95% CI 1.22–5.26) for baseline CD4+ cell counts of > 200 cells/mm3 (Table 2). and women was 1.31 (95% CI 0.95–1.8) for baseline CD4+ cell counts < 50 cells/mm3, p = 0.098; 1.42 (95% CI 1.08–1.87) for baseline CD4+ cell counts of 50–200 cells/mm3, p = 0.012 and 2.1 (95% CI 1.22–5.26) for baseline CD4+ cell counts of > 200 cells/mm3 (Table 2). Fig 2. Mean CD4+ cell count change from baseline over time. Treatment response Women demonstrated a better immunological response during ART follow-up showing con- sistently higher mean CD4+ cell counts at each clinical assessment (Fig 2). Mean CD4+ cell count increases post-ART initiation were lower in men at all follow-up time points (Fig 2). At 5 / 12 PLOS ONE | https://doi.org/10.1371/journal.pone.0184124 September 13, 2017 https://doi.org/10.1371/journal.pone.0184124.g002 https://doi.org/10.1371/journal.pone.0184124.g002 g 2. Mean CD4+ cell count change from baseline over time. tps://doi.org/10.1371/journal.pone.0184124.g002 Variable at ART initiation Univariate Multivariate HR (95% CI) p-value HR (95% CI) p-value Gender (ref: women) Male 1.41 (1.16–1.72) 0.001 1.29 (1.05–1.58) 0.016 Age (per 5 year increase) 1.03 (0.97–1.08) 0.364 1.04 (0.98–1.10) 0.182 Site (ref: rural) Urban 0.82 (0.67–1.01) 0.061 0.90 (0.70–1.16) 0.410 Baseline CD4+ cell count (ref:>200), cells/mm3 <50 4.10 (2.75–6.10) <.0001 3.86 (2.55–5.84) <.0001 50–200 1.89 (1.28–2.79) 0.001 1.94 (1.30–2.91) 0.001 Viral load (log10 copies/ml) 1.52 (1.31–1.76) <0.001 1.30 (1.11–1.52) 0.001 Tuberculosis (ref: Yes) No 1.26 (0.98–1.61) 0.0711 1.74 (1.33–2.28) <.0001 WHO stage (ref: 1–3) Stage 4 2.49 (1.97–3.15) <.0001 2.32 (1.80–2.99) <.0001 Past history of TB (ref: No) Yes 1.20 (0.97–1.47) 0.092 1.04 (0.81–1.33) 0.759 https://doi.org/10.1371/journal.pone.0184124.t003 Table 4. Impact of different risk factors on mortality. Variable Attributable risk (95% CI) Male 10.2% (2.7–16.9) CD4+ cell count <50cells/mm3 24.3% (17.3–30.5) WHO stage 4 13.2% (9.0–18.1) No TB prevalence 24.2% (7.0–40.8) Table 3. Analysis of factors associated with mortality. Variable at ART initiation Univariate Multivariate HR (95% CI) p-value HR (95% CI) p-value Gender (ref: women) Male 1.41 (1.16–1.72) 0.001 1.29 (1.05–1.58) 0.016 Age (per 5 year increase) 1.03 (0.97–1.08) 0.364 1.04 (0.98–1.10) 0.182 Site (ref: rural) Urban 0.82 (0.67–1.01) 0.061 0.90 (0.70–1.16) 0.410 Baseline CD4+ cell count (ref:>200), cells/mm3 <50 4.10 (2.75–6.10) <.0001 3.86 (2.55–5.84) <.0001 50–200 1.89 (1.28–2.79) 0.001 1.94 (1.30–2.91) 0.001 Viral load (log10 copies/ml) 1.52 (1.31–1.76) <0.001 1.30 (1.11–1.52) 0.001 Tuberculosis (ref: Yes) No 1.26 (0.98–1.61) 0.0711 1.74 (1.33–2.28) <.0001 WHO stage (ref: 1–3) Stage 4 2.49 (1.97–3.15) <.0001 2.32 (1.80–2.99) <.0001 Past history of TB (ref: No) Yes 1.20 (0.97–1.47) 0.092 1.04 (0.81–1.33) 0.759 https://doi.org/10.1371/journal.pone.0184124.t003 72 months of ART, mean CD4+ cell count gain was 378.4 cells/mm3 and 556.6 cells/mm3, p = 0.001 among men and women respectively (Fig 2). Multivariate linear mixed model showed a significant interaction between gender and follow-up time suggesting significantly different CD4+ cell count recovery between men and women; p = 0.001 (Fig 2). 72 months of ART, mean CD4+ cell count gain was 378.4 cells/mm3 and 556.6 cells/mm3, p = 0.001 among men and women respectively (Fig 2). Multivariate linear mixed model showed a significant interaction between gender and follow-up time suggesting significantly different CD4+ cell count recovery between men and women; p = 0.001 (Fig 2). High mortality rates in men initiated on ART Table 2. Mortality rates for men and women stratified by baseline CD4+ cell count and age. Men Women Characteristic No of deaths Person- years Mortality rate (95% CI) No of deaths Person- years Mortality rate (95% CI) Rate ratio (95% CI) p-value Age group (years) <20 1 16.32 6.1 (0.2–34.1) 3 86.31 3.5 (0.7–10.2) 1.76 (0.18–16.92) 0.624 20–24 10 84.42 11.8 (5.7–21.8) 22 568.07 3.9 (2.4–5.9) 3.06 (1.45–6.46) 0.003 25 162 2460.88 6.6 (5.6–7.7) 213 4808.60 4.4 (3.9–5.1) 1.49 (1.21–1.83) <0.001 Baseline CD4+ count (cells/mm3) <50 67 626.59 10.7 (8.3–13.6) 85 1039.12 8.2 (6.5–10.1) 1.31 (0.95–1.8) 0.098 50–200 85 1563.29 5.4 (4.3–6.7) 128 3343.48 3.8 (3.2–4.6) 1.42 (1.08–1.87) 0.012 >200 13 250.01 5.2 (2.8–8.9) 16 777.17 2.1 (1.2–3.3) 2.53 (1.22–5.26) 0.013 https://doi.org/10.1371/journal.pone.0184124.t002 Table 2. Mortality rates for men and women stratified by baseline CD4+ cell count and age. Men Women Characteristic No of deaths Person- years Mortality rate (95% CI) No of deaths Person- years Mortality rate (95% CI) Rate ratio (95% CI) p-value Age group (years) <20 1 16.32 6.1 (0.2–34.1) 3 86.31 3.5 (0.7–10.2) 1.76 (0.18–16.92) 0.624 20–24 10 84.42 11.8 (5.7–21.8) 22 568.07 3.9 (2.4–5.9) 3.06 (1.45–6.46) 0.003 25 162 2460.88 6.6 (5.6–7.7) 213 4808.60 4.4 (3.9–5.1) 1.49 (1.21–1.83) <0.001 Baseline CD4+ count (cells/mm3) <50 67 626.59 10.7 (8.3–13.6) 85 1039.12 8.2 (6.5–10.1) 1.31 (0.95–1.8) 0.098 50–200 85 1563.29 5.4 (4.3–6.7) 128 3343.48 3.8 (3.2–4.6) 1.42 (1.08–1.87) 0.012 >200 13 250.01 5.2 (2.8–8.9) 16 777.17 2.1 (1.2–3.3) 2.53 (1.22–5.26) 0.013 https://doi.org/10.1371/journal.pone.0184124.t002 Table 2. Mortality rates for men and women stratified by baseline CD4+ cell count and age. Table 2. Mortality rates for men and women stratified b Fig 2. Mean CD4+ cell count change from baseline over time. https://doi.org/10.1371/journal.pone.0184124.g002 6 / 12 PLOS ONE | https://doi.org/10.1371/journal.pone.0184124 September 13, 2017 PLOS ONE | https://doi.org/10.1371/journal.pone.0184124 September 13, 2017 6 / 1 Adjusted proportional hazards regression analysis showed that men had a 29% higher risk of death compared to women (adjusted hazard ratio (aHR): 1.29, 95% CI: 1.05–1.58, p = 0.016 (Table 3). Risk of mortality varied by pre-ART CD4+ cell count. Compared to patients with baseline CD4+ cell counts >200 cells/mm3, there was a 4-fold higher risk of death among patients with a baseline CD4+ cell count <50 cells/mm3 (aHR: 3.86, 95% CI: 2.55–5.84), p<0.0001, and a two-fold higher risk of death among patients with a baseline CD4+ cell count between 50–200 cells/mm3, (aHR: 1.94, 95%CI: 1.30–2.91), p = 0.001, (Table 3). In addition, patients presenting with no TB at ART initiation or a higher mean log viral load or WHO stage 4 disease had significantly higher risk of death (Table 3). We found that the attributable risk of death among patients initiating ART was 24.3% (95% CI 17.3–30.5) for patients with a low baseline CD4+ cell count and 10.2% (95% CI 2.7–16.9) for male patients (Table 4). Of note, sensitivity analysis evaluating the impact of ART programme maturity on the effect of gender on mortality showed that mortality rates between men and women was consistently two-fold higher and significantly different at all follow-up time-points between 2009–2013. This finding did not hold for the period 2004–2008 during the period of ART initiation (Table 5). High mortality rates in men initiated on ART 72 months of ART, mean CD4+ cell count gain was 378.4 cells/mm3 and 556.6 cells/mm3, p = 0.001 among men and women respectively (Fig 2). Multivariate linear mixed model showed a significant interaction between gender and follow-up time suggesting significantly different CD4+ cell count recovery between men and women; p = 0.001 (Fig 2). Adjusted proportional hazards regression analysis showed that men had a 29% higher risk of death compared to women (adjusted hazard ratio (aHR): 1.29, 95% CI: 1.05–1.58, p = 0.016 (Table 3). Risk of mortality varied by pre-ART CD4+ cell count. Compared to patients with baseline CD4+ cell counts >200 cells/mm3, there was a 4-fold higher risk of death among patients with a baseline CD4+ cell count <50 cells/mm3 (aHR: 3.86, 95% CI: 2.55–5.84), p<0.0001, and a two-fold higher risk of death among patients with a baseline CD4+ cell count between 50–200 cells/mm3, (aHR: 1.94, 95%CI: 1.30–2.91), p = 0.001, (Table 3). In addition, patients presenting with no TB at ART initiation or a higher mean log viral load or WHO stage 4 disease had significantly higher risk of death (Table 3). We found that the attributable risk of death among patients initiating ART was 24.3% (95% CI 17.3–30.5) for patients with a low baseline CD4+ cell count and 10.2% (95% CI 2.7–16.9) for male patients (Table 4). Of note, sensitivity analysis evaluating the impact of ART programme maturity on the effect of gender on mortality showed that mortality rates between men and women was consistently two-fold higher and significantly different at all follow-up time-points between 2009–2013. This finding did not hold for the period 2004–2008 during the period of ART initiation (Table 5). Discussion We provide evidence of gender disparities between HIV infected men and women in access and response to antiretroviral therapy. Clinical outcomes differed by gender; with men dem- onstrating higher overall mortality rates and suboptimal immunologic recovery. Table 3. Analysis of factors associated with mortality. PLOS ONE | https://doi.org/10.1371/journal.pone.0184124 September 13, 2017 Mortality The bulk of deaths in both men and women occurred in the first 24 months of follow-up con- curring with findings from other studies [18,19]. Despite ART access, all patients showed unacceptably high mortality rates with men having 50% higher mortality rate compared women throughout follow-up and their mortality was consistently higher irrespective of base- line CD4+ cell counts. Results from sensitivity analyses, which assumed that all patients who were lost to follow-up died, and that all patients who were lost to follow-up within six months of ART initiation died, were consistent with the main findings (S1 Table). After removing the confounding effect of age, mortality rates observed in men and women in our study was lower than the age-standardised mortality rates. Interestingly, mortality rates among men and women in the study population closely approximated age-standardised mor- tality when we assumed that patients who were loss to follow-up within 6 months of ART initi- ation died. In contrast to the epidemiological data of the evolving epidemic in southern Africa wherein HIV acquisition does not occur in men until their late twenties [20,21] and we found that the highest mortality rates were in young men between the ages of 20–24 years. However, we had a very small number of men less than 24 years of age enrolled into the programme. Conversely, studies from developed countries show differing results, finding no gender dif- ferences in clinical, virological and immunological outcomes among HIV infected men and women initiating ART [22,23]. A multi-site cohort study found higher rates of virological rebound in women [7]. Studies conducted elsewhere in SSA also demonstrate poor outcomes among men on ART with respect to higher rates of mortality and loss-to-follow-up [24,25]. Published literature from South Africa support our findings and demonstrates that while life expectancy was not significantly different pre-ART between genders; in 2011 women were 27% less likely to die from HIV than men [26]. High mortality rates in men initiated on ART Table 5. Sensitivity analysis: Mortality rate by gender. Men Women Follow- up No of deaths Person- years Mortality rate (95% CI) No of deaths Person- years Mortality rate (95% CI) Mortality Rate ratio (95% CI) p-value Scenario† 334 2566.73 13.0 (11.7–14.5) 478 5466.98 8.7 (8.0–9.6) 1.49 (1.30–1.71) <0.001 Scenario‡ 236 2566.73 9.2 (8.1–10.4) 316 5466.98 5.8 (5.2–6.5) 1.59 (1.34–1.88) <0.001 † Assumes all patients who were loss to follow-up died ‡ Assumes all patients who were loss to follow-up within 6 months of ART initiation died htt //d i /10 1371/j l 0184124 t005 Table 5. Sensitivity analysis: Mortality rate by gender. † Assumes all patients who were loss to follow-up died ‡ Assumes all patients who were loss to follow-up within 6 months of ART initiation died https://doi.org/10.1371/journal.pone.0184124.t005 Discussion We provide evidence of gender disparities between HIV infected men and women in access and response to antiretroviral therapy. Clinical outcomes differed by gender; with men dem- onstrating higher overall mortality rates and suboptimal immunologic recovery. Table 4. Impact of different risk factors on mortality. Variable Attributable risk (95% CI) Male 10.2% (2.7–16.9) CD4+ cell count <50cells/mm3 24.3% (17.3–30.5) WHO stage 4 13.2% (9.0–18.1) No TB prevalence 24.2% (7.0–40.8) https://doi.org/10.1371/journal.pone.0184124.t004 Table 4. Impact of different risk factors on mortality. Variable Attributable risk (95% CI) Male 10.2% (2.7–16.9) CD4+ cell count <50cells/mm3 24.3% (17.3–30.5) WHO stage 4 13.2% (9.0–18.1) No TB prevalence 24.2% (7.0–40.8) https://doi.org/10.1371/journal.pone.0184124.t004 Table 4. Impact of different risk factors on mortality. Table 4. Impact of different risk factors on mortality. 7 / 12 PLOS ONE | https://doi.org/10.1371/journal.pone.0184124 September 13, 2017 PLOS ONE | https://doi.org/10.1371/journal.pone.0184124 September 13, 2017 https://doi.org/10.1371/journal.pone.0184124.t005 Risk factors for poor clinical outcomes Poor prognostic markers of ART response were apparent in men at baseline, who were older, had lower CD4+ cell counts and higher viral loads compared to women. This finding concurs with published South African literature demonstrating that compared to men, women were more likely to access HIV testing, and among those testing HIV positive, women were younger with higher CD4+ cell counts than their male counterparts [27]. This study also showed that the late presentation of men was associated with poor immuno- logical response to ART with higher risk of morbidity and mortality [27]. Our findings of significant correlation between baseline characteristics, virological suppression rates, immuno- logic recovery and mortality concur with previously published studies conducted in South 8 / 12 PLOS ONE | https://doi.org/10.1371/journal.pone.0184124 September 13, 2017 High mortality rates in men initiated on ART Africa [28,29]. There were no significant differences in virologic suppression in follow-up between men and women. Notwithstanding the greater than 90% virologic suppression rate on ART, men remained at higher risk of dying, emphasising the impact of advanced HIV dis- ease at baseline on subsequent antiretroviral treatment response. We found significantly differ- ent CD4+ cell count recovery between men and women with men consistently demonstrated significantly smaller CD4+ cell count increases compared to women during longitudinal fol- low-up. Given that viral load suppression was similar between sexes, women however had higher CD4+ cell counts indicating that there may be underlying genetic differences in ART response. We show that during follow up overall mortality rate halved among men and women with baseline CD4+ cell counts of 50–200 cells/mm3 in comparison with those patients that pre- sented with baseline CD4+ cell counts of less than 50 cells/mm3. Furthermore, we found that the mortality risk among patients initiating ART was attributable to both low baseline CD4+ cell count and male gender, corroborating published literature showing that baseline CD4+ cell count is an important factor impacting CD4+ cell count increase and recovery on treat- ment [30]. In settings that are still guided by CD4+ cell count thresholds for ART initiation consideration should be given for men to access ART at higher CD4+ cell counts. Our findings demonstrate that the absence of a diagnosis of prevalent TB increased risk of mortality. Risk factors for poor clinical outcomes Published literature has cited that undiagnosed TB is higher among patients access- ing ART than in the general population; with the majority of incident TB diagnosed in the early weeks of ART initiation being TB prevalent but missed at baseline screening [19,31,32]. It is possible that undiagnosed TB at ART initiation also contributed to higher risk of mortality in our study. The magnitude of the TB epidemic, burden of disease and varied presentation of TB in HIV infected individuals may have contributed to missed TB diagnosis. Programmatic outcomes Men had higher lost to follow-up rates in our cohort in keeping with reports from published literature [33,34]. Adverse clinical outcomes notably death can be masked by LTFU resulting in the underestimation of actual mortality rates within ART programmes [35]. Previous South African studies have documented gender differences in service uptake within the HIV care cas- cade; women have higher rates of HIV testing [27] and linkage to care than men [13]. Once initiated on ART, men have poorer adherence [36] and lower retention in care [28,35]. Govin- dasamy et al in a systematic review of 42 publications from 12 countries showed high attrition of men from pre-ART and ART programmes [37]. These studies did not however describe mortality differentials between the genders. Overall, the design and results of this article are similar to those found by Cornell et al [28]. PLOS ONE | https://doi.org/10.1371/journal.pone.0184124 September 13, 2017 Acknowledgments This work was supported by the US President’s Emergency Plan for AIDS Relief (PEPFAR), the Global Fund to fight AIDS, Tuberculosis and Malaria and the National Institutes of Health Comprehensive International Program of Research on AIDS (UM1AI069469). K.N. and N.P. were supported by the Columbia University-South Africa Fogarty AIDS International Train- ing and Research Program (AITRP, Grant D43 TW000231). KN is currently an Executive Director, Board of Control, Centre for the AIDS Program of Research in South Africa (CAPRISA). NP is currently Deputy Director, Centre for the AIDS Program of Research in South Africa (CAPRISA) and QAK and SAK are directors for Centre for the AIDS Program of Research in South Africa (CAPRISA). SAK is a Director at the DST- NRF Centre of Excellence in HIV Prevention. Supporting information S1 Table. (DOCX) S2 Table. (DOCX) Supporting information S1 Table. (DOCX) S2 Table. (DOCX) Limitations As this was a secondary analysis of routinely collected programme data, missing data limited our ability to comment on various correlations e.g. the impact of socio-economic and demo- graphic variables on clinical outcomes. Treatment adherence was not measured and its impact on outcomes could be underestimated. We have not investigated other biological and beha- vioural factors that may have contributed to poor ART outcomes in men. While the vast majority of patients enrolled into this cohort were women, we had very few pregnancies and are therefore unable to comment on the impact of pregnancy in women on mortality in this cohort. Additional studies that explore potential barriers that South African men experience when seeking HIV care, and optimal strategies to link and engage men in ART services, is warranted. 9 / 12 PLOS ONE | https://doi.org/10.1371/journal.pone.0184124 September 13, 2017 High mortality rates in men initiated on ART Conclusion While heterosexual transmission is the primary mode of HIV transmission in sub Saharan Africa, the impact of free ART provision on access and clinical outcomes in men and women is unequal. Men presented late for ART initiation with more advanced disease and a higher mortality rate compared to women in this six-year ART program. The increased feminization of the HIV epidemic in sub Saharan Africa has rightfully demanded that greater focus be given to women with respect to HIV prevention, transmission, treatment and care. However, in a predominantly heterosexual epidemic, greater programmatic effort should be directed toward encouraging men to test early and frequently. As we prepare to scale-up “Test and Treat” in all HIV infected patients, innovative strategies are needed to ensure that men and women that test HIV positive have access to effective linkage to care for early ART initiation to achieve the goal of reducing mortality and increasing AIDS free survival. PLOS ONE | https://doi.org/10.1371/journal.pone.0184124 September 13, 2017 References 1. STATISTICS SOUTH AFRICA, ‘Midyear population estimates 2015’; online: https://www.statssa.gov. za/publications/P0302/P03022015.pdf acessed: 17 May 2016. 2. UNAIDS report 2015 accessed online: http://www.unaids.org/sites/default/files/media_asset/JC2702_ GARPR2015guidelines_en.pdf accessed 01 April 2016 3. Norheim OF, Jha P, Admasu K, Godal T, Hum RJ, et al. (2015) Avoiding 40% of the premature deaths in each country, 2010–30: review of national mortality trends to help quantify the UN Sustainable Devel- opment Goal for health. The Lancet 385: 239–252. 4. Takarinda KC, Harries AD, Shiraishi RW, Mutasa-Apollo T, Abdul-Quader A, et al. (2015) Gender- related differences in outcomes and attrition on antiretroviral treatment among an HIV-infected patient cohort in Zimbabwe: 2007–2010. International Journal of Infectious Diseases 30: 98–105. https://doi. org/10.1016/j.ijid.2014.11.009 PMID: 25462184 5. Menzaghi B, Ricci E, Vichi F, De Sociod G, Carenzi L, et al. (2014) Gender differences in HIV infection: is there a problem? Analysis from the SCOLTA cohorts. Biomedicine & Pharmacotherapy 68: 385– 390. 6. Rosin C, Elzi L, Thurnheer C, Fehr J, Cavassini M, et al. (2015) Gender inequalities in the response to combination antiretroviral therapy over time: the Swiss HIV Cohort Study. HIV Med 16: 319–325. https://doi.org/10.1111/hiv.12203 PMID: 25329751 7. Cescon A, Patterson S, Chan K, Palmer AK, Margolese S, et al. (2013) Gender differences in clinical outcomes among HIV-positive individuals on antiretroviral therapy in Canada: a multisite cohort study. PLoS One 8: e83649. https://doi.org/10.1371/journal.pone.0083649 PMID: 24391803 8. Saunders P, Goodman A, Smith C, Marshall N, O’Connor J, et al. (2016) Does gender or mode of HIV acquisition affect virological response to modern antiretroviral therapy (ART)? HIV medicine 17: 18–27. https://doi.org/10.1111/hiv.12272 PMID: 26140659 9. Lee MP, Zhou J, Messerschmidt L, Honda M, Ditangco R, et al. (2015) Impact of Gender on Long-Term Treatment Outcomes of Highly Active Antiretroviral Therapy (HAART) in the TREAT Asia HIV Observa- tional Database. AIDS patient care and STDs 29: 229–231. https://doi.org/10.1089/apc.2014.0232 PMID: 25774867 10. Karim QA, Sibeko S, Baxter C (2010) Preventing HIV Infection in Women: A Global Health Imperative. Clinical Infectious Diseases 50: S122–S129. https://doi.org/10.1086/651483 PMID: 20397940 11. Muula AS, Ngulube TJ, Siziya S, Makupe CM, Umar E, et al. (2007) Gender distribution of adult patients on highly active antiretroviral therapy (HAART) in Southern Africa: a systematic review. BMC public health 7: 1. 12. Cornell M, McIntyre J, Myer L (2011) Men and antiretroviral therapy in Africa: our blind spot. Trop Med Int Health 16: 828–829. https://doi.org/10.1111/j.1365-3156.2011.02767.x PMID: 21418449 13. Author Contributions Conceptualization: Kogieleum Naidoo, Salim Abdool-Karim. Data curation: Dhineshree Govender, Nesri Padayatchi, Rochelle Nicola Adams, Aveshen Govender, Tilagavathy Chinappa. Formal analysis: Nonhlanhla Yende-Zuma. Methodology: Kogieleum Naidoo, Dhineshree Govender. Methodology: Kogieleum Naidoo, Dhineshree Govender. Project administration: Kogieleum Naidoo. Supervision: Salim Abdool-Karim, Quarraisha Abdool-Karim. Supervision: Salim Abdool-Karim, Quarraisha Abdool-Karim. Validation: Kogieleum Naidoo. Writing – original draft: Kogieleum Naidoo, Razia Hassan-Moosa. Writing – original draft: Kogieleum Naidoo, Razia Hassan-Moosa. 10 / 12 PLOS ONE | https://doi.org/10.1371/journal.pone.0184124 September 13, 2017 High mortality rates in men initiated on ART Writing – review & editing: Razia Hassan-Moosa, Dhineshree Govender, Nesri Padayatchi, Halima Dawood, Rochelle Nicola Adams, Aveshen Govender, Tilagavathy Chinappa, Salim Abdool-Karim, Quarraisha Abdool-Karim. References Bassett IV, Regan S, Chetty S, Giddy J, Uhler LM, et al. 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(2006) Scaling up of highly active anti- retroviral therapy in a rural district of Malawi: an effectiveness assessment. The Lancet 367: 1335– 1342. 19. Etard J-F, Ndiaye I, Thierry-Mieg M, Gueye NFN, Gueye PM, et al. (2006) Mortality and causes of death in adults receiving highly active antiretroviral therapy in Senegal: a 7-year cohort study. Aids 20: 1181–1189. https://doi.org/10.1097/01.aids.0000226959.87471.01 PMID: 16691070 PLOS ONE | https://doi.org/10.1371/journal.pone.0184124 September 13, 2017 11 / 12 High mortality rates in men initiated on ART 20. Harling G, Newell M-L, Tanser F, Kawachi I, Subramanian S, et al. (2014) Do age-disparate relation- ships drive HIV incidence in young women? Evidence from a population cohort in rural KwaZulu-Natal, South Africa. Journal of acquired immune deficiency syndromes (1999) 66: 443. 21. 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Expanding the primary health care workforce through contracting with nongovernmental entities: the cases of Bahia and Rio de Janeiro
Human resources for health
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© 2016 Ireland et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Expanding the primary health care workforce through contracting with nongovernmental entities: the cases of Bahia and Rio de Janeiro Megan Ireland1, Luciana Cavalini2, Sabado Girardi3, Edson C. Araujo4 and Magnus Lindelow5* Abstract Background: Brazil has experienced difficulties in attracting health professionals (especially doctors and nurses) to practice at the primary health care (PHC) level and in rural and remote areas. This study presents two case studies, each a current initiative in contracting for primary health services in Brazil: one for the state of Bahia and the other for the city of Rio de Janeiro. The two models differ considerably in context, needs, modalities, and outcomes. This article does not attempt to evaluate the initiatives but to identify their strengths and weaknesses. Methods: Analysis was based on indicators produced by the Brazilian health care information systems, a review of literature and other documentation, and key informant interviews. Results: In the case of Bahia, the state and municipalities decided to create a State Foundation, a new institutional public entity acting under private law that centralizes the hiring of health professionals in order to offer stable positions with career plans and mobility within the state. Results have been mixed as a lower than expected municipal involvement resulted in relatively high administrative costs and consequent default on municipal financial contributions. In the case of Rio de Janeiro, the municipality opted to contract not-for-profit Social Organizations as it made a push to expand access to primary health care in the city. The approach has been successful in expanding coverage, but evidence on cost and performance is weak. Conclusions: Both cases highlight that improvements in cost and performance data will be critical for meaningful comparative evaluation of delivery arrangements in primary care. Despite the different institutional and implementation arrangements of each model, which make comparison difficult, the analysis provides important lessons for contracting out health professionals for PHC within Brazil and elsewhere. Keywords: Primary health care, Contracting, Human resources, Health services * Correspondence: mlindelow@worldbank.org 5Health, Education, Social Protection and Poverty in Brazil, World Bank Group, Brasilia, Brazil Full list of author information is available at the end of the article Ireland et al. Human Resources for Health (2016) 14:6 DOI 10.1186/s12960-016-0101-3 Ireland et al. Human Resources for Health (2016) 14:6 DOI 10.1186/s12960-016-0101-3 Background on long-term contracts, has been seen as insufficiently responsive or dynamic due to problems of low-powered incentives, lack of management flexibility, and a bureau- cratic institutional culture. In contrast, private sector en- tities are often smaller and unconstrained by the myriad of administrative rules and constraints of the public sector. Hence, at least in principle, contracting can help improve both quality and efficiency by combining man- agement flexibility with more high-powered incentives. Today, both for-profit and not-for-profit providers play an important role in many health systems, in part be- cause of the historic roots of these systems but also as a Over the last couple of decades, international experience with contracting in the health sector has been growing. Although there are significant differences in context and processes behind these experiences, they have often been motivated by a desire to improve quality and efficiency of service delivery. The “traditional” model of service de- livery through public sector providers, with salaried staff * Correspondence: mlindelow@worldbank.org 5Health, Education, Social Protection and Poverty in Brazil, World Bank Group, Brasilia, Brazil Full list of author information is available at the end of the article Page 2 of 11 Page 2 of 11 Ireland et al. Human Resources for Health (2016) 14:6 social security coverage in favor of a “collective public health model” built on the premise of universal access, equity, integrality (comprehensiveness), decentralization, and social participation. result of policy decisions to “autonomize” providers or contract with the private sector as a means to improve the performance of service delivery. As the experience with contracting has grown, so has the associated literature. Part of this literature is con- cerned with the conditions under which contracting the delivery of public services is likely to be effective, given the uncertainty and information asymmetry that is per- vasive in the health sector [3, 4, 6, 14]. There is also a growing body of evidence on specific experiences, in- cluding efforts to evaluate to what extent the perform- ance of contracted service providers is superior to other models for delivering services (see, e.g., [10–12, 15, 16]). The creation of the SUS unified health financing under one integrated public system that increased and stabi- lized public financing for health, while at the same time decentralizing the responsibility of service delivery to state and municipal levels. The health system is orga- nized across the three levels of government: federal, state, and municipal. Background Each level of government has a health fund. At the federal level, the health fund is fi- nanced by federal taxes and social security contributions and is a major contributor (45 % in 2009) to the state and municipal funds which are also funded by local rev- enues. As of 2000, the state and the municipal govern- ment are required to allocate a minimum of 12 % and 15 %, respectively, of their overall budgets to health care, although no such provision exists at the federal level [9]. g ( g [ ]) The globalization phenomena have changed signifi- cantly the organization of health care systems on a glo- bal basis. There is a concern about underperformance, and many countries have undertaken reforms as an at- tempt to remedy those situations. One of the major con- cerns is related to health care personnel contracting modalities, and experiences are very heterogeneous re- garding design, implementation, and outcomes. In sum- mary, the empirical evidence has shown the emergence of three major strategies related to this issue: (1) con- tractual relations based on delegation of responsibility, including contracts delegating responsibility to private actors, such as contracts for the devolution of a public service; contracts relating to the concession of a geo- graphical area, or public-private partnerships; contracts binding the state and its autonomous institutions, in- cluding internal contracting; (2) contractual relations based on an act of purchase, such as the relations be- tween fundholders and health service providers and health service providers’ production processes; and (3) contractual relations based on cooperation, such as weak organizational interpenetration agreements (franchising, collaboration between health care establishments and voluntary associations, or strategic planning at the level of the local health system and health networks) or strong organizational interpenetration agreements (joint man- agement and alliances). The political decision to reorient and reorganize health care delivery toward a more comprehensive primary health care approach resulted in significant expansion of services, particularly the outpatient network at the mu- nicipal level. This was achieved by the creation of the Family Health Strategy (FHS) in 1994, which was de- signed to expand the coverage of primary health care (PHC) with an emphasis on whole-person care and the social context, and to provide a first point of contact with the broader health system. Background In practice, the FHS is based on Family Health Teams (FHT) composed of a doctor, nurse, nurse assistant, and four to six community health workers, organized by geo- graphic regions, with each team providing PHC to around 1000 families (about 3500 people). The teams are either based in Basic Care Units or operate from purpose-built Family Health Clinics that host several FHT. The FHT are expected to provide comprehensive and integrated primary health care to the target popula- tion, through services provided at the facility and out- reach activities. Since its launch, coverage of the FHS has expanded rapidly across the country. There are currently over 35 000 FHT, present in 96 % of municipalities, and with an estimated national population coverage of 57 % accord- ing to the Department for Basic Care (Ministry of Health, DAB database).1 Priority expansion of services into more rural, poorer municipalities, and to poorer communities within them, has enhanced equity of ac- cess. Utilization rates have risen across all states and particularly in those with lower levels of income. The FHS has also affected the way Brazilians use public ser- vices, reducing the role of hospitals as the “usual source of care” (from 35 % in 1998 to 21 % in 2008) and in- creasing reliance on PHC facilities (from 42 % to 57 % New contracting modalities in the health sector New contracting modalities in the health sector Recognizing the need to increase flexibility in public contracting and service provision, the Programa Nacio- nal de Publicização was approved as part of the State Reform process in 1998. The law authorized the transfer of responsibility for running public services and manage- ment of public goods and personnel to a specific set of qualified entities, including Organizações Sociais or OS, civil society organizations (Organização da Sociedade Civil de Interesse Público—OSCIP), nongovernmental or- ganizations, philanthropic organizations, cooperatives, and private companies. The objective of the reform was to create a mechanism for facilitating the transfer of cer- tain activities from the state to the private sector. It would be a new form of partnership that called upon the “third sector” (i.e., neither public nor private) to provide services of social interest and public use, but that do not necessarily need to be undertaken by public bodies. As part of the decentralization of the SUS, municipalities have responsibility for management and delivery of the FHS and are required to adhere to constitutional norms and public law on employment (recruitment, contracting, and payment of professionals) and services. Direct employ- ment options are limited to three types of contracts (as defined by the constitution and administrative law):  Public civil servant contracts are permanent positions that must be contracted through public competitions (merit-based).  Temporary contracts are allowed under circumstances of “exceptional public interest,” the definition of which is contested and malleable. One form of contracting that has emerged as particu- larly important for the health sector is that of OS. For- mally, OS are legal entities under private law, operate on a not-for-profit basis, carry out activities of social value, and operate in partnership with the state. They are pri- marily financed by public funds and must adopt govern- ance arrangements that allow for state representation. They are subject to public audit (by the Tribunal de Contas) and ministerial supervision.  Commissioned positions are reserved for directorial or high-level managerial functions. In all cases, levels of remuneration are defined by the municipality. However, salaries are harmonized within municipalities, and there is limited scope for variation across specialization, geographic location, or perform- ance. Brazilian health care system Brazil formally embarked on its path to universal health care some 25 years ago with the creation of the Unified Health System or SUS (Sistema Único de Saúde). Health care as the right of the individual and duty of the state was written into the 1988 constitution and was the cul- mination of a broad-based reform process throughout the 1980s that sought democratization and improved so- cial rights. In particular, the “Sanitary Reform Movement” (Movimento da Reforma Sanitária), an informal coalition of health professionals, academics, and other civil society actors, strove for a fundamental break from the prevailing “curative privatizing model” that promoted expanded Ireland et al. Human Resources for Health (2016) 14:6 Page 3 of 11 rigidities that hampered expansion of primary health care coverage. Contracting out the provision of health services has emerged as one option, and initiatives have been adopted throughout the country. over the same period) [13]. Several studies have also demonstrated that the FHS has had a significant impact on outcomes, including infant mortality [9]. After a rapid expansion over the first 10 years of imple- mentation, coverage started to stagnate around 2006, in particular in larger municipalities and metropolitan areas. In addition, there has been growing concern with the quality of care. One of the key constraints to expanding coverage and improving quality are the human resource management arrangements in the public sector. New contracting modalities in the health sector In addition to the direct constraints on public hir- ing, other federal laws, such as the Law of Fiscal Responsibility (Lei de Responsabilidade Fiscal) which limit municipal spending on personnel to a maximum of 60 % of the municipal budget, also indirectly present barriers to expanding social services. Furthermore, the geographic and sector distribution of medical doctors is marked by inequalities, as many physicians work in urban areas in the private sector or in specialized care. In 2010, 1304 municipalities had a shortage of physi- cians, especially those in rural, peri-urban, or difficult- to-access areas [5, 8]. There is also an overall shortage of medical doctors with an interest and appropriate qualifi- cations for working in primary care, particularly in underserved parts of the country, due to the relatively lower salaries and the mainly public hiring of primary health care positions, as compared with other specialties. This results in limited attraction for medical graduates to specialize in PHC specialties as there is, for example, a persistent surplus of family health medical residence positions [1]. The first experiences with the contracting of OS was in science and technology, with some laboratory services contracted out immediately after the new legislation in 1998. The state of São Paulo was also an early adopter of the OS model as a more flexible alternative for hiring professionals while incorporating private sector manage- ment practices, initially focusing on the health sector. The state contracted the management of some hospital services in 1998, and today, OS are involved in most as- pects of health service delivery. Over the last decade, some states in Brazil have also pursued other options for improving the delivery of health services. Specifically, the possibility of using State Foundations started in 2005, when the federal govern- ment, through the Ministry of Planning, Budget and Management and the Ministry of Health, and aided by the National School of Public Health and a group of lawyers, began studying broader legal and institutional options for overcoming the rigidities in the health sys- tem [7]. State Foundations are decentralized administra- tive institutions that carry out public activities and provide social services. A key difference between a State Given these challenges, many states and municipalities started searching for ways to circumvent public sector Page 4 of 11 Ireland et al. New contracting modalities in the health sector Human Resources for Health (2016) 14:6 different, as were the results (see Table 1 for a compari- son of the two models). Foundation and an OS is that a State Foundation is a public (state-owned) institution, albeit operating under private law (including for contracting and managing staff), whereas an OS is a privately owned institution. Foundation and an OS is that a State Foundation is a public (state-owned) institution, albeit operating under private law (including for contracting and managing staff), whereas an OS is a privately owned institution. Methods Third-party contracting of medical doctors in PHC is still limited, accounting for fewer than 7 % of all contracts, with OS being the most popular alternative (Fig. 1). Per- manent public civil servant contracts or other forms of public contract (temporary or commissioned positions) are the predominant form of contracting of medical doc- tors in PHC (nearly 80 % of all doctors), but irregular forms of contracting through stipends and other means are also significant in many states. The two case studies were chosen for their importance in defining new milestones in human resource contract- ing for health in Brazil. Although they aim to overcome the same constraints, the two models represent different modalities of contracting and were implemented in dif- ferent contexts. The selection of case studies was based on the recent trends in the contracting for PHC in Brazil as the OS and the State Foundations are the current dominant modalities of contracting models in the coun- try. The paper does not aim, however, to compare the two experiences given the differences in the contexts, in- stitutional framework, and, particularly, lack of adequate data to control for these differences. The objective is to provide a detailed analysis of each experience to high- light their strengths and weakness that may have influ- enced the observed outcomes. This paper looks at two particularly noteworthy expe- riences: that of creating a State Foundation (Fundação Estatal) in the state of Bahia and contracting with OS (Organizações Sociais) in the city of Rio de Janeiro. In both cases, the aim was to expand PHC in underserved areas and improve performance through enhanced ac- countability and more flexible management, but other- wise, the experiences from the two locations were very Fig. 1 Contracting modalities for doctors in primary care (2013, by state) Fig. 1 Contracting modalities for doctors in primary care (2013, by state) Ireland et al. Methods Human Resources for Health (2016) 14:6 Page 5 of 11 Table 1 Comparison of contracting models Bahia Rio de Janeiro When started 2009 2009 Contracting entity Participating municipalities and the state government of Bahia Municipality of Rio de Janeiro Contracted entity/entities State Foundation Social Organizations Stated aims To formalize and expand employment in primary care and improve quality Provide primary health services Main responsibilities of contracted entity Hiring and training of professional and management support to primary care Hiring and managing complete Family Health Teams and the services they provide in facilities provided by the municipality Full-service management No Yes Legal regime Private law Private law Employment regime Consolidation of Labor Laws (CLT)a CLT Ownership Public Private Supervision Office of the Comptroller Generalb Office of the Comptroller General Funding Public Public Source: Compiled by the authors aCLT is the main piece of legislation relating to Brazilian labor law and procedural labor law. It was created in 1943, unifying all the existing labor legislation in Brazil. Its main aim is the regulation of individual and collective labor relations. Other labor laws, such as for those working as legal entities (Pessoa Jurídica), independent/freelance contract workers, or public civil servants, are covered under a federal statutory legal regime bCGU (Controladoria-Geral da União) or “Comptroller General” is the federal agency responsible for technical supervision, internal control, and public audit Table 1 Comparison of contracting models Sou ce: Co p ed by t e aut o s aCLT is the main piece of legislation relating to Brazilian labor law and procedural labor law. It was created in 1943, unifying all the existing labor legislation in Brazil. Its main aim is the regulation of individual and collective labor relations. Other labor laws, such as for those working as legal entities (Pessoa Jurídica), independent/freelance contract workers, or public civil servants, are covered under a federal statutory legal regime bCGU (Controladoria-Geral da União) or “Comptroller General” is the federal agency responsible for technical supervision, internal control, and public audit regarded as free from conflict of interest. In the case of the Rio de Janeiro Secretariat, the three informants were part of the team responsible for managing the FHS in the municipality. This study used a mixed-method study design that in- cluded both quantitative and qualitative data collection and analysis. Data collection processes included three stages: firstly, a literature review was undertaken. Methods This review included published literature as well as legislation (laws, decrees, ordinances, resolutions, and operational norms defined by different levels of government). This literature review followed the narrative methodology, searching from scientific publication databases (PubMed, Scopus, and ISI Web of Knowledge) and the gray litera- ture retrieved from searches in generic search engines such as Google. The main search keywords were “pri- mary care” and “contracting.” Data analysis consisted of (i) qualitative descriptive analysis for the document research and in-depth inter- views. Particular attention was paid to management, fi- nancing, and service provision issues such as human resources, performance monitoring, and management (For the in-depth interviews, content analysis was car- ried out by repeated reading of the transcribed inter- views in order to codify and analyze the data by theme.) and (ii) quantitative analysis of the secondary data in order to construct a statistical diagnosis of the current state of primary health care in the State of Bahia and the Municipality of Rio de Janeiro. Secondly, secondary data on different dimensions of health care provision and outcomes were collected. The secondary data originated from the Brazilian official health care information systems, which are routinely col- lected, recorded, and stored by the Informatics Depart- ment at the Ministry of Health (Departamento de Informática do SUS—DATASUS). Those are the only sources of information that cover the entire period con- sidered in this study, for the indicators chosen. All data were collected from August to September 2013, covering the period 2007–2013. A complete list of the research themes, indicators, data sources, and type of analysis can be found in Additional file 1. Bahia case study: the State Foundation experience Second, professionals hired by the FESF underwent 6 months of compulsory (and remunerated) training. All FESF professionals were offered continuing education for specialization or masters programs. Third, health professionals contracted by the FESF were also offered performance incentives. This was a bonus of 25–50 % of base salary, paid monthly, dependent on meeting the goals set by the FESF. These goals were monitored quantitatively and qualitatively through a dedicated PHC Monitoring System, with pay- ment based on the number of days worked and results. The most important function of the FESF was to con- tract health professionals, in particular doctors, nurses, and dentists, for PHC on behalf of participating munici- palities. All recruitment by the FESF was governed by the Plan for Employment, Careers and Salaries (Plano de Empregos, Carreiras e Salários), which established public competitive processes and formal labor contracts that allow for mobility across health teams, for career ad- vancement by merit, for continuing education, and for employment stability. This was a pioneering feature of the FESF that was explicitly designed to address the challenges faced by many municipalities in attracting staff by offering both financial and career incentives for professionals to accept positions in underserved areas. The FESF was also expected to provide support to participating municipalities for the management and organization of FHT and the development of primary health care, including training, supervision, and the introduction of management practices supporting quality improvement in primary care. Fourth, all contracts between municipalities and/or the state with the FESF included targets and goals, to which a 10 % variable portion of payments from municipalities to the FESF was linked. The evaluation of the targets and goals was based on trimestral reports drafted and sent by the FESF to the Monitoring and Evaluation Commission. Bahia case study: the State Foundation experience Bahia case study: the State Foundation experience In January 2007, after an extensive consultative process with stakeholders from the executive and judiciary, as well as civil society, the Bahia State Health Secretariat or SESAB (Secretaria de Estado da Saúde da Bahia) pro- posed the State Foundation as the most appropriate so- lution for attracting and retaining health professionals that would reduce the proportion of temporary and ir- regular work contracts and overcome public sector rigidities. The third step consisted of in-depth unstructured in- terviews with key informants from the Secretariat of Health (Rio de Janeiro) and the Secretariat of Health and State Foundation (Bahia). The key informants were re- cruited based on their participation in the process of de- fining new contracting rules. As civil servants, with no financial interests in contracting modalities, they were Page 6 of 11 Page 6 of 11 Page 6 of 11 Ireland et al. Human Resources for Health (2016) 14:6 2012, the default rate on management contracts with mu- nicipalities reached 80 % of revenues. A State Foundation is a state-owned, not-for-profit in- stitution that integrates indirect public administration, but operates within private law, with a mix of private- and public sector governance mechanisms, such as employment contracts. Its legal basis is similar to a state-owned company, except that it functions in the so- cial rather than economic domain, and hence, it may not commercialize its services on the market. Beyond trying to centralize and coordinate the con- tracting of health professionals for FHT, the FESF intended to increase the quality of services through vari- ous strategies. First, the professionals hired by FESF were expected to be better qualified for their positions as a result of a more rigorous hiring process. Moreover, the conditions offered by the FESF (stability, social security, career de- velopment, etc.) were also expected to help attract quali- fied professionals. The Family Health State Foundation or FESF (Funda- ção Estatal Saúde da Família) was designed as a strategy for municipalities with the greatest problems in attract- ing and retaining health professionals and improving the quality of PHC in a coordinated manner. It entailed a tripartite contract between the participating municipal- ities, the state, and the foundation. Municipal participa- tion was optional, but the benefits of greater stability and quality of services provided were expected to en- courage mayors to participate. Rio de Janeiro case study: the Social Organization experience The contracts defined a fixed payment based on number of health teams and an estimate of re- sources required to cover the Portfolio of Basic Services, as well as a variable, performance-based payment linked to three sets of indicators: incentives paid to the OS based on productivity and quality of service targets, in- centives paid to health units based on specific agreed in- dicators, and incentives paid to health workers. Health units were required to produce trimestral reports relat- ing to agreed indicators. salaries. The significantly higher salaries for the more qualified positions facilitated hiring and hence created the conditions for the expansion that has been observed. The new Family Health Clinics were successful in rap- idly increasing coverage (Fig. 3), in part due to the char- acteristics of Rio de Janeiro whereby large populations live in favelas. The clinics were implanted in under- served areas with a high population density. They were in most cases large and included multiple FHT and in some cases incorporated laboratories, x-ray facilities, and other services. In May 2009, the law paving the way for the contract- ing of OS in Rio de Janeiro was enacted and tendering opened immediately. To date, six OS have won tenders to manage FHT in 10 geographical areas. However, only five are still in operation as one of the contracts was canceled due to not meeting minimum standards, and the contract for that territory was passed to another OS. Since contracting with OS started, Family Health Clinics have been implanted all over Rio de Janeiro, primarily by the construction of new clinics (85 new units since 2009) but also by converting Basic Health Units into Family Clinics (49 units that existed before 2009). The expansion of the FHS in Rio de Janeiro was ex- pected to result in improved coverage of priority inter- ventions (antenatal care, management of patients with chronic diseases, etc.) and, ultimately, improved out- comes. It is still early to assess the extent to which these goals have been achieved. Nonetheless, available data suggest that relative to 2007, the municipality has seen a near doubling of PHC consultations, a large increase in the number of chronic disease patients under active management, and a reduction in avoidable admission from diabetes and associated complications. However, there has only been a modest increase in the share of pregnant women with seven or more prenatal consulta- tions (Table 2). Rio de Janeiro case study: the Social Organization experience Until recently, health care in the city of Rio de Janeiro re- lied heavily on an extensive hospital network, including fa- cilities under federal, state, and municipal management, but had very limited primary health care provision. In 2009, the municipality launched the Saúde Presente pro- gram aimed at expanding FHS coverage. In order to es- chew cumbersome public procedures, the municipality constructed purpose-built clinics for several FHT but contracted out the staffing and clinical management to OS. The municipality continued to promote and exercise strategic control over the social actions through entering into management contracts with OS, with which perform- ance targets were agreed to ensure the quality and effect- iveness of services. The FESF was formally created in July 2009. At that time, a total of 256 mayors (61 %) signed Terms of Com- mitment and Adherence to the FESF and 110 (26 %) pro- ceeded to pass authorizing laws. However, when the contracting process began in September 2009, which entailed signing technical cooperation agreements and management contracts between the municipalities and FESF, only 40 municipalities (10 %) actually contracted the FESF to hire health professionals. Of the 40 municipalities contracted in 2009, only 12 (3 %) still had contracts in 2014, accounting for 22 doc- tors, 38 nurses, and 28 dentists. Due to municipalities’ low participation, the FESF contracted only around 180 FHT, well below the earlier expectation of 1000. The number of staff contracted by the FESF peaked in late 2011 and started declining in mid- to late 2012 (Fig. 2). By late All contracts between OS and the municipality of Rio de Janeiro were standardized. The contracts contained a defined set of health services that were to be managed, maintained, and equipped with human resources by the OS. The only difference between contracts was the geo- graphic area covered. Contracts were signed for 2 years and were renewable if at least 80 % of the objectives and Ireland et al. Human Resources for Health (2016) 14:6 Page 7 of 11 Fig. 2 Number of health care workers contracted by the FESF goals were met. Rio de Janeiro case study: the Social Organization experience The financial reports of the OS were only available as from 2012. They showed a concentration in the hiring of community health agents, doctors, nurses, nurse techni- cians, and administrators. This was to be expected as the FHS assumes a large number of community health agents working over small territories in direct contact with the families within them. Equally, doctors, nurses, and nurse technicians form the base of FHT, as are ad- ministrators that help manage them. The OS did not en- counter any problems in attracting professionals. In fact, a critical feature of the OS model was the simplified hir- ing and the flexibility to pay differentiated (and higher) The contracting of OS was also intended to improve performance due to their increased management cap- acity and flexibility as well as the introduction of per- formance incentives for both professionals and health facilities. By 2013, however, the performance incentive modality had not operated as planned, since the pay- ment of the variable part of the funding appeared dis- connected to achieving the targets: the data available indicate that targets had not been met, while the variable Ireland et al. Human Resources for Health (2016) 14:6 Page 8 of 11 Fig. 3 Estimates of FHS coverage, in Rio de Janeiro, 2006–2013 funding had been paid. This was likely to do with prob- lems of information collection rather than poor quality care, and increased reporting of targets in 2013 indicated that reporting was improving. In addition, there also ap- peared to be distortions with respect to the financing of incentives. The municipality transferred the variable part of the funding to the OS regardless of their performance. The OS, on the other hand, were able to retain these funds if the Family Clinics did not attain their targets and goals. While any retained incentive funds were fore- seen to be invested in the health facilities most in need, the fact that the OS stood to capitalize on the poor per- formance of the Family Clinics they ran represented dis- incentives for performance and quality. retaining health professionals, as well as competition between municipalities for scarce human resources. Rio de Janeiro case study: the Social Organization experience More- over, as a result of contractual arrangements, accountabil- ity for performance (e.g., complying with the 40 h a week requirement for doctors) has often been weak and there has been little systematic effort to ensure opportunities for continuing education for health professionals in PHC fa- cilities [7]. By centralizing the hiring of professionals for primary health across the state, the FESF intended to create economies of scale and help reduce competi- tion between municipalities and the high rotation of professionals. funding had been paid. This was likely to do with prob- lems of information collection rather than poor quality care, and increased reporting of targets in 2013 indicated that reporting was improving. In addition, there also ap- peared to be distortions with respect to the financing of incentives. The municipality transferred the variable part of the funding to the OS regardless of their performance. There are a number of reasons for the lower than ex- pected participation by municipalities in the FESF. One concerned costs. The fact that FESF offered stable con- tracts with social security benefits and competitive salar- ies meant that the cost of hiring professionals through the FESF was comparatively expensive. In addition, the expected economies of scale did not materialize. As a re- sult, administrative costs for participating municipalities were high, and the FESF was not able to achieve bar- gaining power in the state labor market. Some muni- cipalities were also reluctant to surrender their autonomy to hire and manage their publicly employed health professionals. Bahia case study Implementation of the FHS in Bahia started in 1997 and since then has been the principal strategy for strengthen- ing PHC in the state. Since its introduction, FHS coverage has increased continuously, reaching all municipalities and 60 % population coverage by 2011 [2]. Nevertheless, Bahia, like other states in the Northeast, lags behind the rest of Brazil on socioeconomic development, and many municipalities have faced challenges in attracting and Table 2 Trends in key indicators in Rio de Janeiro municipalities (2007–2013) 2007 2013 % change Population 6 178 762 6 429 923 4 Primary health care consultations 3 566 747 6 846 453 92 Pregnant women with 7 or more prenatal consultations 68.5 % 70.8 % 3 Number of diabetics under management 125 317 231 960 85 Number of hypertensive patients under management 544 414 791 951 45 Avoidable admission: diabetes and complications 3.6 % 1.0 % −71 Source: Official administrative data provided by the Health Secretariat of Rio de Janeiro Municipality Page 9 of 11 Ireland et al. Human Resources for Health (2016) 14:6 Ireland et al. Human Resources for Health (2016) 14:6 Ireland et al. Human Resources for Health (2016) 14:6 One of the main reasons for the decline in participa- tion of the FESF has been the high proportion of municipality-FESF contract defaults. Many municipal- ities were not transferring funds to the FESF, partly due to a perception that the costs were too high, and yet retained the staff until their contracts were terminated thus defaulting on management contracts. In addition, municipal elections were held at the end of 2012 that changed management in some municipalities, to the det- riment of participation with FESF. In practice, the FESF was obliged to diversify its activ- ities from contracting the workforce to include a con- tract with the State Health Secretariat-SESAB to hire professionals to develop its home care services linked to the state hospital network, regulatory activities, institu- tional support to PMAQ, and other services. Hence, al- though the FESF continues to engage on primary health and work with municipalities, staff for the FHS only accounted for 6 % of employees in 2013, and only 25 % of foundation revenues come from municipalities. Other external factors also impinged upon the appeal of the FESF, such as the simultaneous implementation of other national programs aimed at supplying medical staff to underserved areas. Rio de Janeiro case study Rio de Janeiro is the second largest city in Brazil (6 mil- lion inhabitants). For many years, the hospital network ensured access to basic care through outpatient depart- ments and emergency rooms, as well as critical inpatient services and specialist care. However, access to the poorer segments of the population has long been prob- lematic and has become increasingly so as the popula- tion and health care needs have grown. Moreover, the hospital network does not provide preventive services or health promotion, and integration and coordination of care has been weak. In light of the remarkable expansion of FHS coverage from around 10 % in 2008 to 41 % in 2013, the contract- ing of OS can be considered a great success, especially taking into account that many of the Family Health Clinics were constructed from scratch. It can also be said that this reform in the provision of primary health care has succeeded in attracting significant amounts of government funding to the health sector and in particu- lar a huge increase in primary health spending. The impact of the FESF on the quality of primary health care is difficult to assess, in part due to the recent and limited scope of implementation and because con- tracting with the FESF was voluntary and the participat- ing municipalities are not necessarily comparable to other municipalities. However, the recent performance evaluation of PHC under the National Program for the Improvement in Access and Quality of Primary Health Care or PMAQ (Programa Nacional de Melhoria do Acesso e da Qualidade) found that teams with current FESF staff have consistently better ratings. The differ- ences cannot be causally linked to the FESF, but inter- viewees attributed the better results to the institutional support offered by the FESF to the municipalities [2]. Contracting with OS is an interesting model that is based on longer term relationships between the organi- zations and the municipality. The model requires a high- level of cooperation given that they share responsibilities in primary care, with the municipality responsible for policy and providing the infrastructure, while the OS manage the provision of care and supply of basic drugs and some equipment. The close relationship has provided conditions for close monitoring and problem-solving and may have contributed to improving performance in ways that are hard to measure. Bahia case study In particular, the Program for the Enhancement of Professionals in PHC or PROVAB (Pro- grama de Valorização dos Profissionais na Atenção Básica) that offers a practical 1-year postgraduate course in family health by placing doctors in underserved loca- tions and, more recently, the “More Doctors” program (Mais Médicos) that attracts Brazilian and foreign doc- tors to underserved areas and requires medical students to undertake 2 years in medical residency in PHC facil- ities as a precondition to graduation, both of which led some municipalities to dismiss FESF-hired professionals in preference for professionals hired under these pro- grams at lower cost. Rio de Janeiro case study On the other hand, the harder forms of performance accountability envisaged in the contract, in particular performance-based variable payments, still need to be fine-tuned in order to establish a mechanism that delivers on its intended aims and is workable, with- out inadvertently giving rise to distortions of incentives. The future of the FESF However, despite a high level of initial support, the number of municipalities that actually established a contract with the FESF when it was created was very low and has fallen further since. As a result, the intended economies of scale were not achieved, undermining the appeal of the model for municipalities. There are some indications that the FESF has contrib- uted to improved performance where it is operating, but data are limited and by no means conclusive [2]. y In assessing contracting experiences and comparing them against other approaches, performance in terms of coverage and quality is important, but the cost implica- tions of the approach also need to be considered. It is often claimed that contracting will improve the technical efficiency of service delivery, although such gains may be partially offset by increases in administrative and trans- action costs. However, evidence on these dimensions of contracting experiences if often scant, as is also the case for these two Brazilian cases. There is some limited evi- dence that the productivity of health providers has in- creased due to performance incentives and new forms of management. In both models, personnel costs have, however, increased, although this would perhaps have been required in the alternative models as well in order to attract staff. Although there is currently very little in- formation on administrative costs associated with the re- spective models, it is clear that both the FESF and the OS incur nontrivial administrative costs and there are also transaction costs to be considered in state and mu- nicipal administration related to monitoring and over- sight of contracts. Although it is probably safe to say that the municipal- ity would not have been able to expand coverage to the extent it has based on the traditional model, some of the gains in terms of quality and effectiveness of PHC may have as much to do with the Family Clinic model (com- bining multiple teams in purpose-built facilities) as with the OS model. In other words, the ability to hire and manage staff in a flexible way has been an important gain, but the extent to which the contracting out of management of facilities as opposed to simply providing staff is an important aspect of the model is not clear. There is also insufficient data on costs and efficiency to assess the merit of the OS model relative to alterna- tives. The future of the FESF The FESF model depended on a significant share of mu- nicipalities in the state contracting out human resource management in PHC to the foundation, but for a range of reasons, this did not happen, and over time, the num- ber of municipalities doing so declined even further. The leadership of the FESF undertook an administrative and governance reform to reduce administrative costs, seek- ing to strengthen the focus on quality, productivity, and efficiency by finding a balance between administrative costs for the number of employees and ensuring an ap- propriate administrative structure for strategic planning and management for results. The model has also introduced a potentially problem- atic dynamic between health workers with regard to compensation. Currently, despite the standardized con- tracts between the municipality and the OS, there is no apparent link between the resources received by OS per Page 10 of 11 Page 10 of 11 Page 10 of 11 Ireland et al. Human Resources for Health (2016) 14:6 contract and the salaries they pay. In fact, wage inequal- ity exists across the whole primary health network in Rio de Janeiro. Salary disparities exist between public of- ficials assigned to OS and professionals directly hired by them and between professionals hired by different OS but within the same professional category, as well as be- tween professionals working in different geographical lo- cations. OS covering more remote and often poorer areas of the city tend to pay lower salaries. They are therefore more prone to hiring the less qualified, youn- ger professionals for shorter periods of time. Thus, argu- ably, the introduction of OS has increased the inequality within the primary health system from the traditional management model that had standardized levels of pay. Financial data, however, has only been available since 2012, and therefore, the effects of such wage differentials have yet to be determined but may negatively impact the provision of care. between the municipality and the OS. At this point, there is limited basis on which to assess the quality of OS performance and to compare services provided by OS with those delivered based on alternative models. In the case of Bahia, the FESF managed to contract a number of health professionals for participating munici- palities, contributing to an expansion of coverage in these locations. The future of the FESF This is partly an issue of the costs of increased sal- aries, but also, administrative costs must also be taken into account, both within the OS as well as within the municipality. Assessments of efficiency and perform- ance, however, must take a comprehensive analysis of performance. For instance, higher salary and administra- tive costs may be justifiable if associated with increased productivity and quality of care. g Given the differences in the institutional and imple- mentation arrangements, as well as the different socio- economic context, it is not possible to make a direct comparison between the two models. On the other hand, it is possible to compare with the situation before the contracting arrangements were implemented in Bahia and Rio de Janeiro. In both cases, the experi- ences managed to achieve expansion of PHC cover- age; however, the FESF model did not sustain this expansion given the reduction in the number of mu- nicipalities with contracts. The analysis provides some plausible hypothesis to explain this, as, for example, the fact that FESF was more vulnerable to changes in the political scenarios or the loss of autonomy in hir- ing and managing the workforce by the municipalities contracting with FESF. Endnotes 1 1Data from Jan 2014, available at: http://dab.saude. gov.br/portaldab/historico_cobertura_sf.php. Coverage is determined by a person’s residence within the defined catchment area of a Family Health Team. 11. Liu X, Hotchkiss DR, Bose S. The impact of contracting-out on health system performance: a conceptual framework. Health Policy. 2007;82:200–11. 12. Loevinsohn B, Harding A. Buying results? Contracting for health service delivery in developing countries. Lancet. 2005;366:676–81. 13. Macinko J. A preliminary assessment of the Family Health Strategy (FHS) in Brazil. Consultant Report World Bank. Washington DC: World Bank; 2011. 14. Mills A. To contract or not to contract? Issues for low and middle income countries. Health Policy Plan. 1998;13:32–40. Conclusions to the writing and revision of the article, and they have all read and approved the final manuscript. to the writing and revision of the article, and they have all read and approved the final manuscript. The OS model has a relatively long history in Brazil and is increasingly used for staffing and managing health care provision in different parts of Brazil. Experiences to date have shown that it takes time for the approach to mature, with the need for significant capacity on both the OS and government side (management of services, design of con- tracts, monitoring of costs and performance, oversight, etc.). Nonetheless, as OS with a focus on health care be- come more plentiful and experienced, and with continued pressures to contain civil service wage bills at the state and municipal level, the approach will likely continue to grow, in particular in larger municipalities. Discussion of both studies In terms of achieving the goals of expanding coverage and improving the quality of PHC services, the picture emerging from the two cases is mixed. The municipality of Rio de Janeiro successfully managed to expand the coverage of primary health care through the FHS. This expansion, in turn, has been associated with a large in- crease in the utilization of PHC services, which is ex- pected to contribute to improved outcomes over the longer term. Inevitably, a number of implementation is- sues remain, in particular concerning the collection and compilation of data on costs and performance, which is a critical element of the system of performance-related financial incentives that form part of the contract Page 11 of 11 Page 11 of 11 Ireland et al. Human Resources for Health (2016) 14:6 References 1. Almeida-Filho N. Higher education and health care in Brazil. Lancet. 2011; 4(377(9781)):1898–900. 2. Araujo E, Cavalini L, Girardi S, Ireland M, Lindelow M. Contracting for primary health care in Brazil: the cases of Bahia and Rio de Janeiro. Health, Nutrition and Population Discussion Paper, September 2014. Washington, DC: The World Bank; 2014. 3. Bennett S, Mills A. Government capacity to contract: health sector experience and lessons. Public Adm Dev. 1998;18:307–26. 4. Besley T, Ghatak M. Incentives, choice, and accountability in the provision of public services. Oxf Rev Econ Policy. 2003;19:235–49. 5. Dal Poz MR. The health workforce crisis. Cad Saude Publica. 2013;29(10): 1924–6. 6. Eggleston K, Zeckhauser R. Government contracting for health care. In: Donahue JD, Nye Jr JS, editors. Market-based governance: supply side, demand side, upside, and downside. Washington, D.C.: Brookings Institution Press; 2002. p. 29–65. In both cases, there is a need to carry out rigorous as- sessments. However, this will require the implementa- tion of reliable and accurate information systems that, as of yet, are not in place. Medium-term indicators are ne- cessary for the measurement of the effectiveness of the system while long-term indicators are needed for the as- sessment of the impact on the morbidity and mortality of the population covered. 7. Fundação Estatal Saúde da Família. A Bahia unida por um novo caminho para a Saúde da Família. Salvador, Bahia: Fundação Estatal Saúde da Família; 2009. 8. Girardi, S.N., C.L. Carvalho, J.M. Farah, L.W.D. Maas, and L.A.B. Campos. 2011. Índice de escassez de médicos no Brasil: estudo exploratório no âmbito da Atenção Primária. In: Celia Regina Pierantoni; Mario Roberto Dal Poz, Tania França. (Org.). O trabalho em saúde: abordagens quantitativas e qualitativas. 1st ed.: Cepesc. IMS/UERJ. Observatorio Recursos Humanos, p. 171-186. 8. Girardi, S.N., C.L. Carvalho, J.M. Farah, L.W.D. Maas, and L.A.B. Campos. 2011. Índice de escassez de médicos no Brasil: estudo exploratório no âmbito da Atenção Primária. In: Celia Regina Pierantoni; Mario Roberto Dal Poz, Tania França. (Org.). O trabalho em saúde: abordagens quantitativas e qualitativas. 1st ed.: Cepesc. IMS/UERJ. Observatorio Recursos Humanos, p. 171-186. 9. Gragnolati M, Lindelow M, Couttolenc B. 2013. Twenty years of health system reform in Brazil: an assessment of the Sistema Único de Saúde. Available at: http://hdl.handle.net/10986/15801 10. Liu X, Hotchkiss DR, Bose S. The effectiveness of contracting-out primary health care services in developing countries: a review of the evidence. Health Policy Plan. 2008;23:1–13. Competing interests The authors declare that they have no competing interests. Acknowledgements h k This work was supported by the Japan-World Bank Policy and Human Resources Development Fund (PHRD) Partnership Grant for Universal Health Coverage. Received: 12 May 2015 Accepted: 4 February 2016 The State Foundation model brings some important advantages but has suffered from significant implemen- tation problems. Some of these are related to the com- plex governance and contracting arrangements that arise between the foundation, the state, and the municipal- ities. However, the foundation model has also, for better or worse, been undermined by federal initiatives such as Mais Médicos, which emerged to address some of the same problems that the FESF was expected to help solve. The future of the foundation model for PHC in Bahia and elsewhere hence depends in large part on how it re- lates to federal human resource initiatives and on how efforts to diversify into other areas will permit the FESF to maintain a significant focus on primary care. Additional file 15. Perrot J. Different approaches to contracting in health systems. Bull World Health Organ. 2006;84:859–66. Additional file 1: Data analysis of the two case studies. The file contains a complete list of the research themes, indicators, data sources, and type of analysis 16. Schlesinger M, Gray BH. How nonprofits matter in American medicine, and what to do about it. Health Aff. 2006;25:W287–303. 16. Schlesinger M, Gray BH. How nonprofits matter in American medicine, and what to do about it. Health Aff. 2006;25:W287–303. Author details 1 1Consultant, Health, Nutrition and Population Global Practice, The World Bank Group, Brasilia, Brazil. 2Medical Sciences College, Rio de Janeiro State University, Rio de Janeiro, Brazil. 3Medical School, Federal University of Minas Gerais, Minas Gerais, Brazil. 4Health, Nutrition and Population Global Practice, The World Bank Group, Washington, DC, USA. 5Health, Education, Social Protection and Poverty in Brazil, World Bank Group, Brasilia, Brazil. Received: 12 May 2015 Accepted: 4 February 2016 Received: 12 May 2015 Accepted: 4 February 2016 Received: 12 May 2015 Accepted: 4 February 2016 Received: 12 May 2015 Accepted: 4 February 2016 Authors’ contributions MI drafted the article based on the case study reports. LC and SG conducted the research and authored the study reports for Rio de Janeiro and Bahia, respectively. EA and ML contributed to the design and implementation of both case studies. ML supervised both case studies. All authors contributed
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MEMPERTAHANKAN DAN MEMPERTANGGUNGJAWABKAN DOKTRIN TRINITAS DALAM KONTEKS ASIA
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MEMPERTAHANKAN DAN MEMPERTANGGUNGJAWABKAN DOKTRIN TRINITAS DALAM KONTEKS ASIA Dr. Noh Ruku Dosen STT Arrabona Kata Kunci : Trinitas, Agama, dan Gereja. Abstrak Penulisan yang memaparkan pandangan beberapa agama yang ada di Asia tentang Trinitas dan bagaimana gereja mempertahankan dan mempertanggungjawabkan doktrin tersebut dalam konteks Asia. Allah adalah “Satu dalam hakekat,dibedakan dalam tiga persona” (Mia ousia, tres hypostates). Doktrin Tritunggal sangat vital bagi teologi dan juga bagi pengalaman dan hidup Kristen. Allah Tritunggal adalah keutuhan dan kepenuhan hidup, berada dalam hubungan yg kekal, dan dalam persekutuan yg tak pernah putus atau berhenti. Hal ini membuat penyataan dan pengungkapan diri Allah dapat dimengerti. Persekutuan yang mengikat Trinitas, menjadi dasar bagi persekutuan lingkungan umat manusia, lingkungan keluarga, lingkungan masyarakat, dan secara istimewa dalam lingkungan gereja, karena di situ Roh Kudus menjadi Pengantara persekutuan itu. Rekomendasi dari penulis adalah untuk gereja dan jemaatnya agar mempertahankan dan mempertanggungjawabkan doktrin Trinitas dalam konteks Asia, khususnya dalam konteks masyarakat yang ada di sekitarnya. Allah adalah “Satu dalam hakekat,dibedakan dalam tiga persona” (Mia ousia, tres hypostates). Doktrin Tritunggal sangat vital bagi teologi dan juga bagi pengalaman dan hidup Kristen. Allah Tritunggal adalah keutuhan dan kepenuhan hidup, berada dalam hubungan yg kekal, dan dalam persekutuan yg tak pernah putus atau berhenti. Hal ini membuat penyataan dan pengungkapan diri Allah dapat dimengerti. Persekutuan yang mengikat Trinitas, menjadi dasar bagi persekutuan lingkungan umat manusia, lingkungan keluarga, lingkungan masyarakat, dan secara istimewa dalam lingkungan gereja, karena di situ Roh Kudus menjadi Pengantara persekutuan itu. Kata Kunci : Trinitas, Agama, dan Gereja. Kata Kunci : Trinitas, Agama, dan Gereja. 131 132 This article describes the idea of some religions in Asia about the teaching of Trinity and how the church can maintain and give account for this doctrine in the context of Asia. This article describes the idea of some religions in Asia about the teaching of Trinity and how the church can maintain and give account for this doctrine in the context of Asia. God is one in essence, and differs in three persona (mia ousia, tres hypostates). the doctrine of Trinity is very vital both in theology and in the life and experience of the Christianity. The Triune God is the wholeness and the fullness of live, exists in eternal relation, and in an uninterrupted and unstoppable fellowship. This enables the revelation of God understandable. The fellowship that binds the Trinity is the basis for the fellowship among human kinds, families, communities, and especially in the church communities because the HS is the Mediator in this fellowship. Abstrak recommend church and her members to maintain and to give account for the doctrine of Trinity in the context of Asia, especially in surrounding communities. Key words: Trinity, religion and church. 1 Dede Wijaya, “Allah Tritunggal.”www.in-christ.net. pp. 3-4. Online. Internet. Accesed August 24,2008. 2 Band, Erick Sudharma, Mengenal Satu-Satunya Allah Yang Benar (Bandung: Mitra Pustaka, 2003)hlm. 101-105 1. Doktrin Trinitas Istilah Trinitas tidak terdapat dalam Alkitab, namun konsepnya dengan jelas diajarkan oleh Alkitab. Satu sisi, Alkitab dengan tegas menyatakan keesaan Allah (Ulangan 6:4), di sisi lain, Alkitab dengan tegas menyatakan keilahian tiga pribadi Bapa, Anak dan Roh Kudus. Gereja menolak ajaran-ajaran bidat modalisme dan triteisme. Modalisme adalah ajaran yang menyangkali perbedaan Pribadi-Pribadi yang ada di dalam keesaan Allah, dan menyatakan bahwa Bapa, Anak, dan Roh Kudus 133 hanyalah merupakan tiga cara Allah di dalam mengekspresikan diri- Nya. Dipihak lain, hanyalah merupakan tiga cara Allah di dalam mengekspresikan diri- Nya. Dipihak lain, triteisme mengungkapkan pernyataan yang salah, ya triteisme mengungkapkan pernyataan yang salah, ya itu ada tiga keberadaan yang menjadi Allah.1 Allah adalah “Satu dalam hakekat,dibedakan dalam tiga persona” (Mia ousia, tres hypostates), untuk menyatakan realitas Allah yang sesungguhnya menurut Alkitab, dan juga menegaskan bahwa kesatuan dan ketigaan dalam diri Allah harus dipahami dalam arti yang berbeda satu sama lain,dalam hal hakekat, Allah adalah satu. Namun dalam hal kepribadian, Allah ada sejak kekekalan sebagai tiga persona.2 Dilihat dari peran-peran yang dipunyai oleh Allah Tritunggal, maka peranan ketiga oknum Allah dapat dilihat dari sisi soterio logis atau rencana keselamatan manusia. Seluruh tindakan Allah seperti karya penciptaan (oleh Allah Bapa), karya penebusan ( oleh Yesus Kristus), dan pewahyuan (oleh Roh Kudus) merupakan “isi”dari sejarah keselamatan yang telah dirancangkannya sejak kekekalan. Apabila doktrin Allah Tritunggal ini runtuh, ataupun bergeser, maka dapat dipastikan bahwa semua doktrin Kristen yang dibangun di atasnya akan ikut ambruk, karena doktrin ini merupakan fondasi dari semua doktrin tersebut. 3 Daud Ali al-Fadhili, Ushulul Masihiyyah Kama Yushowwiruhal Qur’an, terbitan Dar Zahron, tahun 2008. 2. Pandangan Agama-agama di Asia tentang Trinitas 2. Pandangan Agama-agama di Asia tentang Trinitas 2. Pandangan Agama-agama di Asia tentang Trinitas 134 Pada bagian ini tidak diuraikan pandangan semua agama yang ada di Asia tentang Trinitas, melainkan hanya beberapa agama saja, yakni Islam, Hindu, Buddha dan Yahudi (Yudaisme). 4 Yusuf Syalabi, Adhwa’ alal Masihiyyah, terbitan Dar Al- Kuwaitiyyah, cetakan ketiga tahun 1973. 5 Ustadz Wahid Hasyim Asyrofi, Lc Muhammad Abduh Tuasikal Artikel Muslim.Or.Id 6 Sukarji K, , Agama- agama yang Berkembang di Dunia dan Pemeluknya PT. PANAKOM, Denpasar , 1993. 7 Shalaby, Ahmad, Agama-Agama Besar India (Hindu– Jaina – Budha)Terjemahan, Departemen Agama RI, Jakarta, 1998 hlm. 83. 8 Titib, I Made, 2003, Tri Sandhya, Sembahyang dan Berdoa Penerbit Paramita, Surabaya 1996, hlm. 73. a. Agama Islam Menurut Islam, di dalam kamus kitab muqoddas, Trinitas didefinisikan sebagai berikut: beriman pada Tuhan yang satu Bapa, anak (Yesus Kristus) dan Roh Kudus, Tuhan yang satu, zat yang satu pula, dan kesemuanya adalah sama dalam hal kekuasaan, kekuatan dan kemulyaan. Doktrin Trinitas bukanlah ajaran asli Nashrani, karena ajaran asli Nashrani adalah ajaran tauhid yang dibawa oleh Nabi Isa ‘alaihis salam yang menyeru bani Israel untuk beribadah hanya kepada Allah semata dan tidak menyekutukannya, sama seperti ajaran yang dibawa oleh para Nabi dan Rasul sebelumnya.3 Dalam ajaran Islam, yang menjadi pertanyaan besar tentang Trinitas adalah bagaimana mungkin dasar utama aqidah keimanan yang seharusnya menjadi wewenang Allah untuk menjelaskannya ditetapkan berdasarkan hasil musyawarah manusia? Karena itu Al-Quran membantah dengan tegas ajaran Trinitas. Trinitas adalah ajaran syirik dan kufur, banyak sekali ayat-ayat Al-Qur’an yang membantah ajaran sesat Nashrani, dan menyeru mereka 135 untuk kembali kefitrah dan mengikuti agama yang haq agama Islam.4 Selanjutnya menurut Islam, di dalam Perjanjian Lama (Taurat), tidak ada satu pun nash yang jelas yang menjelaskan atau menyeru kepada ajaran Trinitas, bahkan Perjanjian Lama tidak pernah mengajarkan doktrin Trinitas, bukti yang paling jelas adalah tidak adanya satu pun orang Yahudi yang beriman pada doktrin ini. Sedangkan dalam Perjanjian Baru (Injil), doktrin Trinitas kebanyakan hanya berupa isyarat-isyarat tidak secara langsung, adapun bukti tentang Trinitas yang paling jelas ada dalam Injil Matius 28: 19 “Karena itu pergilah, jadikanlah semua bangsa murid-Ku dan baptislah mereka dalam nama Bapa dan Anak dan Roh Kudus”. Akan tetapi, bila diamati secara seksama kutipan Injil ini bukan menjelaskan bahwa Allah, Kristus dan Roh Kudus membentuk satu keilahian atau ketuhanan yang satu, yaitu ketiganya sama dalam bentuk kekekalan dan kekuasaan sebagaimana maksud dari doktrin Trinitas. Justru malah sebaliknya ayat ini menunjukkan tiga wujud yang berlainan, sama halnya ketika kita menyebut tiga orang: Budi, Andi dan Bambang, misalnya.5 b. Agama Hindu 136 Agama Hindu menyatakan pengertian Dewa secara etimologis, berasal dari bahasa Sansekerta yaitu Dev, yang berarti sinar.6 Dev juga diartikan sebagai terang, karena pengertian dewa adalah benda yang terang dan dianggap sebagai kekuatan alam yang mempunyai person.7 Di dalam weda, Tuhan Yang Maha Esa dan para dewa disebut Dewata. a. Agama Islam Kata ini berarti cahaya berkilauan, sinar gemerlapan yang semuanya ditujukan kepada manivestasi- Nya, juga ditujukan kepada matahari atau langit, termasuk api, petir atau fajar.8 Arti dan pengertian dewa menurut konsepsi itu adalah sesuai dengan pemujaan dan penyembahan yang dilakuk an terhadap sesuatu yang dianggap sebagai Dewa atau Tuhan dalam rangka memperoleh manfaat, keuntungan, dan perlindungan dari mereka. Selanjutnya dalam Agama Hindu, konsep panteisme menanamkan pandangan bahwa Tuhan tidak memiliki wujud tertentu maupun tempat tertentu, melainkan Tuhan berada dan menyatu pada setiap ciptaan-Nya, dan terdapat dalam setiap benda apapun. Konsep panteisme disebut dengan istilah Wyapi Wyapaka. Upanisad menyebutkan bahwa Tuhan memenuhi alam semesta tanpa wujud 137 tertentu, tidak berada di surga atau dunia tertinggi melainkan ada pada setiap ciptaan-Nya9 Dalam perkembangannya, tersebar pengajaran yang mengupas dan membandingkan antara paham Tri-Murti (three form) dari agama Hindu dengan Trinitas (Satu Allah dalam tiga pribadi) dari agama Kristen. Argumentasi dari artikel ini dapat disarikan sebagai berikut:10 1) Karena Alkitab ditulis dalam bahasa Yunani dan kebudayaan Yunani dipengaruhi oleh agama Hindu, maka Trimurti dari agama Hindu mempengaruhi paham Trinitas dari agama Kristen. 2) Karena agama Hindu lebih tua dari agama Kristen, maka kalau ada kesamaan, agama Kristenlah yang meniru agama Hindu. 3) Kemudian penulis berusaha untuk mengambil beberapa ayat dari Kitab Suci agama Hindu dan mencoba melihat kesamaannya dengan Alkitab, seperti: Brahma sebagai bapa, Wisnu sebagai anak Tuhan, dan Shiwa sebagai Roh Kudus. 9 Suhardana, KM, Upadsa Tentang Ajaran-Ajaran Agama Hindu ,PARAMITA, Surabaya 2006. 10 http://www.katolisitas.org/membandingkan-trinitas-dengan- , y 10 http://www.katolisitas.org/membandingkan-trinitas-denga 9 Suhardana, KM, Upadsa Tentang Ajaran-Ajaran Agama Hindu ,PARAMITA, Surabaya 2006. trimurti/ 11 http://members.tripod.com. c. Agama Buddha Sang Buddha mengajarkan Ketuhanan Yang Maha Esa, sejak khotbah pertamanya. Pernyataan di atas menerima penolakan karena banyak orang yang Sang Buddha mengajarkan Ketuhanan Yang Maha Esa, sejak khotbah pertamanya. Pernyataan di atas menerima penolakan karena banyak orang yang trimurti/ 138 menyatakan bahwa agama Buddha tidak ber-Tuhan, bahkan menyangkal adanya Tuhan. Menurut agama Buddha, anggapan demikian sebenarnya adalah suatu kesalahan semantik, salah paham bahasa, karena orang secara bebas menterjemahkan istilah-istilah dari literatur Barat ke dalam bahasa Indonesia, seperti misalnya: 'god' dengan 'Tuhan, 'theisme' dengan 'percaya Tuhan'. Tuhan adalah Yang Mutlak, Yang Tertinggi, Yang Maha Suci, dan akhir tujuan semua mahluk. Yang Mutlak adalah istilah falsafah, bukan istilah yang biasa dipakai dalam kehidupan keagamaan. Dalam kehidupan keagamaan, “Yang Mutlak” disebut dengan Tuhan Yang Maha Esa. Tuhan dalam Buddha Mahayana disebut Dharmakaya atau Adhi Buddha. 11 Agama Buddha percaya bahwa Buddha-Buddha adalah pancaran Adhi Buddha dan kemudian dari sinilah terbentuklah tiga aspek Buddha yang disebut dengan Trikaya. Trikaya adalah pandangan kaum Mahayana mengenai tiga tubuh Buddha, yakni: a. Inti dari Dharma itu sendiri yang tidak terbayangkan b. Kemampuan yang tidak terbatas dan tidak bermanifestasi dalam tubuh. c. Buddha yang bermanifestasi, yakni kebuddhaan yang berwujud dalam duniawi. Dari tiga aspek tersebut muncul tiga oknum Buddha yang terdiri dari Dharmakaya yaitu tubuh halus Buddha dan sari dari segala alam fenomena dan kehidupan tidak berbentuk. Sambhogakaya adalah tubuh berkah atau sinar dari Buddha 139 yang memancarkan sinar berkah. Nirmanakaya adalah tubuh perwujudan dari Buddha yang dapat dilihat oleh manusia, seperti yang dapat dijumpai pada Buddha Sakyamuni saat membabarkan Dharma pada umat manusia.12 Perbandingan Konsep Trikaya Dengan Trinitas: adalah Jiwa Alam Dharmakaya, Adhi Buddha Bodhisattva, Sambogyakaya Nirmanakaya yang sama dengan Allah Bapa, Roh Kudus, Logos Yesus Kristus. Menurut Buddha Mahayana : 1) Alam diciptakan oleh Adhi Buddha dan Adhi Buddhalah sumber dari segala yang ada. Pandangan tentang 81 Tuhan dapat dilihat pada Sutta-sutta yang berasal dari Sang Buddha Sang . Buddha sebagaimana tercantum dalam Kitab Suci Pali dan berbagai ekstrak dari literatur Mahayana. Dari teks-teks ini akan terlihat ide Sang pencipta yang kekal dan sangat berkuasa. Kesaksian Alkitab dalam kejadian 1 dan 2 memuat kesaksian bahwa Allah telah menciptakan langit dan bumi. 2) Bodhisattva dan Roh Kudus, dalam Trikaya dalam tingkatan Buddha yang kedua yaitu Sambogya kaya, ia pancaran dari Dharmakaya atau Adhi-Buddha adalah Buddha-Buddha yang ada di langit atau Bodhisattva yang berkewajiban untuk menyelamatkan manusia. 12 Pokok-Pokok Dasar Mahayana, Yayasan Yasodhara Puteri, Jakarta, 1994, hlm. 48 c. Agama Buddha Dan di antara Bodhisattva tersebut ada yang turun ke bumi menjelma sebagai Manushi Buddha Roh kudus adalah Pribadi ketiga dalam Tritunggal. Begitu juga menurut iman 2) Bodhisattva dan Roh Kudus, dalam Trikaya dalam tingkatan Buddha yang kedua yaitu Sambogya kaya, ia pancaran dari Dharmakaya atau Adhi-Buddha adalah Buddha-Buddha yang ada di langit atau Bodhisattva yang berkewajiban untuk menyelamatkan manusia. Dan di antara Bodhisattva tersebut ada yang turun ke bumi menjelma sebagai Manushi Buddha Roh kudus adalah Pribadi ketiga dalam Tritunggal. Begitu juga menurut iman 140 Kristiani menyakini bahwa Roh Kudus adalah roh yang mempersatukan Kristus dengan Allah Bapa. 3) Manushi Buddha Dan Yesus Kristus, dalam tradisi Buddha Mahayana Gautama dianggap jelmaan yang ilahi (Adhi Buddha). Adhi Buddha mengalir Menjadi Sambogyakaya (Bodhisattva), karena Bodhisattva mempunyai kewajiban untuk menyelamatkan manusia dari penderitaan maka ada salah satu Bodhisattva tersebut menjelma sebagai Buddha manusia. Menurut Kristologi, karya penyelamatan yang direncanakan Allah sejak semula itu sudah berlangsung pada waktu Perjanjian Lama, tetapi mencapai sepenuhnya dalam Perjanjian Baru Roh Kudus menjelma dalam diri Yesus Kristus. Ini berarti Yesus Kristus adalah penjelmaan dari Roh Kudus. 3) Manushi Buddha Dan Yesus Kristus, dalam tradisi Buddha Mahayana Gautama dianggap jelmaan yang ilahi (Adhi Buddha). Adhi Buddha mengalir Menjadi Sambogyakaya (Bodhisattva), karena Bodhisattva mempunyai kewajiban untuk menyelamatkan manusia dari penderitaan maka ada salah satu Bodhisattva tersebut menjelma sebagai Buddha manusia. Menurut Kristologi, karya penyelamatan yang direncanakan Allah sejak semula itu sudah berlangsung pada waktu Perjanjian Lama, tetapi mencapai sepenuhnya dalam Perjanjian Baru Roh Kudus menjelma dalam diri Yesus Kristus. Ini berarti Yesus Kristus adalah penjelmaan dari Roh Kudus. Kebohongan ajaran Paulus di atas bertentangan dengan tugas kerasulan Yesus yang tertera dalam Injil. Ini menunjukkan bahwa doktrin Yesus anak Tuhan hanyalah karangan Paulus, dan doktrin Trinitas hanyalah ciptaan gereja.13 13 Baandingkan, A. G. Honig, J. R., Ilmu Agama, di Indonesiakan oleh Soesastro dan Soegiarto, Gunung Mulia, Jakarta, 1992, hlm. 252. 14 https://id.wikipedia.org/wiki/Dasar-dasar_iman_Yahudi 3. Mempertahankan Doktrin Trinitas dalam Konteks Asia 15 Bandingkan Ibid, h. 115 d. Yudaisme Kepercayaan Judaisme secara ketat didasarkan pada Unitarian Monoteisme. Doktrin ini mengekspresikan kepercayaan kepada satu Tuhan. Konsep Tuhan yang mengambil beberapa bentuk (misalnya Trinitas) dianggap bidaah dalam Judaisme. Dalam doa secara utuh dalam hal mendefinisikan Tuhan adalah Shema Yisrael, awalnya 141 muncul di dalam Alkitab Ibrani: "Dengarkan O Israel, Tuhan adalah Allah kita, Tuhan adalah satu", juga diterjemahkan sebagai "Dengarkan O Israel, Tuhan kami adalah Allah, Tuhan adalah yang tunggal ". Allah adalah disusun sebagai zat yang kekal, pencipta alam semesta, dan sumber moralitas. Allah mempunyai kuasa untuk campur tangan di dunia. Istilah Allah sehingga terkait dengan kenyataan sebenarnya, dan bukan hanya proyeksi dari jiwa manusia. Allah dijelaskan dalam pengertian seperti: "Ada satu Zat, sempurna dalam segala cara, yang merupakan penyebab utama dari semua keberadaan. Semua tergantung pada keberadaan Allah dan semua berasal dari Allah. " Dalam Alkitab Bahasa Ibrani klasik yang berhubungan dengan sastra Talmud menegaskan theisme dan menolak deism. Namun, dalam tulisan-tulisan dari abad filosof Yahudi kemudian terlihat adanya pengaruh dari neo-filosofi Aristotel, diistilahkan kemahatahuan yang terbatas.14 Titik perbedaan berikut terletak pada Alkitab yakni Keristenan menerima Perjanjian Lama dan Perjanjian Baru adalah Firman Allah, sedangkan bagi umat Yahudi Perjanjian Baru bukanlah Firman Allah. Sehingga beberapa uraian pemahaman menjadi berbeda secara khusus berkaitan tentang Trinitas. Yudaisme tidak menerima Yesus sebagai salah satu pribadi dalam Allah Trinitas. Bagi Yudaisme, Mesias dipahami sebagai Raja keturunan Daud yang sah yang akan memulihkan kedaulatan Yahudi dan membebaskan rakyat dari dominasi asing. Karena Yesus 142 tidak dapat mencapai tujuan politik yang dapat dilihat (visible), Yudaisme masih terus mengharapkan kedatangan Mesias. Yudaisme tidak akan pernah menganggap Yesus sebagai Allah atau sebagai inkarnasi Allah. Dari sudut pandang Yahudi, sangat peka terhadap pengilahian atas objek materil apa pun. Yudaisme juga mengajarkan tentang Allah yang mempunyai dua atribut – adil dan berbelas kasih. Kadang Ia bertindak penuh belas kasih dengan mengampuni pelanggaran manusia dan menahan hukuman yang seharusnya dijatuhkannya. Tidak aturan yang jelas menentukan kapan belas kasihan menggantikan keadilan..15 a. Asal mula Meskipun pengajaran ini tidak 'berkibar' dalam PL, Trinitas itu sudah tersirat dalam penyataan diri Allah sejak semula. Ajaran ini tersirat bukan hanya dalam bagian- bagian tersendiri, tapi terajut di sepanjang bentangan penyataan dalam PL. Siratan paling tua ialah yang terdapat dalam riwayat penciptaan, di mana Trinitas terungkap di sana (Kej 1:3). Jadi dari sejak pada mulanya, Trinitas sudah dinyatakan suatu pusat kegiatan dari tiga yang satu seutuhnya. Kej 1:26 pernah dianggap menyatakan secara tidak langsung, bahwa penyataan Trinitas telah diberikan kepada manusia saat penciptaannya. Setiap ayat PL yang mengandung ungkapan ini merujuk kepada diri Allah, 2 143 Sam 24:16; 1 Raj 19:5; 2 Raj 19:35, Kej 16:7; 24:7 dan 48:16. Roh Allah juga diberi tempat khas dalam sejarah penyataan dan penebusan. Roh memperlengkapi Mesias untuk pekerjaan-Nya (Yes 11:2; 42:1; 61:1) dan memperlengkapi umat-Nya untuk menanggapi Mesias dengan iman dan ketaatan (Yl 2:28; Yes 32:15; Yeh 36:26- 27). Ucapan Berkat Imam (Bil 6:24) juga dapat disimak mungkin sebagai bentuk pertama dari berkat dalam 2 Kor 13:14. Sebelum Kristus datang, Roh Kudus datang memasuki hati orang-orang yang takut akan Allah, dengan cara yang belum pernah dikenal sejak akhir pelayanan nabi Maleakhi. Yohanes Pembaptis -- secara khusus dan khas -- menyadari kehadiran dan panggilan Roh Kudus. Pemberitaannya menggambarkan Trinitas, yang tersirat dalam hal ia memanggil orang supaya bertobat kepada Allah, supaya percaya kepada Mesias yang sedang datang, dan tuturannya tentang baptisan oleh Roh Kudus. Pada peristiwa lain kepada Maria dinyatakan bahwa Roh Kudus akan mengambil bagian dalam penjelmaan anak-Nya, Yesus (Luk 1:35), pada baptisan Yesus di sungai Yordan ketiga Oknum itu dapat dibedakan: Anak, Bapa dan Roh. Pengertian para rasul tentang Roh Kudus dan hubungan- Nya dengan Allah Bapa dan Allah Anak disajikan jelas dalam peristiwa Pentakosta, menggambarkannya sebagai pekerjaan Trinitas (Kis 2:32-33). Tepat jika dikatakan bahwa gereja zaman rasul dibangun beralaskan kepercayaan kepada Allah Bapa, Anak dan Roh Kudus. 'Kasih karunia Tuhan Yesus Kristus, dan kasih Allah, dan 144 persekutuan Roh Kudus...' (2 Kor 13:13) tidak hanya menyimpulkan seluruh ajaran para rasul, tapi juga menerangkan makna yang lebih dalam dan hakiki dari Trinitas. Argumentasi kedua yang digunakan untuk memojokkan konsep Trinitas adalah dengan mengatakan bahwa karena Agama Hindu dan agama Buddha lebih tua dari pada agama Kristen, dan ada persamaan antara trimurti, atau tridharma dan Trinitas, maka konsep agama Hindu atau Buddhalah yang benar. a. Asal mula Alasan ini sebenarnya tidaklah dapat dipertanggungjawabkan, dengan alasan:  Agama Kristen harus dilihat satu kesatuan dalam Perjanjian Lama dan Perjanjian Baru. Oleh karena itu, sebetulnya agama Kristen lebih tua dari agama Hindu. Dengan menggunakan analogi yang sama, yakni Kitab Suci agama Kristen lebih tua dari Kitab Suci agama Islam, dan keduanya menyebut tentang Yesus, maka dapat disimpulkan bahwa Yesus, seperti yang disebutkan dalam Kitab Suci agama Kristenlah yang benar dan agama Islam telah memodifikasi Yesus seperti yang dijelaskan di dalam Alkitab agama Kristen.  Agama Kristen harus dilihat satu kesatuan dalam Perjanjian Lama dan Perjanjian Baru. Oleh karena itu, sebetulnya agama Kristen lebih tua dari agama Hindu. Dengan menggunakan analogi yang sama, yakni Kitab Suci agama Kristen lebih tua dari Kitab Suci agama Islam, dan keduanya menyebut tentang Yesus, maka dapat disimpulkan bahwa Yesus, seperti yang disebutkan dalam Kitab Suci agama Kristenlah yang benar dan agama Islam telah memodifikasi Yesus seperti yang dijelaskan di dalam Alkitab agama Kristen.  Dikatakan oleh penganut Hindu bahwa Alkitab seharusnya ditulis dalam bahasa Ibrani dan bukan Yunani. Mungkin yang dimaksud adalah Alkitab Perjanjian Baru, karena Perjanjian Lama ditulis dalam bahasa Ibrani, dan kita tidak perlu mendiskusikan hal ini. Perjanjian Baru (kecuali Matius) memang ditulis dalam bahasa Yunani, karena memang pada waktu itu, bahasa yang dipakai adalah Aramaic dan Koine Greek  Dikatakan oleh penganut Hindu bahwa Alkitab seharusnya ditulis dalam bahasa Ibrani dan bukan Yunani. Mungkin yang dimaksud adalah Alkitab Perjanjian Baru, karena Perjanjian Lama ditulis dalam bahasa Ibrani, dan kita tidak perlu mendiskusikan hal ini. Perjanjian Baru (kecuali Matius) memang ditulis dalam bahasa Yunani, karena memang pada waktu itu, bahasa yang dipakai adalah Aramaic dan Koine Greek 145 (Yunani). Dan bahasa Yunani ini adalah bahasa sehari- hari yang dipakai pada abad-abad awal di seluruh provinsi Roma di mediterania Timur. Dengan demikian kalau Perjanjian Baru ditulis dalam bahasa Yunani tidaklah aneh, karena ditulis dalam bahasa yang dipakai pada waktu itu. Kalau Perjanjian Baru ditulis dalam bahasa Sangsekerta barulah aneh. Selanjutnya, Kekristenan telah dipersiapkan dari Perjanjian Lama, maka sebenarnya zaman Abraham lebih tua dari Hinduism dan Budhism, karena Abraham adalah 10 generasi setelah nabi Nuh dan 20 generasi setelah Adam, dan diperkirakan sekitar abad 19 SM. Oleh karena itu, argumentasi yang mengatakan bahwa kebudayaan Hindu mempengaruhi kebudayaan Yunani dan kemudian kebudayaan Yunani mempengaruhi Kekristenan, tidak dapat dibuktikan. Termasuk di dalamnya pemahaman Budhism yang menyampaikan bahwa Trinitas terpengaruh Budhism melalui keberadaan Rasul Paulus tidak dapat dibuktikan. a. Asal mula Dari fakta sejarah pun, tidaklah terbukti pengaruh Hinduism dan Budhism terhadap Yunani dan tidak ada pengaruh Yunani terhadap agama Yahudi, dan apalagi mempengaruhi Kekristenan. Mungkin yang dapat didiskusikan adalah bagaimana filosofi Yunani membantu untuk menerangkan misteri iman Kristen. Namun, agama Kristen tidaklah mendasarkan kebenarannya pada filosofi Yunani, sebaliknya filosofi melayani Teologi. Agama Kristen bukanlah agama berdasarkan akal budi, namun berdasarkan wahyu Allah 146 (yang juga tidak bertentangan dengan akal budi yang benar), yang dilakukan secara terus-menerus melalui para nabi di Perjanjian Lama, dan mendapatkan kepenuhannya dalam diri Yesus, Allah yang menjelma menjadi manusia.  Titik perbedaan berikut terletak pada Alkitab yakni Keristenan menerima Perjanjian Lama dan Perjanjian Baru adalah Firman Allah, sedangkan bagi umat Yahudi Perjanjian Baru bukanlah Firman Allah. Sehingga beberapa uraian pemahaman menjadi berbeda secara khusus berkaitan tentang Trinitas. Yudaisme tidak menerima Yesus sebagai salah satu pribadi dalam Allah Trinitas. Bagi Yudaisme, Mesias dipahami sebagai Raja keturunan Daud yang sah yang akan memulihkan kedaulatan Yahudi dan membebaskan rakyat dari dominasi asing. Karena Yesus tidak dapat mencapai tujuan politik yang dapat dilihat (visible), Yudaisme masih terus mengharapkan kedatangan Mesias. Yudaisme tidak akan pernah menganggap Yesus sebagai Allah atau sebagai inkarnasi Allah. Dari sudut pandang Yahudi, sangat peka terhadap pengilahian atas objek materil apa pun. b. Perumusan Trinitas Dalam pengajaran Islam dan Yudaisme, konsep Trinitas sama sekali mendapatkan penolakan, namun bagi penganut Hindu dan Buddha terdapat asumsi kesamaan antara Trinitas, Trimurti dan Tridharma. Apakah ada kesamaan perumusan di antara ketiganya? Tentu saja 147 berbeda pengertiannya. Perbedaaan antara Trimurti, Tridharma dengan Trinitas : Secara prinsip Hinduisme dan Buddhism sebenarnya lebih mirip dengan ajaran Pantheism, di mana mempercayai bahwa semua ciptaan adalah mempunyai percikan Ilahi. Kita juga melihat bahwa ada begitu banyak dewa-dewi lain yang disembah selain trimurti dan tridharma di dalam Hinduisme dan Budhisme. Di satu sisi, agama Kristen tidak menganggap manusia sebagai percikan Ilahi, namun berpartisipasi dalam Allah. Dan tidak ada dewa-dewi yang disembah, kecuali Allah Trinitas. Dan dari prinsip dasar ini sebenarnya telah membuktikan bahwa tidak ada kemiripan antara Hinduisme, Budhisme dan Kekristenan. Perbedaan yang menyolok, khususnya tentang trimurti adalah dikatakan bahwa Brahma adalah pencipta, Wisnu pemelihara, dan Shiwa perusak. Bandingkan dengan konsep Trinitas, di mana Allah Bapa adalah pencipta, Allah Putera adalah penebus, dan Allah Roh Kudus adalah pengudusan. Appropriation (pembedaan) ini dibuat untuk menangkap ketiga pribadi dari Trinitas, namun penciptaan, penebusan dan pengudusan dilakukan bersama-sama oleh ketiga pribadi Trinitas. Lebih lanjut di dalam Trinitas tidak ada elemen Shiwa atau perusak, namun sebaliknya pengudusan, yaitu Roh Kudus. Trinitas atau satu Allah dalam tiga pribadi adalah merupakan wahyu Allah, yang gambarannya dapat dilihat di dalam Perjanjian Lama, dan mencapai puncaknya dengan Inkarnasi, di mana pribadi ke dua (Allah Putera) masuk ke dalam sejarah manusia, yaitu Yesus Kristus. Dan kedatangan-Nya, keberadaan-Nya dapat dilihat dalam 148 sejarah manusia, dan dicatat dalam sejarah manusia, seperti oleh Yosefus, sejarahwan berkebangsaan Yahudi. Umat Kristen mengerti akan kesetaraan antara Allah Bapa, Allah Putera dan Allah Roh Kudus, karena ketiga-Nya adalah satu Allah. Oleh karena itu, penyembahan kepada semua Pribadi atau masing-masing Pribadi adalah merupakan penyembahan kepada setiap Pribadi. Dan hal ini dilakukan secara konsisten sepanjang sejarah Kekristenan. Bandingkan dengan pemujaan trimurti. Kita dapat menemukan begitu banyak kuil pemujaan terhadap Wisnu dan Shiwa (mungkin ada sekitar 10,000 kuil pemujaan di India), namun jarang sekali kita menemukan kuil pemujaan terhadap Brahma di India. 16 Bandingkan Ibid, h. 115 c. Implikasi Teologis Implikasi-implikasi doktrin Tritunggal sangat vital bagi teologi dan juga bagi pengalaman dan hidup Kristen. Berkaitan dengan ke-Allah-an, doktrin ini menyatakan bahwa Allah benar-benar HIDUP. Dan bahwa Allah jauh sama sekali dari apa pun yg disebut berhenti atau pasif. Allah Tritunggal adalah keutuhan dan kepenuhan hidup, berada dalam hubungan yg kekal, dan dalam persekutuan yg tak pernah putus atau berhenti. Hal ini membuat penyataan dan pengungkapan diri Allah dapat dimengerti. Allah, dalam arti mutlak, dapat mengungkapkan diri-Nya sendiri melalui tindakan penyataan diri sendiri antara ketiga Oknum itu. Dia dapat juga dalam arti terbatas, mengungkapkan diri-Nya ke luar melalui penyataan diri sendiri berkomunikasi terhadap ciptaan-Nya. 149 Yudaisme juga mengajarkan tentang Allah yang mempunyai dua atribut – adil dan berbelas kasih. Sedangkan dalam Kekristenan belas kasihan dinyatakan dalam diri Yesus, pribadi kedua Trinitas.16 Sesungguhnya kesamaan pengajaran ini dapat dijadikan pintu masuk pemberitaan kabar baik kepada umat Yahudi, bahwa melalui pengorbanan Yesuslah, kasih dan keadilan Allah tergenapi bagi seluruh umat manusia. Jembatan penginjilan ini dapat dilakukan dengan mencari kesamaan di antara Kekristenan, Islam, Hindu, Buddha dan Yudaisme. Namun tentu saja dengan pendekatan yang hati-hati agar tidak jatuh kepada penafsiran yang salah sehingga dapat mengakibatkan Kekristenan yang sinkretis. Bagian terakhir dari implikasi teologis adalah persekutuan yang mengikat Trinitas, menjadi dasar bagi persekutuan lingkungan umat manusia, lingkungan keluarga, lingkungan masyarakat, dan secara istimewa dalam lingkungan gereja, karena di situ Roh Kudus menjadi Petugas dan Pengantara persekutuan itu. Sebagaimana tertera dalam Yohanes 17:22, “Dan Aku telah memberikan kepada mereka kemuliaan, yang Engkau berikan kepada- Ku, supaya mereka menjadi satu, sama seperti Kita adalah satu”. 150 Kepustakaan A. G. Honig, J. R., Ilmu Agama, di Indonesiakan oleh Soesastro dan Soegiarto, Gunung Mulia, Jakarta, 1992. Daud Ali al-Fadhili, Ushulul Masihiyyah Kama Yushowwiruhal Qur’an, terbitan Dar Zahron, tahun 2008. Dede Wijaya, “Allah Tritunggal.”www.in-christ.net. pp. 3-4. Online. Internet. Accesed August 24,2008. Erick Sudharma, Mengenal Satu-Satunya Allah Yang Benar, Bandung: Mitra Pustaka, 2003 Ezra Alfred Soru, Tritunggal yang Kudus, Bandung: Lembaga Literatur Baptis, 2002. 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Ustadz Wahid Hasyim Asyrofi, Lc Muhammad Abduh Tuasikal Artikel Muslim.Or.Id Yusuf Syalabi, Adhwa’ alal Masihiyyah, terbitan Dar Al- Kuwaitiyyah, cetakan ketiga tahun 1973. Timothy Ware, “Allah Tritunggal Maha Kudus.” www.sarapanpagi.org no.pp.4. Online. Internet.Accesed August 24, 2008. Timothy Ware, “Allah Tritunggal Maha Kudus.” www.sarapanpagi.org no.pp.4. Online. Internet.Accesed August 24, 2008. Titib, I Made, 2003, Tri Sandhya, Sembahyang dan Berdoa Penerbit Paramita, Surabaya 1996. Ustadz Wahid Hasyim Asyrofi, Lc Muhammad Abduh Tuasikal Artikel Muslim.Or.Id Yusuf Syalabi, Adhwa’ alal Masihiyyah, terbitan Dar Al- Kuwaitiyyah, cetakan ketiga tahun 1973. Titib, I Made, 2003, Tri Sandhya, Sembahyang dan Berdoa Penerbit Paramita, Surabaya 1996. Ustadz Wahid Hasyim Asyrofi, Lc Muhammad Abduh Tuasikal Artikel Muslim.Or.Id Yusuf Syalabi, Adhwa’ alal Masihiyyah, terbitan Dar Al- Kuwaitiyyah, cetakan ketiga tahun 1973.